A cell-specific transgenic approach in Xenopus intermediate pituitary:

the role of p24 in the early secretory pathway

 

 

 

 

A cell-specific transgenic approach in Xenopus intermediate pituitary: the role of p24 in the early secretory pathway

 

 

Een wetenschappelijke proeve op het gebied van de Natuurwetenschappen, Wiskunde en Informatica

 

 

Proefschrift

Ter verkrijging van de graad van doctor aan de Katholieke Universiteit Nijmegen, op gezag van de Rector Magnificus prof. dr. C.W.P.M. Blom, volgens besluit van het College van Decanen in het openbaar te verdedigen op vrijdag 2 april 2004 des namiddags om 13.30 uur precies

door

 

 

Gerrit Bouw

geboren op 10 april 1973 te Dordrecht

 

 

Promotor

Prof. Dr. G.J.M. Martens

 

Manuscriptcommissie

Prof. Dr. I.L.C. Braakman

Dr. F.J.M. van Kuppeveld

 

 

 

“Shoot for the moon. Even if you miss it you will land among the stars…..”

Les Brown, renowned public speaker

 

 

Contents

 

Chapter 1 9

General Introduction

 

Chapter 2 30

Cell-type specific and selectively induced expression of members

of the p24 family of putative cargo receptors

 

Chapter 3 52

The localization of p24 putative cargo receptors in the early secretory

pathway depends on the biosynthetic activity of the cell

 

Chapter 4 75

Cell-specific Xp24δ2 transgene expression in the intermediate pituitary

melanotrope cells of Xenopus laevis

 

Chapter 5 95

A cell-specific transgenic approach in Xenopus reveals the importance

of a functional p24 system for a secretory cell

 

Chapter 6 122

Differential effect of transgenic overexpression of two closely related

Xp24δ subfamily members on the endogenous p24 proteins and prohormone processing in the Xenopus intermediate pituitary cell

 

Chapter 7 141

General Discussion

Summary / Samenvatting 160

 

 

Chapter 1: General introduction

 

GENERAL INTRODUCTION

In order to function properly, most eukaryotic cells are equipped with a secretory system that delivers newly synthesized proteins to the plasma membrane. Upon secretion, the proteins can then function in cell-cell communication and as extracellular stimuli to direct target cells to carry out their specialized function. In turn, these target cells use the secretory pathway to expose transmembrane receptor proteins at the cell surface, which can bind extracellular ligands leading to an intracellular response. The secretory pathway consists of a number of distinct membrane-bounded compartments, which have specialized functions in the process of protein biosynthesis (Palade, 1975). One of the key steps in the secretory process is packaging of cargo from the endoplasmic reticulum (ER) into vesicles destined for the subsequent secretory compartments such as the Golgi apparatus. This step involves numerous proteins, including chaperones and coat proteins, but the actual recruitment of cargo into secretory vesicles is thought to be mediated by the p24 family of putative cargo receptors. This family of type I transmembrane proteins may play an important role in the first steps of the biosynthetic process and is the main focus of this thesis. In this introductory chapter, a number of aspects relevant to the studies described (e.g. components of the secretory pathway, the p24 family, the intermediate pituitary cells of the amphibian model system Xenopus laevis and the recently developed technique of Xenopus transgenesis) will be dealt with.

 

The secretory pathway

Most newly synthesized secretory proteins, en route to the plasma membrane in order to undergo secretion, enter the secretory pathway by virtue of the translocation machinery in the ER (reviewed by Rapoport et al., 1996). In the ER, many of the newly synthesized proteins are modified in such a way that they become properly folded and subsequently directed to specialized regions of the ER, also called ER exit sites (Bannykh and Balch, 1997; Bannykh et al., 1996). Here, the cargo proteins are packaged into transport vesicles, which eventually fuse to form vesicular tubular clusters (VTCs; Balch et al., 1994; Tisdale et al., 1997), commonly known as the ER-Golgi intermediate compartment (ERGIC; Schweizer et al., 1990). Transport from the ER to the VTCs depends on the recruitment of cytoplasmic coat proteins, called coatomer protein (COP) II, to ER exit sites where they initiate vesicle budding (Barlowe, 1998; Barlowe et al., 1994; Kuehn et al., 1998). Once the budding event is completed, COP II proteins dissociate and transport vesicles fuse with or together form the VTCs, which then travel to the Golgi apparatus along microtubules (Presley et al., 1998; Saraste and Svensson, 1991; Scales et al., 1997). The Golgi apparatus consists of a series of polarized stacks of compacted cisternae. The cis-Golgi stacks face the nucleus whereas the trans-side of the Golgi is oriented towards the plasma membrane. Bi-directional membrane traffic in both the secretory pathway and the endocytic pathway depends on the Golgi apparatus (Rothman and Wieland, 1996). While travelling through the Golgi, cargo proteins often undergo various covalent modifications (e.g. O-linked glycosylation and sulfation; Huttner, 1988; Kornfeld and Kornfeld, 1985; Mellman and Simons, 1992; Rottger et al., 1998), finally reaching the trans-Golgi Network (TGN). In this compartment, secretory proteins are sorted and distributed to various post-Golgi destinations (Griffiths and Simons, 1986). After sorting into vesicles, secretory proteins can be targeted to the plasma membrane in two ways: some vesicles move in a constitutive manner, delivering their cargo in an ongoing fashion, independent of extracellular stimuli. This is called the constitutive secretory pathway. In highly specialized secretory cells (e.g. endocrine, neuronal and exocrine cells) a second secretory pathway (the regulated secretory pathway) exists, in which secretory vesicles are stored as secretory granules, releasing their contents only upon an extracellular signal (Kelly, 1985). The secretory pathway consists of a number of separate compartments with unique functions. This compartmentalization provides the cell with environments in which several steps in the process of protein biosynthesis and processing can be tightly regulated. To maintain these microenvironments, the cell has to firmly control incoming and outgoing transport vesicles (reviewed by Mellman and Warren, 2000) (Fig. 1). The process of cargo transport and release starts in the ER with the molecular sorting of the right cargo into the right transport vesicles. The actual inclusion of cargo proteins into COP II-coated vesicles is thought to be mediated by transmembrane proteins that interact with cargo in the ER lumen and with coat proteins at the cytoplasmic site. These transmembrane proteins include the glycoprotein cargo receptor ERGIC-53 (Appenzeller et al., 1999), the Erv41p-Erv46p complex (Otte et al., 2001) and a family of ~24-kDa putative cargo receptors, the p24 proteins (Muñiz et al., 2000; Schimmöller et al., 1995). COP II-dependent export of these proteins from the ER is regulated by distinct and well-characterized ER-exit signals in the transmembrane proteins (reviewed by Barlowe, 2003). The cargo receptors are proposed to recognize and bind specific cargo molecules in the ER, which are released in post-ER compartments.

Figure 1: Schematic representation of the secretory pathway. After synthesis in the ER, cargo molecules are packaged into COP II-coated transport vesicles at ER exit sites. These vesicles then fuse to form the ER-Golgi intermediate compartment (ERGIC) that are transported to the Golgi. COP I-coated vesicles recycle components of the vesicular machinery of ER resident proteins back to the ER. As the Golgi stacks mature, resident Golgi proteins are shuttled back to earlier stacks. Finally, cargo vesicles originating from the trans-Golgi network (TGN) fuse with the plasma membrane to release their contents into the extracellular environment.

The p24 family of putative cargo receptors

In the mid-90s, in studying the role of COP I using in vitro budding assays, the group of Jim Rothman discovered a highly abundant membrane protein that was isolated from reconstituted transport vesicles (Stamnes et al., 1995). This protein appeared to belong to a family of structurally related 24-kDa type I transmembrane proteins that was termed the p24 family. According to their amino acid sequences the p24 proteins could be classified into four main subfamilies, named p24α, -β, -γ and -δ (Dominguez et al., 1998). Among subfamily members there is only a low degree of amino acid sequence homology, but all p24 proteins share structural characteristics such as a large lumenal domain with two conserved cysteine residues that form a disulfide bridge, a transmembrane domain and a short cytosolic tail that contains coat protein binding motifs (Fig. 2). Studies in cell lines have revealed that p24 proteins can bind to both COP II and COP I coat proteins, which allows them to shuttle from the ER to the Golgi and back (Dominguez et al., 1998; Fiedler et al., 1996; Nickel et al., 1997). Removal of the COP I binding motif resulted in a cell surface staining of p24 proteins, whereas mutation of the COP II binding motif showed retention of p24 proteins in the ER (Dominguez et al., 1998). Experimental data indicate that p24 proteins can form functional heterotetrameric complexes, containing one representative of each subfamily (Belden and Barlowe, 1996; Füllekrug et al., 1999; Marzioch et al., 1999), and that complex formation is necessary for proper trafficking of p24 proteins (Emery et al., 2000; Füllekrug et al., 1999). Mutational analysis of a small part of the lumenal domain of p24β1 (=Emp24p) in yeast has revealed a stretch of amino acids close to the transmembrane domain that is a putative coiled-coil domain and is involved in the assembly of this protein in to a p24 protein complex (Ciufo and Boyd, 2000). Further evidence for functional complex formation was provided by the fact that inactivation of one p24δ1 allele in mice led not only to reduced levels of p24δ1 itself but also to reduced levels of other family members, which indicates that p24 proteins oligomerize into heteromeric complexes (Denzel et al., 2000). However, contradictory results indicate that individual p24 members exist predominantly as monomers and dimers, depending on the subcellular localization of the p24 protein involved, and that p24 proteins can cycle differentially (Jenne et al., 2002). Recently, it was shown that certain p24 family members are able to bind to the Golgi matrix proteins GRASP55 and GRASP65. Binding could only occur when the p24 proteins were assembled into a complex; monomeric p24 tails could not bind the matrix proteins, suggesting functional p24 complex formation (Barr et al., 2001).

 

Figure 2: Schematic representation of a p24 protein. (A) N-terminal domain, possibly involved in cargo binding and also called GOLgi Dynamics (GOLD) domain. Two conserved cysteine residues form a putative loop structure. (B) Coiled-coil domain involved in functional multimerization of p24 proteins. (C) Double phenylalanine (FF) motif that is used for COP II binding. (D) Double lysine motif (KK) involved in COP I binding.

A detailed computational sequence analysis has revealed that p24 proteins exhibit a lumenal globular domain that is also present in other Golgi and lipid-trafficking proteins (Golgi Dynamics (GOLD) domain; (Anantharaman and Aravind, 2002) (Fig. 2). The role of this domain, if any, is at present not clear. Besides descriptive data, functional approaches have been used to reveal the role of the p24 family. For instance, the effect of yeast p24 knockouts on protein transport has been studied. Single deletions of four p24 members in S. cerevisiae resulted in defects of the secretion of Gas1p and caused secretion of the ER-resident protein BiP. Knocking out additional p24 members or all members of a p24 complex did not have an additional effect. The yeast cells were viable, showed no growing defects and transport mediated by COP I- and COP II-coated vesicles was normal (Marzioch et al., 1999; Springer et al., 2000). In contrast to this, targeted disruption of both p24δ1 alleles in knockout mice resulted in early embryonic lethality (Denzel et al., 2000). The yeast work has been extended by the group of Barlowe, who showed that, besides the accumulation of a subset of secretory proteins (Gas1p, invertase) and the secretion of BiP, deletion of yeast p24 genes activates the unfolded protein response (Belden and Barlowe, 2001). This response involves an upregulation of ER chaperones and attenuation of protein biosynthesis due to ER stress caused by unfolded protein accumulation (reviewed by Ma and Hendershot, 2001). Most functional approaches involve in vitro reconstitution of vesicle budding. Such studies have shown that p24 proteins are responsible for de novo formation of cargo exit sites, since antibodies against the C-tail of p24α2 reduced exit site formation (Lavoie et al., 1999). Others state that p24 proteins are involved in vesicle budding events due to the bimodal interaction of coatomer with the GTPase ADP-ribosylation factor (ARF) and with p24δ1 (=p23) tails (Bremser et al., 1999). Studies closer to the in vivo situation have been performed in cell lines in which overexpression of p24δ1 resulted in the accumulation of p24δ1 in smooth ER membranes and a relocalization of endogenous p24δ1 to these structures. However, anterograde and retrograde transport of vesicular stomatitis virus glycoprotein (VSV-G) and Vero toxin B was not affected, indicating that p24δ1 is not necessary for proper transport of cargo proteins (Rojo et al., 2000). A mutation in p24β1 (=SEL-9) in C. elegans resulted in the trafficking of a mutant protein to the plasma membrane that would otherwise accumulate within the cell. This suggests a role for p24 proteins in quality control in the ER (Wen and Greenwald, 1999). Using the restriction mediated integration (REMI) method on P. Pallidum, the group of Tanaka found that deletion of the p24β1 (coding for LbrA) inhibited branching in the fruiting body of this animal. They suggest that p24 proteins play a role in morphogenesis and more particularly in the intracellular transport of proteins that are involved in this process. Distortion of transport would then result in impaired branching. Their next step will be to search for cargo proteins that are selectively packages in vesicles containing p24β1 (=Emp24p) (Kawabe et al., 1999). Finally, the group of Riezman was able to directly cross-link p24β1 (=Emp24p) and p24δ1 (=Erv25p) to the cargo protein Gas1p in ER-derived vesicles in yeast cells. These results suggest that p24 proteins act as cargo receptors during vesicle biogenesis from the ER (Muñiz et al., 2000). Altogether, the results presented so far on the role of p24 proteins in the early secretory pathway have not been satisfactory and provide no conclusive answers. In order to tackle this difficult cell biological problem, the choice for an in vivo approach is obvious.

 

The intermediate pituitary melanotrope cells of Xenopus laevis as a model system to study protein transport

The intermediate pituitary melanotrope cells of the amphibian Xenopus laevis provide an ideal model system to study protein transport in vivo. Xenopus is capable of adapting its skin colour to the background, which is caused by the dispersion of pigment in skin melanophores. Pigment dispersion is regulated by the α-melanophore-stimulating hormone (α-MSH), which is a cleavage product of the prohormone proopiomelanocortin (POMC), a major biosynthetic product in the melanotrope cells of the intermediate pituitary of black-background-adapted Xenopus laevis (Jenks et al., 1977). In the active melanotrope cell, POMC constitutes about 80% of the newly synthesize. Upon black-background adaptation, the melanotrope cell is thus highly activated, which is reflected by an increased cell size and a highly developed biosynthetic machinery (reviewed by Roubos, 1997). The sole function of the melanotrope cell therefore appears to be the production and processing of vast amounts of POMC. This highly specialized task makes the melanotrope cell a powerful and easy to manipulate model system for studying cargo protein transport in vivo. The amount of cargo proteins that have to be transported through the secretory pathway can be dramatically changed by placing the animal on a black- or a white background, since in the melanotrope cells of a white-adapted animal, the amount of newly synthesized POMC is 20-30 fold lower than in those of black animals. In order to synthesize and process high amounts of POMC in a black-adapting animal, a number of other proteins need to be upregulated in a similar fashion as POMC. Using a differential screening approach on melanotrope cells of a black-adapted versus white-adapted animal, several types of proteins have been found to be coordinately expressed with POMC (Holthuis et al., 1995a). These proteins thus likely participate in a number of steps in the biosynthesis, processing and transport of POMC. Among the differentially expressed proteins were both secretory and transmembrane proteins that were upregulated at least ten times upon stimulation of the melanotrope cells. Interestingly, a member of the p24 family (X1262) was also found to be coordinately expressed with POMC (Holthuis et al., 1995b). X1262 is a member of the p24δ subfamily (named Xp24δ2) and has been further characterized in the melanotrope cell (Kuiper et al., 2000). From this study, it became clear that during black-background adaptation only this member of the p24δ family is upregulated (Xp24δ2), whereas the expression of the structurally highly related Xp24δ1 subfamily member was not induced. To examine the role of the p24 protein family, we chose to generate and analyze Xenopus transgenic for p24 proteins and mutants thereof. The use of a tissue-specific promoter allowed us to target expression of transgenes specifically to the melanotrope cell. The cellular functions and regulations (neuronal input) of the melanotrope cell have been studied in detail in the past (Roubos, 1997) and provides us with a well-defined platform to perform functional studies. One of the major advantages of the melanotrope cell for its use in our studies is that this cell type is dedicated to make vast amounts of one single cargo molecule, namely the prohormone POMC. We can thus study the function of p24 proteins in POMC cargo transport in detail, a role in which this protein family has been implicated before (Kuiper et al., 2001; Kuiper et al., 2000; Rötter et al., 2002).

 

Stable transgenesis of Xenopus laevis

The recently developed technique of Xenopus transgenesis provides a powerful tool to study the functional role of proteins close to the in vivo situation. Initially, Amaya and co-workers developed this technique (Kroll and Amaya, 1996). Later, the technique was simplified by the group of Mohun, who showed that without the use of interphase egg extract and restriction-mediated integration (REMI) of the transgene, the generation of transgenic Xenopus is a convenient system for studying protein function (Sparrow et al., 2000). Briefly, Xenopus sperm nuclei are mixed with linearized DNA encoding the transgene of interest and subsequently, the suspension is microinjected into unfertilized eggs (Fig. 3); details of this procedure are described in chapter 4. In our experiments, we used a Xenopus POMC gene A promoter fragment to drive transgene expression specifically to the melanotrope cells of the intermediate pituitary (Jansen et al., 2002) and the p24 protein fused to the jellyfish green fluorescent protein (GFP) as a tag for direct screening of transgenic animals. In this way, we combined stable Xenopus transgenesis with the unique properties of the Xenopus melanotrope cells to study the function of p24.

Figure 3: Flow diagram of the stable Xenopus transgenesis procedure and the generation of F1 transgenic animals. This scheme represents the modified transgenesis technique described by the group of Mohun (Sparrow et al., 2000). For the generation of F1 transgenic animals, we use either transgenic sperm and wild-type eggs or transgenic eggs in combination with wild-type sperm.

 

Aim and outline of this thesis

Many research groups have attempted to unravel the highly debated role of p24 proteins in the early secretory pathway. However, the results obtained so far have not provided conclusive answers. The aim of this thesis was to shed light on the p24 protein family in the highly specialized Xenopus intermediate pituitary melanotrope cell and to gain more insight into the role of p24 proteins in the transport of POMC cargo molecules in these cells. In chapter 2, we identified, in addition to Xp24δ1 and -δ2, all other p24 family members that are expressed in the Xenopus melanotrope cell. Expression studies revealed that, similar to Xp24δ2, Xp24α3, -β1 and -γ3 but not -γ2 and -δ1 were upregulated with POMC upon black-background adaptation. Chapter 3 describes the relationship between the biosynthetic activity of the melanotrope cell, and the subcellular localization and recruitment of p24 proteins. In chapter 4, we used the recently developed technique of stable Xenopus transgenesis to cell-specifically express transgenes in the Xenopus melanotrope cell and to study the in vivo role of p24 proteins in this highly specialized secretory cell. Chapter 5 reports the functional analysis of Xenopus transgenic for Xp24δ2-GFP. We found that distortion of the endogenous p24 system in the melanotrope cells by overexpressing Xp24δ2-GFP resulted in a decreased number and pigment content of skin melanophores, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect appeared to be linked to the observed affected profile of POMC-derived peptides produced in the transgenic melanotrope cells. Chapter 6 describes preliminary data concerning the functional analysis of transgenic Xenopus overexpressing Xp24δ1-GFP in the melanotrope cells. In contrast to the physiological effect of overexpressing Xp24δ2-GFP, these animals do not show an impaired adaptation system and synthesize a normal profile of POMC-derived peptides. Finally, in chapter 7 we discuss the results described in this thesis and propose a model for the function of p24 proteins in the early secretory pathway.

 

REFERENCES

Anantharaman, V. and Aravind, L. (2002). The GOLD domain, a novel protein module involved in Golgi function and secretion. Genome Biol 3, research0023.

Appenzeller, C., Andersson, H., Kappeler, F. and Hauri, H. P. (1999). The lectin ERGIC-53 is a cargo transport receptor for glycoproteins. Nat Cell Biolog 1, 330-334.

Balch, W. E., McCaffery, J. M., Plutner, H. and Farquhar, M. G. (1994). Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell 76, 841-852.

Bannykh, S. I. and Balch, W. E. (1997). Membrane dynamics at the endoplasmic reticulum-Golgi interface. Journal of Cell Biology 138, 1-4.

Bannykh, S. I., Rowe, T. and Balch, W. E. (1996). The organization of endoplasmic reticulum export complexes. J Cell Biol 135, 19-35.

Barlowe, C. (1998). COPII and selective export from the endoplasmic reticulum. Biochimica et Biophysica Acta 1404, 67-76.

Barlowe, C. (2003). Signals for COPII-dependent export from the ER: what's the ticket out? Trends Cell biol 13, 295-300.

Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M. and Schekman, R. (1994). COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895-907.

Barr, F. A., Preisinger, C., Kopajtich, R. and Korner, R. (2001). Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus. Journal of Cell Biology 155, 885-91.

Belden, W. J. and Barlowe, C. (1996). Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J Biol Chem 271, 26939-26946.

Belden, W. J. and Barlowe, C. (2001). Deletion of yeast p24 genes activates the unfolded protein response. Molecular Biology of the Cell 12, 957-69.

Bremser, M., Nickel, W., Schweikert, M., Ravazzola, M., Amherdt, M., Hughes, C. A., Sollner, T. H., Rothman, J. E. and Wieland, F. T. (1999). Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors. Cell 96, 495-506.

Ciufo, L. F. and Boyd, A. (2000). Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway. Journal of Biological Chemistry 275, 8382-8.

Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J. J., Solari, R. C. and Owen, M. J. (2000). The p24 family member p23 is required for early embryonic development. Current Biology 10, 55-8.

Dominguez, M., Dejgaard, K., Füllekrug, J., Dahan, S., Fazel, A., Paccaud, J. P., Thomas, D. Y., Bergeron, J. J. and Nilsson, T. (1998). gp25l/emp24/p24 protein family members of the cis-Golgi network bind both Cop I and II coatomer. Journal of Cell Biology 140, 751-65.

Emery, G., Rojo, M. and Gruenberg, J. (2000). Coupled transport of p24 family members. Journal of Cell Science 113 ( Pt 13), 2507-16.

Fiedler, K., Veit, M., Stamnes, M. A. and Rothman, J. E. (1996). Bimodal interaction of coatomer with the p24 family of putative cargo receptors. Science 273, 1396-1399.

Füllekrug, J., Suganuma, T., Tang, B. L., Hong, W., Storrie, B. and Nilsson, T. (1999). Localization and Recycling of gp27 (hp24gamma3): Complex Formation with Other p24 Family Members. Molecular Biology of the Cell 10, 1939-1955.

Griffiths, G. and Simons, K. (1986). The trans Golgi network: sorting at the exit site of the Golgi complex. Science 234, 438-443.

Holthuis, J. C., Jansen, E. J. R., van Riel, M. C. and Martens, G. J. M. (1995a). Molecular probing of the secretory pathway in peptide hormone-producing cells. Journal of Cell Science 108, 3295-305.

Holthuis, J. C., van Riel, M. C. and Martens, G. J. M. (1995b). Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary. Biochem J 312, 205-213.

Huttner, W. B. (1988). Tyrosine sulfation and the secretory pathway. Annual Review of Physiology 50, 363-76.

Jansen, E. J. R., Holling, T. M., van Herp, F. and Martens, G. J. M. (2002). Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis. FEBS Letters 516, 201-7.

Jenks, B. G., Overbeeke, A. P. and McStay, B. F. (1977). Synthesis, storage and release of MSH in the pars internmedia of the pituitary gland of Xenopus laevis during background adaptation. Canadian Journal of Zoology 55, 922-927.

Jenne, N., Frey, K., Brugger, B. and Wieland, F. T. (2002). Oligomeric state and stoichiometry of p24 proteins in the early secretory pathway. Journal of Biological Chemistry 277, 46504-11.

Kawabe, Y., Enomoto, T., Morio, T., Urushihara, H. and Tanaka, Y. (1999). LbrA, a protein predicted to have a role in vesicle trafficking, is necessary for normal morphogenesis in Polysphondylium pallidum. Gene 239, 75-9.

Kelly, R. B. (1985). Pathways of protein secretion in eukaryotes. Science 230, 25-32.

Kornfeld, R. and Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54, 631-64.

Kroll, K. L. and Amaya, E. (1996). Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173-83.

Kuehn, M. J., Herrmann, J. M. and Schekman, R. (1998). COPII-cargo interactions direct protein sorting into ER-derived transport vesicles. Nature 391, 187-90.

Kuiper, R. P., Bouw, G., Janssen, K. P., Rötter, J., van Herp, F. and Martens, G. J. M. (2001). Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell. Biochemical Journal 360, 421-9.

Kuiper, R. P., Waterham, H. R., Rötter, J., Bouw, G. and Martens, G. J. M. (2000). Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell. Molecular Biology of the Cell 11, 131-40.

Lavoie, C., Paiement, J., Dominguez, M., Roy, L., Dahan, S., Gushue, J. N. and Bergeron, J. J. (1999). Roles for alpha(2)p24 and COPI in Endoplasmic Reticulum Cargo Exit Site Formation. Journal of Cell Biology 146, 285-300.

Ma, Y. and Hendershot, L. M. (2001). The unfolding tale of the unfolded protein response. Cell 107, 827-30.

Marzioch, M., Henthorn, D. C., Herrmann, J. M., Wilson, R., Thomas, D. Y., Bergeron, J. J., Solari, R. C. and Rowley, A. (1999). Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex. Molecular Biology of the Cell 10, 1923-1938.

Mellman, I. and Simons, K. (1992). The Golgi complex: in vitro veritas? Cell 68, 829-840.

Mellman, I. and Warren, G. (2000). The road taken: past and future foundations of membrane traffic. Cell 100, 99-112.

Muñiz, M., Nuoffer, C., Hauri, H. P. and Riezman, H. (2000). The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles. Journal of Cell Biology 148, 925-30.

Nickel, W., Sohn, K., Bunning, C. and Wieland, F. T. (1997). p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway. Proc Natl Acad Sci U S A 94, 11393-8.

Otte, S., Belden, W. J., Heidtman, M., Liu, J., Jensen, O. N. and Barlowe, C. (2001). Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex. Journal of Cell Biology 152, 503-18.

Palade, G. (1975). Intracellular aspects of the process of protein synthesis. Science 189, 347-358.

Presley, J. F., Smith, C., Hirschberg, K., Miller, C., Cole, N. B., Zaal, K. J. M. and Lippincott Schwartz, J. (1998). Golgi membrane dynamics. Molecular Biology of the Cell 9, 1617-26.

Rapoport, T. A., Rolls, M. M. and Jungnickel, B. (1996). Approaching the mechanism of protein transport across the ER membrane. Current Opinion in Cell Biology 8, 499-504.

Rojo, M., Emery, G., Marjomaki, V., McDowall, A. W., Parton, R. G. and Gruenberg, J. (2000). The transmembrane protein p23 contributes to the organization of the Golgi apparatus. Journal of Cell Science 113, 1043-57.

Rothman, J. E. and Wieland, F. T. (1996). Protein sorting by transport vesicles. Science 272, 227-34.

Rötter, J., Kuiper, R. P., Bouw, G. and Martens, G. J. M. (2002). Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors. Journal of Cell Science 115, 1049-58.

Rottger, S., White, J., Wandall, H. H., Olivo, J. C., Stark, A., Bennett, E. P., Whitehouse, C., Berger, E. G., Clausen, H. and Nilsson, T. (1998). Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus. Journal of Cell Science 111 ( Pt 1), 45-60.

Roubos, E. W. (1997). Background adaptation by Xenopus laevis: a model for studying neuronal information processing in the pituitary pars intermedia. Comparative Biochemistry and Physiology. Part A, Physiology 118, 533-50.

Saraste, J. and Svensson, K. (1991). Distribution of the intermediate elements operating in ER to Golgi transport. Journal of Cell Science 100 ( Pt 3), 415-30.

Scales, S. J., Pepperkok, R. and Kreis, T. E. (1997). Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 90, 1137-48.

Schimmöller, F., Singer Krüger, B., Schröder, S., Krüger, U., Barlowe, C. and Riezman, H. (1995). The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO Journal 14, 1329-39.

Schweizer, A., Fransen, J. A., Matter, K., Kreis, T. E., Ginsel, L. and Hauri, H. P. (1990). Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. European Journal of Cell Biology 53, 185-96.

Sparrow, D. B., Latinkic, B. and Mohun, T. J. (2000). A simplified method of generating transgenic Xenopus. Nucleic Acids Research 28, E12.

Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S. and Schekman, R. (2000). The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 97, 4034-9.

Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P. and Rothman, J. E. (1995). An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding [published erratum appears in Proc Natl Acad Sci U S A 1995 Nov 7;92(23):10816]. Proc Natl Acad Sci U S A 92, 8011-5.

Tisdale, E. J., Plutner, H., Matteson, J. and Balch, W. E. (1997). p53/58 binds COPI and is required for selective transport through the early secretory pathway. Journal of Cell Biology 137, 581-93.

Wen, C. and Greenwald, I. (1999). p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans. Journal of Cell Biology 145, 1165-75.

 

 

 

Chapter 2: Cell-type specific and selectively induced expression of members of the p24 family of putative cargo receptors

Cell-type specific and selectively induced expression of members of the p24 family of putative cargo receptors

 

Jutta Rötter, Roland P. Kuiper, Gerrit Bouw, and Gerard J. M. Martens

Journal of Cell Science, 115(5): 1049-58 (2002)

 

 

ABSTRACT

Members of the p24 family of type I transmembrane proteins are highly abundant in transport vesicles and are thought to be involved in selective protein transport between the endoplasmic reticulum and the Golgi complex. The p24 proteins have been grouped into four subfamilies (α, β, γ, and δ) and appear to assemble into tetrameric complexes that contain only one representative from each subfamily. Here we molecularly dissected the p24 family in a single cell type, namely in the intermediate pituitary melanotrope cells of the amphibian Xenopus laevis. The biosynthetic activity of these cells for production of their major cargo protein proopiomelanocortin (POMC) can be physiologically manipulated via the process of background adaptation (~30-fold induction, with highly active cells in black toads and virtually inactive cells in white animals). Extensive cDNA library screening revealed the identity of six p24 proteins expressed in the Xenopus melanotrope cells, namely one member of the p24α (α3), one of the p24β (β1), two of the p24γ (γ2, γ3) and two of the p24δ (δ1, δ2) subfamily. Two other Xenopus p24 proteins, Xp24α2 and -γ1, were not expressed in the melanotrope cells, pointing to cell-type specific p24 expression. Of the six melanotrope p24 proteins, the expression of four (Xp24α3, -β1, -γ3 and -δ2) was 20- to 30-fold induced in active versus inactive melanotropes, whereas that of the other two members (Xp24γ2 and -δ1) had not or only slightly increased. The four proteins were induced only in the intermediate melanotrope cells and not in the anterior pituitary cells, and displayed similar overall tissue distributions that differed from those of Xp24γ1, -γ2 and -δ1. Together, our results reveal that p24 expression can be cell-type specific and selectively induced, and suggest that in Xenopus melanotrope cells an α3132 p24 complex is involved in POMC transport through the early stages of the secretory pathway.

 

 

INTRODUCTION

Proteins that are synthesized in the endoplasmic reticulum (ER) are subsequently transported along the secretory pathway by coated vesicular carriers. Thus far, two types of coats, termed COPI and COPII, have been identified which mediate transport between the ER and the Golgi complex. Cargo proteins exit the ER in COPII-coated vesicles that bud from specialized regions, called ER exit sites (Aridor et al., 1995). After budding, COPII vesicles quickly shed their coat and fuse to form vesicular-tubular clusters (VTCs; Balch et al., 1994; Scales et al., 1997), also referred to as ER-to-Golgi intermediate compartment ERGIC; (Schweizer et al., 1990). VTCs are transported as a whole along microtubules to the Golgi complex where they appear to fuse and form the cis-Golgi network (Saraste and Svensson, 1991). Retrograde transport of components of the vesicle targeting/fusion machinery as well as escaped ER-resident proteins back to the ER is mediated by COPI-coated vesicles (Aridor et al., 1995; Letourneur et al., 1994; Scales et al., 1997). Furthermore, the COPI coat may be involved in intra-Golgi vesicular transport (Nickel et al., 1998; Orci et al., 1997). Cargo proteins can leave the ER without prior concentration (Martinez-Menarguez et al., 1999; Warren and Mellman, 1999), but several studies have demonstrated that the cell has mechanisms for concentration of cargo in ER-derived vesicles and for accelerated transport out of the ER (Kuehn et al., 1998; Mizuno and Singer, 1993; Nishimura and Balch, 1997), suggesting a selective mechanism of cargo transport, presumably via cargo receptors. Thus far, three evolutionarily conserved families of integral membrane proteins have been proposed to facilitate ER-to-Golgi transport. Representatives of these protein families are fairly abundant and in the cytoplasmic tail of these proteins, binding sites for COPI and/or COPII coat subunits are found, which enable these proteins to cycle constantly within the early secretory pathway. The BAP family seems to regulate trafficking of certain membrane proteins out of the ER (Adachi et al., 1996; Kim et al., 1994; Terashima et al., 1994). BAP31, a representative of this family, has been shown to bind with high specificity to the endosomal membrane protein cellubrevin and to control its export out of the ER (Annaert et al., 1997). ERGIC-53/p58, a mannose-specific membrane lectin, belongs to another class of receptors involved in the transport of a number of glycoproteins from the ER to the ERGIC (Hauri et al., 2000). The third group of putative cargo receptors is a family of structurally related 24-kDa type I transmembrane proteins, collectively termed p24 proteins. Based on their amino acid sequences, these proteins have been classified into four main subfamilies, designated p24α, -β, -γ, and -δ (Dominguez et al., 1998). Members of the various p24 subfamilies exhibit only a low degree of amino acid sequence identity (17-30%) but all p24 proteins have certain structural characteristics in common, such as a relatively large lumenal domain with two conserved cysteine residues forming a disulfide bridge, a C-terminally located transmembrane stretch and a short cytoplasmic tail with sequence motifs known to specify interactions with vesicle coat proteins. Experimental evidence indicates that members of the various subfamilies can interact and tetrameric complexes are formed containing one representative of each subfamily (Belden and Barlowe, 1996; Füllekrug et al., 1999; Marzioch et al., 1999). Consistent with this view is that in yeast mutants and knockout mice deficient in the expression of a single p24 member, the stability of other family members is compromised (Denzel et al., 2000; Marzioch et al., 1999). A function for p24 proteins in cargo transport has been proposed on the basis of the observation that in yeast, deletion of certain p24 members slows ER export of a set of secretory proteins, whereas the export rate of a number of other cargo proteins is normal (Schimmöller et al., 1995). More recently, it was shown that two of these yeast p24 members, Emp24 (yp24β) or Erv25p (yp24δ), which coexist in a heteromeric complex, could be directly cross-linked to the lumenal cargo protein Gas1p in ER-derived vesicles. Efficient packaging of Gas1p was reduced when vesicles were generated from membranes lacking Emp24p activity (Muñiz et al., 2000). Furthermore, genetic experiments in yeast and Caenorhabditis elegans indicated that loss of p24 protein activity affects the fidelity of ER sorting (Elrod Erickson and Kaiser, 1996; Wen and Greenwald, 1999). Although in yeast p24 proteins are not essential for vesicular transport (Springer et al., 2000), deleting a single p24 member (p23) leads to early embryonic death in mice (Denzel et al., 2000).

In this study, we identified the members of the p24 family that are expressed in the intermediate pituitary of the South-African clawed toad Xenopus laevis. The intermediate pituitary consists of a homogenous population of melanotrope cells that are involved in the process of background adaptation of the animal. The central function of the melanotrope cells is the production of proopiomelanocortin (POMC) and in an active cell this prohormone constitutes over 80% of all newly synthesized proteins (Holthuis et al., 1995). The processing of POMC yields a number of bioactive peptides of which the α-melanophore stimulating hormone (α-MSH) stimulates the dispersion of the black pigment melanin in skin melanophores, causing darkening of the animal (Jenks et al., 1977). In the melanotrope cells, the expression levels of POMC can be manipulated in a physiological way simply by changing the background color of the animal. On a black background, the POMC gene is highly active, whereas on a white background the gene is virtually inactive. The high levels of POMC production in black-adapted animals cause an enormous increase in cargo transport in the melanotrope cells, reflected by an extremely well-developed biosynthetic and secretory pathway, and a melanotrope cell size about twice as large as that in white-adapted animals (reviewed in Roubos, 1997). One would thus expect that the p24 proteins, which have a presumed function in POMC transport, be coordinately expressed with this prohormone. We could demonstrate that the expression of a selective set of p24 proteins is induced in the melanotrope cells of black-adapted animals, whereas others are not or only slightly induced. The coordinate expression of Xp24α3, -β1, -γ3 and -δ2 with POMC suggests that these p24 proteins assemble into a tetrameric complex involved in the ER-to-Golgi transport of the prohormone.

 

 

MATERIALS AND METHODS

Animals

Adult South African clawed toads (Xenopus laevis) were obtained from laboratory stock and kept under constant illumination in water of 22°C. Animals were allowed to adapt to the background by placing them in either white or black tanks for at least 3 weeks.

 

Library screening and DNA sequencing

The nonredundant GenBank/EMBL/DDBJ database at NCBI was searched for p24 sequences from mouse with the BLAST program. Expressed sequence tags (ESTs) containing the entire ORFs of mouse p24α3, -γ1, -γ2, -γ3 and -γ4 were identified and received from the I.M.A.G.E. Consortium (Zehetner and Lehrach, 1994). PCR products, constituting parts of the ORFs, were generated using the mouse EST clones as templates. Degenerated oligonucleotides encoding conserved sequences in vertebrate p24β proteins were used to amplify a 313-bp fragment of Xp24β1 (nucleotides 286 - 599 in Xp24β1 ORF) from cDNA obtained through standard reverse-transcriptase reactions (Sambrook et al., 1989) on RNA isolated from Xenopus brain by the Trizol isolation method (Life Technologies-BRL). Fragments were gel purified, labelled with [32P]-dATP by random primer extension (Ausubel et al., 1989), and unincorporated nucleotides were removed using NucTrap Probe purification columns (Stratagene, Cedar Creek, TX, USA). The probes were used to screen an oligo dT-primed cDNA library of neurointermediate lobes of the pituitary gland of black-adapted X. laevis (Kuiper et al., 2000). In addition, ZapII-cDNA libraries made from whole Xenopus embryos (stage 42; Michael King, Indiana University, USA) or embryo heads (stage 28-30; Richard Harland, University of California, Berkeley, USA) were used. Plaques (density 400/cm2) were replicated on duplicate nylon membrane filters by standard procedures (Sambrook et al., 1989). Filters were prehybridized for at least one hour at 50°C in hybridization solution (10% dextrane sulfate, 1% SDS, 1 M NaCl, 0.1% sodiumpyrophosphate, 0.2% bovine serum albumin, 0.2% polyvinylpyrollidone K90, 0.2% Ficoll 400, 50 mM Tris pH 7.5) and hybridized under conditions of low stringency (at 50°C) with a labelled 524-bp probe for mp24α3 (nucleotides 150-674 in accession number AA109932), a 313-bp probe for Xp24β1 (nucleotides 286 - 599 in Xp24β1 ORF), a 537-bp probe for mp24γ1 (nucleotides 107-644 in accession number W08294), a 462-bp probe for mp24γ2 (nucleotides 248-710 in accession number W58982), a 301-bp probe for mp24γ3 (nucleotides 5-306 in accession number AA020489), and a 453-bp probe for mp24γ4 (nucleotides 274-727 in accession number AA060892). Filters were washed twice for 40 min at 50°C in 2×SSPE/0.1% SDS (where 1×SSPE is 0.15 M NaCl, 10 mM sodium phosphate, 1 mM EDTA; pH 7.4) and exposed to X-ray films between two intensifying screens at -70°C. Positive plaques were purified, in vivo excised and analyzed by DNA sequencing, using the ABI-PRISM DNA sequencing kit and the ABI-PRISM automatic sequencer (Perkin Elmer-Cetus Applied Biosystems, Foster City, CA). The cDNA libraries were screened under high-stringency hybridization conditions with the 3'-untranslated sequences of isolated cDNA clones encoding the various Xenopus p24 proteins. These sequences were amplified in a PCR reaction yielding for Xp24α3 a 0.43-kb fragment, for Xp24β1 ~1.8 kb, for Xp24γ2 ~0.6 kb and for Xp24γ3 ~1.7 kb. The high-stringency hybridizations were performed at 63°C and filters were washed to a final stringency of 0.1×SSPE/0.1% SDS at 63°C. Standard procedures, such as PCR and single clone in vivo excision of l-phage, were performed according to Ausubel et al. (Ausubel et al., 1989).

 

RNA isolation and Northern blot analysis

Total RNA was isolated with RNAgents isolation system according to the instructions of the manufacturer (Promega, Madison, WI, USA) and quantified by spectrophotometry. Aliquots of 5 µg per lane were separated by electrophoresis on 2.2 M formaldehyde-containing 1.2% agarose gels in MOPS buffer (Sambrook et al., 1989) and blotted onto nylon membranes using downward capillary transfer. Hybridizations were performed for 18 hours at 42°C in ULTRAhyb hybridization solution (Ambion, Austin, TX, USA) with probes comprising the entire ORFs of Xp24α3, -γ1, -γ2, and -γ3, a 364-bp fragment of Xp24β1 (nucleotides +77 to +364) or a 230-bp of XGAPDH (nucleotides +266 to +496), as a control for RNA loading and integrity. Blots were washed at 60°C to a final stringency of 0.1×SSPE/0.1% SDS (twice for 30 min) and exposures were taken using a PhosphorImager (BioRad Personal FX).

 

Antibodies

For antibody production, a region in the lumen of Xp24α3 (residues 31-128) was cloned into the expression vector pQE30 (Qiagen, Chatsworth, CA, USA), the recombinant protein was produced in E. coli and purified by Ni2+-NTA agarose affinity chromatography. The lumenal domains of Xp24γ1 (residues 18-194) and Xp24γ2 (residues 32-191) were cloned as GST fusion proteins into the bacterial expression vector pGEX-2T (Pharmacia Biotech Benelux, NL). The expressed GST fusions were largely insoluble. Therefore, the aggregated fusion proteins were isolated as inclusion bodies from E. coli (Nagai and Thogersen, 1987). A synthetic peptide against the cytoplasmic tail sequence of Xp24γ3 (C-FSDKRTTTTRVGS) was coupled to keyhole limpet hemocyanin (Pierce, Rockford, IL, USA). All antigens described were injected into rabbits and the immunization was done as described (Kuiper et al., 2000). Polyclonal antibodies against amino acid sequences in the lumenal part of Xp24δ1 (C-FDSKLPAGAGRVP; anti-δ1) and -δ2 (residues 72-150; anti-δ2), as well as the C-terminally directed p24δ antibody (anti-δC) have been described previously (Kuiper et al., 2000). The p24β1 and -γ3 peptide antibodies (anti-β1L and anti-γ3) recognize orthologs in human and Xenopus, and were kindly provided by Dr. T. Nilsson (EMBL, Heidelberg, Germany; (Dominguez et al., 1998). Affinity purifications of antisera using antigen-sepharose 4B columns were performed according to standard protocols (Harlow and Lane, 1988).

 

Immunological characterization

Extraction of proteins from different tissues of X. laevis, SDS-PAGE gel electrophoresis, Western blotting, antibody detection and immunocytochemistry were performed as described (Kuiper et al., 2000).

 

 

RESULTS

Cloning of cDNAs encoding the p24 proteins in the Xenopus intermediate pituitary

The melanotrope cells in the intermediate pituitary of the amphibian Xenopus laevis are primarily dedicated to the production of POMC and the expression levels of this prohomone can be readily manipulated by changing the background color of the animal. On a black background, the POMC gene is actively transcribed and the prohormone represents ~80% of all newly synthesized proteins, whereas on a white background the gene is nearly inactive. Therefore, the Xenopus melanotrope cell is an attractive model system to study the role of p24 proteins in POMC transport. The degree of amino acid sequence identity between vertebrate p24 orthologs is usually above 68%, allowing us to search for Xenopus p24 proteins expressed in the melanotrope cells by low-stringency hybridization of a neurointermediate pituitary (NIL) cDNA library of black-adapted toads with [32P]-labeled mouse p24 cDNA fragments. We identified the Xenopus members of the four p24 subfamilies p24α, -β, -γ, and -δ.

 

The p24α subfamily

Within the p24α subfamily two branches of p24 proteins can be distinguished, namely the α1-branch and the α23-branch (Dominguez et al., 1998) (Fig. 1C). Thus far, the only isolated representative of the α1-branch is from dog pancreatic microsomes (Wada et al., 1991). Two closely related representatives of the α23-branch are expressed in mouse, with mouse p24α2 (mp24α2; GMP25/mp25) being 80.8% identical to mp24α3 (GMP25iso; (Dominguez et al., 1998). In our screening for p24α subfamily members, we used a 524-bp fragment of a mouse EST p24α3 clone as a probe, yielding 12 hybridization-positive plaques from 2×105 plaques of the Xenopus NIL cDNA library screened. Nucleotide sequence analysis revealed that the positive clones all contained overlapping cDNAs. The insert size of two full-length clones was approximately 1.2 kb with a 687-bp open reading frame (ORF). Since the deduced Xenopus p24α protein (excluding the signal peptide region) was more closely related to mp24α3 (92,4% identity) than to mp24α2 (81,3% identity), the protein was named Xp24α3 (Fig. 1). The low degree of amino acid sequence conservation between the α1- and the α23-branch (identity <60%) does not allow the isolation of a p24>α1-related protein using mouse Xenopus p24α3 cDNA as a probe. However, the degree of homology within the α23-branch (identity >80%) should be high enough for the identification of a p24α2 ortholog in Xenopus. We therefore performed a low-stringency hybridization using the coding region of Xp24α3 cDNA as a probe. From a total number of 4.2×105 plaques, 51 hybridization-positive plaques were obtained. All clones positive in this screening were also recognized by the 0.43-kb 3'-untranslated region of Xp24α3 cDNA on a duplicate filter under stringent hybridization conditions, indicating that only p24α3, and not p24α2, is expressed in the Xenopus intermediate pituitary. Nevertheless, p24α2 does exist in X. laevis, because recently performed database searches revealed two Xenopus EST clones isolated from embryo and liver cDNA libraries, of which the deduced amino acid sequences were more similar to mp24α2 than to mp24α3 (Fig. 1).

Figure 1: Xenopus p24 proteins and their relationship with p24 proteins from other species.

(A) Alignment of members of the p24 protein family in X. laevis. Aligned are the amino acid sequences deduced from cDNA clones, the EST database entry BF611875 (representing Xp24α2) and the two p24δ subfamily members identified previously (Kuiper et al., 2000). The cDNAs of the Xenopus p24 proteins have been isolated from a neurointermediate pituitary cDNA library, except for those of Xp24α2 and -γ1 (isolated from an embryo library). Amino acids that are conserved in at least five sequences are in black boxes. The putative signal peptidase cleavage sites of the N-terminal signal sequences (incomplete for Xp24α2) are indicated by an arrow. Asterisks indicate the two conserved cysteine residues present in the lumenal domains of all p24 proteins. The predicted transmembrane region (TM) is underlined. (B) Amino acid sequence identity (%) between p24 proteins of mouse and X. laevis. (C) Phylogenetic tree of the p24 proteins from mouse (m), Xenopus (X), Drosophila melanogaster (d), Caenorhabditis elegans (c) and p24α1 from dog, and the classification in the four proposed p24 subfamilies. For sequence comparisons and phylogenetic tree construction, the p24 proteins without their signal sequences were aligned by the Clusteral W algorithm using default parameters (AlignX program in Vector NTI Suite 6; InforMax, North Bethesda, MD, USA). The mp24α1 protein lacks part of the amino terminal region (indicated with an asterisk). Sequences are mostly compilations of several data base entries and accession numbers are available upon request.

 

The p24β subfamily

Only one member of the p24β subfamily exists in higher vertebrates and its high degree of sequence conservation enabled us to amplify a 313-bp fragment of Xp24β1 in a PCR reaction using Xenopus brain-derived cDNA and degenerate primers corresponding to two conserved domains in vertebrate p24β1 proteins. This fragment was used to screen the Xenopus NIL cDNA library, and two full-length clones of Xp24β1 with an insert size of 2.4 kb were isolated. A comparison of the deduced primary sequence of Xp24β1 with mouse p24β1 revealed that, when the signal peptide sequence is excluded, the two proteins share a sequence identity of 99.4% (Fig. 1). To assess if other representatives of the p24β subfamily exist and are expressed in the library, duplicate filters were prepared, of which one filter was hybridized under low-stringency conditions with a probe comprising the entire ORF of Xp24β1, whereas the second filter was hybridized under high stringency conditions with a probe directed against the ~1.8-kb 3'-untranslated sequence of Xp24β1. All 345 hybridization-positive plaques from 6.7×105 plaques screened were recognized by both probes, indicating that Xenopus melanotrope cells express only one member of the p24β subfamily.

 

The p24γ subfamily

The p24γ subfamily is more diverse than the other p24 subfamilies, and database searches revealed four members in mouse. An evolutionary tree showed that two groups of p24γ proteins could be distinguished; one represented by p24γ1 and -γ2, and the other one by p24γ3 and -γ4 (Fig. 1C). Low-stringency hybridization of the Xenopus NIL cDNA library (3.4×105 plaques) with a 462-bp mouse p24γ2 fragment allowed the identification of 9 hybridization-positive cDNA clones, which carried an insert that coded for a p24γ2 ortholog in X. laevis. The deduced protein sequence of Xp24γ2 displays 72.4% identity with mouse p24γ2 and 52.9% with mouse p24γ1 (excluding signal peptides; Fig. 1). Out of 7.3×105 NIL cDNA plaques screened under stringent conditions, 30 plaques were recognized by a ~0.6-kb probe corresponding to the 3'-untranslated region of Xp24γ2. A subsequent low-stringency screening of the same library filters with the coding sequence of Xp24γ2 yielded no additional hybridizing plaques. Thus, under the conditions used, no cross hybridization with other Xp24γ sequences was found.

A low-stringency hybridization of the NIL cDNA library detected no positive clones when a mouse p24γ1 fragment was used. However, screening of two Xenopus embryo cDNA libraries with the mouse p24γ1 probe resulted in the identification of several overlapping cDNA clones, encoding the Xp24γ1 protein (Fig.1). Therefore, Xp24γ1 does not appear to be expressed in the Xenopus intermediate pituitary, but is expressed in other Xenopus tissues. In its mature form (without signal peptide), the Xp24γ1 protein shares an overall sequence identity of 68.5% with mouse p24γ1, 56.4% with mouse p24γ2 and 56.2% with Xp24γ2. Moreover, nucleotide sequence analysis revealed that 2 out of the 17 Xp24γ1 clones isolated from the embryo head library showed an insertion of 30 nucleotides at position +442 in Xp24γ1 cDNA. Consequently, the protein encoded by the two cDNAs contained an in-frame insertion of 10 amino acid residues following amino acid 148, with the amino acid sequence VRFCPLTFEE (single-letter code). This finding suggests that in a low incident of cases the transcripts of Xp24γ1 are subject to alternative splicing, giving rise to two structurally distinct Xp24γ1 proteins that differ in size by ~1.1 kDa. The in-frame insertion was not found in other known p24γ1 proteins and alternative splicing has so far not been described for other p24 proteins. In conclusion, since under identical hybridization conditions Xp24γ1 could be isolated from an embryo cDNA library but not from a NIL cDNA library, only Xp24γ2 but not -γ1 is expressed in the melanotrope cells of the Xenopus intermediate pituitary.

For the isolation of Xenopus orthologs of p24γ3 and p24γ4, the NIL cDNA library was screened with a 453-bp fragment derived from a mouse p24γ4 cDNA. Out of the 4×105 plaques screened, 25 were positive on a duplicate set of filters. Nucleotide sequence analysis of 10 of these clones revealed a single ORF and the corresponding amino acid sequence (without signal peptide) was 93.2% identical to mp24γ3 and 69.1% to mp24γ4. Thus, the cDNA clones isolated with the mp24γ4 probe encode a Xenopus ortholog of p24γ3 (Fig. 1). The NIL cDNA library was rescreened and 175 hybridization-positive plaques were observed when the filters were first hybridized with a 3'-untranslated probe of Xp24γ3 under stringent hybridization conditions (6×105 plaques screened). A subsequent low-stringency hybridization with the coding sequence of Xp24γ3 revealed no differences in the hybridization pattern, suggesting that only Xp24γ3 and not Xp24γ4 is expressed in Xenopus melanotrope cells. Besides the NIL cDNA library we also screened the whole-embryo cDNA library with a 306-bp mp24γ3 probe under conditions of low-stringency. This led to the identification of 66 positive plaques (4×105 plaques screened), which also remained positive after a more stringent wash (0.1×SSC/0.1% SDS; 50°C). Together with the fact that from 20 of these clones a specific Xp24γ3 fragment was amplified in a PCR reaction, the hybridization-positive clones most likely contain an Xp24γ3 cDNA insert. From these experiments we conclude that a p24γ4 ortholog is not expressed in the Xenopus intermediate pituitary and embryos.

 

The p24δ subfamily

In Xenopus, the p24δ subfamily has already been characterized in our laboratory, leading to the identification of two p24δ proteins, Xp24δ1 and -δ2, expressed in the melanotrope cells (Kuiper et al., 2000). Up to then, only one representative of the p24δ subfamily had been described in vertebrates, with mouse p24δ being more related to Xp24δ1 than to Xp24δ2 (amino acid sequence identities of 82.2% and 70%, respectively).

 

Analysis of p24 sequences

A multiple amino acid sequence alignment of all known Xenopus p24 proteins revealed a low degree of overall amino acid sequence identity between the members of the different subfamilies (Fig. 1A). However, the topology common to all p24 proteins is also preserved in the Xenopus proteins (an N-terminal signal sequence, a large lumenal domain followed by a transmembrane stretch, and a cytoplasmically exposed C-terminal region of 10-16 residues), as are conserved amino acid residues such as the two cysteine residues that form a disulfide bridge in the N-terminal region, a glutamine residue within the transmembrane domain and a phenylalanine residue which constitutes part of a COPII-binding motif (Dominguez et al., 1998). Furthermore, heptad repeats of aliphatic amino acids are found in the membrane proximal parts of the lumenal domains of Xp24α2, -α3, -β1, -γ1, -δ1 and -δ2 (but not in Xp24γ2 and -γ3) that have a medium to high propensity to form coiled-coil structures (>0.4 by coils algorithm (version 2.2; Lupas, 1996). Coiled-coil interactions between members of different p24 subfamilies are involved in the formation of hetero-oligomeric complexes (Ciufo and Boyd, 2000). The alternatively spliced form of Xp24γ1 has in this particular region an insertion of ten amino acids (following residue +148), which reduces its propensity to form coiled-coil structures. The cytoplasmic tail sequences are highly conserved between the two Xenopus p24α subfamily members, as they are in the p24δ subfamily and the p24γ12 subgroup. A classical ER retrieval/COPI-binding motif in the C-terminal region (K(X)KXX) is present only in the Xenopus p24α proteins, whereas members of the other subfamilies show variations of this motif. Binding studies with the cytoplasmic domains of human p24 proteins have revealed efficient COPI binding for hp24α2 and also for hp24δ1 (despite of an imperfect COPI-binding motif), while all human p24 proteins analyzed (α2, β1, γ3, γ4, δ1) have been found to interact with COPII (Dominguez et al., 1998). Since the critical amino acid residues in the cytoplasmic tails are conserved between the human and Xenopus p24 proteins, similar binding properties are to be expected for the Xenopus p24 proteins.

During evolution, the genome of Xenopus laevis underwent a genome duplication event, causing this species to be tetraploid (Graf and Kobel, 1991). As a consequence, two highly conserved genes (paralogs) are usually found. This was also the case for every novel p24 protein isolated in this study. The nucleotide sequence identities over the entire ORFs were found to be in the order of 94 to 95.5%, and the nucleotide substitutions were either neutral or led to conservative amino acid substitutions in the deduced primary sequences of the respective p24 proteins. For clarity, the primary sequence of only one paralog is shown in Fig. 1.

In summary, the melanotrope cells in the intermediate pituitary of Xenopus express one member of the p24α (α3), one of the p24β (β1), two of the p24γ 2, γ3) and two of the p24δ (δ1, δ2) subfamily. Two members, Xp24α2 and -γ1, show a tissue-specific distribution in that they were expressed in Xenopus embryos but not in the intermediate pituitary.

 

Expression of the various Xenopus p24 proteins in the pituitary gland

To study the expression of the various Xenopus p24 proteins, polyclonal antisera directed against sequences in the lumenal domains of these p24 proteins were generated. Western blot analysis revealed that the various antisera recognize specifically the corresponding Xp24 proteins of the melanotrope cells (Kuiper et al., 2000) (data not shown), except for the anti-δC antiserum, which recognizes both Xp24δ proteins (δ1 and δ2). To study the expression of the p24 proteins in the pituitary, lobes from black- and white-adapted Xenopus were manually dissected into the NIL and the anterior lobe (AL). Together with tissue extracts from brain and hypothalamus, NIL and AL lysates were separated with SDS-PAGE and analyzed by immunoblotting (three times more protein was loaded for brain and hypothalamus). In NIL lysates of black-adapted animals, the antibodies directed against epitopes in the lumenal domains of Xp24α3, -β1, -γ2, -γ3, -δ1, and -δ2 recognized proteins with a molecular mass of ~25, 21, 24, 25.5, 19, and 21 kDa, respectively (Fig. 2, data not shown for anti-δ1 and anti-δ2). With an antibody against Xp24γ1 (residues 18-194), a 22-kDa protein was identified in tissue lysates from brain and hypothalamus, while no protein was detected in the NIL (Fig. 2), in line with the finding that a p24γ1 cDNA could be isolated only from a Xenopus embryo but not from a NIL cDNA library. At present it is not clear if the 23-kDa product in the anterior lobe of the pituitary detected with the anti-γ1L antiserum corresponds to the alternatively spliced form of Xp24γ1, or is due to nonspecific binding to the similarly sized and highly expressed anterior pituitary hormones prolactin/growth hormone (judged by staining of the Western blot with Ponceau S). Interestingly, immunoblot analysis of cellular extracts showed that the protein levels of Xp24α3, -β1, -γ3, and -δ2 were highly upregulated in NILs of black-adapted Xenopus when compared to that in white-adapted animals (~20-30 fold). In contrast, the level of Xp24δ1 expression was only slightly induced (~3 fold) in NIL lysates of black-adapted animals, and no change was observed for Xp24γ2 (Fig. 2). As previously demonstrated for POMC (Holthuis et al., 1995), the physiologically induced changes in the expression levels of Xp24α3, -β1, -γ3, and -δ2 were strictly confined to the melanotrope cells of the NIL and did not occur in cells from the AL of the pituitary (Fig. 2). Semi-quantitative reverse transcription (RT)-PCR revealed a 3-5 fold increase in the levels of Xp24α3, -β1, -γ3, and -δ2 mRNA in the melanotropes of black-adapted animals while the levels of Xp24δ1 and -γ2 mRNA were similar (data not shown).

Figure 2: p24 protein expression in Xenopus pituitary.

The neurointermediate lobe (NIL) was manually dissected from the anterior lobe (AL) of the pituitary of black- or white-adapted Xenopus. 20 µg of NIL and AL extracts were resolved together with tissue extracts from brain and hypothalamus (~60 µg each) by SDS-PAGE. For immunoblotting, antisera directed against sequences in the lumenal domains of the respective p24 proteins or the anti-δC antiserum recognizing Xp24δ1 and -δ2 were used. The asterisk indicates an AL protein band presumably resulting from cross-reactivity of the anti-Xp24γ1 antiserum with an abundant 23 kDa protein (likely growth hormone/prolactin).

 

Next, we tried to confirm our results using immunocytochemistry. The p24 proteins Xp24α3, -β1, -γ3, and -δ2, shown by Western blot analysis to be differentially regulated in the NIL, displayed intense staining in the melanotrope cells of the intermediate pituitary of a black-adapted animal, whereas their expression in a white-adapted animal was indeed low (Fig. 3). Conversely, for Xp24γ2 and -δ1 a low degree of expression was observed in the melanotrope cells, independent of background adaptation. The differential regulation of the various p24 proteins was again confined to the melanotrope cells, because no differences in the expression patterns were observed in the anterior lobes of black- and white-adapted animals. Furthermore, the homogenous distribution of the p24 proteins throughout the intermediate lobe suggests that they are expressed in all melanotrope cells. Antisera directed against Xp24γ1 showed no immunoreactive staining in the melanotrope cells while in the anterior pituitary some immunoreactive material was detected, again likely due to

Figure 3: Immunocytochemical analysis visualizing the expression of p24 proteins in the pituitary gland of Xenopus laevis adapted to a black or white background.

Shown are sagittal sections stained with the peroxidase-anti-peroxidase method for Xp24α3 (affinity-purified anti-α3L, 1:300 dilution), Xp24β1 (anti-β1L, 1:200 dilution), Xp24γ1 (anti-γ1L, 1:600 dilution), Xp24γ2 (affinity-purified anti-γ2L, 1:40 dilution), Xp24γ3L (anti-γ3L, 1:200 dilution), Xp24δ1 (affinity-purified anti-δ1, 1:50 dilution), and Xp24δ2 (anti-δ2, 1:1500 dilution). NL, neural lobe; IL, intermediate lobe; AL, anterior lobe. Bar 200 µm.

 

non-specific cross-reactivity as was observed during Western blot analysis. In conclusion, we find that during background adaptation only a subset of the p24 proteins (Xp24α3, -β1, -γ3, and -δ2) expressed in the melanotrope cells of the intermediate pituitary is coordinately expressed with the prohormone POMC.

 

p24 expression in Xenopus tissues

The distribution of the various Xp24 proteins in tissues other than the pituitary was studied at the level of RNA (Northern blot analysis) and at the protein level (Western blotting). For Northern blot analysis, total RNA was isolated from a number of Xenopus tissues and hybridized with [32P]-labelled probes covering the entire ORF of Xp24α3, -γ1, -γ2, -γ3 or with a 364-bp fragment of Xp24β1 (nucleotides +77 to +364). The applied method was not sensitive enough for the detection of Xp24γ1 and -γ2 transcripts, whereas more than one transcript was found for Xp24α3, -β1 and -γ3 (Fig. 4A). Xp24α3, -β1 and -γ3 mRNAs were found to be expressed in brain, liver, kidney, spleen, heart and lung, as was previously found for Xp24δ1 and -δ2 mRNAs (Holthuis et al., 1995) (our unpublished results). Due to the tetraploid nature of the Xenopus genome, a pair of closely related genes (paralogs) is expressed that often gives rise to transcripts with different sizes. Xp24α3 is represented by two transcripts of about 1.2 and 2.5 kb, ubiquitously expressed in all tissues examined. The size of the 1.2-kb transcript corresponds to the insert sizes of two full-length cDNA clones encoding paralog A of Xp24α3. The transcript length expected for paralog B of Xp24α3 is not known and may be represented by the 2.5-kb transcript. With an Xp24β1 probe, a predominant mRNA transcript of 2.5 kb and a weak one of 1.2 kb were detected. The Xp24β1-encoding cDNAs for the two paralog genes were derived from a 2.5-kb transcript and thus the 1.2-kb transcript may arise from alternative splicing of nuclear RNA or from the utilization of an alternative polyadenylation signal. Three transcripts (2.3, 2.5 and >4 kb) with similar intensities were observed for Xp24γ3 mRNAs. Since the sizes of two transcripts (2.3 and 2.5 kb) correspond to full-length Xp24γ3 cDNA clones, the presence of the >4-kb transcript indicates that Xp24γ3 gene expression may also include alternative splicing and/or alternative usage of polyadenylation signals.

Figure 4: p24 expression in Xenopus tissues.

(A) Northern blot analysis of Xenopus p24α3, -β1, and -γ3 mRNAs. Equal amounts of RNA were loaded and Xenopus GAPDH was used as a control for RNA loading and integrity. The positions of the 18S and 28S Xenopus ribosomal RNAs are indicated by arrows. (B) Western blot analysis of the Xenopus p24 proteins using antibodies against lumenal epitopes of the respective p24 proteins.

The ubiquitous tissue expression of Xp24 proteins observed at the mRNA level was also seen at the protein level (Fig. 4B). With the anti-α3L antibody, expression of Xp24α3 was found to be high in ovarian and liver, intermediate in hypothalamus, brain, kidney and spleen, and low in heart and lung. This antibody recognized a second protein band in liver and lung that was slightly larger in size and represents the only protein detected in heart tissue. The two protein bands (~25 and ~26 kDa) may reflect different glycosylation states of Xp24α3, which is N-linked glycosylated at a single site (our unpublished observation). Tissue-dependent variations in the glycosylation state have also been described for hp24α2 (Füllekrug et al., 1999). Alternatively, the anti-α3L antibody may recognize with low affinity the highly related Xp24α2 protein, which at least in liver is known to be expressed. The overall tissue distributions of Xp24β1, -γ3, and -δ2 were similar to that obtained for Xp24α3 and the four proteins are predominantly expressed in liver and ovarian. Except for the intermediate pituitary, the Xp24γ1 protein was expressed in all tissues examined (Fig. 4). When compared to the levels obtained for Xp24α3, -β1, -γ3 and -δ2, the relatively low levels of Xp24γ1 expression in ovarian and liver are remarkable. The tissue distribution of Xp24γ1 is similar to those of Xp24γ2 and -δ1 (the two proteins that are not differentially regulated in the intermediate pituitary), although both Xp24γ2 and -δ1 show higher levels of protein expression in ovarian. Taken together, these experiments indicate that the members of the p24 family are widely expressed but they may display a cell-type specific expression, as was found for Xp24α2 and -γ1 in the melanotrope cells of the intermediate pituitary.

 

 

DISCUSSION

We set out to examine the expression profiles of members of the p24 family in a single cell type, namely in the intermediate pituitary melanotrope cell of X. laevis. The primary function of this neuroendocrine transducer cell is the production of POMC and the release of POMC-derived melanophore-stimulating peptides during adaptation of the animal to a black background (Holthuis et al., 1995). The melanotrope cells are highly active in black-adapted Xenopus and produce ~30-fold higher POMC mRNA levels than the biosynthetically virtually inactive melanotrope cells of white-adapted animals. A set of genes has been isolated whose transcripts are coordinately expressed with POMC (differential induction is >10-fold; (Holthuis et al., 1995), including POMC processing enzymes and members of the so-called granin family (reviewed in (Kuiper et al., 2000). Other gene products identified reflect the dynamic changes in the secretory apparatus observed between active and inactive melanotrope cells. Included in this category are the molecular chaperone BiP and TRAPδ (translocon-associated protein subunit δ) as components of the ER translocation machinery, and the cysteine protease ER60 and the chaperone calreticulin as part of the quality control system in the ER. Another differentially expressed gene product was Xp24δ2 (X1262), a member of the p24 family of putative cargo receptors (Holthuis et al., 1995) (our unpublished observations). Multiple members of the p24 family have been found in eukaryotes (eight in yeast, five in C. elegans, seven in Drosophila melanogaster, and nine in human and mouse; Fig. 1C), which prompted us to search for Xenopus p24 proteins other than Xp24δ2 and to study their expression in the melanotrope cells. Our extensive Xenopus intermediate pituitary cDNA library screening led to the identification of six p24 proteins, namely one member of the p24α (α3), one of the p24β (β1), two of the p24γ (γ2, γ3) and two of the p24δ (δ1, δ2) subfamily. Two other identified Xenopus p24 proteins, Xp24α2 and -γ1, were expressed in embryos, but not in the melanotrope cells. A phylogenetic tree constructed from the identified Xenopus p24 proteins and from the p24 proteins of mouse, D. melanogaster and C. elegans revealed that each of these species has at least one representative in each subfamily (Fig. 1C). Members of the p24α subfamily have evolved into two separate branches (α1 and α2/3). The only representative of the α1-branch is gp25L (dog p24α1), but recent database searches indicate that orthologs of dog p24α1 may also exist in mouse and human. Invertebrates seem to have only one representative of the p24α subfamily that belongs to the α2/3-branch. Two closely related but distinct p24 proteins of the α2/3-branch were identified in human and mouse, and two representatives were also found in Xenopus (Xp24α2 and -α3). Vertebrate proteins of the α2/3-branch may have a cell-type specific expression pattern, since in Xenopus only Xp24α3 but not -α2 was found to be expressed in the melanotrope cells. Thus far, only one representative of the p24β subfamily has been described, but database searches revealed that two p24β subfamily members exist in D. melanogaster. A single member was found to be expressed in Xenopus melanotrope cells. The primary sequences of human and mouse p24β1 (excluding the signal peptide) are identical, and mouse p24β1 and Xp24β1 share 99.4% identity. Such exceptionally high sequence conservation was not found between the vertebrate orthologs of the other p24 subfamilies and thus may point to an evolutionarily conserved function for the p24β protein. The phylogenetic tree shows that members of the p24γ subfamily of C. elegans, D. melanogaster, mouse and human can be assigned to two distinct subgroups: the γ12-subgroup and the γ34-subgroup (Fig. 1C). Since in vertebrates the two subgroups are only distantly related (amino acid sequence similarity <50% and identity ><35%), perhaps a reclassification of these subgroups into separate subfamilies should be considered. The resulting classification into five subfamilies would imply that >C. elegans has a single representative in each subfamily, whereas higher-developed organisms have mostly more than one. In mouse and human, the p24γ subfamily has four members, two of the p24γ12-subgroup (γ1 and γ2) and two of the p24γ34-subgroup (γ3 and γ4). We found that in the Xenopus melanotrope cells one member of the p24γ12-subgroup (Xp24γ2) and one member of the p24γ34-subgroup (Xp24γ3) is expressed. A second member of the Xenopus p24γ12-subgroup (Xp24γ1) was found in an embryo cDNA library, while screening for a p24γ4 member of the p24γ34-subgroup remained negative, despite of the fact that the probe used (mouse p24γ4) cross-hybridized even with the Xp24γ3 sequence. Therefore, a Xenopus p24γ4 orthologue appears not to be present in the melanotrope cells and in embryos. The restricted expression of Xp24γ1, namely in embryos but not in the melanotrope cells, was confirmed by immunoblotting. A cell-type specific expression pattern has also been described for the p24γ1 orthologues in human and mouse (T1/ST2 receptor; (Gayle et al., 1996). Thus far, with the exception of Xenopus, only one representative of the p24δ subfamily has been reported in species of the animal kingdom. The two Xenopus p24δ subfamily members, Xp24δ1 and Xp24δ2, are both expressed in the melanotrope cells, although only Xp24δ2 but not Xp24δ1 is coexpressed with POMC in these cells (Kuiper et al., 2000). Taken together, these findings show that the complexity of the p24 family is species dependent with certain organisms having multiple members in distinct subfamilies whereas others have only one representative.

In the physiologically manipulated Xenopus melanotrope cells, we could demonstrate that during black background adaptation the protein levels of Xp24α3, -β1, -γ3, and -δ2 were increased 20 to 30 times, whereas the expression of the Xp24γ2 and -δ1 proteins remained unchanged or was increased only ~3 times, respectively. In yeast and mammals, p24 proteins form tetrameric complexes with a defined complex composition, in which one member from each subfamily is present (Füllekrug et al., 1999; Marzioch et al., 1999). Furthermore, the steady-state protein level and the intracellular localization of a p24 protein are dependent on the presence or absence of other p24 members that participate in the oligomeric complex (Denzel et al., 2000; Emery et al., 2000; Füllekrug et al., 1999; Marzioch et al., 1999). Since Xp24α3, -β1, -γ3, and -δ2 show similar dynamics in protein expression in the melanotrope cells and also in other tissues, they may well form a tetrameric p24 complex, whereas Xp24γ2 and -δ1, which are not differentially regulated in the melanotrope cells, could be constituents of other p24 complexes. Interestingly, the apparent composition of the main tetrameric melanotrope p24 complex (Xp24α3, -β1, -γ3, and -δ2) is different from the one previously identified in HeLa cells, where a p24α2131 p24 complex exists (GMP25/p24/gp27/p23; (Füllekrug et al., 1999). Variations in p24 complex formation are likely to occur, e.g. because of the observed cell-type specific expression of the various Xenopus p24 proteins. Furthermore, in yeast an Erp1p (yp24α)/Erp2p (yp24γ)/Emp24p (yp24β)/Erv25p (yp24δ) complex is present, in which Erp1p can be substituted by another p24α subfamily member (Erp5p and/or Erp6p) if Erp1p is not expressed (Marzioch et al., 1999). Also, hp24γ4 has been found to be excluded from the HeLa cell α2131 p24 complex (Füllekrug et al., 1999), while mp24γ4 may well participate in a p24 complex (Denzel et al., 2000). Therefore, the composition of a tetrameric p24 complex appears to be cell-type specific.

The exact role of the p24 proteins is still elusive, but p24 proteins have been implicated in a number of functions which are all linked to vesicular and protein transport, such as regulation of cargo inclusion in ER vesicles (Muñiz et al., 2000; Schimmöller et al., 1995), quality control mechanisms in the ER (Belden and Barlowe, 1996; Wen and Greenwald, 1999), recruitment and regulation of COPI/II vesicle coat assembly (Bremser et al., 1999; Kaiser, 2000; Kuehn et al., 1998), and generation of vesicular tubular clusters (Lavoie et al., 1999; Rojo et al., 2000). In yeast, an interaction between the cargo protein Gas1p and p24 proteins has been demonstrated, and loss of function of certain p24 proteins reduces the kinetics of ER-to-Golgi transport of a subset of secretory proteins, whereas resident ER proteins (Kar2p and Pdi1p) are less efficiently retained in the ER (Elrod Erickson and Kaiser, 1996; Marzioch et al., 1999; Muñiz et al., 2000; Schimmöller et al., 1995). In C. elegans, reducing the activity of certain p24 proteins restores at least partially the ER transport block of a mutant protein to the plasma membrane (Wen and Greenwald, 1999). Proper p24 function may thus facilitate the transport of certain cargo molecules and restrict the entry of ER proteins and incorrectly folded proteins into COPII vesicles. Along this line, in the activated Xenopus melanotrope cells a Xp24α3132 complex could be involved in the inclusion of POMC into transport vesicles, as the four p24 members are coordinately expressed with this prohormone. Unfortunately, extensive cross-linking and co-immunoprecipitation studies using the Xenopus intermediate pituitary cells have not allowed us to establish a direct physical interaction between the Xenopus p24 proteins or between p24 and POMC.

In conclusion, we isolated and characterized the set of p24 proteins expressed in a single cell type (the Xenopus intermediate pituitary melanotrope cell), and revealed that their expression is cell-type specific and can be selectively induced. In the melanotropes, four of the six p24 members are coexpressed and these representatives of the four subfamilies may form a complex that is involved in the efficient ER to Golgi transport of its major cargo protein POMC. Together, our results thus point to an involvement of p24 proteins in the process of selective protein transport within the early secretory pathway.

 

 

ACKNOWLEDGEMENTS

We thank T. Nilsson for antibodies (see Material and Methods), M. King and R. Harland for providing us with Xenopus embryo cDNA libraries, A.J.M. Coenen for technical assistance and R. Engels for animal care. This work was supported by grant ERB-FMRX-CT960023 from the European Union for Training and Mobility of Researchers (EU-TMR).

 

 

REFERENCES

Adachi, T., Schamel, W. W., Kim, K. M., Watanabe, T., Becker, B., Nielsen, P. J. and Reth, M. (1996). The specificity of association of the IgD molecule with the accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence. EMBO Journal 15, 1534-41.

Annaert, W. G., Becker, B., Kistner, U., Reth, M. and Jahn, R. (1997). Export of cellubrevin from the endoplasmic reticulum is controlled by BAP31. Journal of Cell Biology 139, 1397-410.

Aridor, M., Bannykh, S. I., Rowe, T. and Balch, W. E. (1995). Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport. Journal of Cell Biology 131, 875-93.

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (1989). Current Protocols in Molecular Biology. John Wiley and Sons, New York.

Balch, W. E., McCaffery, J. M., Plutner, H. and Farquhar, M. G. (1994). Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell 76, 841-852.

Belden, W. J. and Barlowe, C. (1996). Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J Biol Chem 271, 26939-26946.

Bremser, M., Nickel, W., Schweikert, M., Ravazzola, M., Amherdt, M., Hughes, C. A., Sollner, T. H., Rothman, J. E. and Wieland, F. T. (1999). Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors. Cell 96, 495-506.

Ciufo, L. F. and Boyd, A. (2000). Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway. Journal of Biological Chemistry 275, 8382-8.

Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J. J., Solari, R. C. and Owen, M. J. (2000). The p24 family member p23 is required for early embryonic development. Current Biology 10, 55-8.

Dominguez, M., Dejgaard, K., Füllekrug, J., Dahan, S., Fazel, A., Paccaud, J. P., Thomas, D. Y., Bergeron, J. J. and Nilsson, T. (1998). gp25l/emp24/p24 protein family members of the cis-Golgi network bind both Cop I and II coatomer. Journal of Cell Biology 140, 751-65.

Elrod Erickson, M. J. and Kaiser, C. A. (1996). Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations. Molecular Biology of the Cell 7, 1043-1058.

Emery, G., Rojo, M. and Gruenberg, J. (2000). Coupled transport of p24 family members. Journal of Cell Science 113 ( Pt 13), 2507-16.

Füllekrug, J., Suganuma, T., Tang, B. L., Hong, W., Storrie, B. and Nilsson, T. (1999). Localization and Recycling of gp27 (hp24gamma3): Complex Formation with Other p24 Family Members. Molecular Biology of the Cell 10, 1939-1955.

Gayle, M. A., Slack, J. L., Bonnert, T. P., Renshaw, B. R., Sonoda, G., Taguchi, T., Testa, J. R., Dower, S. K. and Sims, J. E. (1996). Cloning of a putative ligand for the T1/ST2 receptor. Journal of Biological Chemistry 271, 5784-9.

Graf, J. D. and Kobel, H. R. (1991). Genetics of Xenopus laevis. Methods in Cell Biology 36, 19-34.

Harlow, E. and Lane, D. (1988). Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.

Hauri, H. P., Kappeler, F., Andersson, H. and Appenzeller, C. (2000). ERGIC-53 and traffic in the secretory pathway. Journal of Cell Science 113, 587-96.

Holthuis, J. C., van Riel, M. C. and Martens, G. J. M. (1995). Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary. Biochem J 312, 205-213.

Jenks, B. G., Overbeeke, A. P. and McStay, B. F. (1977). Synthesis, storage and release of MSH in the pars internmedia of the pituitary gland of Xenopus laevis during background adaptation. Canadian Journal of Zoology 55, 922-927.

Kaiser, C. (2000). Thinking about p24 proteins and how transport vesicles select their cargo. Proc Natl Acad Sci U S A 97, 3783-3785.

Kim, K. M., Adachi, T., Nielsen, P. J., Terashima, M., Lamers, M. C., Kohler, G. and Reth, M. (1994). Two new proteins preferentially associated with membrane immunoglobulin D. EMBO Journal 13, 3793-800.

Kuehn, M. J., Herrmann, J. M. and Schekman, R. (1998). COPII-cargo interactions direct protein sorting into ER-derived transport vesicles. Nature 391, 187-90.

Kuiper, R. P., Waterham, H. R., Rötter, J., Bouw, G. and Martens, G. J. M. (2000). Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell. Molecular Biology of the Cell 11, 131-40.

Lavoie, C., Paiement, J., Dominguez, M., Roy, L., Dahan, S., Gushue, J. N. and Bergeron, J. J. (1999). Roles for alpha(2)p24 and COPI in Endoplasmic Reticulum Cargo Exit Site Formation. Journal of Cell Biology 146, 285-300.

Letourneur, F., Gaynor, E. C., Hennecke, S., Demolliere, C., Duden, R., Emr, S. D., Riezman, H. and Cosson, P. (1994). Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell 79, 1199-1207.

Lupas, A. (1996). Coiled coils: new structures and new functions. Trends Biochem Sci 21, 375-382.

Martinez-Menarguez, J. A., Geuze, H. J., Slot, J. W. and Klumperman, J. (1999). Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles. Cell 98, 81-90.

Marzioch, M., Henthorn, D. C., Herrmann, J. M., Wilson, R., Thomas, D. Y., Bergeron, J. J., Solari, R. C. and Rowley, A. (1999). Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex. Molecular Biology of the Cell 10, 1923-1938.

Mizuno, M. and Singer, S. J. (1993). A soluble secretory protein is first concentrated in the endoplasmic reticulum before transfer to the Golgi apparatus. Proc Natl Acad Sci U S A 90, 5732-6.

Muñiz, M., Nuoffer, C., Hauri, H. P. and Riezman, H. (2000). The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles. Journal of Cell Biology 148, 925-30.

Nagai, K. and Thogersen, H. C. (1987). Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods in Enzymology 153, 461-81.

Nickel, W., Malsam, J., Gorgas, K., Ravazzola, M., Jenne, N., Helms, J. B. and Wieland, F. T. (1998). Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTP(&ggr ;)S in vitro. Journal of Cell Science 111, 3081-3090.

Nishimura, N. and Balch, W. E. (1997). A di-acidic signal required for selective export from the endoplasmic reticulum. Science 277, 556-8.

Orci, L., Stamnes, M., Ravazzola, M., Amherdt, M., Perrelet, A., Sollner, T. H. and Rothman, J. E. (1997). Bidirectional transport by distinct populations of COPI-coated vesicles. Cell 90, 335-49.

Rojo, M., Emery, G., Marjomaki, V., McDowall, A. W., Parton, R. G. and Gruenberg, J. (2000). The transmembrane protein p23 contributes to the organization of the Golgi apparatus. Journal of Cell Science 113, 1043-57.

Roubos, E. W. (1997). Background adaptation by Xenopus laevis: a model for studying neuronal information processing in the pituitary pars intermedia. Comparative Biochemistry and Physiology. Part A, Physiology 118, 533-50.

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York.

Saraste, J. and Svensson, K. (1991). Distribution of the intermediate elements operating in ER to Golgi transport. Journal of Cell Science 100 ( Pt 3), 415-30.

Scales, S. J., Pepperkok, R. and Kreis, T. E. (1997). Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 90, 1137-48.

Schimmöller, F., Singer Krüger, B., Schröder, S., Krüger, U., Barlowe, C. and Riezman, H. (1995). The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO Journal 14, 1329-39.

Schweizer, A., Fransen, J. A., Matter, K., Kreis, T. E., Ginsel, L. and Hauri, H. P. (1990). Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. European Journal of Cell Biology 53, 185-96.

Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S. and Schekman, R. (2000). The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 97, 4034-9.

Terashima, M., Kim, K. M., Adachi, T., Nielsen, P. J., Reth, M., Kohler, G. and Lamers, M. C. (1994). The IgM antigen receptor of B lymphocytes is associated with prohibitin and a prohibitin-related protein. EMBO Journal 13, 3782-92.

Wada, I., Rindress, D., Cameron, P. H., Ou, W. J., Doherty, J. J., 2d, Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y. and Bergeron, J. J. (1991). SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J Biol Chem 266, 19599-19610.

Warren, G. and Mellman, I. (1999). Bulk flow redux? Cell 98, 125-7.

Wen, C. and Greenwald, I. (1999). p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans. Journal of Cell Biology 145, 1165-75.

Zehetner, G. and Lehrach, H. (1994). The Reference Library System-sharing biological material and experimental data. Nature 367, 489-91.

 

 

 

Chapter 3: Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell

The localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell

 

 

Roland P. Kuiper, Gerrit Bouw, Karel P. C. Janssen, Jutta Rötter, François van Herp, and Gerard J. M. Martens

Biochemical Journal, 360(2): 421-9 (2001)

 

 

ABSTRACT

Members of the p24 family of putative cargo receptors (subdivided into p24α, -β, -γ, and -δ) are localized to the intermediate and cis-Golgi compartments of the early secretory pathway, and are thought to play an important role in protein transport. In this study, we wondered what effect increased biosynthetic cell activity with resulting high levels of protein transport would have on the subcellular localization of p24. We examined p24 localization in Xenopus intermediate pituitary melanotrope cells that in black- and white-adapted animals are biosynthetically highly active and virtually inactive, respectively. In addition, p24 localization was studied in Xenopus anterior pituitary cells whose activity is not changed during background adaptation. Using organelle fractionation, we found that in the inactive melanotropes and moderately active anterior pituitary cells of white-adapted animals, the p24α, -β, -γ, and -δ proteins are all located in the Golgi compartment. In the highly active melanotropes, but not in the anterior cells of black animals, the steady-state distribution of all four p24 members changed towards the intermediate compartment and subdomains of the endoplasmic reticulum (ER), most likely the ER exit sites. In the active melanotropes, the major cargo protein proopiomelanocortin was mostly localized to ER subdomains and partially colocalized with the p24 proteins. Furthermore, in the active cells, in vitro blocking of protein biosynthesis by cycloheximide or dispersion of the Golgi complex by brefeldin A led to a redistribution of the p24 proteins, indicating their involvement in ER-to-Golgi protein transport and extensive cycling in the early secretory pathway. We conclude that the subcellular localization of p24 proteins is dynamic and depends on the biosynthetic activity of the cell.

 

 

INTRODUCTION

Proteins destined to leave the endoplasmic reticulum (ER) are transported to specialized regions, named ER exit sites (Bannykh and Balch, 1997; Bannykh et al., 1996), where they are packaged in Coat protein (COP) II-coated vesicles (Barlowe, 1998; Rothman and Wieland, 1996; Schekman and Orci, 1996). These vesicles form the Vesicular Tubular Clusters (VTCs), which fuse to form the ER-Golgi Intermediate Compartment (ERGIC) (Hauri et al., 2000; Schweizer et al., 1990), and are subsequently transported to the Golgi complex. The mechanism that underlies the inclusion of cargo in the ER-derived vesicles is still poorly understood, but several studies have pointed out that this is a selective process that may involve transmembrane proteins acting as cargo receptors (Balch et al., 1994; Kuehn et al., 1998; Mizuno and Singer, 1993). These receptors, which continuously cycle between ER and Golgi complex, thereby facilitating efficient ER-to-Golgi transport, may include ERGIC-53/p58 (which selectively and transiently interacts with glycoproteins) (Hauri et al., 2000) and the p24 protein family (subdivided into p24α, -β, -γ, and δ) (Dominguez et al., 1998). The putative role of p24 proteins as cargo receptors was based on the finding that in yeast deletion of certain members of the p24 family caused a selective defect in the transport of invertase and Gas1p, but left other cargo protein transport unaffected (Belden and Barlowe, 1996; Marzioch et al., 1999; Schimmöller et al., 1995). The recent finding that two of these members, Emp24p and Erv25p, which coexist in a heteromeric complex, can be directly cross-linked to the cargo protein Gas1p in ER-derived vesicles strongly supports the hypothesis that p24 proteins play a direct role in cargo inclusion at the level of the ER (Muñiz et al., 2000). In addition, based on a genetic study in Caenorhabditis elegans, p24 proteins have been suggested to be involved in the quality control of newly synthesized proteins in the ER (Wen and Greenwald, 1999), and in mammalian cells they may play an important role in vesicular transport as well (Rojo et al., 1997). Although p24 proteins are not essential for vesicular transport in yeast (Springer et al., 2000), they are necessary for mice viability (Denzel et al., 2000).

In general, the steady-state localization of the p24 proteins has been found to be in the intermediate- and cis-Golgi compartments (Blum et al., 1999; Dominguez et al., 1998; Füllekrug et al., 1999; Lin et al., 1999; Rojo et al., 1997; Sohn et al., 1996). However, considering the role p24 proteins may play in cargo transport, one could imagine that p24 localization is different in biosynthetically active and inactive cells. To test this hypothesis, we have now investigated the subcellular localization of p24 family members in the melanotrope cell of the intermediate pituitary of the South-African clawed toad Xenopus laevis. This cell type has a number of interesting features. First, melanotrope cells are involved in the process of background adaptation of the animal, and their biosynthetic activity can therefore be manipulated in a physiological manner from virtually inactive (when the animal is adapted to a white background) to highly active (in a black-adapted animal). Changing the background color of the animal from white to black leads to an enormous increase of cargo transport in the melanotrope cells. Second, the melanotrope cells are primarily focused on the biosynthesis and processing of the prohormone proopiomelanocortin (POMC), the precursor protein of α-melanophore stimulating hormone that is responsible for pigment dispersion in the skin. POMC is by far the major cargo protein in the melanotrope cells, representing ~80% of all newly synthesized proteins, and thus most of the p24 proteins coexpressed in these cells are expected to be linked to POMC transport. Third, the Xenopus intermediate pituitary can be easily dissected and consists of a homogenous population of melanotrope cells. Together, these characteristics make the melanotrope cell an interesting physiological model system to study the subcellular localization of p24 proteins during different states of biosynthetic cell activity. We found that depending on the biosynthetic activity of the melanotrope cell the p24 family members redistribute between the cis-Golgi and pre-Golgi compartments.

 

 

MATERIALS AND METHODS

Animals

South-African clawed toads, Xenopus laevis, were adapted to their background by keeping them in either white or black buckets under constant illumination for at least three weeks at 22ºC.

 

Antibodies

The affinity-purified rabbit polyclonal antibody against the lumenal domain of Xp24δ2 (anti-δ2) and the C-terminally-directed p24δ antibody (anti-δC) have been described previously (Kuiper et al., 2000). Two other rabbit polyclonal antibodies (anti-αC and anti-γC) were raised against synthetic peptides that comprised the carboxyl-terminal 12 amino acids of Xp24α3 (CRHLKSFFEAKKL) or Xp24γ3 (CFSDKRTTTTRVGS), respectively. Both peptides were coupled to keyhole limpet hemocyanin (Pierce, Rockford, USA) and used for immunization as described (Kuiper et al., 2000). The generation of the polyclonal rabbit anti-rat adrenocorticotropic hormone (ACTH) and anti-Xenopus secretogranin III antibodies has been described previously (Holthuis et al., 1996; Van Eys and Van Den Oetelaar, 1981). The following antibodies were kindly provided by others: guinea pig polyclonal serum against the precursor of POMC (ST62; Dr. S. Tanaka, Shizuoka University, Shizuoka, Japan) (Berghs et al., 1997), rabbit polyclonal sera against human p24β (Dr. T. Nilsson, EMBL, Heidelberg, Germany) (Dominguez et al., 1998), bovine α/γCOP (Dr. F. Wieland, EMBL, Heidelberg, Germany) (Gerich et al., 1995), rat p58 (Dr. J. Saraste, University of Bergen, Bergen, Norway) (Saraste and Svensson, 1991), yeast Sec23p (Dr. R. Schekman, University of California, Berkeley, CA), human calnexin (Dr. J. Bergeron, Mc Gill University, Montreal, Canada), murine ERp72 (Dr. M. Green, St. Louis University, St. Louis, MO), and bovine PDI (Dr. N Bulleid, University of Manchester, Manchester, UK) (John et al., 1993).

Subcellular fractionation of organelles from Xenopus pituitary tissues

Organelle fractionation of Xenopus pituitary tissues was performed using a 10-30% linear iodixanol gradient (OptiprepTM, Nycomed Pharma AS, Oslo, Norway) according to the manufacturer's procedure. In such a fractionation gradient, Golgi membranes can be expected to appear in the lowest-density fractions, whereas higher-density ER membranes migrate further into the gradient. Twenty-five neurointermediate lobes (NILs) or anterior lobes (ALs) were homogenized in 500 µl homogenization buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Hepes, pH 7.4) in a glass-glass homogenizer (10 µm inner space), and the homogenate was centrifuged at 3,000g for 10 minutes at 4°C. When indicated, NILs were incubated in Xenopus culture medium (XL15: 67% Leibovitz's-15 medium; Life Technologies-BRL) in the presence of 50 µg/ml cycloheximide for one hour, or 5 µg/ml brefeldin A for two hours at 22°C prior to homogenization. Following rehomogenization of the pellet in 500 µl homogenization buffer and centrifugation (3,000g for 10 minutes at 4°C), supernatants were pooled and loaded on top of a preformed 11-ml 10-30% iodixanol gradient and centrifuged for 1.5 hours at 26,000 g in an SW40-rotor (Beckman Instruments, Palo Alto, CA). One-ml fractions were collected, with the first fraction being the top of the gradient. All fractions were analyzed by enzymatic assays, Western blotting, or ELISA. For the gradients presented in Fig. 5, the total volume of NIL homogenate loaded on the gradient was 500 µl instead of 1 ml, which resulted in a relative shift of the fraction contents towards the top of the gradient. To allow comparisons between the various subcellular fractionation experiments, the distributions of α- and γ-COP were determined for each experiment.

 

Enzyme assays

The fractions obtained by subcellular fractionation were tested in enzyme assays for the presence of α-mannosidase II activity, which is found in the Golgi complex and to some extent in the lysosomes, and acid phosphatase (a lysosomal enzyme). The α-mannosidase-II activity was measured according to Storrie and Madden (Storrie and Madden, 1990) and the acid phosphatase assay was performed as described by Graham (Graham, 1993).

 

Immunodetection of marker proteins in the subcellular fractions

To localize marker proteins in the subcellular fractions, Western blotting was performed. Gradient fractions were loaded on SDS-polyacrylamide gels and proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) by electroblotting. Blots were blocked for 1 hour (blocking buffer: 5% non-fat dried milk in PBS containing 1% Tween-20, 0.3% Triton-X100) and incubated overnight with specific antibody in blocking buffer. Bound antibodies were detected with peroxidase-conjugated goat anti-rabbit followed by chemiluminescence (Lumilight, Roche Diagnostics, Mannheim, Germany). In an alternative procedure, the subcellular fractions were sonicated for 10 seconds in the presence of 0.1% Triton X-100, coated on microtiter plates, and analyzed by ELISA.

 

Deglycosylation with endoglycosidase H

Proteins in the subcellular gradient fractions were deglycosylated using endoglycosidase H (EndoH, Roche Diagnostics, Mannheim, Germany), which cleaves glycoproteins that contain high-mannose N-glycan chains. Fractions were boiled for 10 minutes in 50 mM Na-citrate buffer, pH 5.5 containing 0.1% SDS, supplemented with 0.5% NP40, 40 µg/ml soybean trypsin inhibitor, and 40 µM PMSF, and incubated in the presence or absence of 40 mU/ml endoH for 18 h at 37°C.

 

Primary culture of Xenopus melanotrope cells

To perform immunofluorescence localization studies on the melanotrope cells, a primary culture was made of NILs of X. laevis that were adapted to a black background. Lobes were dissected, washed several times in sterile Xenopus culture medium (XL15: 67% Leibovitz's-15 medium; Life Technologies-BRL), and transferred to Ringer's solution (112 mM NaCl, 2 mM KCl, 2 mM CaCl2, 15 mM Hepes, pH 7.4, and 2 mg/ml glucose) containing 0.25% (wt/vol) trypsin. After incubating for 45 minutes at 20°C, trypsin activity was blocked by adding XL15 medium supplemented with 10% (vol/vol) FCS. The lobes were suspended by 15 passes through a siliconized Pasteur's pipet, transferred to a syringe, and filtered through a nylon filter (pore size 60 µm). Cells were collected by centrifugation, resuspended in a small volume of serum-free XL15 medium, and seeded on poly-L lysine-coated coverslips. After 1 hour, XL15/10% FCS was added and the cells were cultured overnight at 22°C before using them for experiments.

 

Immunofluorescence microscopy

Primary cultures of melanotrope cells were fixed in 2% paraformaldehyde/XPBS, pH 7.4 (XPBS: 67% PBS) for 1 hour on ice. All the subsequent steps were performed at room temperature. The melanotropes were rinsed in XPBS, incubated in 100 mM glycine/XPBS for 30 minutes, and permeabilized by three 5 minutes washes in 0.1% Triton X-100/XPBS (XPBS-T). Antibody incubations were performed sequentially for 1 hour in XPBS-T containing 2% (wt/vol) bovine serum albumin. Texas red-conjugated donkey anti-rabbit antibodies (1:500; Jackson's Laboratories, Pennsylvania) were used to visualize the first antibody-antigen complex. The second antibody-antigen complex (guinea pig anti-POMC) was visualized using FITC-conjugated donkey anti-guinea pig (1:300; Jackson's Laboratories). Finally, coverslips were mounted in Mowiol (10% [wt/vol]; CalBiochem, La Jolla, CA); 15% glycerol; 100 mM Tris-HCl, pH 8.5; 2.5% NaN3) and analyzed with a DM RB/E microscope (Leica Instruments, Nussloch, Germany). Digital images were obtained using a confocal laser-scanning microscope (MRC 1000, Bio-Rad).

 

 

RESULTS

Redistribution of p24 proteins from cis- to pre-Golgi compartments upon activation of Xenopus melanotropes

To establish the distribution of the p24 proteins in biosynthetically active and inactive cells, we performed subcellular fractionation on homogenates of neurointermediate lobes (NILs) and anterior lobes (ALs) of pituitaries of black- and white-adapted Xenopus laevis. In Xenopus adapting to a black background, the intermediate pituitary melanotrope cells are activated and start to produce high amounts of the prohormone POMC, whereas in white animals these cells are virtually inactive (Jenks et al., 1993). The biosynthetic activity of anterior pituitary cells is not influenced by changing the background of the animal, and these cells were used as a control. The subcellular fractionation was accomplished with an iodixanol density gradient and Western blotting was used to determine the migration of the p24 proteins on the gradient. We initially started our p24 studies with members of the p24δ family, of which two (δ1 and δ2) are expressed in the Xenopus melanotrope cells (Kuiper et al., 2000). In the gradient of the active melanotrope cells of black animals, we found both δ1 and δ2 predominantly in fractions 2, 3, and 4, and to a much lesser extent in fraction 5 (Fig. 1A). The ratio between δ1 and δ2 was always ~1:10, as was previously found in unfractionated NIL lysates (Kuiper et al., 2000). Interestingly, in the analysis of the virtually inactive melanotropes of white-adapted animals, the p24δ doublet was found only in fraction 2 (Fig. 1A), again with a ratio similar to what was found for the unfractionated tissues (Kuiper et al., 2000). Also for the moderately active cells of the ALs of both black- and white-adapted animals, the distribution of the p24δ proteins was restricted to fraction 2 (Fig. 1A). Apparently, in less active secretory cells, the p24δ proteins have a restricted steady-state localization (fraction 2), whereas in the biosynthetically active melanotrope cells of black animals these proteins are distributed over a much broader range (fractions 2-5).

To establish which subcellular compartments are present in the various fractions of our gradient, we determined the composition of the fractions by performing both enzymatic assays and Western blot analysis. The lumenal ER marker protein PDI (Hauri and Schweizer, 1992; Sitia and Meldolesi, 1992) was restricted to fractions 7-10 (Fig. 1B). The COPII-subunit Sec23p, which is localized to the ER exit sites and the intermediate compartment (Barlowe et al., 1994; Scales et al., 1997), was found only in fraction 3, and the intermediate compartment marker protein p58 (Saraste and Svensson, 1991) in fractions 3 and 4 (Fig. 1C). Since the first two fractions of the gradient were positive for the Golgi-localized COPI subunits α-COP and γ-COP (Fig. 1B), and displayed α-mannosidase II activity (Fig. 1C), these fractions are likely to contain Golgi membranes. Moreover, using an antibody directed against the neuroendocrine secretory protein secretogranin III (SgIII) (Holthuis et al., 1996), we detected the 61/63 kDa precursor forms of this protein in fractions 1-10, whereas the first cleavage product of SgIII (48 kDa), which was found to be formed only after the precursor form is Tyr-sulfated in the trans-Golgi (Holthuis et al., 1996), was most prominent in fractions 1 and 2, and to a lesser extent in fractions 8-10 (Fig. 1D). Furthermore, other cleavage products of SgIII were found in fractions 1-2 and 8-11 (28 kDa), and in fractions 8 and 9 (20 kDa; Fig. 1D). Together, these findings indicate that the Golgi membranes migrate to fractions 1 and 2 of our gradient, whereas the high-density trans-Golgi network (TGN) and/or the post-TGN compartments (immature and mature secretory granules) are distributed to fractions 8-11 of our gradient. The positions of the marker proteins in the gradient of melanotrope cells of white animals or AL cells of black- or white animals were not different from those in the gradient obtained with melanotropes of black animals, and thus were not influenced by the biosynthetic activity of the cell. Together, these findings suggest that in the inactive intermediate pituitary cells and in the cells of the AL, the steady-state distribution of p24δ is restricted to a single Golgi membrane-containing fraction, whereas in the biosynthetically active melanotrope cells ~60% of the δ proteins has shifted to the higher density fractions that contain subcompartments of the ER (ER exit sites) and/or the intermediate compartment.

Figure 1: Subcellular fractionation of Xenopus pituitary tissues in a 10-30% iodixanol gradient

Neurointermediate lobes (NILs) and anterior lobes (ALs) of pituitary glands of 20 animals were used for fractionation as described in Materials and Methods.

(A) Western blot analysis of the p24δ proteins in the organellar fractions of the NILs and anterior lobes ALs of black- and white-adapted (BA and WA, respectively) Xenopus was performed using the anti-δC antibody. An equivalent of 0.5 NIL/AL was loaded on the gel, except for the inactive NIL of white toads where an equivalent of 2.5 NILs was loaded. The data presented are representative examples of the results obtained from three independent experiments. (B) Western blot analysis demonstrating the localization of the ER marker protein PDI and the Golgi marker α/γCOP in the gradient fractions of BA NIL homogenate. (C) Distribution of marker proteins in the gradient fractions of BA NIL homogenate indicating the position of the ER exit sites and intermediate compartment (Sec23p, ♦; p58, ), as determined by ELISA, and the Golgi apparatus (mannosidase II, ), as obtained using an enzyme assay. (D) Western blot analysis of the neuroendocrine protein secretogranin III (SgIII) in fractions 1-11 (NIL of black-adapted Xenopus), showing the 61/63 kDa precursor form and the 48-, 28-, and 20 kDa processing products of SgIII.

 

The members of the p24 family are assembled into hetero-oligomeric complexes (Belden and Barlowe, 1996; Ciufo and Boyd, 2000; Füllekrug et al., 1999; Gommel et al., 1999; Marzioch et al., 1999), and influence the localization of each other (Dominguez et al., 1998). We therefore investigated whether in the gradient, in addition to p24δ, also other p24 members expressed in the NIL were redistributed to pre-Golgi fractions upon adaptation of the animal to a black background. To identify the Xenopus representatives of the p24α, -β, and -γ subfamilies that are expressed in the NIL, we screened a NIL cDNA library of black-adapted toads, and found p24α3, p24β1, and p24γ3 (Rötter et al., 2002). Western blot analysis of the gradient fractions also analyzed for p24δ (see above) revealed that in the fractionated melanotrope cell homogenates from white-adapted Xenopus, the p24α3, -β1, and -γ3 proteins had a distribution that was restricted to fraction 2, whereas in the analysis of the melanotropes of black-adapted animals ~60% of these proteins migrated to fractions 3 and 4 (Fig. 2), similar to what was observed for the p24δ proteins (Fig. 1A). Thus, the dynamics in the distribution upon physiological activation of the melanotrope cells is similar for the members of the four p24 subfamilies.

Figure 2: Subcellular distribution of Xp24α3, -β1, and -γ3 in Xenopus neurointermediate lobes

Western blot analysis of Xp24α3, -β1, and -γ3 in the first eight subcellular fractions of the neurointermediate lobe (NIL) homogenates of black-adapted and white-adapted Xenopus. The remaining fractions 9-13 were negative for the presence of the respective p24 proteins. An equivalent of 0.5 NIL was loaded on the gel, except for the inactive NIL of white toads where an equivalent of 2.5 NILs was loaded. Antibodies were against the C-terminal region of Xp24α3 or -γ3, or against the lumenal part of human p24β1. The data presented are representative examples of the results obtained from three independent experiments.

 

Distribution of POMC in Xenopus pituitary cells

The major task of the melanotrope cells of the Xenopus intermediate pituitary is the production of the prohormone POMC. This is reflected by an increase in POMC mRNA levels of ~30-fold in the biosynthetically activated cells of black-adapted animals and the fact that POMC represents ~80% of the total of newly synthesized proteins. Thus, in these active melanotropes, POMC is by far the major cargo protein to be transported through the secretory pathway. Since the p24 proteins have been proposed to fulfil an important role in cargo transport between ER and Golgi complex, we analyzed the steady-state localization of POMC in the pituitary cells and compared this localization with that of the p24 proteins. For immunodetection, we used an antibody raised against adrenocorticotropic hormone (ACTH), one of the POMC-derived peptide hormones, recognizing POMC and several of its cleavage products. As expected, the amount of POMC that could be detected in the fractions of the NIL-gradient of black-adapted animals was much higher than in that of white-adapted toads. Moreover, the former gradient showed a number of cleavage intermediates that were also recognized by the ACTH-antibody.

Figure 3: Distribution of the prohormone POMC in the subcellular fractions of Xenopus pituitary homogenates

Western blot analysis of neurointermediate lobe (NIL) homogenates of both black- and white-adapted (BA and WA, respectively) Xenopus, and the anterior lobe (AL) of BA toads. For immunolabeling of POMC, an antibody directed against ACTH was used. Blots of the WA NIL and BA AL were exposed considerably longer than that of the BA NIL. The data presented are representative examples of the results obtained from threeindependent experiments.

 

These POMC cleavage intermediates, which, as for the cleavage products of SgIII (Fig. 1D), indicate the presence of late-Golgi and post-Golgi compartments, were found mainly in fractions 2 and 3 (23- and 16 kDa) and fractions 7-10 (<16 kDa), and could not be observed in the gradient of the NIL of white-adapted animals (our unpublished results). In the gradient of active melanotropes, the 37-kDa precursor form of POMC was abundantly present in fractions 2-7, with highest amounts in fraction 5, whereas in the gradient of inactive melanotropes, POMC was found almost exclusively in fraction 2 (Fig. 3). In cells of the AL, POMC had a somewhat broader distribution than in the melanotropes of white toads (fractions 2-4), but also in this case, POMC migrated predominantly to fraction 2 (Fig. 3). Thus, the high rate of POMC biosynthesis in the melanotropes of black animals clearly results in a steady-state distribution of POMC that is different from that in the melanotropes of white animals and the cells of the AL, an observation similar to what was found for the p24 proteins. Although the composition of fractions 5 and 6 could not be determined with marker proteins, the POMC molecules present in these fractions, which are N-linked glycosylated, were sensitive to endoH (our unpublished results), indicating that these POMC molecules did not pass the medial-Golgi (Kornfeld and Kornfeld, 1985). On >the basis of this observation, together with the finding that the cis- and medial-Golgi, and the intermediate compartment were localized to fractions 1-4, we conclude that the major pool of POMC in fractions 5/6 may be located to the ER. It therefore appears that the high levels of POMC that are produced in the melanotrope cells of black-adapted animals can be found almost exclusively in the early compartments of the secretory pathway, with highest amounts in the ER. Although the distribution of POMC in the gradient of melanotropes of black animals does not completely overlap with that of the p24 proteins, there was a considerable amount of codistribution of these proteins (Figs. 1-3). To further investigate the degree of overlap in the localization of the p24 proteins and POMC, we performed double-labelling immunolocalization experiments with anti-δ2 and anti-POMC antibodies (Fig. 4). To circumvent any interference of POMC-derived cleavage products present in the late secretory pathway, we used for the immunodetection of POMC an antibody that recognizes only the precursor form of POMC. In the melanotropes of black toads, part of the POMC-staining was present in distinct perinuclear structures that completely overlapped with the staining pattern of δ2, whereas a substantial amount of POMC could be detected as a diffuse staining throughout the melanotrope cell that was not overlapping with δ2 (Fig. 4). In the melanotropes of white-adapted animals, hardly any POMC could be detected, except for a limited number of cells that showed some POMC-staining in perinuclear structures (our unpublished results). This latter finding is in line with previous results showing that a small subpopulation of melanotropes of white animals is active (De Rijk et al., 1990). In the POMC-producing corticotrope cells of the AL (~10% of the total population of AL cells), the immunolabeling of POMC often showed a typical Golgi-staining that, despite their comigration on the density gradient, had only a limited overlap with δ2 and in some cases the staining patterns of the two proteins were totally different (Fig. 4). In this connection it is important to note that the diffuse staining pattern of POMC that was observed throughout the active melanotrope cells was not found in the POMC-producing cells of the AL (Fig. 4), suggesting that this diffuse pattern is not background staining, but rather represents POMC molecules localized to the ER. Thus, a complete overlap between POMC and δ2 was observed only in the perinuclear structures in the melanotropes of black toads. Together, these data indicate that in the biosynthetically active melanotrope cells of the NIL, the localization of POMC partly overlaps with that of the p24 proteins in early secretory pathway compartments, while an additional pool of POMC can be found in p24-negative subdomains of the ER.

 

The localization of p24 is linked to POMC biosynthesis

The observed differences in subcellular localization of the p24 proteins between the melanotropes of black- and white-adapted animals may be directly related to the very different biosynthetic activities of these cells. To explore this possibility, we investigated whether blocking protein synthesis would affect p24 localization in active melanotropes. NILs of black-adapted toads were incubated for one hour in the presence of cycloheximide, and subjected to subcellular fractionation and Western blot analysis. The distributions of α- and γ-COP were determined to establish the reproducibility among the various fractionation experiments (see Materials and Methods). In this experiment α- and γ-COP were found to be restricted to fraction 1. Upon cycloheximide treatment, the biosynthesis of POMC was decreased (our unpublished results).

Figure 4: Double-immunofluorescence labeling of Xp24δ2 and POMC in primary cultured pituitary cells

Melanotrope cells of the neurointermediate lobe (NIL) and a number of POMC-producing cells of the anterior lobe (AL) of black-adapted Xenopus were used. The amount of POMC-producing cells in a cell suspension of the AL is ~10%. Antibodies used were the affinity-purified anti-δ2 antibody, and the anti-POMC antibody ST-62 that specifically recognizes the precursor form and not the cleavage products of POMC. Bar: 10 µm.

 

Furthermore, at steady-state levels, POMC had shifted towards the top of the gradient (Fig. 5). Thus, cycloheximide treatment of biosynthetically active NILs resulted in a significant decrease of POMC levels in the higher-density ER-containing fractions. As shown in Fig. 5, the p24 proteins δ1 and δ2 distributed in the gradient of the untreated NILs to fractions 1-5, with most immunoreactivity in fraction 2. Upon treatment of the biosynthetically active NILs with cycloheximide, δ1 and δ2 redistributed towards lower-density fractions, with the highest levels in fraction 1. Again, as was found in the case of the physiologically manipulated NILs (Figs. 1 and 2), this redistribution of the δ proteins was accompanied by a similar shift in the distribution of γ3 (our unpublished observations). Thus, blocking of protein synthesis in the active melanotropes results in a redistribution of the subcellular localization of the p24 proteins.

 

Effect of brefeldin A on p24 localization in Xenopus melanotropes

The difference in the steady-state localization of the p24 proteins between active and inactive melanotrope cells could be the result of a true redistribution of p24 to other subcellular compartments or, alternatively, may reflect activity-dependent differences in p24 cycling behavior. To be able to distinguish between these two possibilities, we tried to interfere with any continuous p24 cycling in the active melanotropes using the fungal metabolite brefeldin A (BFA), and then analyzed the subcellular distribution of the p24 proteins. BFA is known to block COPI-mediated transport, resulting in a dispersion of the Golgi complex and a block in forward transport (Lippincott-Schwartz et al., 1989; Orci et al., 1991). Several cycling components of the ER-Golgi interface, including members of the p24 family, have been described to accumulate in structures localized to the periphery of the BFA-treated cell (Blum et al., 1999; Füllekrug et al., 1999; Rojo et al., 1997; Saraste and Svensson, 1991).

Figure 5: The effect of cycloheximide on the subcellular localization of p24δ1 and δ2 in biosynthetically active melanotrope cells

NILs of black-adapted Xenopus were incubated in the absence or presence of 50 µg/ml cycloheximide for one hour at 22°C, the homogenates were subjected to subcellular fractionation and were analyzed by Western blotting (the first five fractions are shown). The distributions of α- and γ-COP were determined to enable comparisons between the various subcellular fractionation experiments (see Materials and Methods). Higher exposures revealed that POMC again migrated further into the gradient (as far as fraction 9) compared to the p24δ proteins (fraction 1-5). The experiment was carried out in duplicate with similar results.

 

Therefore, redistribution of p24 proteins after treatment with BFA serves as a good indication that these proteins are recycling from the Golgi apparatus (Füllekrug et al., 1999). In Xenopus melanotrope cells, BFA effectively inhibits transport and processing of POMC (Braks et al., 1996). Immunofluorescence analysis of BFA-treated Xenopus melanotrope cells revealed a drastic redistribution of p24δ2 towards peripheral structures (Fig. 6A), similar to what was found for other members of the p24 family (Blum et al., 1999; Füllekrug et al., 1999; Rojo et al., 1997). Moreover, subcellular fractionation of BFA-treated NILs revealed that the p24δ proteins were predominantly present in fraction 2 of the gradient, clearly different from the broader distribution observed in untreated cells (compare Figs. 6B and 1A). Thus, upon BFA-treatment, the majority of the p24δ proteins that originally appeared in the pre-Golgi compartments (fractions 3 and 4) redistributed towards lower-density structures, suggesting that this pool of p24 proteins is indeed actively cycling between ER and Golgi complex.

Figure 6: The effect of brefeldin A on the subcellular localization of p24δ1 and δ2 in biosynthetically active melanotrope cells

(A) Immunofluorescence analysis of primary cultured melanotrope cells of black-adapted Xenopus after a two-hour incubation with 5 µg/ml BFA using the affinity-purified Xp24δ2 antibody. Bar: 5 µm. (B) Western blot analysis of subcellular fractions of BFA-treated NILs; experimental conditions as under A. The distributions of α- and γ-COP were determined to enable comparisons between the various subcellular fractionation experiments. The experiment was carried out in duplicate with similar result.

 

 

DISCUSSION

We have analyzed the steady-state subcellular distribution of members of the four subfamilies of p24 proteins (p24α, -β, -γ, and -δ) during different states of cellular biosynthetic activity. For this study, we used the physiologically inducible POMC-producing melanotrope cells of the Xenopus intermediate pituitary. These cells regulate background adaptation of this animal and their biosynthetic activity can vary from virtually inactive (in white-adapted toads) to highly active (in black animals). The analysis of the two physiological states of the melanotropes revealed an interesting correlation between the subcellular sites of p24 localization and biosynthetic cell activity. Using subcellular fractionation, the p24 proteins of the biosynthetically active melanotropes distributed to fractions 2-5, including to those containing the intermediate compartment and ER exit sites. In contrast, the p24 proteins of the inactive melanotrope cells were found in the low-density Golgi-containing fraction 2, similar to the situation for the non-induced, moderately active anterior pituitary cells that were used as a control. Furthermore, blocking of protein synthesis in the active melanotropes also caused a redistribution of the p24 proteins to lower-density fractions. From these results we conclude that the distribution of the p24 proteins is varying between cis-Golgi and pre-Golgi compartments, depending on the biosynthetic activity of the cells.

Melanotrope cell activation is accompanied by a drastic increase in the amount of the cargo protein POMC that has to be transported through the secretory pathway. This extremely high level of cargo transport requires an increase in the capacity of the whole transport machinery, which results in the coordinate upregulation of the expression of a number of proteins, including members of the p24 family (Holthuis et al., 1995a; Holthuis et al., 1995b; Kuiper et al., 2000; Rötter et al., 2002). We therefore considered the possibility that the appearance of the higher-density p24-containing compartments in the biosynthetically active melanotropes could be caused simply by an increase in the amount of cargo leading to higher densities of these structures, rather than reflecting a true shift in the steady-state localization of the p24 proteins to other compartments. However, our subcellular fractionation data indicate that the localization of the p24 proteins changes relative to markers of the early secretory pathway, which suggests that the density of these compartments is not influenced by the high amounts of POMC produced in the active melanotropes. Earlier studies on the localization of p24 family members in transfected tumor cells in culture have revealed that overexpression of p24 proteins can result in the appearance of artificial membranous structures, especially when individual members are overexpressed (Dominguez et al., 1998; Emery et al., 2000; Füllekrug et al., 1999; Rojo et al., 2000). We believe that the change in distribution of the p24 proteins observed here is not caused by such a phenomenon, since the biosynthetic activation of the melanotrope cells is a physiological process that is necessary for efficient, high-level transport and processing of POMC. Together, we conclude that Xenopus melanotrope cell activation leads to the localization of p24 proteins in a broad spectrum of compartments, including the cis-Golgi, the intermediate compartment and, most likely, the ER exit sites.

Similar to what was observed for the p24 proteins, the subcellular localization of POMC was found to be dependent on the biosynthetic activity of the cell. In the gradient containing the NIL compartments of white toads, POMC comigrated with the p24 proteins to the low-density fraction 2. In the subcellular fractions containing the highly active NIL compartments of black toads, however, only low amounts of the prohormone were localized to the low-density fraction, while the majority was found in a number of higher-density fractions. Hence, both the p24 family members and the cargo protein POMC are distributed over a broader range of subcellular compartments when the melanotrope cells become active. However, POMC and the p24 proteins were not completely overlapping in the active melanotrope cells, since a substantial amount of POMC migrated further into the density gradient (mainly to fraction 5; Fig. 3). This finding is in line with our immunofluorescence data with primary melanotrope cells in culture, showing that structures containing p24δ2 were always positive for POMC, while a substantial amount of POMC was not colocalizing with δ2 and was present as a diffuse staining throughout the melanotrope cell. Thus, two pools of POMC could be identified in the melanotrope cells of black toads. The first pool, which distributed to fractions 2-4 of the density gradient in Fig. 3 and colocalized with the p24 proteins, is likely present in the ER exit sites, and the intermediate- and cis-Golgi compartments. The second, p24-negative pool of POMC is, although only partially overlapping with the ER marker PDI, most likely localized to (subdomains of) the ER, since i) it was sensitive to endoH and thus did not pass the medial-Golgi, and ii) migrated to fractions 5-7 (in Fig. 3), while the Golgi- and intermediate compartments are in the first four fractions of the gradient. Together, these data indicate that the high expression of POMC in the melanotropes of black animals leads to a steady-state localization of this prohormone in the early compartments of the secretory pathway. The p24-negative pool of POMC may represent freshly-made molecules that just entered the ER lumen, which may suggest that in these cells ER exit is a rate-limiting step in POMC biosynthesis.

The very high level of POMC biosynthesis in the melanotropes of black toads requires a highly active and efficient ER-to-Golgi transport machinery. Our observation that the p24 proteins have a broader subcellular distribution in the highly active melanotrope cells may be a direct result of this increased vesicular transport. This assumption was confirmed by our experiments with BFA, a metabolite that is known to interfere with the cycling of components between ER and Golgi complex (Füllekrug et al., 1997; Füllekrug et al., 1999). BFA-treatment of active melanotropes led to a redistribution of pre-Golgi-localized p24 molecules towards low-density Golgi-like peripheral structures. Thus, the observed change in steady-state p24 localization actually indicates that the dynamics of p24 cycling has changed, and that in the highly active melanotropes the time period that the p24 proteins reside in the ER and intermediate compartment is longer than in less active cells.

The hypothesis that p24 proteins play a role in protein transport has been well accepted (Kaiser, 2000). Our finding that the sites of localization of p24 proteins in the secretory pathway of Xenopus melanotropes are linked to the biosynthetic activity of the cell is in line with this hypothesis. How can this notion be correlated to existing models for p24 function? The ability of some members of the p24 family to interact with coatomer and their enrichment in COPI-coated vesicles led to the hypothesis that p24 proteins could act as coatomer receptors, driving and regulating the formation of COPI-coated vesicles (Goldberg, 2000; Nickel and Wieland, 1997; Sohn et al., 1996). However, the pre-Golgi compartments that accumulate high amounts of p24 proteins in the biosynthetically active Xenopus melanotropes do not contain COPI, suggesting that an additional role for the p24 proteins may exist in these compartments. Several functional models have been proposed in which the p24 proteins fulfil a regulatory role during the inclusion of cargo in transport vesicles at the ER membrane. For instance, p24 proteins could act as cargo receptors, directly interacting with cargo and thereby facilitating cargo inclusion in COPII-coated vesicles at the ER membrane (Muñiz et al., 2000; Schimmöller et al., 1995). Furthermore, since in yeast the p24 proteins are not essential for COP-mediated vesicular transport in the early secretory pathway, they may be indirectly involved in a selection mechanism during vesicle formation through the active exclusion of misfolded cargo proteins (as part of the quality control mechanism) or ER resident proteins (Elrod Erickson and Kaiser, 1996; Kaiser, 2000; Springer et al., 2000; Wen and Greenwald, 1999). Finally, p24 proteins have been proposed to delay the budding process, enabling correct packaging of cargo in vesicle buds, or to create membrane rafts that define nucleation sites for the generation of vesicles and tubules (Kaiser, 2000; Lavoie et al., 1999; Rojo et al., 2000). All of these models have in common that the p24 proteins would control the selectivity during cargo packaging at the ER membrane. In the biosynthetically active melanotropes of black-adapted Xenopus, the extremely high level of POMC biosynthesis may increase the need for regulation at this stage, which would explain the shift in steady-state distribution of the p24 proteins in these cells during the process of background adaptation of the animal.

 

 

ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. J. Bergeron, N. Bulleid, M. Green, T. Nilsson, J. Saraste, R. Schekman, S. Tanaka, and F. Wieland who generously provided us with antibodies (see Materials and Methods). Furthermore, we thank Ron Engels for animal care and Maarten van den Hurk for technical assistance. This work was supported by grant 805-33-150 from the Netherlands Organization for Scientific Research-Earth and Life Sciences (NWO-ALW) and by European Union-Training and Mobility of Researchers (EU-TMR) research network ERB-FMRX-CT960023.

 

 

REFERENCES

Balch, W. E., McCaffery, J. M., Plutner, H. and Farquhar, M. G. (1994). Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell 76, 841-852.

Bannykh, S. I. and Balch, W. E. (1997). Membrane dynamics at the endoplasmic reticulum-Golgi interface. Journal of Cell Biology 138, 1-4.

Bannykh, S. I., Rowe, T. and Balch, W. E. (1996). The organization of endoplasmic reticulum export complexes. J Cell Biol 135, 19-35.

Barlowe, C. (1998). COPII and selective export from the endoplasmic reticulum. Biochimica et Biophysica Acta 1404, 67-76.

Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M. and Schekman, R. (1994). COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895-907.

Belden, W. J. and Barlowe, C. (1996). Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J Biol Chem 271, 26939-26946.

Berghs, C. A., Tanaka, S., Van Strien, F. J., Kurabuchi, S. and Roubos, E. W. (1997). The secretory granule and pro-opiomelanocortin processing in Xenopus melanotrope cells during background adaptation. Journal of Histochemistry and Cytochemistry 45, 1673-82.

Blum, R., Pfeiffer, F., Feick, P., Nastainczyk, W., Kohler, B., Schafer, K. H. and Schulz, I. (1999). Intracellular localization and in vivo trafficking of p24A and p23. Journal of Cell Science 112, 537-548.

Braks, J. A., van Horssen, A. M. and Martens, G. J. M. (1996). Dissociation of the complex between the neuroendocrine chaperone 7B2 and prohormone convertase PC2 is not associated with proPC2 maturation. European Journal of Biochemistry, 505-510.

Ciufo, L. F. and Boyd, A. (2000). Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway. Journal of Biological Chemistry 275, 8382-8.

De Rijk, E. P., Jenks, B. G. and Wendelaar Bonga, S. E. (1990). Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation. General and Comparative Endocrinology 79, 74-82.

Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J. J., Solari, R. C. and Owen, M. J. (2000). The p24 family member p23 is required for early embryonic development. Current Biology 10, 55-8.

Dominguez, M., Dejgaard, K., Füllekrug, J., Dahan, S., Fazel, A., Paccaud, J. P., Thomas, D. Y., Bergeron, J. J. and Nilsson, T. (1998). gp25l/emp24/p24 protein family members of the cis-Golgi network bind both Cop I and II coatomer. Journal of Cell Biology 140, 751-65.

Elrod Erickson, M. J. and Kaiser, C. A. (1996). Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations. Molecular Biology of the Cell 7, 1043-1058.

Emery, G., Rojo, M. and Gruenberg, J. (2000). Coupled transport of p24 family members. Journal of Cell Science 113 ( Pt 13), 2507-16.

Füllekrug, J., Sonnichsen, B., Schafer, U., Nguyen Van, P., Soling, H. D. and Mieskes, G. (1997). Characterization of brefeldin A induced vesicular structures containing cycling proteins of the intermediate compartment/cis- Golgi network. FEBS Lett 404, 75-81.

Füllekrug, J., Suganuma, T., Tang, B. L., Hong, W., Storrie, B. and Nilsson, T. (1999). Localization and Recycling of gp27 (hp24gamma3): Complex Formation with Other p24 Family Members. Molecular Biology of the Cell 10, 1939-1955.

Gerich, B., Orci, L., Tschochner, H., Lottspeich, F., Ravazzola, M., Amherdt, M., Wieland, F. and Harter, C. (1995). Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth. Proc Natl Acad Sci U S A 92, 3229-33.

Goldberg, J. (2000). Decoding of sorting signals by coatomer through a GTPase switch in the COPI coat complex. Cell 100, 671-9.

Gommel, D., Orci, L., Emig, E. M., Hannah, M. J., Ravazzola, M., Nickel, W., Helms, J. B., Wieland, F. T. and Sohn, K. (1999). p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway. FEBS Letters 447, 179-85.

Graham, J. M. (1993). Biomembrane Protocols (Graham, J.M. and Higgins J.A., eds), Humana Press, Totowa, NJ 19, 1-18.

Hauri, H. P., Kappeler, F., Andersson, H. and Appenzeller, C. (2000). ERGIC-53 and traffic in the secretory pathway. Journal of Cell Science 113, 587-96.

Hauri, H. P. and Schweizer, A. (1992). The endoplasmic reticulum-Golgi intermediate compartment. Current Opinion in Cell Biology 4, 600-8.

Holthuis, J. C., Jansen, E. J. R. and Martens, G. J. M. (1996). Secretogranin III is a sulfated protein undergoing proteolytic processing in the regulated secretory pathway. Journal of Biological Chemistry 271, 17755-60.

Holthuis, J. C., Jansen, E. J. R., van Riel, M. C. and Martens, G. J. M. (1995a). Molecular probing of the secretory pathway in peptide hormone-producing cells. Journal of Cell Science 108, 3295-305.

Holthuis, J. C., van Riel, M. C. and Martens, G. J. M. (1995b). Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary. Biochem J 312, 205-213.

Jenks, B. G., Leenders, H. J., Martens, G. J. M. and Roubos, E. W. (1993). Adaptation physiology: the functioning of pituitary melanotrope cells during background adaptation of the amphibian Xenopus laevis. Zool. Science 10, 1-11.

John, D. C., Grant, M. E. and Bulleid, N. J. (1993). Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit. EMBO Journal 12, 1587-95.

Kaiser, C. (2000). Thinking about p24 proteins and how transport vesicles select their cargo. Proc Natl Acad Sci U S A 97, 3783-3785.

Kornfeld, R. and Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54, 631-64.

Kuehn, M. J., Herrmann, J. M. and Schekman, R. (1998). COPII-cargo interactions direct protein sorting into ER-derived transport vesicles. Nature 391, 187-90.

Kuiper, R. P., Waterham, H. R., Rötter, J., Bouw, G. and Martens, G. J. M. (2000). Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell. Molecular Biology of the Cell 11, 131-40.

Lavoie, C., Paiement, J., Dominguez, M., Roy, L., Dahan, S., Gushue, J. N. and Bergeron, J. J. (1999). Roles for alpha(2)p24 and COPI in Endoplasmic Reticulum Cargo Exit Site Formation. Journal of Cell Biology 146, 285-300.

Lin, C. C., Love, H. D., Gushue, J. N., Bergeron, J. J. and Ostermann, J. (1999). ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro. Journal of Cell Biology 147, 1457-72.

Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S. and Klausner, R. D. (1989). Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56, 801-13.

Marzioch, M., Henthorn, D. C., Herrmann, J. M., Wilson, R., Thomas, D. Y., Bergeron, J. J., Solari, R. C. and Rowley, A. (1999). Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex. Molecular Biology of the Cell 10, 1923-1938.

Mizuno, M. and Singer, S. J. (1993). A soluble secretory protein is first concentrated in the endoplasmic reticulum before transfer to the Golgi apparatus. Proc Natl Acad Sci U S A 90, 5732-6.

Muñiz, M., Nuoffer, C., Hauri, H. P. and Riezman, H. (2000). The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles. Journal of Cell Biology 148, 925-30.

Nickel, W. and Wieland, F. T. (1997). Biogenesis of COP I-coated transport vesicles. FEBS Letters 413, 395-400.

Orci, L., Tagaya, M., Amherdt, M., Perrelet, A., Donaldson, J. G., Lippincott Schwartz, J., Klausner, R. D. and Rothman, J. E. (1991). Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae. Cell 64, 1183-1195.

Rojo, M., Emery, G., Marjomaki, V., McDowall, A. W., Parton, R. G. and Gruenberg, J. (2000). The transmembrane protein p23 contributes to the organization of the Golgi apparatus. Journal of Cell Science 113, 1043-57.

Rojo, M., Pepperkok, R., Emery, G., Kellner, R., Stang, E., Parton, R. G. and Gruenberg, J. (1997). Involvement of the transmembrane protein p23 in biosynthetic protein transport. Journal of Cell Biology 139, 1119-35.

Rothman, J. E. and Wieland, F. T. (1996). Protein sorting by transport vesicles. Science 272, 227-34.

Rötter, J., Kuiper, R. P., Bouw, G. and Martens, G. J. M. (2002). Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors. Journal of Cell Science 115, 1049-58.

Saraste, J. and Svensson, K. (1991). Distribution of the intermediate elements operating in ER to Golgi transport. Journal of Cell Science 100 ( Pt 3), 415-30.

Scales, S. J., Pepperkok, R. and Kreis, T. E. (1997). Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 90, 1137-48.

Schekman, R. and Orci, L. (1996). Coat proteins and vesicle budding. Science 271, 1526-33.

Schimmöller, F., Singer Krüger, B., Schröder, S., Krüger, U., Barlowe, C. and Riezman, H. (1995). The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO Journal 14, 1329-39.

Schweizer, A., Fransen, J. A., Matter, K., Kreis, T. E., Ginsel, L. and Hauri, H. P. (1990). Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. European Journal of Cell Biology 53, 185-96.

Sitia, R. and Meldolesi, J. (1992). Endoplasmic reticulum: a dynamic patchwork of specialized subregions. Molecular Biology of the Cell 3, 1067-72.

Sohn, K., Orci, L., Ravazzola, M., Amherdt, M., Bremser, M., Lottspeich, F., Fiedler, K., Helms, J. B. and Wieland, F. T. (1996). A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding. Journal of Cell Biology 135, 1239-48.

Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S. and Schekman, R. (2000). The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 97, 4034-9.

Storrie, B. and Madden, E. A. (1990). Isolation of subcellular organelles. Methods in Enzymology 182, 203-25.

Van Eys, G. J. and Van den Oetelaar, P. (1981). Cytological localization of alpha-MSH, ACTH and beta-endorphin in the pars intermedia of the cichlid teleost Sarotherodon mossambicus. Cell and Tissue Research 215, 625-33.

Wen, C. and Greenwald, I. (1999). p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans. Journal of Cell Biology 145, 1165-75.

 

 

 

Chapter 4: Targeting Xp24δ2 transgene expression specifically to the intermediate pituitary melanotrope cells of Xenopus laevis

Targeting Xp24δ2 transgene expression specifically to the intermediate pituitary melanotrope cells of Xenopus laevis

 

 

Gerrit Bouw, Eric J.R. Jansen, Rick van Huizen and Gerard J.M. Martens

Part of this chapter was published (November 2003) in a Hot Topic issue on Xenopus Genomics in Current Genomics

 

 

ABSTRACT

Members of the p24 family of type I transmembrane proteins (designated p24α, -β, -γ and -δ) share several characteristics, such as an N-terminal putative cargo binding domain, a coiled-coil region and a short cytoplasmic tail, which for some family members can bind to coatomer. These proteins are highly abundant in transport vesicles cycling between the endoplasmic reticulum and the Golgi apparatus, and are thought to be involved in cargo-selective transport. In our model system, the intermediate pituitary melanotrope cell of the amphibian Xenopus laevis, a number of members of the p24 family, but not all, are coordinately expressed with the prohormone proopiomelanocortin (POMC). POMC is highly expressed and the major cargo protein in the melanotrope cells of black-adapted Xenopus. In this study, the technique of stable transgenesis of Xenopus laevis is explored as a tool to manipulate the expression of one of the members of the Xenopus p24 family (Xp24δ2). For this purpose, p24δ2 was tagged with the green fluorescent protein GFP. Expression of the Xp24δ2-GFP fusion protein was targeted to the melanotrope cells using a Xenopus POMC gene promoter fragment. Southern blot analysis revealed ~1-5 sites of integration and ~10-20 copies of the exogenous DNA in the genome that corresponded with transgene expression levels. Western blot analysis showed expression of the Xp24δ2-GFP fusion protein specifically in the melanotrope cells of transgenic Xenopus and a competition of Xp24δ2-GFP expression with endogenous p24δ2 expression. The endogenous p24 proteins were displaced by the overexpression of Xp24δ2-GFP. Immunocytochemistry on brain tissue slices from Xenopus transgenic for Xp24δ2-GFP showed expression of the fusion protein in cells that stained for POMC and α-MSH. Immunoelectron microscopy revealed that the fusion protein was localized to structures that resemble ER and Golgi. Furthermore, two-dimensional gel electrophoretic analysis of proteins extracted from melanotrope cells of transgenic and control animals revealed a difference in protein patterns. In conclusion, we successfully targeted expression of Xp24δ2-GFP specifically to the melanotrope cells and characterized the transgene product in vivo.

 

 

INTRODUCTION

Protein trafficking between the endoplasmic reticulum (ER) and the Golgi involves vesicles and numerous polypeptides that are necessary to maintain anterograde and retrograde transport. The vesicles are composed of lipids and transmembrane proteins, and carry selected cargo from the ER to the Golgi. One of the major constituents of transport vesicles is the p24 family of putative cargo receptors (Nickel et al., 1997; Sohn et al., 1996). These proteins have been classified into four main subfamilies, designated p24α, -β, -γ and -δ (Dominguez et al., 1998). Between subfamilies, members have only a low degree of amino acid sequence identity (17-30%). The p24 proteins are type I transmembrane proteins that share several characteristics such as a large lumenal domain with two conserved cysteine residues that form a disulfide bridge, a transmembrane region located at the C-terminal end and a short cytoplasmic tail containing COP I and COP II binding motifs (Fiedler et al., 1996). Extensive studies on the coatomer-binding motifs have shown that p24 proteins largely depend on these motifs for traveling from the ER to the Golgi and back (Barlowe, 1998; Dominguez et al., 1998; Sohn et al., 1996). In yeast and mammalian cells, it was shown that p24 proteins form functional heterotetrameric complexes containing one representative of each subfamily (Belden and Barlowe, 1996; Füllekrug et al., 1999; Marzioch et al., 1999). Additional proof for functional complex formation came from studies on yeast mutants and knockout mice lacking a single p24 member (Denzel et al., 2000; Marzioch et al., 1999). Furthermore, co-immunoprecipitation and immunofluorescence studies have shown that certain subfamily members are specifically excluded from functional complexes indicating that these complexes are involved in specialized sorting or trafficking events in the early secretory pathway (Emery et al., 2000). Since p24 proteins are highly abundant in both COP I- and COP II-coated transport carriers, these proteins have been suggested to play a role in cargo selective protein transport at the ER / Golgi interface. Several models explaining the role of p24 in vivo have evolved in the last few years. First, p24 proteins may act as cargo receptors, selecting the cargo that has to be transported to the Golgi or retrieved from the Golgi back to the ER. Since p24 proteins have divergent lumenal domains and have the ability to form oligomeric complexes, a combination of p24 complexes could exhibit a variety of binding sites for selected cargo (Stamnes et al., 1995). Other evidence for this model came from the group of Riezman who showed by cross-linking experiments that Emp24p (=p24β1) is directly required for efficient packaging of a lumenal cargo protein, Gas1p, into ER-derived vesicles (Muñiz et al., 2000). Finally, deletion of two yeast p24 genes independently caused activation of the unfolded protein response and accumulation of cargo in the ER (Belden and Barlowe, 2001), which supports a function for p24 proteins in cargo transport. Secondly, the fact that p24 proteins are very abundant in membrane compartments of the early secretory pathway and their ability to form complexes led to the idea that they play a role in membrane dynamics. Recent studies showed that p23 (=p24δ1) is necessary for early embryonic development in mice (Denzel et al., 2000) and that p24 proteins are not required for vesicular transport in yeast (Springer et al., 2000). Furthermore, the group of Denzel also showed that inactivation of one p23 allele in mice induced structural changes in Golgi morphology. This observation was supported by the work of Rojo, which showed that overexpression of p23 leads to an accumulation of smooth ER membranes and changes the ultrastructure of the CGN and Golgi membranes. Anterograde and retrograde transport of VSV-G cargo proteins was not affected (Rojo et al., 2000). The group of Bergeron found that p24α2 is involved in ER cargo exit site formation and suggested a structural role for the p24 proteins (Lavoie et al., 1999). Together, these data suggest that p24 proteins are involved in organizing the membranes of the early secretory pathway. Third, p24 proteins have been suggested to play a role in the ER quality control system. Evidence for this model came from studies in C. elegans where sel-9, a member of the p24β subfamily, was found to play a role in the negative regulation of transport of two cargo proteins to the cell surface. A reduced activity of sel-9 caused a mutant cargo protein to reach the plasma membrane that would otherwise accumulate within the cell (Wen and Greenwald, 1999). Furthermore, additional roles for p24 have been suggested: p24 proteins act as negative regulators of vesicle budding, delaying the budding process to allow efficient segregation of cargo away from ER residents (Elrod Erickson and Kaiser, 1996); p24 proteins exclude ER resident proteins from the lumen of vesicles by sterically occupying the space within the lumen (Kaiser, 2000; Springer et al., 2000).

Taken together, the available data do not provide a clear answer with respect to the function of p24 proteins. To tackle this question, several approaches have been used, including many cell biological techniques. To investigate the role of p24 proteins in vivo, the need of a model system may be advantageous for this purpose. We therefore use the South African clawed toad Xenopus laevis. This amphibian is capable of adapting its skin color to the background. The secretion of α-Melanophore Stimulating Hormone into the blood regulates this process. This bioactive peptide is cleaved from the precursor hormone pro-opiomelanocortin (POMC) in the melanotrope cells of the neurointermediate pituitary (NIL). POMC is the major cargo protein in this cell type and during adaptation to a black background the amount of POMC mRNA increases 30-fold and cell activity and cell size increase dramatically. Placing the animal on a white or a black background allows physiological manipulation of biosynthetic and secretory activity of the melanotrope cell, which can be used as a tool to study processes in the early secretory pathway. Using a differential screening approach, we identified a member of the Xenopus p24 family (p24δ2) that is differentially expressed in the melanotrope cell of black- and white-adapted animals (Holthuis et al., 1995b). Additional screening of a NIL cDNA library revealed all members of the p24 family that are expressed in the Xenopus melanotrope cell. During black background adaptation, some of the p24 family members are upregulated together with POMC (Xp24α3, -β1, -γ3 and -δ2), whereas others are not or only slightly induced (Xp24γ2 and -δ1) (Rötter et al., 2002). The coordinate expression of a selective set of p24 proteins suggests that these proteins assemble into a tetrameric complex, which is involved in the transport of POMC from the ER to the Golgi. In order to study the role of p24 proteins in more detail, we utilize the technique of stable Xenopus transgenesis. This technique has been developed by Kroll and Amaya (Kroll and Amaya, 1996) and simplified by the group of Mohun (Sparrow et al., 2000). Since its development, transgenesis in Xenopus has proven useful for several expression experiments and promoter studies. Xenopus transgenesis is a rapid, efficient, simple and inexpensive way of making large amounts of transgenic embryos per day. In the present study, we combine this technique with the unique properties of the Xenopus intermediate pituitary melanotrope cell by using a Xenopus POMC gene promoter fragment to target the expression of the Xp24δ2 protein tagged with the green fluorescent protein GFP to these cells. These transgenic animals were characterized and can now be used for functional studies.

 

 

MATERIALS AND METHODS

Generation of DNA constructs

A 630-bp DNA fragment containing nucleotides –54bp to +561 of the Xenopus p24δ2 open reading frame was amplified by PCR using Xenopus clone X1262 (Holthuis et al., 1995b) as a template. The primers used were: p24δ2 5’:-AACCTCTAAGCCT-CAATACCAGGATT, and X1262 5’:AAGCCTAGGCTACCG. The pPOMC-p24δ2-EGFP fusion construct was generated first by inserting the PCR fragment into the pEGFPN3∆AUG vector (Clontech, modified in this lab) to create the fusion and then by cloning the fusion construct into the pPOMC-pCS2+ vector containing the Xenopus POMC gene A promoter (Jansen et al., 2002). The fusion construct was checked by cycle sequencing using the Big Dye Ready Reaction system (Perkin Elmer) and the primer 5’-AATCCTAGATTCTAAATTCT reading from EGFP into the p24δ2 gene. Figure 1 presents a schematic overview of construct pPOM-p24δ2-EGFP with the Xenopus p24δ2 open reading frame fused to EGFP cDNA (Fig. 1A) and the expression in COS-1 cells to check the reading frame (Fig. 1B).

 

Preparation of Xenopus unfertilized eggs

Mature female Xenopus (Xenopus Express, Cape Town, South Africa) were injected with 375 iU human gonadotrope hormone (HCG; Pregnyl, Organon, The Netherlands) 18 hrs before harvesting the unfertilized eggs. Eggs were then collected from the females, dejellied in 2 % cysteine/1xMMR (pH 8.2) and immediately used for transgenesis. All animal experiments were carried out in accordance with the European Communities Council Directive 86/609/EEC for animal welfare, and permit TRC 99/15072 to generate and house transgenic Xenopus laevis.

 

Generation of Xenopus embryos transgenic for p24δ2-GFP

A 2166-bp SalI/NarI fragment containing the pPOMC-p24δ2-EGFP construct and the SV40 poly-A signal (Fig. 2A) was purified using Qiaex II Gel Extraction Kit (Qiagen). This fragment was mixed with sperm nuclei, incubated for 15’ at room temperature and diluted to 500 µl. About 10 nl was injected per egg. Sperm nuclei were prepared as described previously (Huang et al., 1999; Sparrow et al., 2000). Normally cleaving embryos were selected at the 4-cell stage and cultured in 0.1xMMR/6% Ficoll-400 with 50 µg/ml Gentamycin at 18°C until gastrulation (stage 12) was reached. Then, embryo culturing was continued in 0.1xMMR with 50 µg/µl Gentamycin at 22°C. From stage 45 onwards, tadpoles were raised in tap water at 22 ºC. The presence of GFP fluorescence was examined in living embryos anaesthetized with 0.25 mg/ml MS222 (3-aminobenzoic acid ethyl ester, Sigma) using a Leica MZ FLIII fluorescent stereomicroscope and photographs were taken with a Leica DC200 color camera using the Leica DCviewer software. Staging of Xenopus embryos was carried out according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967).

 

Genomic DNA analysis

Genomic DNA was isolated either from liver of transgenic frogs or from whole tadpoles, as described previously (Ausubel et al., 2001). 5 µg DNA, or the total amount isolated from one tadpole, was digested with HindIII in a volume of 100 µl, precipitated and separated on an 0.7% agarose gel. The gel was depurinated with 0.25 M HCl and the DNA was transferred to Nytran Super Charge nylon transfer membrane (Schleicher and Schuell) in 40 mM NaOH. The blot was then prehybridized in Church (0.5 M Na –P, 7 % SDS, 10 mM EDTA) for 30 min, at 68 °C. Twenty-five ng EcoRI/XbaI fragment of the pPOMC-p24δ2-EGFP construct (corresponding to the open-reading frame of p24δ2-EGFP) or 25 ng BamHI/XbaI fragment of the pPOMC-GFP construct (corresponding to the open-reading frame of GFP) was labelled with 32P using the Random Primers Labeling system (Gibco-BRL) and 32P dCTP (Amersham-Pharmacia Biotech), and used for hybridization at 68 °C in Church. The blot was subsequently washed until 0.5 x SSC/0.1 % SDS at 22 °C and exposed to Kodak X-Omat X-ray film at -70 °C or quantified using a PhosphoImager (Biorad).

 

Antibodies

The rabbit polyclonal antibodies against portions of the lumenal and C-terminal regions of Xp24δ2 (anti-1262N and anti-1262C, respectively), against part of the lumenal region of Xp24δ1 (anti-RH6) and against a region in the lumenal part of Xp24α3 have been described previously (Kuiper et al., 2001; Rötter et al., 2002). A polyclonal antibody directed against Xenopus POMC (ST62, recognizing only the precursor form) was kindly provided by Dr. S. Tanaka (Shizuoka University, Japan; Berghs et al., 1997) and to GFP by Dr. J. Fransen (Nijmegen University, The Netherlands; Cuppen et al., 1999).

 

Cryosectioning and immunohistochemistry

Brain-pituitary preparations were dissected from juvenile transgenic frogs and fixed in 4% paraformaldehyde in PBS. After cryoprotection in 10% sucrose-PBS, sagittal 20 µm cryosection were mounted on poly-L-lysine coated slides and dried for 2 h at 45 ºC. To study GFP expression, sections were directly viewed under the fluorescent microscope. For immunohistochemistry, sections were rinsed for 30 min in 50 µM Tris buffered saline (pH 7.6), containing 150 µM NaCl and 0.1% Triton X100 (TBS-TX). To prevent nonspecific binding, blocking was performed with 0.5 % BSA in TBS-TX. Antisera directed against the POMC-derived adrenocorticotrope hormone (ACTH) (Van Eys and Van den Oetelaar, 1981), the POMC precursor protein, the p24δ1 protein and the p24δ2 proteins were used as primary antibodies, diluted in TBS-Tx containing 0.5% BSA. Primary antibodies were incubated for 16 hrs at 37°C. After rinsing in TBS-TX, the slides were incubated with a second antibody, Goat-anti-Rabbit-TexasRed (Jackson ImmunoResearch Laboratories, Inc, West Grove, USA) at a dilution of 1:50 for 1 h at 37 ºC. Following an additional washing step, the sections were mounted in Citifluor (Agar Scientific Ltd, Stansted, Essex, UK) and coverslipped. Immunofluorescence was viewed under a Leica DM RA fluorescent microscope and digital photographs were taken with a Cohu High Performance CCD Camera using the Leica Q Fluoro software.

 

Metabolic cell labelling and immunoprecipitation analysis

Neurointermediate lobes (NILs) and anterior lobes (ALs) of pituitaries from transgenic and wild type Xenopus were dissected and starved in methionine- and cysteine-free XL-15 medium (67% L-15 medium, Gibco-BRL, Gaithersburg, MD, USA) for 30 minutes at 22 °C. The lobes were subsequently pulsed in methione-and cysteine-free medium containing 5 mCi/ml 35S- Promix (Amersham-Pharmacia Biotech) for 3 h at 22 ºC. The lobes were then homogenized on ice in lysis buffer (50 mM Hepes pH 7.2, 140 mM NaCl, 1% Tween 20, 0,1% Triton X100, 0,1% deoxycholic acid, 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.1 mg/ml soyabean trypsin inhibitor). The lysates were incubated on ice for 15 minutes, cleared by centrifugation (7 min, 13000 g, 4ºC) and supplemented with 0,1 % SDS. Ten percent of the lysates was directly separated on SDS-PAGE (total lysates), while the remainder was used for immunoprecipitation, employing a 1:500 dilution of an anti-GFP antiserum (kindly provided by Dr. B. Wieringa, Department of Cell biology, University of Nijmegen, The Netherlands, Cuppen et al., 1999). Immune complexes were precipitated with protein-A Sepharose (Amersham-Pharmacia Biotech) and resolved by SDS-PAGE, and radiolabelled proteins were visualized by fluorography.

 

Immunoelectron microscopy

Ultrastructural localisation studies were performed on juvenile neurointermediate lobes of control- and transgenic Xenopus expressing Xp24δ2-GFP. Entire lobes were fixed for one hour at room temperature in 2% paraformaldehyde (PFA) + 0.01% glutaraldehyde in PHEM buffer (50 mM MgCl 2 , 70 mM KCl, 10 mM EGTA, 20 mM HEPES, 60 mM PIPES, pH 6.8). Fixed tissue was stored in 1% PFA in 0.1M phosphate buffer until use. Ultrathin cryosectioning was performed as described before (Fransen et al., 1985; Schweizer et al., 1988). Sections were incubated with an antiserum against EGFP at a 1:100 dilution (Cuppen et al., 1999) followed by protein A complexed with 10 nm gold (Fransen et al., 1985). Electron microscopy was performed using a Jeol 1010 electron microscope operating at 80 kV.

 

 

RESULTS

Our goal is to elucidate the role of p24 employing stable Xenopus transgenesis. To examine various steps in the transgenesis procedure (number of copies and sites of DNA integration, level of transgene expression, competition of transgene product with endogenous protein and morphology of transgenic melanotrope cells), we used the Xp24δ2 protein fused to GFP.

 

Generation of the Xp24δ2-GFP fusion construct

We cloned the entire open-reading frame of Xenopus p24δ2 cDNA lacking the stop codon in front of GFP cDNA into the CMV-driven pEGFP-N3 vector. For this purpose, a PCR reaction with specific primers was performed on plasmid DNA harboring Xenopus intermediate pituitary cDNA clone X1262 (=Xp24δ2) (Holthuis et al., 1995a), resulting in a 624-bp PCR-fragment containing the restriction sites EcoRI and BamHI. The EGFP-N3 vector was modified such that the start ATG of GFP cDNA was lacking. The resulting construct contained p24δ2 N-terminally fused to GFP followed by a poly-adenylation signal (construct pEGFPN3-Xp24δ2; Fig. 1A) and was checked by cycle DNA sequencing. This construct was transiently transfected into COS-1 cells to verify the Xp24δ2-GFP open-reading frame and to examine the intracellular localization of the fusion protein using fluorescence microscopy. The fusion protein was found to be localized close to the nucleus and perinuclear in structures that resemble ER and Golgi, respectively (Fig. 1B). Generation of soluble GFP through internal translational initiation did apparently not occur since the cytoplasm was devoid of fluorescence.

Figure 1: Generation and analysis of the Xp24δ2-GFP fusion construct.

(A) The Xp24δ2-GFP fusion construct. CMV indicates the human cytomegalovirus (CMV) immediate early promoter; EGFP is enhanced green fluorescent protein; pA is a SV40 early mRNA polyadenylation signal. (B) Fluorescent microscopy analysis of the fusion protein expressed in transiently transfected COS-1 cells.

 

Generation of the pPOMC-Xp24δ2-GFP transgene construct and of stable and non-mosaic parent (F0) Xenopus transgenic for Xp24δ2-GFP

For transgenic overexpression, we selected one of the Xenopus melanotrope p24 proteins coexpressed with POMC, namely the Xp24δ2 protein, and fused it to the N-terminus of GFP. For this, the entire open-reading frame of Xenopus p24δ2 (clone X1262, (Holthuis et al., 1995a) was cloned in front of GFP in a pEGFP-N3 vector lacking the start ATG of GFP (to prevent internal translational initiation). To verify the open-reading frame, the construct was transiently transfected into COS-1 cells. The fusion protein was found to be localized close to the nucleus and perinuclear in structures that resemble ER and Golgi, respectively (data not shown). For the transgenic studies, the Xp24δ2-GFP insert of the EGFPN3-Xp24δ2 construct was cloned into a Xenopus POMC gene promoter (pPOMC) fragment containing pCS2+ vector resulting in construct pPOMC-pCS-Xp24δ2-GFP (Fig. 2A). In this approach, the 529-bp Xenopus POMC gene A promoter fragment of the pPOMC-CS2+ vector directs transgene expression to the melanotrope cells of the Xenopus intermediate pituitary (Jansen et al., 2002). The DNA fragment used for injection was a linear 2166-bp SalI / NarI fragment of the pPOMC-pCS-Xp24δ2-GFP construct (Fig. 2A). To generate Xenopus transgenic for the pPOMC-Xp24δ2-GFP fragment, the recently described (Kroll and Amaya, 1996) and subsequently modified (Sparrow et al., 2000) method for stable Xenopus transgenesis was used. Healthy looking Xenopus embryos were selected at the 4-cell stage, grown and screened for fluorescence during early development. The first Xp24δ2-GFP expression in the transgenic embryos was observed around stage 25 (i.e. before hatching) in an area around the presumptive eye. This expression became gradually more restricted, eventually leading to specific expression in the pituitary during development to the tadpole stage. A number of injection experiments resulted in animals transgenic for Xp24δ2-GFP and expressing the fusion protein at different levels, which was directly visible in the living embryo under a fluorescence microscope (Fig. 2B).

 

In vitro fertilization of Xenopus eggs using sperm cells transgenic for pPOMC-Xp24δ2-GFP

The testes of male transgenic Xenopus frogs were isolated and used to generate F1 offspring by in vitro fertilization of eggs harvested from wild type Xenopus females. Simply incubating the eggs with pieces of testes generated a few hundred embryos in one round of fertilization. Screening of these embryos for the degree of GFP fluorescence in the pituitary revealed that ~25% of the embryos were negative in that they showed no visible expression of the fusion protein, while among the remainder various levels of Xp24δ2-GFP expression could be detected (Fig. 2C). The embryos were divided into embryos expressing no, a moderate level and a high level of the fusion protein, and the groups were separately grown at 26°C with extensive feeding such that after four months they became frogs sufficiently large to be analyzed. Remaining pieces of testes were used to isolate sperm nuclei that were stored at -80°C for future injection experiments. Injection of these stored nuclei resulted in percentages of Xp24δ2-GFP-expressing embryos similar to those obtained by in vitro fertilization. Using this technique we obtained three independent F1 lines (referred to as #115, #124 and #125) that differed in Xp24δ2-GFP expression levels.

 

Southern blot analysis of transgenic Xenopus

To examine the number of copies and sites of integration of the transgene, we isolated genomic DNA from liver tissue of transgenic F1 lines #115, #124 and #125, and wild-type frogs. HindIII-digested genomic DNA was analyzed on a southern blot using a probe corresponding to the open-reading frame of GFP (Fig. 2D) The number of integration sites were 4, 5 and 1 sites of integration for #115, #124 and #125 F1 transgenic animals, respectively. The presence of the 2166-bp and 3340-bp hybridizing fragments were the result of head-to-tail and head-to-head concatemerization, and indicated that the transgene had integrated into the genome in concatemers of ~25, ~20 and 2 copies per genome for #115, #124 and #125, respectively, as determined by quantitative densitometric analysis of the southern blot. Interestingly, in F1 line #124 both expressing and non-expressing animals showed similar amounts of integrated copies of the transgene. However, in addition to the sites containing multiple copies that were also present in the non-expressing animals, the expressing animals harboured two sites with only one or two transgene copies (Fig. 2D, asterisks). Thus, a single or low amount of integrated copies of the transgene appeared to determine whether the fusion protein was expressed or not, indicating that most of the integrated transgenes were silenced. The silencing of the multiple, in-tandem transgene copies could be the result of DNA methylation, as has been observed previously in transgenic plants (Matzke et al., 2002; Muskens et al., 2000) and higher vertebrates (Henikoff, 1998).

Figure 2: Generation of Xenopus transgenic for Xp24δ2-GFP

(A) Schematic representation of the pPOMC-Xp24δ2-GFP construct and the 2166-bp SalI-NarI linear injection fragment containing the Xenopus POMC gene A promoter fragment (pPOMC) and the Xp24δ2-GFP fusion protein-coding sequence was used to generate transgenic Xenopus tadpoles. (B) Pituitary-specific fluorescence in transgenic Xenopus embryos. Shown are living stage 45 embryos, whereby the arrows indicate the locations of the pituitaries with various levels of transgene expression. Fluorescent pituitaries expressing the transgene fusion product could be detected from stage 25 onwards. Bar equals 0.4 mm. (C) F1 offspring of a male Xenopus transgenic for pPOMC-Xp24δ2-GFP. The sperm of a transgenic male frog was used to fertilize eggs from a wild type female. The degree of fluorescence is indicated by (+). Bar equals 0,5 mm. (D) Southern blot analysis of #124 transgenic animals. Genomic liver DNA was digested with HindIII and analyzed with the open reading frame of GFP as a probe.

 

Microscopy on transgenic Xenopus intermediate pituitary melanotrope cells expressing the Xp24δ2-GFP fusion protein

For microscopy analysis, we dissected the neurointermediate lobe (NIL), consisting of the pars nervosa with the intermediate pituitary attached to it, and the anterior lobe (AL) of the pituitary. We observed a bright fluorescent signal in the POMC-producing melanotrope cells of the intermediate pituitary. No clear GFP signal was detected in the AL in which the POMC-producing corticotrope cells are located (Fig. 3A). To investigate whether the fusion protein was indeed targeted specifically to the melanotrope cells, we performed immunocytochemistry on frozen tissue sections of brains (with the pituitary attached) of Xenopus transgenic for Xp24δ2-GFP and wild-type animals. We used antibodies directed against α-MSH and POMC to stain the melanotrope cells and compared the staining pattern with the Xp24δ2-GFP-induced fluorescent signal. We found that the fluorescence was restricted to the cells that were stained for α-MSH and POMC, indicating that the expression of Xp24δ2-GFP was targeted specifically to the melanotrope cells of the pituitary (data not shown). For the localization of the Xp24δ2-GFP fusion protein in the transgenic melanotrope cells at the ultrastructural level, we performed immunoelectron microscopy using a GFP antibody. Xp24δ2-GFP was localized to structures that resemble the ER and the Golgi (Fig. 3C), in line with previous findings showing that p24 proteins shuttle between the ER and the Golgi (Barlowe, 1998; Dominguez et al., 1998; Sohn et al., 1996).

 

Western blot analysis of Xp24δ2-GFP fusion protein in transgenic Xenopus melanotrope cells

To analyze the steady-state expression level of Xp24δ2-GFP relative to endogenous p24δ expression, we prepared lysates of NILs and Als of transgenic Xenopus expressing the fusion protein. Western blot analysis of the tissue lysates confirmed the restricted expression of the fusion protein in the NIL. Equal amounts of Xp24δ1 and -δ2 were found in the NIL of a wild-type animal (Fig 3B, lane 1). The NIL lysate of a transgenic animal contained a protein of ~49 kDa that likely represented the Xp24δ2-GFP fusion protein, since it was of the expected size (24 kDa for Xp24δ2 and 25 kDa for GFP) and in the wild-type NIL this protein was not detected (Fig. 3B, lanes 1 and 2). In the AL of both transgenic and wild-type animals, no fusion protein was expressed (Fig. 3B, lanes 3 and 4). In the transgenic NIL, the expression of the Xp24δ2-GFP protein led to reduced amounts of the endogenous Xp24δ1 and -δ2 proteins (Fig. 3B, lane 2). No effect on endogenous levels of Xp24δ1 and -δ2 in the AL of transgenic Xenopus could be observed (Fig. 3B, lanes 3 and 4).

Figure 3: Analysis of Xp24δ2-GFP fusion protein expression in transgenic Xenopus intermediate pituitary cells

(A) Fluorescence is specific to the intermediate pituitary of transgenic Xenopus. Ventro-caudal view on the brain that was lifted to reveal the bright fluorescence caused by the Xp24δ2-GFP fusion protein and observed in the intermediate lobe (IL), but not in the anterior lobe (AL), of the pituitary of a black-adapted transgenic frog of 6 months. Bar equals 0,9 mm. (B) Tissue lysates of Nils and Als from wild-type and transgenic animals were dissected and subjected to SDS-PAGE. Western blot analysis was performed using a mixture of Xp24δ2- and Xp24δ1-specific antibodies. (C) Pituitary glands from transgenic frogs (F1 #224, expressing high levels of Xp24δ2-GFP) were subjected to immunoelectron microscopical analysis. Left panel shows an overview of a melanotrope cell of a black-adapted transgenic animal, bar equals 1 µm. For immunodetection, the GFP antibody was used in combination with protein-A-gold to visualize the Xp24δ2-GFP fusion protein, bars equal 0,1 µm.

 

 

DISCUSSION

In the past few years, several important steps in fundamental biological science have been taken. The various genome projects helped to understand the molecular basis of proteins encoded by the DNA. However, to fully understand the function of the proteins encoded by the DNA, the need for model systems in which functional analysis of proteins can be performed close to the in vivo situation is obvious. We use the melanotrope cells of the South African clawed toad frog Xenopus laevis as a model for studying the function of proteins in vivo. In contrast to problems observed with transgenic zebra fish and mice, the Xenopus transgenics animals have stable, non-mosaic integration of the transgene in a one-cell stage. An additional unique feature of Xenopus is its background adaptation. By placing the animal on a black background the melanotrope cells start to produce large amounts of the POMC-derived hormone α-MSH which is responsible for dispersion of the melanin in skin melanophores. Together with the POMC gene, a number of genes that play a role in the early secretory pathway are switched on during black-background adaptation. In this way, cell activity can be manipulated very easily. We use these unique features together with stable Xenopus transgenesis to study the function of the p24 family of putative cargo receptors in vivo. The technique of stable Xenopus transgenesis has been developed recently and allows stable insertion of a transgene into the genome of Xenopus laevis and targeting the expression of the transgenes to specific tissues using tissue specific promoters.

This study shows that the technique of stable Xenopus transgenesis can be a powerful technique to manipulate in vivo the expression of a protein of unknown function. We show that a member of the p24 family of putative cargo receptors tagged with GFP is specifically targeted to, the melanotrope cells of the pituitary in Xenopus laevis, our model system. Overexpression of this fusion protein resulted in a down regulation of endogenous p24δ1 and -δ2. In order to study the role of p24 proteins, we tagged the Xp24δ2 protein with GFP and targeted the expression of this fusion protein specifically to the melanotrope cells by using a 529-bp fragment of the POMC promoter to drive its expression. We cloned the open reading frame of Xp24δ2 in front of GFP using the EGFPN-3 vector without the start codon of GFP. The start codon was removed to prevent internal initiation of translation, which would result in expression of GFP only and not of the fusion protein. Initial attempts to clone the open reading frame into the EGFP-N1 vector and express this construct in the pituitary of Xenopus failed due to reasons that remain unclear. The N1 vector differs from the N3 vector in that the reading frame of the multiple cloning site is shifted two bases. Furthermore, in this vector the start codon of GFP was still present. Transfection of the N1-based construct in COS-1 cells resulted in expression of the fusion protein in the ER and Golgi area. However, no fluorescence was observed in the pituitary when the N1-based fusion constructs were used in transgenesis (Bouw, G., Wels, M., unpublished observations). Although the localization of Xp24δ2-GFP was not checked with appropriate markers, we conclude that the fusion protein is present in structures throughout the cell and in the perinuclear area, reminiscent of ER and Golgi structures, respectively. Furthermore, localization studies in COS-1 cells generally show that the degree of overexpression influences steady state localization of the protein.

In order to make Xenopus transgenic for Xp24δ2-GFP, we subcloned the fusion construct into the pPOMC-CS2+ vector containing the Xenopus POMC promoter fragment driving expression of the transgene. We observed that injection of a linear fragment containing the promoter, the open reading frame and a poly-A translation termination signal instead of circular DNA or linearized vector was the most efficient way to obtain transgenic Xenopus. Injection of linearized vector dramatically increased the number of transgenic animals per injection round which is in line with previous results published (Sparrow et al., 2000). We found the transgene to be successfully integrated into the genome of Xenopus. The number of copies of the integrated transgene varied among transgenic animals and the degree of expression of the Xp24δ2-GFP fusion protein correlated with the number of integrations.

The expression patterns observed for Xp24δ2-GFP in early embryonic development of transgenic Xenopus resemble those seen in Xenopus transgenic for POMC promoter-driven GFP (Jansen et al., 2002). Furthermore, these patterns are in line with the expression of endogenous POMC-derived α-MSH in developing Xenopus (Kramer et al., 2003). In some cases in the tadpole stage expression of the transgene Xp24δ2-GFP was observed in brain areas other than the pituitary. This could be due to the integration of the transgene in regions of the genome that harbor highly active brain genes. Microscopical examination of transgenic melanotrope cells revealed a clear colocalization of Xp24δ2-GFP with both α-MSH and POMC, suggesting that the POMC promoter fragment indeed targeted the expression of the fusion protein to the melanotrope cells. Furthermore, immunoelectron microscopy using a GFP antibody showed staining in structures that resemble ER, Golgi and phagosomes. The latter can be explained by the fact that in highly active cells, Xp24δ2-GFP containing membranous transport intermediates are rapidly turned over. We have not been successful in proving that the fusion protein detected by the GFP antibody is indeed Xp24δ2-GFP (by using Xp24δ2 specific antibodies). However we could not detect any cytoplasmic or nuclear staining using the GFP antibody suggesting that generation of soluble GFP through internal translational initiation did apparently not occur.

Overexpression of the Xp24δ2-GFP fusion protein had several effects on the endogenous levels of p24 proteins. First, the endogenous level of Xp24δ2 in the melanotrope cells was lower in transgenic animals than in control animals. Possibly, the overexpression of the POMC promoter fragment causes the utilization of a major portion of transcription factors such that endogenous proteins are expressed less. However, experiments involving the use of other transgenes driven by the same promoter fragment have shown that expression of endogenous POMC was not affected (Jansen et al., 2002). In addition, driving transgene expression by using the POMC promoter fragment did not result in differences in POMC levels between wild-type and transgenic NILs of black-adapted animals (data not shown), indicating that the amount of transcription factors interacting with the POMC promoter is not limiting. Therefore, a more likely explanation is that the expression of p24 proteins is very tightly regulated and that the cell allows only a certain amount of p24 proteins to be present. In such a case, the Xp24δ2-GFP fusion protein would compete with and take over the function of the endogenous protein, whereby, due to the GFP tag at its C-terminus, the fusion protein may act as a dominant negative mutant (e.g. possible interference with COP I / II binding). In future experiments, we will investigate the effect of overexpression of the fusion protein on ER to Golgi transport of the major cargo protein POMC in the Xenopus melanotrope cells. Interestingly, the p24δ1 protein was found to be down regulated as well, although to a much lesser extent than the p24δ2 protein. This down regulation of δ1 may be explained by the high degree of amino acid sequence identity between these two p24 family members. Excessive amounts of p24δ2 would then lead to a decrease in the level of both endogenous p24δ1 and p24δ2 proteins in the cell.

In order be able to perform a number of experiments with animals transgenic for Xp24δ2-GFP, an F1 generation of Xenopus transgenic for this transgene was made using testes from male transgenic animals. The differences in expression of the fusion protein in animals of the same offspring could be due to the different inheritance of the parental chromosomes with the integrated transgene. Since these integrations were random, all kinds of combinations of parental genes could cause these differences, dependent on the number of integration sites on different chromosomes. We also injected nuclei, which were harvested from the transgenic testes after one round of in vitro fertilization with these testes, and obtained similar numbers of transgenics that expressed Xp24δ2-GFP in the pituitary. To check whether the negatively scored animals did indeed not express Xp24δ2-GFP, we analyzed these animals together with the positive animals on southern blot and concluded that to our surprise, there was integration of the transgene. Interestingly, in the expressing animals a low amount of copies was integrated in a site separate from sites in non-expressing animals. This suggests that a single additional integration site appeared to determine whether the fusion protein was expressed or not, indicating that most integrated transgenes were silenced. The silencing of the transgenes could be the result of DNA methylation and has been observed in transgenic plants (Matzke et al., 2002; Muskens et al., 2000) and vertebrates (Henikoff, 1998). Using in vitro fertilization we obtained three independent lines of F1 animals from three different parental transgenic animals. The three lines showed different levels of Xp24δ2-GFP expression in the pituitary that corresponded with the number of integrations analyzed by southern blotting and levels Xp24δ2-GFP expression seen on western blot analysis of the pituitaries. In the case of one line, the offspring animals showed a high level of expression of Xp24δ2-GFP in other areas than the pituitary, unlike the parental animal. A likely explanation for this phenomenon is that the transgene initially integrated in silenced parts of the genome in the parental animal and became active in the F1 offspring animals.

 

 

ACKNOWLEDGEMENTS

We would like to thank Ron Engels for animal care, Tony Coenen, Coen van der Meij and Huib Croes for technical assistance with confocal- and (immuno)electronmicroscopy, respectively, and Michiel Wels for cloning and transgenesis. We also thank Drs Irene Schulz, Tommy Nilsson, Wim Van de Ven, Jack Fransen and Shige Tanaka for providing antibodies. This work was supported by grant 811.38.002 from the Netherlands Organization for Scientific Research Earth and Life Sciences (NWO-ALW).

 

 

REFERENCES

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (2001). in: Current Protocols in Molecular Biology. 2.2.1-2.2.3.

Barlowe, C. (1998). COPII and selective export from the endoplasmic reticulum. Biochimica et Biophysica Acta 1404, 67-76.

Belden, W. J. and Barlowe, C. (1996). Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J Biol Chem 271, 26939-26946.

Belden, W. J. and Barlowe, C. (2001). Deletion of yeast p24 genes activates the unfolded protein response. Molecular Biology of the Cell 12, 957-69.

Berghs, C. A., Tanaka, S., Van Strien, F. J., Kurabuchi, S. and Roubos, E. W. (1997). The secretory granule and pro-opiomelanocortin processing in Xenopus melanotrope cells during background adaptation. Journal of Histochemistry and Cytochemistry 45, 1673-82.

Cuppen, E., Wijers, M., Schepens, J., Fransen, J., Wieringa, B. and Hendriks, W. (1999). A FERM domain governs apical confinement of PTP-BL in epithelial cells. Journal of Cell Science 112 ( Pt 19), 3299-308.

Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J. J., Solari, R. C. and Owen, M. J. (2000). The p24 family member p23 is required for early embryonic development. Current Biology 10, 55-8.

Dominguez, M., Dejgaard, K., Füllekrug, J., Dahan, S., Fazel, A., Paccaud, J. P., Thomas, D. Y., Bergeron, J. J. and Nilsson, T. (1998). gp25l/emp24/p24 protein family members of the cis-Golgi network bind both Cop I and II coatomer. Journal of Cell Biology 140, 751-65.

Elrod Erickson, M. J. and Kaiser, C. A. (1996). Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations. Molecular Biology of the Cell 7, 1043-1058.

Emery, G., Rojo, M. and Gruenberg, J. (2000). Coupled transport of p24 family members. Journal of Cell Science 113 ( Pt 13), 2507-16.

Fiedler, K., Veit, M., Stamnes, M. A. and Rothman, J. E. (1996). Bimodal interaction of coatomer with the p24 family of putative cargo receptors. Science 273, 1396-1399.

Fransen, J. A., Ginsel, L. A., Hauri, H. P., Sterchi, E. and Blok, J. (1985). Immuno-electronmicroscopical localization of a microvillus membrane disaccharidase in the human small-intestinal epithelium with monoclonal antibodies. European Journal of Cell Biology 38, 6-15.

Füllekrug, J., Suganuma, T., Tang, B. L., Hong, W., Storrie, B. and Nilsson, T. (1999). Localization and Recycling of gp27 (hp24gamma3): Complex Formation with Other p24 Family Members. Molecular Biology of the Cell 10, 1939-1955.

Henikoff, S. (1998). Conspiracy of silence among repeated transgenes. BioEssays 20, 532-5.

Holthuis, J. C., Jansen, E. J. R., van Riel, M. C. and Martens, G. J. M. (1995a). Molecular probing of the secretory pathway in peptide hormone-producing cells. Journal of Cell Science 108, 3295-305.

Holthuis, J. C., van Riel, M. C. and Martens, G. J. M. (1995b). Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary. Biochem J 312, 205-213.

Huang, K. M., D'Hondt, K., Riezman, H. and Lemmon, S. K. (1999). Clathrin functions in the absence of heterotetrameric adaptors and AP180-related proteins in yeast. EMBO Journal 18, 3897-3908.

Jansen, E. J. R., Holling, T. M., van Herp, F. and Martens, G. J. M. (2002). Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis. FEBS Letters 516, 201-7.

Kaiser, C. (2000). Thinking about p24 proteins and how transport vesicles select their cargo. Proc Natl Acad Sci U S A 97, 3783-3785.

Kramer, B. M., Claassen, I. E., Westphal, N. J., Jansen, M., Tuinhof, R., Jenks, B. G. and Roubos, E. W. (2003). Alpha-melanophore-stimulating hormone in the brain, cranial placode derivatives, and retina of Xenopus laevis during development in relation to background adaptation. Journal of Comparative Neurology 456, 73-83.

Kroll, K. L. and Amaya, E. (1996). Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173-83.

Kuiper, R. P., Bouw, G., Janssen, K. P., Rötter, J., van Herp, F. and Martens, G. J. M. (2001). Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell. Biochemical Journal 360, 421-9.

Lavoie, C., Paiement, J., Dominguez, M., Roy, L., Dahan, S., Gushue, J. N. and Bergeron, J. J. (1999). Roles for alpha(2)p24 and COPI in Endoplasmic Reticulum Cargo Exit Site Formation. Journal of Cell Biology 146, 285-300.

Marzioch, M., Henthorn, D. C., Herrmann, J. M., Wilson, R., Thomas, D. Y., Bergeron, J. J., Solari, R. C. and Rowley, A. (1999). Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex. Molecular Biology of the Cell 10, 1923-1938.

Matzke, M. A., Aufsatz, W., Kanno, T., Mette, M. F. and Matzke, A. J. (2002). Homology-dependent gene silencing and host defense in plants. Advances in Genetics 46, 235-75.

Molloy, M. P., Herbert, B. R., Walsh, B. J., Tyler, M. I., Traini, M., Sanchez, J. C., Hochstrasser, D. F., Williams, K. L. and Gooley, A. A. (1998). Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19, 837-44.

Muñiz, M., Nuoffer, C., Hauri, H. P. and Riezman, H. (2000). The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles. Journal of Cell Biology 148, 925-30.

Muskens, M. W., Vissers, A. P., Mol, J. N. and Kooter, J. M. (2000). Role of inverted DNA repeats in transcriptional and post-transcriptional gene silencing. Plant Molecular Biology 43, 243-60.

Nickel, W., Sohn, K., Bunning, C. and Wieland, F. T. (1997). p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway. Proc Natl Acad Sci U S A 94, 11393-8.

Nieuwkoop, P. D. and Faber, J. (1967). in: Normal Table of Xenopus laevis (Daudin). 2nd edn. Elsevier Amsterdam.

Rojo, M., Emery, G., Marjomaki, V., McDowall, A. W., Parton, R. G. and Gruenberg, J. (2000). The transmembrane protein p23 contributes to the organization of the Golgi apparatus. Journal of Cell Science 113, 1043-57.

Rötter, J., Kuiper, R. P., Bouw, G. and Martens, G. J. M. (2002). Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors. Journal of Cell Science 115, 1049-58.

Schweizer, A., Fransen, J. A., Bachi, T., Ginsel, L. and Hauri, H. P. (1988). Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. Journal of Cell Biology 107, 1643-53.

Sohn, K., Orci, L., Ravazzola, M., Amherdt, M., Bremser, M., Lottspeich, F., Fiedler, K., Helms, J. B. and Wieland, F. T. (1996). A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding. Journal of Cell Biology 135, 1239-48.

Sparrow, D. B., Latinkic, B. and Mohun, T. J. (2000). A simplified method of generating transgenic Xenopus. Nucleic Acids Research 28, E12.

Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S. and Schekman, R. (2000). The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 97, 4034-9.

Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P. and Rothman, J. E. (1995). An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding [published erratum appears in Proc Natl Acad Sci U S A 1995 Nov 7;92(23):10816]. Proc Natl Acad Sci U S A 92, 8011-5.

Van Eys, G. J. and Van den Oetelaar, P. (1981). Cytological localization of alpha-MSH, ACTH and beta-endorphin in the pars intermedia of the cichlid teleost Sarotherodon mossambicus. Cell and Tissue Research 215, 625-33.

Wen, C. and Greenwald, I. (1999). p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans. Journal of Cell Biology 145, 1165-75.

 

 

 

Chapter 5: A cell-specific transgenic approach in Xenopus reveals the importance of a functional p24 system for a secretory cell

A cell-specific transgenic approach in Xenopus reveals the importance of a functional p24 system for a secretory cell

 

 

Gerrit Bouw, Rick Van Huizen, Eric J.R. Jansen Karel P.C. Janssen and Gerard J.M. Martens

Molecular Biology of the Cell, 15(3): ….-…. (2004)

 

 

ABSTRACT

The p24α, -β, -γ and -δ proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24δ2 specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24δ2 protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing.INTRODUCTION

Transport of cargo proteins through the early secretory pathway involves cargo selection, transport vesicle formation, quality control to recycle misfolded cargo, and cycling of the COPI- and COPII-coated vesicles between the endoplasmic reticulum (ER) and Golgi (Barlowe, 2000). One of the major constituents of the transport vesicles is the p24 family of type I transmembrane proteins that can be classified into four main subfamilies, designated p24α, -β, -γ and -δ (Dominguez et al., 1998; Nickel et al., 1997; Schimmoller et al., 1995; Sohn et al., 1996; Stamnes et al., 1995). The p24 proteins share a number of structural characteristics, such as a relatively large lumenal putative cargo-binding domain, a coiled-coil region thought to be involved in the formation of multimeric p24 complexes, a transmembrane region, and a short cytoplasmic tail containing COPI- and COPII-binding motifs that are used for p24 travelling from the ER to the Golgi and back (for review, see Kaiser, 2000). In yeast and mammalian cells, p24 proteins form functional heterotetrameric complexes containing one representative of each subfamily, whereby the composition of the complex may differ in various cell types (Belden and Barlowe, 2001; Ciufo and Boyd, 2000; Dominguez et al., 1998; Emery et al., 2000; Füllekrug et al., 1999; Marzioch et al., 1999). Furthermore, the stability of the p24 members appears to be compromised when cells are deficient in the expression of a single p24 protein (Denzel et al., 2000; Marzioch et al., 1999). Recent evidence suggests a complex and dynamic p24 system of mostly monomers and homo-/hetero-dimers, and that the degree of oligomerization constantly alters and largely depends on the subcellular localizations of the p24 subfamily members (Jenne et al., 2002).

The p24 proteins have been suggested to play a key role in cargo-selective protein transport at the ER / Golgi interface (Kaiser, 2000). For the elusive mechanism of action of p24, a number of functional models have been proposed, including a role as cargo receptor, membrane organizer, or regulator of vesicle budding, as well as in the ER quality control system, or excluding ER resident proteins from the vesicular lumen (Belden and Barlowe, 2001; Bremser et al., 1999; Denzel et al., 2000; Elrod Erickson and Kaiser, 1996; Kaiser, 2000; Lavoie et al., 1999; Muñiz et al., 2000; Rojo et al., 1997; Schimmöller et al., 1995; Springer et al., 2000; Wen and Greenwald, 1999). Defining the importance of a functional p24 system for proper cell physiology has however turned out to be difficult. For instance, deletion of all p24 proteins resulted in viable yeast (Marzioch et al., 1999; Springer et al., 2000), whereas genetic ablation of a single p24 family member caused early lethality in mice (Denzel et al., 2000). To investigate the significance of the p24 system in a highly specialized secretory cell, we decided to use a physiological model (background adaptation of the South-African clawed frog Xenopus laevis) with a well-defined secretory cell (the intermediate pituitary melanotrope cell) and its single major soluble cargo protein proopiomelanocortin (POMC) (Roubos, 1997). In the trans-Golgi network (TGN) / immature secretory granules of Xenopus melanotrope cells, endoproteolytic cleavage of POMC results in a number of bioactive peptides, including α-melanophore-stimulating hormone (α-MSH). This hormone mediates adaptation of the animal to a black background by causing dispersion of melanin pigment granules (melanosomes) in skin melanophores. On a black background the melanotrope cell is dedicated to produce vast amounts of POMC such that this prohormone represents ~80% of all newly synthesized melanotrope proteins. On a white background POMC mRNA levels are decreased ~30-fold (Holthuis et al., 1995a) and α-MSH secretion from the melanotropes into the blood stream is inhibited by neurons of hypothalamic origin that directly innervate the cells (Jenks et al., 1993; Tuinhof et al., 1994), leading to melanosome aggregation and, consequently, pallor of the skin. Placing Xenopus on a black or a white background therefore allows physiological manipulation of the biosynthetic and secretory activities of the melanotrope cell. Using a differential screening approach, we have identified a number of proteins coexpressed with POMC and thus differentially expressed in the melanotrope cells of black- and white-adapted Xenopus, including the POMC-cleavage enzyme prohormone convertase PC2 and a member of the p24 family, namely Xp24δ2 (Holthuis et al., 1995b). Subsequent extensive cDNA library screening resulted in the identification of all members of the p24 family that are expressed in the Xenopus melanotrope cell (Xp24α3, -β1, -γ2,3 and -δ1,2) (Rötter et al., 2002). Of these, Xp24α3, -β1, -γ3 and -δ2 constitute the major representatives and are highly upregulated with POMC in the melanotropes during black background adaptation (at least 20-fold), whereas the two low-abundant ones (Xp24γ2 and -δ1) are not or only slightly induced (Kuiper et al., 2001; Rötter et al., 2002). The coordinate and induced expression of a selective set of Xenopus p24 proteins (Xp24α3, -β1, -γ3 and -δ2) in the melanotrope cell suggests that these p24 members are somehow involved in POMC biosynthesis. To explore the importance of p24 in the Xenopus melanotrope cell, we combined the unique properties of this cell with the technique of stable Xenopus transgenesis by using a Xenopus POMC gene promoter fragment to target transgene expression specifically to the melanotrope cell, leaving the integrity of the regulation by the hypothalamic neurons intact. For transgenic overexpression, we selected one of the Xenopus melanotrope p24 proteins coexpressed with POMC, namely the Xp24δ2 protein, and fused it to the N-terminus of the green fluorescent protein GFP. Here we report the effect of this transgenic manipulation of the endogenous p24 system on the functioning of the Xenopus melanotrope cells.

 

 

MATERIALS AND METHODS

Animals

Xenopus laevis were reared in the Central Animal Facility of the University of Nijmegen. For the transgenesis experiments, female Xenopus laevis were obtained directly from South Africa. For background adaptation, the animals were kept in either white or black containers under constant illumination for at least three weeks. All animal experiments were carried out in accordance with the European Communities Council Directive 86/609/EEC for animal welfare, and permit TRC 99/15072 to generate and house transgenic Xenopus.

 

Antibodies

The rabbit polyclonal antibodies against portions of the lumenal and C-terminal regions of Xp24δ2 (anti-1262N and anti-1262C, respectively), against part of the lumenal region of Xp24δ1 (anti-RH6) and against a region in the lumenal part of Xp24α3 have been described previously (Kuiper et al., 2001; Rötter et al., 2002). A polyclonal antibody to human p24β1 (p24A) was kindly provided by Dr. I. Schulz (University of the Saarland, Homburg, Germany; Blum et al., 1999), to human p24γ3 by Dr. T. Nilsson (EMBL, Heidelberg, Germany; Dominguez et al., 1998), against recombinant mature human PC2 by Dr. W.J.M. Van de Ven (University of Leuven, Belgium; Van Horssen et al., 1998), to GFP by Dr. J. Fransen (Cuppen et al., 1999) and against Xenopus POMC (ST62, recognizing only the precursor form) by Dr. S. Tanaka (Shizuoka University, Japan; Berghs et al., 1997).

 

Generation of Xenopus transgenic for Xp24δ2-GFP

A linear 2166-bp SalI/NarI DNA fragment encoding the Xenopus p24δ2 protein with the enhanced GFP protein fused in frame to its C terminus (Xp24δ2-GFP fusion protein) and cloned behind a 529-bp Xenopus POMC gene A promoter (pPOMC; Jansen et al., 2002) fragment (construct pPOMC-Xp24δ2-GFP) was used for stable Xenopus transgenesis (Kroll and Amaya, 1996) (Sparrow et al., 2000). A number of injection rounds resulted in animals transgenic for Xp24δ2-GFP and expressing the fusion protein at various levels (animals #115 and #125 with moderate and #124 with high expression levels). The number of integration sites and integrated copies of the transgene were determined by southern blot analysis of genomic DNA isolated from transgenic livers (Ausubel et al., 2001) revealing 4, 1 and 3 sites of integration, and ~25, 2 and ~20 integrated copies of the transgene in animals #115, #125 and #124, respectively. To generate F1 offspring, the testes of male transgenic Xenopus frogs were isolated and for in vitro fertilization pieces of testis were incubated with eggs harvested from wild-type Xenopus females.

 

Microscopy

For ultrastructural analysis, electron microscopy was performed as described previously (De Rijk et al., 1990). Ultrastructural (immuno)localization studies were performed on neurointermediate lobes of wild-type Xenopus, and #124 and #224 transgenic animals expressing Xp24δ2-GFP at high levels. Entire lobes were fixed for one hour at room temperature in 2% paraformaldehyde (PFA) + 0.01% glutaraldehyde in PHEM buffer (50 mM MgCl2 , 70 mM KCl, 10 mM EGTA, 20 mM HEPES, 60 mM PIPES, pH 6.8). Fixed tissue was stored in 1% PFA in 0.1M phosphate buffer until use. Ultrathin cryosectioning was performed as described before (Fransen et al., 1985; Schweizer et al., 1988). Sections were incubated with an antiserum against EGFP at a 1:100 dilution followed by protein A complexed with 10 nm gold (Fransen et al., 1985). Electron microscopy experiments using the anti-Xp24δ antibodies were not successful. Electron microscopy was performed using a Jeol 1010 electron microscope operating at 80 kV. For confocal microscopy, brains with the pituitaries attached were dissected and fixed in 4% paraformaldehyde in PBS. After cryoprotection in 10% sucrose-PBS, sagittal 20-µm cryosections were mounted on poly-L-lysine coated slides, dried for 2 hr at 45ºC and studied with a MRC 1024 confocal laser scanning microscope (Biorad). To examine direct fluorescence as a result of GFP fusion protein expression, cryosections were directly viewed under a Leica DM RA fluorescent microscope and photographs were taken with a Cohu High Performance CCD Camera using the Leica Q Fluoro software. Immunohistochemistry for POMC and α-MSH was performed as described previously (Jansen et al., 2002). For light microscopy analysis of the webs and melanophores of wild-type and transgenic animals, webs were cut out, mounted on slides and cover slipped. Digital images were obtained using a Leica MZFLIII microscope mounted with a DC200 digital colour camera. Equal integration intervals and magnifications were used to capture images with Leica DC viewer software.

 

Western blot analysis

Western blot analysis was performed as described previously (Kuiper et al., 2000). For quantification, detection was performed using a BioChemi imaging system and signals were analyzed using the Labworks 4.0 program (UVP BioImaging systems, Cambridge, UK).

 

Pulse and pulse-chase analysis

For metabolic labelling, neurointermediate lobes (NILs) from wild-type and transgenic Xenopus were preincubated for 30 min, pulse labelled in the presence of 5 mCi/ml Tran35S-label (ICN Radiochemicals) and chased with 0.5 mM L-methionine for the indicated time periods, and homogenized as described previously (Braks and Martens, 1994). Parts of the lysates and incubation media were analyzed directly on SDS-PAGE, while the remainder was used for immunoprecipitation, western blot and/or HPLC analysis.

 

Immunoprecipitation analysis

For immunoprecipitation analysis, NIL lysates were diluted with lysis buffer to 1 ml, and supplemented with SDS (final concentration of 0.075%) and the respective antibodies. Precipitation was performed overnight at 4°C while rotating the samples. Immune complexes were precipitated with protein-A Sepharose (Amersham-Pharmacia Biotech) and resolved by SDS-PAGE. Radiolabeled proteins were detected using autoradiography at -70°C or a PhosphoImager (Personal FX, Biorad).

 

HPLC analyis

For the separation of the small newly synthesized end products of POMC processing, radiolabelled NIL lysates were subjected to HPLC analysis as described previously (Martens et al., 1982a).

 

 

RESULTS

Generation of Xenopus transgenic for Xp24δ2-GFP

To generate Xenopus transgenic for the Xenopus p24δ2 protein with GFP fused to its C-terminus (Xp24δ2-GFP), we first made a DNA construct (pPOMC-Xp24δ2-GFP) containing a 529-bp Xenopus POMC gene promoter fragment in front of the sequence encoding the fusion protein. The GFP-moiety was fused to the C-terminus of the Xp24δ2-protein to avoid interference with a possible binding of cargo to the N-terminal loop domain of Xp24δ2. The pPOMC-Xp24δ2-GFP DNA was mixed with Xenopus sperm nuclei and the mixture was microinjected into unfertilized Xenopus eggs. The different levels of expression of the fusion protein among the various transgenic animals could be readily and directly established by visual inspection of the living embryos under a fluorescence microscope (Fig. 1A). Lifting the brain of the transgenic animal showed that the expression of the Xp24δ2-GFP fusion protein was restricted to cells located in the intermediate lobe of the pituitary, and no fluorescence was observed in the anterior lobe of the pituitary or in any other brain structures (Fig. 1B). An immunocytochemical analysis revealed that the fusion protein was coexpressed in the melanotrope cells with POMC and α-MSH (data not shown). Adaptation of the transgenic animals to a black- or a white background resulted in high and low levels of fluorescence in the intermediate pituitary, respectively (Fig. 1C), suggesting that the level of Xp24δ2-GFP transgene expression was dependent on the colour of the background of the animal and coregulated with POMC expression. Thus, the 529-bp Xenopus POMC gene promoter fragment was sufficient to drive melanotrope cell-specific expression of the transgene and give different levels of transgene expression depending on background colour.

 

Steady-state p24 protein levels in the pituitary of Xenopus transgenic for Xp24δ2-GFP

From the pituitary (consisting of the pars nervosa, and the anterior and intermediate lobes), the anterior part can be dissected but the pars nervosa (biosynthetically not active nerve terminals of hypothalamic origin) is intimately associated with the intermediate pituitary (the neuroendocrine melanotrope cells). For our studies, we therefore used the anterior lobe (AL) and neurointeremediate lobe (NIL; pars nervosa plus intermediate lobe) of the pituitary. Western blot analysis of p24 steady-state protein levels was performed on lysates of NILs and ALs of wild-type and transgenic Xenopus employing specific anti-p24 antibodies. We first used the anti-Xp24δ antibody 1262C directed against the C-terminal region of Xp24δ2 that recognizes endogenous Xp24δ1 and -δ2 with comparable affinities (Kuiper et al., 2000). With this antibody we detected ~8 and ~3 times more of the ~24-kDa Xp24δ2 protein than ~23-kDa Xp24δ1 in the wild-type NIL and AL, respectively (Fig. 1D, upper panel). However, the C-terminally directed antibody 1262C hardly recognized the Xp24δ2-GFP fusion protein (Fig. 1D, compare lanes 2 of the upper and lower panels), presumably because of the fusion of GFP to the C terminus of Xp24δ2. For the simultaneous detection of the transgene and endogenous Xp24δ products, we therefore used in all subsequent experiments a mixture of anti-Xp24δ1 and anti-Xp24δ2 antibodies (RH6 and 1262N, respectively), each directed against a portion of the respective N-terminal region and specifically recognizing the corresponding Xp24δ protein. This antibody mix showed in the wild-type NIL about equal amounts

of the endogenous Xp24δ1 and Xp24δ2 proteins (Fig. 1D, lower panel, lane 1). In the

Figure 1: Xp24δ2-GFP transgene expression is specific to Xenopus intermediate pituitary and dependent on background colour.

(A) Pituitary-specific fluorescence in transgenic Xenopus embryos. Shown are living stage 45 embryos, whereby the arrows indicate the locations of the pituitaries with various levels of transgene expression. Fluorescent pituitaries expressing the transgene fusion product could be detected from stage 25 onwards. Bar equals 0.4 mm. (B) Fluorescence is specific to the intermediate pituitary of transgenic Xenopus. Ventrocaudal view on the brain that was lifted to reveal the bright fluorescence caused by the Xp24δ2-GFP fusion protein and observed in the intermediate lobe (IL), but not in the anterior lobe (AL), of the pituitary of a black-adapted transgenic frog of 6 months. Bar equals 0.5 mm. (C) Fluorescence in the intermediate lobe of black- and white-adapted transgenic (tr) Xenopus. Ventrocaudal view with the anterior part of the pituitary removed. Bar equals 0.5 mm. (D) Western blot analysis of p24δ protein expression in the neurointermediate lobe (NIL) and anterior lobe (AL) of black-adapted wild-type (wt) and transgenic (tr) Xenopus. (E) Western blot analysis of p24δ protein expression in the NIL of black-adapted (BA) and white-adapted (WA) wild-type and transgenic Xenopus using the p24δ1/-δ2 antibody mix. (F) Newly synthesized proteins produced in NILs of black-adapted (BA) and white-adapted (WA) wild-type and transgenic Xenopus. NILs were pulse labelled for 1 hr, part of the total cell lysates was analyzed directly on SDS-PAGE and radiolabelled proteins were visualized by fluorography.

 

transgenic NIL, the antibodies revealed an additional product of ~52 kDa, presumably corresponding to the transgene Xp24δ2-GFP fusion protein (~24 kDa for Xp24δ2 and ~28 kDa for GFP) (Fig. 1D, lower panel, lane 2). The fusion protein was found only in the NIL and not AL (Fig 1D, lower panel) again indicating that the expression of the transgene product is melanotrope cell specific. In the transgenic cells, the fusion protein was about ~15-fold higher in black than in white animals (Fig. 1E), in line with the data obtained by direct fluorescence analysis (Fig. 1C). Furthermore, metabolic labelling of wild-type and transgenic NILs revealed an ~9-fold higher level of newly synthesized Xp24δ2-GFP fusion protein in black- than in white-adapted #124 transgenic animals, similar to the ~10-fold difference in radiolabelled POMC precursor levels (Fig. 1F). Having established that transgene expression is coregulated with POMC and specific for the melanotrope cells, we then wondered what the effect of the overexpression of the Xp24δ2-GFP fusion protein would be on the levels of the endogenous p24 proteins. For this and subsequent analyses, two male transgenic Xenopus that differed in Xp24δ2-GFP expression levels (animals #124 and #125) were selected and used to generate F1 offspring by in vitro fertilization. Western blot analysis revealed that the expression of the transgene product was ~4-fold higher in #124 than in #125 transgenic melanotrope cells (Fig. 2A). The overexpresssion of the Xp24δ2-GFP fusion protein resulted in reduced levels of the endogenous Xp24δ1 and -δ2 proteins in #125 cells (~40% and ~54% reduction, respectively), whereas in #124 cells the two endogenous Xp24δ proteins were even nearly completely displaced (~87% and ~95% reduction of Xp24δ1 and -δ2, respectively) (Fig. 2A). These findings indicate that high levels of the transgene product cause low levels of the endogenous Xp24δ proteins. To examine whether the degree of competition between the exogenous and endogenous Xp24δ proteins was correlated with the level of newly synthesized Xp24δ2-GFP produced in the #124 and #125 transgenic NILs, we performed metabolic cell labelling experiments. Direct SDS-PAGE analysis of newly synthesized NIL proteins revealed an ~52-kDa radiolabelled product in #124 transgenic but not in #125 transgenic or wild-type cells (Fig. 2B, left panel). The ~52 kDa product comigrated with a radiolabelled protein immunoprecipitated with the anti-δ12 antibody mix from newly synthesized proteins produced by transgenic NILs (Fig. 2B), indicating that it represents the newly synthesized Xp24δ2-GFP fusion protein. The #125 melanotrope cells produced ~5-fold and the #124 cells at least 15-fold more newly synthesized transgene Xp24δ2-GFP product than newly synthesized endogenous Xp24δ1 protein. The lower level of immunoprecipitated newly synthesized endogenous Xp24δ2 in the #125 cells was likely due to the high amount of competing radiolabelled transgene δ2 fusion product, since in cells from the independent line #115 with less transgene expression the amount of immunoprecipitated endogenous Xp24δ2 was not affected (Fig. 2B, right panel). Therefore, the biosynthesis of the endogenous Xp24δ1 and -δ2 proteins does not appear to be affected by the transgene expression. Together, the above findings indicate that in the #124 melanotrope cells the high level of Xp24δ2-GFP protein biosynthesis resulted in lower amounts of the endogenous Xp24δ1 and -δ2 proteins than in the #125 cells, and thus that the level of transgene expression is correlated with the degree of displacement of the endogenous Xp24δ proteins by the exogenous fusion product.

Figure 2: Xp24δ2-GFP protein levels in transgenic Xenopus intermediate pituitary determine the degree of displacement of the endogenous p24 proteins.

(A) Western blot analysis of Xp24δ protein expression in the neurointermediate lobe (NIL) of wild-type (wt) and transgenic (#125 and # 124) Xenopus using an anti-Xp24δ1/-δ2 antibody mix. (B) Newly synthesized proteins produced in NILs of wild-type and transgenic Xenopus. NILs were pulse labelled for 1 hr, and parts of the total cell lysates were analyzed directly on SDS-PAGE (left panel) or immunoprecipitated using an anti-Xp24δ1/-δ2 antibody mix followed by resolving the immunoprecipitates on SDS-PAGE (right panel). Radiolabelled proteins were visualized by fluorography. Asterisks indicate POMC- and PC2-related proteins binding non-specifically to the antibodies. (C) Western blot analysis of Xp24α3, -β1 and -γ3 protein expression in the NIL of wild-type and transgenic Xenopus.

We next examined what the consequences of the expression of the Xp24δ2-GFP protein were on the steady-state expression levels of the major endogenous Xenopus melanotrope p24 members other than the Xp24δ proteins. Overexpression of the fusion protein in #124 transgenic melanotrope cells led to a more than 5-fold reduction in the amounts of the endogenous Xp24α3, -β1 and -γ3 proteins, whereas these levels were essentially unchanged in the #125 cells (Fig. 2B). The level of expression of the transgene product therefore appears to determine the degree of displacement not only of the endogenous Xp24δ proteins but also of the other endogenous p24 members. Taken together, we conclude that in the #124 transgenic melanotrope cells the exogenous Xp24δ2-GFP fusion protein caused a drastic reduction in the amounts of the endogenous p24 members, resulting in a p24 system predominantly consisting of the transgene product.

Figure 3: Electron microscopy on transgenic Xenopus intermediate pituitary cells.

(A) Electron micrographs of melanotrope cells of wild-type (wt) and #124 transgenic (tr) Xenopus adapted to a black (BA) or white (WA) background. N, nucleus; ER, endoplasmic reticulum; sg, secretory/storage granule. Bar equals 2 ˜m. (B) Pituitary glands from wild-type (wt) and transgenic (tr) frogs (F1 #224, expressing high levels of Xp24δ2-GFP) were subjected to immunoelectron microscopical analysis. For immunodetection, the anti-GFP antibody was used in combination with protein-A-gold to visualize the Xp24δ2-GFP fusion protein. Immunoreactivity was found in structures that resemble the ER and the Golgi, bars equal 0,1 µm.

 

Microscopy analyses of Xenopus melanotrope cells transgenic for Xp24δ2-GFP

In transfected mammalian cells in culture, overexpression of p24δ1 (p23) or p24β1 (p24) caused the induction of an expansion of smooth ER membranes (Blum et al., 1999; Rojo et al., 1997). We therefore wondered what in the #124 transgenic Xenopus melanotrope cells the effect of the overexpression of the Xp24δ2-GFP protein would be on the morphology of subcellular structures. Electron microscopy analyses were performed on intermediate pituitaries of both black- and white-adapted wild-type and #124 transgenic animals. Despite the severely affected p24 system in the #124 transgenic melanotrope cells, at the ultrastructural level no gross morphological differences were observed between the wild-type and transgenic cells (Fig. 3A). As expected, the melanotrope cells of black-adapted animals showed extensive ER structures as these cells are highly active in synthesizing large amounts of POMC. The melanotropes of white-adapted animals showed virtually no ER structures but many storage granules, reflecting their biosynthetic and secretory inactivity (Fig. 3A). We can thus conclude that the structural changes occurring in the melanotrope cells during background adaptation of the animal are similar in the wild-type and #124 transgenic cells, and consistent with previous electron microscopy studies on wild-type Xenopus melanotrope cells (De Rijk et al., 1990; Weatherhead et al., 1971). The Xp24δ2-GFP fusion protein was found to be capable of reaching the Golgi, since confocal microscopy revealed that both ER- and Golgi-regions displayed fluorescence (data not shown) and immunoelectron microscopy confirmed that the Xp24δ2-GFP fusion protein was localized to structures that resemble the ER and the Golgi (Fig. 3B). These results are in line with previous findings showing that endogenous Xp24δ2 localizes to the ER and the Golgi in wild-type Xenopus melanotrope cells (Kuiper et al., 2001) and that p24 proteins shuttle between the ER and the Golgi (Barlowe, 1998; Dominguez et al., 1998; Sohn et al., 1996). Together, these observations suggest that the overexpression of the transgene product was to such an extent that in the transgenic cells the early secretory pathway was not destroyed and that the transgene product was localized to the proper secretory pathway subcompartments.

 

Background adaptation of Xenopus transgenic for Xp24δ2-GFP

In Xenopus, the intermediate pituitary melanotrope cells to which we specifically targeted transgene expression are involved in the process of background adaptation (Jenks et al., 1977). This fact together with the disrupted p24 machinery in the #124 Xenopus transgenic melanotrope cells prompted us to examine the physiological consequence of this situation for background adaptation of the transgenic animal. Following their metamorphosis, animals were placed on a black background for four months and thus the melanotrope cells were biosynthetically very active during a relatively long time period. As expected, wild-type Xenopus were black and contained many completely dispersed pigment-filled granules in the dermal melanophores of their webs. Following the long adaptation to a black background, the skin colour of the #124 transgenic animal was lighter than those of wild-type and #125 animals. Upon closer inspection of the webs, only in the vicinity of blood vessels pigment-containing web melanophores were observed, and the number and sizes of melanophores were clearly reduced in the #124 transgenic animal (~5- and ~3-fold reduction, respectively) (Fig. 4). These results indicate that the transgenic manipulation of the p24 system exclusively in the Xenopus melanotrope cells led to a physiological effect regarding morphological changes in skin melanophores.

 

Steady-state protein levels of POMC and PC2 in Xenopus melanotrope cells transgenic for Xp24δ2-GFP

Since the process of background adaptation is mediated by α-MSH, a cleavage product of POMC, we next examined by western blot analysis whether the altered p24 system had affected the steady-state level of the 37-kDa POMC precursor in the transgenic melanotrope cells. No differences in POMC levels were observed between wild-type, and #124 transgenic NILs of black-adapted animals (Fig. 5). Upon white-background adaptation of the transgenic animals the amount of the POMC protein decreased to similar levels as observed in wild-type melanotrope cells of white animals (at least 10-fold reduction; Fig. 5). Likewise, in the #124 transgenic cells of black-adapted animals, the steady-state amounts of both the proenzyme and mature forms of the POMC cleavage enzyme PC2 (75-kDa proPC2 and 69-kDa PC2, respectively) were not affected when compared to those in the wild-type situation. Furthermore, in both the inactive wild-type and transgenic cells of white animals, the expression of the proPC2 protein was greatly reduced (at least 15-fold), whereas the level of mature PC2 remained essentially the same as in black-adapted animals (Fig. 5). These results indicate that the steady-state POMC and proPC2 protein levels, and the changes in these levels induced by the process of background adaptation were not affected by the introduction of the transgene into the melanotrope cells.

Figure 4: Wild-type and transgenic Xenopus adapted to a black background. Wild-type (wt) and #125 and #124 transgenic animals were placed on a black background for 4 months. Shown below are the pigment-containing dermal melanophores in the webs; bars equal 1 mm and 250 µm for the upper- and lower panel, respectively.

 

Biosynthesis and processing of newly synthesized POMC and proPC2 in Xenopus melanotrope cells transgenic for Xp24δ2-GFP

We next studied the dynamics of protein synthesis by performing in vitro pulse- and pulse-chase analyses of newly synthesized proteins produced in wild-type and transgenic NILs. Since besides the melanotrope cells, the Xenopus NIL consists of nerve terminals of hypothalamic origin that are biosynthetically not active (the pars nervosa), the radiolabelled proteins are synthesized by the melanotropes. During the 10-min pulse incubation of wild-type NILs, the 37-kDa POMC precursor protein was clearly the major newly synthesized protein (Fig. 6A and Fig. 6B, lane 1). During the subsequent 1,5-hr and 2,5-hr chase incubations, 37-kDa POMC was gradually processed to an 18-kDa cleavage product (Fig. 6A, upper panel, left and Fig. 6B, lane 2). This product represents the N-terminal portion of 37-kDa POMC, is generated by the first endoproteolytic cleavage step during POMC processing and contains the only N-linked glycosylation site present in the POMC molecule (Martens, 1986). The amount of the 18-kDa POMC protein was lower for the 2,5-hr than for the 1,5-hr time point, since during the chase period this newly synthesized product is processed further (to γ-MSH; Martens et al., 1982) (Fig. 6A, upper panel, left). During the 10-min pulse, the POMC-cleavage enzyme PC2 was synthesized as a 75-kDa proenzyme form that in the course of the subsequent 1,5-hr and 2,5-hr chase incubations was processed to a 69-kDa mature form of PC2 that represents the end product of proPC2 processing (Fig. 6A, upper panel, right and Fig. 6B, lanes 1 and 2). Within the time frame of these pulse-chase experiments, virtually no newly synthesized 18-kDa POMC and mature PC2 was released into the incubation medium (<10% of the cellular content). In the #124 transgenic melanotrope cells, similar amounts of 37-kDa POMC were synthesized during the 10-min pulse incubation as in wild-type cells. However, the amounts of 18-kDa POMC that were produced in the transgenic cells following 1,5 hr and 2,5 hr of chase were less than those synthesized in the wild-type cells (Fig. 6A). Moreover, reloading of the samples on a higher-percentage polyacrylamide gel revealed that a substantial portion of the newly synthesized 18-kDa product produced in the #124 transgenic cells migrated slower than the majority of 18-kDa POMC synthesized in the wild-type cells (Fig. 6C). We refer to this slower-migrating product as 18-kDa POMC* and the normal product as 18-kDa POMC without an asterisk. The nature of the difference between the two 18-kDa POMC products is presently unknown. During the 10-min pulse, the amount of newly synthesized 75-kDa proPC2 produced in the transgenic melanotrope cells was similar to that synthesized by the wild-type cells. In contrast, as for the reduced rate of 37-kDa POMC processing, during the 1,5-hr and 2,5-hr chase periods the rate of conversion of newly synthesized proPC2 into mature PC2 was lower in the transgenic than in the wild-type cells (Fig. 6A). >

Following conversion of 37-kDa POMC into the 18-kDa N-terminal POMC cleavage product, the remaining C-terminal half of the POMC molecule was processed further to a number of peptides that were analyzed by HPLC, namely des-Nα-acetyl-α-MSH (the nonacetylated form of α-MSH), two corticotropin-like intermediate lobe peptides (CLIPs) and two endorphins (Fig. 6D). In the Xenopus melanotrope cells, des-Nα-acetyl-α-MSH is the major form of α-MSH and its acetylation occurs just prior to release, thereby making the acetylated form (α-MSH) the released (and more bioactive) product (Martens et al., 1981). HPLC analysis revealed that, following a 10-min pulse/2,5-hr chase and relative to the peptides produced in wild-type melanotrope cells, the amounts of the small POMC cleavage products (des-Nα-acetyl-αMSH, CLIPs and endorphins) were reduced in the #124 cells (Fig. 6E). Thus, besides the production of a lower amount of 18-kDa POMC and the additional form of the 18-kDa POMC cleavage product (18-kDa POMC*), reduced amounts of the POMC-derived peptides were synthesized in the #124 transgenic cells.

Figure 5: POMC and PC2 protein levels are similar in the intermediate pituitary of wild-type and transgenic Xenopus and dependent on background colour.

Western blot analysis of NIL proteins of wild-type and #124 transgenic Xenopus adapted to a black (BA) or white (WA) background.

 

Manipulation of POMC biosynthesis in Xenopus melanotrope cells transgenic for Xp24δ2-GFP

To test whether the processing machinery, in particular the supply of (re)folding enzymes, was indeed affected in the #124 transgenic melanotrope cells with the disrupted p24 system, we decided to challenge the transgenic cells with improperly folded newly synthesized 37-kDa POMC. For this purpose, we incubated NILs during the 10-min pulse period in the presence of either the thiol-reducing agent dithiothreitol (DTT) or the proline analogue L-azetidine 2-carboxylic acid (AZC), agents that directly interfere with the folding of newly synthesized proteins in the ER (Braakman et al., 1992; Trotter et al., 2001). DTT and AZC were not present during the 150-min chase period. We then compared the handling of the improperly folded radiolabelled 37-kDa POMC by the wild-type and transgenic cells. Considering the possibility that p24 proteins are involved in such handling, the #124 transgenic cells are expected to have more problems to cope with this situation and may allow more improperly folded POMC to leave the ER than the wild-type cells with a functional p24 system. In wild-type Xenopus NILs, DTT prevents disulfide bond formation of newly synthesized proteins, blocks POMC transport out of the early secretory pathway and strongly inhibits POMC processing. When the reducing agent is removed during the chase the disulfide bonds are allowed to be formed, and in the melanotrope cells newly synthesized POMC refolds and is properly processed, indicating that the treatment with DTT is reversible (Van Horssen et al., 1998). Analysis of newly synthesized proteins produced in DTT-treated wild-type and transgenic NILs revealed a lower amount of POMC and related proteins than in untreated cells (~70% reduction; data not shown). HPLC analysis revealed that in wild-type, and #125 and #124 cells the DTT treatment resulted in the production of a lower amount of POMC-derived peptides than in untreated cells. However, in the wild-type cells the recovery from the treatment, as reflected by the HPLC peptide profiles, was ~30 % relative to untreated cells, and ~25 % and only ~8 % for the #125 and #124 cells, respectively (Fig. 7). It therefore appears that improperly folded POMC was not efficiently processed to its end products.

Figure 6: Biosynthesis and processing of newly synthesized POMC and proPC2 in wild-type and transgenic Xenopus intermediate pituitary cells.

(A) Neurointermediate lobes (NILs) of black-adapted wild-type (wt) and #124 transgenic animals were pulse labelled for 10 min or pulse labelled for 10 min and chased for 1,5 hr or 2,5 hr. Newly synthesized proteins were extracted from the lobes, resolved by SDS-PAGE directly (for POMC analysis) or following immunoprecipitation (for PC2 analysis) and visualized by fluorography. The amounts of newly synthesized 37-kDa POMC, 18-kDa POMC cleavage product, 75-kDa proPC2 and 69-kDa mature PC2 were quantified by densitometric scanning and are presented in arbitrary units (AU), relative to the amount of 37-kDa POMC or of 75-kDa proPC2 produced during the pulse. Shown are the means ± SEM (n=3, except n=5 for the 2,5-hr chase values). (B) Newly synthesized proteins produced by NILs of black-adapted wild-type (wt) and #124 transgenic animals during a 10-min pulse (lane 1) or during a 10-min pulse/2,5-hr chase (lanes 2 and 3) were extracted from the lobes, resolved by SDS-PAGE on 12,5% gels and visualized by fluorography. (C) Samples corresponding to (B), lanes 2 and 3, were reloaded for the separation of newly synthesized 18-kDa POMC and 18-kDa POMC* by SDS-PAGE on 15% gels. (D) Samples corresponding to (B), lanes 2 and 3, were subjected to HPLC analysis to separate the newly synthesized POMC-derived peptides des-Nα-acetyl-α-MSH (des-α-MSH), corticotropin-like intermediate lobe peptides (CLIPs) and endorphins. (E) The amounts of the five peptides (des-α-MSH, two CLIPs and two endorphins) produced in the #124 transgenic cells were calculated on the basis of the HPLC profiles and are presented in arbitrary units (AU), relative to the corresponding peaks in the wild-type profile.

 

AZC is a synthetic analogue of proline and is incorporated into newly synthesized proteins competitively with endogenous proline causing protein misfolding (Trotter et al., 2001). Unlike the DTT treatment, the treatment of NILs with AZC is likely not reversible in that newly synthesized POMC molecules that have incorporated AZC during the pulse labelling are not expected to be properly folded when AZC is removed during the chase period. For both wild-type and transgenic cells, the treatment with AZC resulted in reduced levels of newly synthesized POMC and related proteins (~70% reduction; data not shown). HPLC analysis revealed that within the time frame of the experiments the AZC-treated wild-type and transgenic cells produced virtually no POMC-derived peptides (Fig. 7).

Figure 7: Newly synthesized proteins produced in wild-type and transgenic Xenopus intermediate pituitary cells exposed to agents directly interfering with protein folding. NILs of black-adapted wild-type (wt) and #125 and #124 transgenic animals were pulse labelled for 10 min in the presence of the thiol-reducing agent DTT or the proline analogue AZC and cell lysates were analyzed by HPLC. Newly synthesized POMC-derived peptides des-Nα-acetyl-α-MSH, CLIPs and endorphins were separated and quantified as described in Figure 6E legend. The results represent the means ± SEM from four independent experiments.

Taken together, the results of the biosynthetic studies with the misfolding agents suggest that the p24 system in the #124 transgenic melanotrope cells can handle improperly folded, newly synthesized POMC less efficiently than wild-type cells and thus that the machinery for secretory protein (re)folding may indeed be damaged in these cells.
 
 
DISCUSSION

The type I transmembrane p24 proteins are abundantly present in ER- and Golgi-derived transport vesicles, and are therefore thought to play an important role in some aspect of cargo-selective transport through the early secretory pathway. The complex and dynamic behaviour of this protein family has hampered functional analyses. In this study, we used the Xenopus laevis intermediate pituitary melanotrope cell with one major secretory cargo protein (the prohormone POMC) and melanotrope cell-specific transgene expression of a GFP-tagged Xenopus p24 family member as a model to explore the importance of a functional p24 complex for a highly specialized secretory cell. Of the four abundant melanotrope p24 members upregulated with POMC (Xp24α3, -β1, -γ3 and -δ2) and thus likely somehow involved in the biosynthesis of the prohormone, Xp24δ2 was chosen for transgenic expression. The microscopy analyses revealed that the GFP-tag did not prevent the Xp24δ2-GFP fusion protein from reaching the Golgi. The two selected, independent transgenic lines #125 and #124 displayed moderate and high expression levels of the Xp24δ2-GFP fusion protein, respectively. From the western blot and biosynthetic studies on the Xenopus p24δ proteins, we conclude that the level of newly synthesized Xp24δ2-GFP produced in the transgenic cells determined the degree of displacement of the endogenous Xp24δ2 and -δ1 proteins by the fusion protein. Thus, high levels of newly synthesized fusion protein, as produced in the #124 transgenic melanotrope cells, caused the near-absence of endogenous Xp24δ1 and -δ2. Due to the lower level of transgene expression in the #125 cells, substantial amounts of the endogenous Xp24δ proteins were still present, albeit at lower steady-state levels than in the wild-type cells. In the #124 cells, the high level of Xp24δ2-GFP effectively displaced not only the endogenous Xp24δ proteins, but also the normally abundant Xp24α3, -β1 and -γ3 family members such that the resulting p24 system consisted mainly of the transgene product. It therefore appears that the number of ER/Golgi subcompartments that can harbour p24 proteins is limited and that the relative amounts of the various newly synthesized p24 family members expressed in a cell determine the final composition of the p24 machinery in the early secretory pathway (by a “displacement effect”). In transiently transfected cells in culture, overexpression of a single p24 member resulted in aberrant ER structures (Rojo et al., 2000). Since our ultrastructural analysis did not reveal gross morphological changes, the level of transgene expression may have been relatively less than the amount of exogenous p24 produced in the transfected cells and thus to an extent that did not destroy the early secretory pathway in the transgenic Xenopus melanotrope cells.

Of special interest was that the number and sizes of the melanophores in the skin of the #124 transgenic animals were clearly reduced and as a result, these animals were not able to fully adapt to a black background. Because in Xenopus the intermediate pituitary melanotrope cells regulate skin melanophores, the phenotype of the #124 animal urged us to investigate in detail the functioning of the transgenic melanotropes. From the western blot analyses of POMC and the POMC cleavage enzyme PC2, it appeared that the steady-state levels of these proteins were similar in the wild-type and transgenic melanotrope cells. We then examined the dynamics of protein biosynthesis and for this study we focused on the major newly synthesized secretory cargo protein POMC, its well-defined processing products and PC2. The results of the in vitro metabolic cell labelling studies suggested that in the #124 transgenic melanotrope cells the distortion of the endogenous p24 complex did not affect the level of POMC and proPC2 biosynthesis. However, relative to the wild-type melanotrope cells, the transgenic cells produced lower amounts of newly synthesized 18-kDa POMC, of the newly synthesized peptides derived from POMC (des-Nα-acetyl-αMSH, CLIPs and endorphins) and of newly synthesized mature PC2. This effect may have been caused by a lower rate of transport of newly synthesized POMC and proPC2 through the secretory pathway, resulting in a lower rate of precursor protein processing and the observed reduced amounts of the newly synthesized precursor-derived peptides produced within the time frame of the pulse-chase experiments. Alternatively, the distorted p24 system may have exerted a more direct effect on the POMC processing event itself, e.g. because it failed to provide the proper processing conditions in the various secretory pathway subcompartments. In considering such a role in processing, a recently proposed model for ER-to-Golgi cargo transport is of special interest (Mironov et al., 2003). According to this model, that was based on the results of high-resolution morphological studies, secretory proteins would exit the ER by bulk flow in large transport carriers emerging from specialized ER exit sites, and this process would not involve budding and fusion of COPII-coated vesicles. In adjacent COPII-coated exit sites, a specific set of ‘machinery proteins’ would be recruited and subsequently incorporated into the outgoing secretory cargo-containing carrier, e.g. for providing the correct lumenal environment in the carrier (Mironov et al., 2003). Because of their well-established ability to bind COPII (Fiedler et al., 1996; Nickel et al., 1997; Dominguez et al., 1998), p24 proteins may be involved in the COPII-dependent targeting of the ‘machinery proteins’ to the secretory cargo transport carriers. In view of the results from our transgenic studies, the proteins recruited by p24/COPII could include components of the biosynthetic machinery that are needed for proper prohormone processing. Thus, in the #124 transgenic Xenopus melanotrope cells with the severely distorted p24 system, the set of ‘machinery proteins’ incorporated into the outgoing POMC-containing carriers may be incomplete and these cells would therefore lack a fully functional POMC processing system. We also investigated how the #124 cells would handle improperly folded POMC produced by the cells because they were exposed to the protein-misfolding agents DTT and AZC. From the results of these experiments it appears that, in contrast to wild-type cells, the #124 cells have indeed an impaired processing system that fails to properly recognize or recycle misfolded POMC. We therefore conclude that an intact p24 system is essential to allow proper POMC processing.

The observation that the #124 pigment-containing skin cells were found only in the vicinity of blood vessels suggests that these transgenic animals have a shortage of the factor(s) responsible for the signalling to these cells. The reduced size and pigment content of the melanophores may be attributed to the lower amount of intermediate pituitary α-MSH, the POMC-derived peptide with a well-established role in background adaptation of amphibians by causing both the dispersion and synthesis of melanin in dermal melanophores (Hadley et al., 1981). An intriguing explanation for the lower number of skin melanophores in the #124 animal concerns the 18-kDa POMC cleavage product. The N-terminal 52 amino acids of the mammalian counterpart of Xenopus 18-kDa POMC (16-kDa POMC, also named pro-γ-MSH), resulting from a post-secretional cleavage of 16-kDa POMC by a serine protease localized on the target cell membrane, has been found to act as a growth factor (Bicknell et al., 2001). Hence, a deficit in normal melanotrope 18-kDa POMC may have resulted in insufficient mitogenic activity to produce normal quantities of skin melanophores in the #124 transgenic animal.

Taken together, our results are most consistent with a role for p24 in the transport of newly synthesized secretory cargo proteins through the early stages of the secretory pathway or in the processing of secretory cargo by recruiting the proper components of the biosynthetic machinery into ER-to-Golgi cargo transport carriers. Furthermore, our transgenic approach in a physiological context has shown that distortion of the complex p24 system results in an affected profile of prohormone-derived bioactive peptides with the eventual consequence at the level of the target cell of the secretory signals.

 

 

ACKNOWLEDGEMENTS

We would like to thank Ron Engels for animal care, Tony Coenen, Huib Croes and Coen Van der Meij for technical assistance, Drs Roland Kuiper and Jutta Rötter for helpful discussions, and Drs Wiljan Hendriks and Bruce Jenks for critical reading of the manuscript. We also thank Drs Irene Schulz, Tommy Nilsson, Wim Van de Ven and Shige Tanaka for providing antibodies. This work was supported by grant 811.38.002 from the Netherlands Organization for Scientific Research - Earth and Life Sciences (NWO-ALW).

 

 

 

Appendix to chapter 5

Chapter 5 describes the physiological effects and the resulting phenotype of the specific overexpression of Xp24δ2-GFP in the melanotrope cell. For these studies, we used the transgenic F1 lines #125 and #124 that express moderate and high levels of the fusion protein, respectively. We recently obtained an additional F1 transgenic line (F1 line #224) expressing very high levels of Xp24δ2-GFP in the melanotrope cell. Examination of these animals showed that the effects of this manipulation (distorted endogenous p24 system, affected POMC-peptide profile, reduced number and pigment content of skin melanophores) were more clear than in F1 line #124 and their ability to adapt to a black background was less. To further support the findings in chapter 5, we therefore present here the results of experiments performed with F1 line #224. Western blot analysis of F1 #224 NIL proteins, using antibodies specific for Xp24δ1 and -δ2, and for Xp24α, -β and -γ revealed high steady-state levels of the Xp24δ2-GFP fusion protein and in addition, similar to what was found in F1 line #124, the endogenous p24 family members were competed away, resulting in a severely distorted endogenous p24 system (Fig. A1 and data not shown). Pulse and pulse-chase labeling of #224 transgenic NILs showed no effect on the level of POMC synthesis and subsequent processing to 18-kDa POMC. However, similar to what was observed in #124 transgenic animals, an aberrant form of 18-kDa POMC was produced in the #224 transgenic melanotrope cells that was more clearly visible on the gel than in the experiments with #124 (Fig. A2). In addition, HPLC analysis of NIL cell lysates of #224 transgenic animals showed lower levels of POMC-derived peptides as compared to lysates of #124 transgenic Xenopus (data not shown). We recently performed two-dimensional electrophoresis to identify proteins that are differentially expressed in the melanotrope cells of wild-type and #224 transgenic animals. Preliminary results suggest that several proteins were differentially expressed in melanotrope cells that overexpress Xp24δ2-GFP. Clearly, the amount of BiP was less in the transgenic melanotrope cells (Fig. A3). Finally, close inspection of the webs and skin melanophores showed more severe effects on the webs and melanophores (decreased number and pigment content) as compared with #124 transgenic animals (compare Fig. A4 and Fig. 4, chapter 5). Therefore, the results of the analysis of transgenic F1 line #224 underline the causal relation between the overexpression of Xp24δ2-GFP and the resulting phenotypic effects observed in these transgenic animals.

Figure A1: Expression of Xp24δ2-GFP in intermediate pituitary cells of F1 transgenic animals.

Western blot analysis of Xp24δ protein expression in the neurointermediate lobes of wild-type (wt) and transgenic (#124 and #224) Xenopus using an anti-Xp24δ1/-δ2 antibody mix.

Figure A2: Newly synthesized proteins produced in intermediate pituitary cells of wild-type and transgenic Xenopus.

Neurointermediate lobes (NILs) were pulse labelled for 30 min and chased for 150 min; newly synthesized proteins extracted from the lobes (10%) or secreted into the incubation medium (20%) were resolved by SDS-PAGE and visualized by fluorography. Exposure times were adjusted for lysates and media such that the signals were similar.

Figure A3: Two-dimensional gel electrophoretic analysis of wild-type and #224 neurointermediate lobes.

2D analysis of protein patterns in wild-type (wt) and transgenic (tr) melanotrope cells was carried out as described (Devreese et al., 2002). Briefly, neurointermediate lobes were dissected and collected dry in a glass-glass potter. Tissues were homogenized in 40 mM Tris base (pH9.5) and 10 mM Pefablock (Roche, Basel, Switzerland). Proteins were TCA precipitated (Molloy et al., 1998) and air-dried before dissolving the proteins in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT and 0.8% IPGphor buffer (pH4-10L; Amersham-Bioscience). Isoelectric focusing was carried out on 90% of the cell lysates using 18 cm gel strips and protein spots were identified as described (Devreese et al., 2002) (BiP; glucose-regulated protein precursor; PDI-A1, protein disulfide isomerase-A1 precursor).

Figure A4: Number and pigment content of dermal melanophores in black-adapted wild-type and #224 transgenic animals.

Wild-type (wt) and #224 transgenic animals were placed on a black background for 4 months. Shown are the pigment-containing dermal melanophores in the webs; bars equal 1 mm and 250 µm for the lower- and upper panel, respectively.

 

 

REFERENCES

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (2001). in: Current Protocols in Molecular Biology. 2.2.1-2.2.3.

Barlowe, C. (1998). COPII and selective export from the endoplasmic reticulum. Biochimica et Biophysica Acta 1404, 67-76.

Barlowe, C. (2000). Traffic COPs of the early secretory pathway. Traffic 1, 371-7.

Belden, W. J. and Barlowe, C. (2001). Deletion of yeast p24 genes activates the unfolded protein response. Molecular Biology of the Cell 12, 957-69.

Berghs, C. A., Tanaka, S., Van Strien, F. J., Kurabuchi, S. and Roubos, E. W. (1997). The secretory granule and pro-opiomelanocortin processing in Xenopus melanotrope cells during background adaptation. Journal of Histochemistry and Cytochemistry 45, 1673-82.

Bicknell, A. B., Lomthaisong, K., Woods, R. J., Hutchinson, E. G., Bennett, H. P., Gladwell, R. T. and Lowry, P. J. (2001). Characterization of a serine protease that cleaves pro-gamma-melanotropin at the adrenal to stimulate growth. Cell 105, 903-12.

Blum, R., Pfeiffer, F., Feick, P., Nastainczyk, W., Kohler, B., Schafer, K. H. and Schulz, I. (1999). Intracellular localization and in vivo trafficking of p24A and p23. Journal of Cell Science 112, 537-548.

Braakman, I., Helenius, J. and Helenius, A. (1992). Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO Journal 11, 1717-22.

Braks, J. A. and Martens, G. J. M. (1994). 7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway. Cell 78, 263-273.

Bremser, M., Nickel, W., Schweikert, M., Ravazzola, M., Amherdt, M., Hughes, C. A., Sollner, T. H., Rothman, J. E. and Wieland, F. T. (1999). Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors. Cell 96, 495-506.

Ciufo, L. F. and Boyd, A. (2000). Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway. Journal of Biological Chemistry 275, 8382-8.

Cuppen, E., Wijers, M., Schepens, J., Fransen, J., Wieringa, B. and Hendriks, W. (1999). A FERM domain governs apical confinement of PTP-BL in epithelial cells. Journal of Cell Science 112 ( Pt 19), 3299-308.

De Rijk, E. P., Jenks, B. G. and Wendelaar Bonga, S. E. (1990). Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation. General and Comparative Endocrinology 79, 74-82.

Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J. J., Solari, R. C. and Owen, M. J. (2000). The p24 family member p23 is required for early embryonic development. Current Biology 10, 55-8.

Dominguez, M., Dejgaard, K., Füllekrug, J., Dahan, S., Fazel, A., Paccaud, J. P., Thomas, D. Y., Bergeron, J. J. and Nilsson, T. (1998). gp25l/emp24/p24 protein family members of the cis-Golgi network bind both Cop I and Ii coatomer. Journal of Cell Biology 140, 751-65.

Elrod Erickson, M. J. and Kaiser, C. A. (1996). Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations. Molecular Biology of the Cell 7, 1043-1058.

Emery, G., Rojo, M. and Gruenberg, J. (2000). Coupled transport of p24 family members. Journal of Cell Science 113 ( Pt 13), 2507-16.

Fiedler, K., Veit, M., Stamnes, M. A. and Rothman, J. E. (1996). Bimodal interaction of coatomer with the p24 family of putative cargo receptors. Science 273, 1396-1399.

Fransen, J. A., Ginsel, L. A., Hauri, H. P., Sterchi, E. and Blok, J. (1985). Immuno-electronmicroscopical localization of a microvillus membrane disaccharidase in the human small-intestinal epithelium with monoclonal antibodies. European Journal of Cell Biology 38, 6-15.

Füllekrug, J., Suganuma, T., Tang, B. L., Hong, W., Storrie, B. and Nilsson, T. (1999). Localization and Recycling of gp27 (hp24gamma3): Complex Formation with Other p24 Family Members. Molecular Biology of the Cell 10, 1939-1955.

Hadley, M. E., Heward, C. B., Hruby, V. J., Sawyer, T. K. and Yang, Y. C. (1981). Biological actions of melanocyte-stimulating hormone. CIBA Foundation Symposium 81, 244-262.

Holthuis, J. C., Jansen, E. J. R., van Riel, M. C. and Martens, G. J. M. (1995a). Molecular probing of the secretory pathway in peptide hormone-producing cells. Journal of Cell Science 108, 3295-305.

Holthuis, J. C., van Riel, M. C. and Martens, G. J. M. (1995b). Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary. Biochem J 312, 205-213.

Jansen, E. J. R., Holling, T. M., van Herp, F. and Martens, G. J. M. (2002). Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis. FEBS Letters 516, 201-7.

Jenks, B. G., de Koning, H. P., Valentijn, K. and Roubos, E. W. (1993). Dual action of GABAA receptors on the secretory process of melanotrophs of Xenopus laevis. Neuroendocrinology 58, 80-5.

Jenks, B. G., Overbeeke, A. P. and McStay, B. F. (1977). Synthesis, storage and release of MSH in the pars internmedia of the pituitary gland of Xenopus laevis during background adaptation. Canadian Journal of Zoology 55, 922-927.

Jenne, N., Frey, K., Brugger, B. and Wieland, F. T. (2002). Oligomeric state and stoichiometry of p24 proteins in the early secretory pathway. Journal of Biological Chemistry 277, 46504-11.

Kaiser, C. (2000). Thinking about p24 proteins and how transport vesicles select their cargo. Proc Natl Acad Sci U S A 97, 3783-3785.

Kroll, K. L. and Amaya, E. (1996). Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173-83.

Kuiper, R. P., Bouw, G., Janssen, K. P., Rötter, J., van Herp, F. and Martens, G. J. M. (2001). Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell. Biochemical Journal 360, 421-9.

Kuiper, R. P., Waterham, H. R., Rötter, J., Bouw, G. and Martens, G. J. M. (2000). Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell. Molecular Biology of the Cell 11, 131-40.

Lavoie, C., Paiement, J., Dominguez, M., Roy, L., Dahan, S., Gushue, J. N. and Bergeron, J. J. (1999). Roles for alpha(2)p24 and COPI in Endoplasmic Reticulum Cargo Exit Site Formation. Journal of Cell Biology 146, 285-300.

Martens, G.J.M., Jenks, B.G. and Van Overbeeke, A.P. (1982) Biosynthesis of a γ3-melanotropin-like peptide in the pars intermedia of the amphibian pituitary gland. Eur. J. Biochem. 126, 23-28

Martens, G. J. M. (1986). Expression of two proopiomelanocortin genes in the pituitary gland of Xenopus laevis: complete structures of the two preprohormones. Nucleic Acids Research 14, 3791-3798.

Martens, G. J. M., Biermans, P. P., Jenks, B. G. and Van Overbeeke, A. P. (1982). Biosynthesis of two structurally different pro-opiomelanocortins in the pars intermedia of the amphibian pituitary gland. European Journal of Biochemistry 126, 17-22.

Martens, G. J. M., Jenks, B. G. and Overbeeke, A. P. (1981). N alpha-acetylation is linked to alpha-MSH release from pars intermedia of the amphibian pituitary gland. Nature 294, 558-560.

Marzioch, M., Henthorn, D. C., Herrmann, J. M., Wilson, R., Thomas, D. Y., Bergeron, J. J., Solari, R. C. and Rowley, A. (1999). Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex. Molecular Biology of the Cell 10, 1923-1938.

Mironov, A. A., Mironove Jr., A. A., Beznoussenko, G. V., Trucco, A., Lupetti, P., Smith, J. D., Geerts, W. J. C., Koster, A. J., Burger, K. N. J., Martone, M. E., Deerinck, T. J., Ellisman, M. H. and Luini, A. (2003). ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains. Developmental Cell 5, 583-594.

Muñiz, M., Nuoffer, C., Hauri, H. P. and Riezman, H. (2000). The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles. Journal of Cell Biology 148, 925-30.

Nakamura, N., Yamazaki, S., Sato, K., Nakano, A., Sakaguchi, M. and Mihara, K. (1998). Identification of potential regulatory elements for the transport of emp24p. Molecular Biology of the Cell 9, 3493-503.

Nickel, W., Sohn, K., Bunning, C. and Wieland, F. T. (1997). p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway. Proc Natl Acad Sci U S A 94, 11393-8.

Rojo, M., Emery, G., Marjomaki, V., McDowall, A. W., Parton, R. G. and Gruenberg, J. (2000). The transmembrane protein p23 contributes to the organization of the Golgi apparatus. Journal of Cell Science 113, 1043-57.

Rojo, M., Pepperkok, R., Emery, G., Kellner, R., Stang, E., Parton, R. G. and Gruenberg, J. (1997). Involvement of the transmembrane protein p23 in biosynthetic protein transport. Journal of Cell Biology 139, 1119-35.

Rötter, J., Kuiper, R. P., Bouw, G. and Martens, G. J. M. (2002). Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors. Journal of Cell Science 115, 1049-58.

Roubos, E. W. (1997). Background adaptation by Xenopus laevis: a model for studying neuronal information processing in the pituitary pars intermedia. Comparative Biochemistry and Physiology. Part A, Physiology 118, 533-50.

Schimmöller, F., Singer Krüger, B., Schröder, S., Krüger, U., Barlowe, C. and Riezman, H. (1995). The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO Journal 14, 1329-39.

Schweizer, A., Fransen, J. A., Bachi, T., Ginsel, L. and Hauri, H. P. (1988). Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. Journal of Cell Biology 107, 1643-53.

Sohn, K., Orci, L., Ravazzola, M., Amherdt, M., Bremser, M., Lottspeich, F., Fiedler, K., Helms, J. B. and Wieland, F. T. (1996). A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding. Journal of Cell Biology 135, 1239-48.

Sparrow, D. B., Latinkic, B. and Mohun, T. J. (2000). A simplified method of generating transgenic Xenopus. Nucleic Acids Research 28, E12.

Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S. and Schekman, R. (2000). The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 97, 4034-9.

Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P. and Rothman, J. E. (1995). An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding [published erratum appears in Proc Natl Acad Sci U S A 1995 Nov 7;92(23):10816]. Proc Natl Acad Sci U S A 92, 8011-5.

Trotter, E. W., Berenfeld, L., Krause, S. A., Petsko, G. A. and Gray, J. V. (2001). Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomycescerevisiae. Proc Natl Acad Sci U S A 98, 7313-8.

Tuinhof, R., Artero, C., Fasolo, A., Franzoni, M. F., Ten Donkelaar, H. J., Wismans, P. G. and Roubos, E. W. (1994). Involvement of retinohypothalamic input, suprachiasmatic nucleus, magnocellular nucleus and locus coeruleus in control of melanotrope cells of Xenopus laevis: a retrograde and anterograde tracing study. Neuroscience 61, 411-420.

Van Horssen, A. M., Van Kuppeveld, F. J. and Martens, G. J. M. (1998). Manipulation of disulfide bonds differentially affects the intracellular transport, sorting, and processing of neuroendocrine secretory proteins. Journal of Neurochemistry 71, 402-9.

Weatherhead, B., Thornton, V. F. and Whur, P. (1971). Effects of change of background colour on the ultrastructure of the 'MSH cells' of the pars intermedia of Xenopus laevis. Journal of Endocrinology 49, xxv-xxvi.

Wen, C. and Greenwald, I. (1999). p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans. Journal of Cell Biology 145, 1165-75.

 

 

 

Chapter 6: Differential effect of transgenic overexpression of two closely related p24δ subfamily members on the endogenous p24 proteins and prohormone processing in the Xenopus intermediate pituitary cell

 

 

Differential effect of transgenic overexpression of two closely related p24δ subfamily members on the endogenous p24 proteins and prohormone processing in the Xenopus intermediate pituitary cell

Gerrit Bouw, Rick Van Huizen, Eric J.R. Jansen and Gerard J.M. Martens

 

 

ABSTRACT

The p24δ proteins comprise a subclass of the p24 family of type I transmembrane proteins (p24α, -β, -γ and -δ) that are thought to play some role in protein transport through the early secretory pathway. Recently, we have identified and characterized two members of the p24δ subfamily (Xp24δ1 and -δ2) in the Xenopus intermediate pituitary melanotrope cell, of which Xp24δ2, but not Xp24δ1, was coordinately expressed with the major secretory cargo protein in this cell type, the prohormone proopiomelanocortin (POMC). Transgenic overexpression of Xp24δ2 specifically in the melanotrope cell nearly completely displaced the endogenous p24 proteins, and affected POMC maturation and processing, indicating that Xp24δ2 is somehow involved in the biosynthesis of POMC. Xp24δ1 is highly similar to Xp24δ2, except for the N-terminal loop domain. In this study, we therefore investigated the effect of overexpressing Xp24δ1 on the biosynthesis of POMC. Intriguingly, we did not observe the phenotypic effects caused by the overexpression of Xp24δ2 (severely distorted p24 system, affected POMC-peptide profile, reduced number and size of skin melanophores). Instead, members of the Xp24α and -γ subfamilies were upregulated, the p24 system appeared to be intact, and no effect on POMC maturation and processing was observed. These results suggest that the N-terminal domains of p24 proteins are involved in selective multimerization and point to a role for p24 in directing the build-up of specific secretory pathway subcompartments, thereby providing the appropriate environment for correct secretory protein processing.

 

 

INTRODUCTION

Cargo proteins are synthesized and subjected to quality control before they enter the early secretory pathway. Inclusion into vesicles generated at ER exit sites and subsequent binding of COP II coat proteins to the vesicles initiate the trafficking of cargo molecules from the ER to the Golgi apparatus (Barlowe, 2000). For several steps of cargo-selective transport, a role for the p24 family of type I transmembrane proteins has been implicated, since p24 proteins are highly abundant membrane proteins in transport intermediates of the early secretory pathway (Stamnes et al., 1995). The p24 family can be classified into four main subfamilies, designated p24α, -β, -γ and -δ (Dominguez et al., 1998; Nickel and Wieland, 1998; Schimmöller et al., 1995; Sohn et al., 1996; Stamnes et al., 1995), and the members share a number of structural characteristics, such as a large lumenal putative cargo-binding domain, a coiled-coil region thought to be involved in the formation of dimeric or multimeric p24 complexes, a transmembrane region, and a short cytoplasmic tail containing COPI- and COPII-binding motifs that are used for p24 travelling from the ER to the Golgi and back (for review, see Kaiser, 2000). In yeast and mammalian cells, p24 proteins form functional heterotetrameric complexes. These complexes are composed of one member of each subfamily, whereby the composition of the complex may differ in various cell types (Belden and Barlowe, 2001; Ciufo and Boyd, 2000; Dominguez et al., 1998; Emery et al., 2000; Füllekrug et al., 1999; Marzioch et al., 1999). Furthermore, in yeast or mice, deficiency of one p24 member affects the stability of other p24 members (Denzel et al., 2000; Marzioch et al., 1999) and in mammalian cells, overexpression of a single member causes proliferation of ER-derived membranous structures, indicating that the stoichiometry between the different p24 family members is very subtle (Rojo et al., 2000). In addition, recent evidence suggests a complex and dynamic p24 system, consisting of mostly monomers and homo-/hetero-dimers, and that the degree of oligomerization can alter and largely depends on the subcellular localizations of the p24 subfamily members (Jenne et al., 2002). Although a number of studies have resulted in various presumed functions for the p24 proteins in the early secretory pathway (reviewed by Kaiser, 2000), the precise molecular action of p24 proteins remains to be elucidated. In order to investigate the role of p24, we used the South-African claw-toed frog Xenopus laevis as a model system, and in particular the intermediate pituitary melanotrope cell. This cell type is a neuroendocrine secretory cell that is dedicated to produce high amounts (~80% of all newly synthesized proteins) of proopiomelanocortin (POMC), a prohormone that mediates the process of background adaptation, when the animal is placed on a black background (Roubos, 1997). POMC is cleaved endoproteolytically to α-Melanophore Stimulating Hormone (α-MSH) as a major bioactive cleavage product that, once released into the blood, causes dispersion of melanin pigment granules (melanosomes) in skin melanophores. Adapting Xenopus to a black- or a white background therefore allows us to physiologically manipulate the biosynthetic and secretory activities of the melanotrope cell. Moreover, the endoproteolytic processing of POMC molecules to smaller products provides an elegant read-out system for protein folding and subsequent transport out of the ER to the late secretory pathway cleavage compartments (trans-Golgi network (TGN) and secretory granules).

In mammalian cells, the p24 family member p24δ1 represents the first identified p24 protein and one of the best-characterized p24 proteins. It was isolated from Golgi-derived COP I-coated transport vesicles (Sohn et al., 1996) and is able to form functional complexes with p24β (Belden and Barlowe, 1996; Gommel et al., 1999). Furthermore, in mouse, genetic deletion of p24δ1 resulted in early embryonic death, revealing the importance of this protein in development (Denzel et al., 2000). In order to investigate the role of p24 proteins in the Xenopus melanotrope cell, we first identified all members of the p24 family expressed in this highly specialized cell type. Analogous to the mammalian p24 protein family, we could subdivide the seven Xenopus p24 family members in four subfamilies, Xp24α, -β, -γ and -δ. Of these subfamily members, we found Xp24α3, Xp24β1, Xp24γ3 and Xp24δ2 to be coordinately expressed with POMC, indicating that these family members may be involved in the transport of the prohormone through the early secretory pathway of the melanotrope cell. Two other members (Xp24γ2 and -δ1) were not or only slightly induced, while one member (Xp24γ1) is not expressed in the Xenopus melanotrope cell (Holthuis et al., 1995; Kuiper et al., 2000; Rötter et al., 2002). Thus, the expression of Xp24δ2, but not of Xp24δ1, is induced during black-background adaptation. We observed that upon stable transgenic overexpression of Xp24δ2 specifically in the Xenopus melanotrope cell, the expression levels of other p24 family members in this cell type were severely reduced (Bouw et al., 2003). This caused no apparent changes in the morphology of the melanotrope cell and the level of newly synthesized POMC was normal, despite the heavily distorted p24 system. However, we found that an aberrant form of 18-kDa POMC (a major cleavage product of POMC) and reduced levels of POMC-derived peptides apparently decreased the number and pigment content of the skin melanophores (chapter 5). These in vivo results underlined the importance of an intact p24 system in the highly specialized melanotrope secretory cell and pointed to a role for p24 in directing cargo to specific secretory pathway subcompartments containing the machinery for proper secretory protein maturation and processing (Bouw et al., 2003). Xp24δ1 and -δ2 are highly related proteins with the highest degree of amino acid sequence identity in the coiled-coil regions, transmembrane domains and the short C-terminal cytoplasmic tails (81% identity, 81% identity and 86% identity, respectively), and the lowest identity in the N-terminal loop domains (61% identity). Although both subfamily members are expressed ubiquitously, the levels of Xp24δ1 and -δ2 are tissue dependent with high levels of Xp24δ2 in neuronal and neuroendocrine tissues, such as the pituitary and the brain. Xp24δ1 is the predominant p24δ protein in non-neuroendocrine tissues. Furthermore, upon black-background adaptation, in Xenopus melanotrope cells the Xp24δ2 protein levels are increased ~25 times, whereas Xp24δ1 protein expression was only slightly elevated (~2.5 times). In addition, the mRNA levels of Xp24δ2 increased 5 times in animals that were adapted to a black background as compared to a white background, whereas for Xp24δ1 no change was observed (Kuiper et al., 2000). Since Xp24δ1 is apparently not coexpressed with POMC, we extended our cell-specific transgenic overexpression studies from Xp24δ2 to Xp24δ1 and wondered which effect Xp24δ1 overexpression would have on the early transport and subsequent processing of POMC in the transgenic Xenopus melanotrope cells. For this purpose, we C-terminally tagged Xp24δ1 with green fluorescent protein (GFP) and overexpressed this fusion protein specifically in the Xenopus melanotrope cell, using the technique of stable Xenopus transgenesis and a small POMC gene promoter fragment, as shown previously for melanotrope cell-specific transgene expression of GFP and Xp24δ2-GFP (Bouw et al., 2003; Jansen et al., 2002). In this study, we combined the unique properties of the melanotrope cell with targeted expression of the Xp24δ1 protein in this cell in order to explore the role of Xp24δ1 close to the in vivo situation. The effect of Xp24δ1-GFP overexpression on the transport and processing of POMC was then examined in the transgenic melanotrope cells. Our results suggest that the N-terminal domains of p24 proteins determine complex formation and that functional p24 complexes are necessary for the build-up of proper secretory pathway subcompartments, thereby providing the appropriate environment for correct secretory protein processing.

 

 

MATERIALS AND METHODS

Animals

Xenopus laevis were reared in the Central Animal Facility of the University of Nijmegen. For the transgenesis experiments, female Xenopus laevis were obtained directly from South Africa. For background adaptation, the animals were kept in either white or black containers under constant illumination for at least three weeks. All animal experiments were carried out in accordance with the European Communities Council Directive 86/609/EEC for animal welfare, and permit TRC 99/15072 to generate and house transgenic Xenopus.

 

Antibodies

The rabbit polyclonal antibodies against a portion of the lumenal region of Xp24δ2 (anti-1262N), against part of the lumenal region of Xp24δ1 (anti-RH6) and against a region in the lumenal part of Xp24α3 have been described previously (Kuiper et al., 2001; Rötter et al., 2002). A polyclonal antibody to human p24β1 (p24A) was kindly provided by Dr. I. Schulz (University of the Saarland, Homburg, Germany; Blum et al., 1996), to human p24γ3 by Dr. T. Nilsson (EMBL, Heidelberg, Germany; Dominguez et al., 1998), against recombinant mature human PC2 by Dr. W.J.M. Van de Ven (University of Leuven, Belgium; Creemers et al., 1996) and against Xenopus POMC (ST62, recognizing only the precursor form) by Dr. S. Tanaka (Shizuoka University, Japan; Berghs et al., 1997).

 

Generation of Xenopus transgenic for Xp24δ1-GFP

A linear 2166-bp SalI/NarI DNA fragment encoding the Xenopus p24δ1 protein with the enhanced GFP protein fused in frame to its C terminus (Xp24δ1-GFP fusion protein) and cloned behind a 529-bp Xenopus POMC gene A promoter (pPOMC; Jansen et al., 2002) fragment (construct pPOMC-Xp24δ1-GFP) was used for stable Xenopus transgenesis (Kroll and Amaya, 1996; Sparrow et al., 2000). A number of injection rounds resulted in animals transgenic for Xp24δ1-GFP and expressing the fusion protein at high levels. To generate F1 offspring, the testes of a male transgenic Xenopus frog, expressing the fusion protein at high level, was isolated and used for in vitro fertilization. For this pieces of testis were incubated with eggs harvested from wild-type Xenopus females. From the remainder of the testes, sperm nuclei were prepared and stored for future injections.

 

Western blot analysis

Western blot analysis was performed as described previously (Kuiper et al., 2000). For quantification, detection was performed using a BioChemi imaging system and signals were analyzed using the Labworks 4.0 program (UVP BioImaging systems, Cambridge, UK).

 

Pulse and pulse-chase analysis

For metabolic labelling, neurointermediate lobes (NILs) from wild-type and transgenic Xenopus were preincubated for 30 min, pulse labelled in the presence of 5 mCi/ml Tran35S-label (ICN Radiochemicals) and chased with 0.5 mM L-methionine for the indicated time periods, and homogenized as described previously (Braks and Martens, 1994).

 

Immunoprecipitation analysis

For immunoprecipitation analysis, NIL lysates were supplemented with 0.1% SDS and the respective antibodies in lysis buffer. Precipitation was performed overnight at 4°C while rotating the samples. Immune complexes were precipitated with protein-A Sepharose (Amersham-Pharmacia Biotech) and resolved by SDS-PAGE. Radiolabelled proteins were detected using autoradiography at -70°C or a PhosphoImager (Personal FX, Biorad).

 

HPLC analysis

For the separation of the small newly synthesized end products of POMC processing, radiolabelled NIL lysates were subjected to HPLC analysis as described previously (Martens et al., 1982).

 

Microscopy

For ultrastructural analysis, electron microscopy was performed as described previously (De Rijk et al., 1990). For immunoelectron microscopy, entire pituitary lobes were fixed for one hour at room temperature in 2% paraformaldehyde (PFA) + 0.01% glutaraldehyde in PHEM buffer (50 mM MgCl 2 , 70 mM KCl, 10 mM EGTA, 20 mM HEPES, 60 mM PIPES, pH 6.8). Fixed tissue was stored in 1% PFA in 0.1M phosphate buffer until use. Ultra-thin cryosectioning was performed as described before (Fransen et al., 1985; Schweizer et al., 1988). Sections were incubated with an antiserum against EGFP at a 1:1000 dilution (Cuppen et al., 1999) followed by protein A complexed with 10 nm gold (Fransen et al., 1985). Electron microscopy was performed using a Jeol 1010 electron microscope operating at 80 kV. To examine direct fluorescence as a result of GFP fusion protein expression, cryosections were directly viewed under a Leica DM RA fluorescent microscope and photographs were taken with a Cohu High Performance CCD Camera using the Leica Q Fluoro software. For confocal microscopy, brains with the pituitaries attached were dissected and fixed in 4% paraformaldehyde in PBS. After cryoprotection in 10% sucrose-PBS, sagittal 20-µm cryosections were mounted on poly-L-lysine coated slides, dried for 2 hr at 45ºC and immunohistochemistry for POMC was performed as described previously (Jansen et al., 2002). Slides were analyzed with a MRC 1024 CLSM. To examine direct fluorescence as a result of GFP fusion protein expression, cryosections were directly viewed under a Leica DM RA fluorescent microscope and photographs were taken with a Cohu High Performance CCD Camera using the Leica Q Fluoro software. For light microscopy analysis of the webs and melanophores of wild-type and transgenic animals, digital images were obtained using a Leica MZFLIII microscope mounted with a DC200 digital colour camera. Equal integration intervals and magnifications were used to capture images with Leica DC viewer software.

 

 

RESULTS

Generation of the Xp24δ1-GFP transgene construct and of Xenopus transgenic for this construct

For transgenic studies, the entire open-reading frame of Xp24δ1 was cloned in front of enhanced green fluorescent protein (EGFP). This construct was then cloned behind a POMC gene promoter fragment in the pCS2+ vector resulting in the transgene construct pPOMC-Xp24δ1-GFP that was used to make transgenic animals. By using the POMC promoter fragment to drive the expression of Xp24δ1-GFP, we targeted the fusion protein specifically to the Xenopus melanotrope cells, analogous to previous studies (Bouw et al., 2003; Jansen et al., 2002). The DNA was mixed with Xenopus sperm nuclei and the mixture was microinjected into unfertilized Xenopus eggs, resulting in healthy transgenic embryos. The different levels of expression of the fusion protein among the various transgenic animals could be directly established by analysis of the living embryos under a fluorescence microscope (Figure 1A). From various rounds of transgenesis, we obtained a male transgenic animal expressing the fusion protein at high levels. Subsequent in vitro fertilization of wild type eggs with transgenic sperm resulted in an F1 generation of Xenopus transgenic for Xp24δ1-GFP (F1 line #252).

Figure 1: Xp24δ1-GFP transgene expression is specific to Xenopus intermediate pituitary and dependent on background colour.

(A) Pituitary-specific fluorescence in transgenic Xenopus embryos. Shown are living stage-45 embryos, whereby the arrows indicate the locations of the pituitaries with different levels of transgene expression. Fluorescent pituitaries expressing the transgene fusion product could be detected from stage 25 onwards. Bar equals 0.4 mm. (B) Fluorescence is specific to the intermediate pituitary of transgenic Xenopus. Ventrocaudal view on the brain that was lifted to reveal the bright fluorescence caused by the Xp24δ1-GFP fusion protein. Fluorescence was observed in the intermediate lobe (IL), but not in the anterior lobe (AL), of the pituitary of a black-adapted transgenic frog of 6 months. Bar equals 0.5 mm. (C) Xp24δ1-GFP is primarily expressed in the melanotrope cells of the intermediate pituitary of transgenic Xenopus. Sagittal brain-pituitary cryosections of an Xp24δ1-GFP transgenic frog were analysed. Sections were stained for POMC using an antibody recognizing entire prohormone and a Texas-Red conjugated second antibody (middle panel), and stained cells that show GFP fluorescence observed by direct visualization (left panel). The right panel shows the merged picture of the direct GFP fluorescent signal and the signal for endogenous POMC. Bar equals 50µm.

 

Xp24δ1-GFP is expressed only in the Xenopus intermediate pituitary melanotrope cells

To examine the specificity of the POMC promoter fragment driving the expression of the Xp24δ1-GFP transgene product, we dissected the neurointermediate lobe (NIL) and anterior lobe (AL) of wild-type and transgenic animals. Direct microscopical analysis of the pituitary of transgenic animals showed that only cells situated in the intermediate lobe (IL) of the pituitary were positive for GFP. No obvious GFP signal was detected in the neural lobe, AL or other parts of the brain (Fig. 1B). In addition, confocal microscopy using a POMC antibody recognizing only intact POMC showed that the Xp24δ1-GFP fusion protein was expressed in the melanotrope cells that produce the prohormone (Fig. 1C). Western blot analysis using Xp24δ1 and -δ2 specific antibodies confirmed the tissue-specific expression (data not shown). In addition to endogenous Xp24δ1 and -δ2 in the NIL, a protein of ~51 kDa was detected in the transgenic cells. This protein most likely represents the Xp24δ1-GFP fusion protein (~23 kDa for Xp24δ1 and ~28 kDa for GFP) (Fig. 2A). In wild-type animals, no immunoreactive protein of ~51 kDa was detected in either the NIL or AL (Fig. 2A, and data not shown).

Figure 2: p24 protein levels in the intermediate pituitary of transgenic Xenopus expressing Xp24δ1-GFP.

(A) Western blot analysis of p24δ protein expression in the neurointermediate (NIL) lobe of black-adapted wild-type (wt) and various transgenic (tr) Xenopus. (B) Western blot analysis of Xp24α3 and -γ3 protein expression in the NIL of wild-type and transgenic Xenopus. Tubulin was used as a control for protein loading.

Steady-state p24 protein levels in the intermediate pituitary of Xenopus transgenic for Xp24δ1-GFP

Western blot analysis was performed on lysates of NILs of wild-type and transgenic animals using a mixture of antibodies specific for Xp24δ1 and -δ2 for simultaneous detection of the transgene and endogenous Xp24δ products. This antibody mix showed in the wild-type NIL about equal amounts of the endogenous Xp24δ1 and Xp24δ2 proteins (Fig. 2A, lane 1). Although the levels of Xp24δ1-GFP in the transgenic NIL varied among animals, in all cases the amount of Xp24δ1-GFP was clearly higher than that of endogenous Xp24δ1 (4,8x ± 2; n=6) and -δ2 (4,4x ± 1,6; n=6) (Fig. 2A, lanes 2-5). Western blot analysis of NILs of white- and black-adapted transgenic animals revealed that the expression of Xp24δ1-GFP was higher in black than in white animals (Fig. 2B). Thus, in addition to the cell-specific expression of transgenes and in line with previous results (Bouw et al., 2003; Jansen et al., 2002), the use of the POMC promoter fragment allowed us to physiologically manipulate the expression level of Xp24δ1-GFP by placing transgenic animals on a black- or white background (Fig. 2B). The amounts of endogenous Xp24δ1 and -δ2 in the transgenic NIL were slightly induced (1,6x ± 0,7; n=9 and 1,6x ± 0,8; n=9, respectively) as compared to those in the wild-type NIL. We next wondered whether other p24 members were affected by the overexpression of Xp24δ1-GFP. Western blot analysis using p24 antibodies specific for Xp24α3 and -γ3 showed that in #252 transgenic melanotrope cells the steady-state levels of both endogenous Xp24α3 and -γ3 were ~3-4x higher (3,8 ± 1,6 and 3,6 ± 1,7, respectively) than in wild-type cells (Fig. 2B). Unfortunately, for Xp24β1 no reliable data could be obtained with the available antibodies.

Figure 3: Xp24δ1-GFP localizes to ER and Golgi, and overexpression does not alter subcellular structures.

Electron micrographs of melanotrope cells of #252 transgenic Xenopus adapted to a black background. Ultra-thin sections of transgenic NILs were incubated with an antiserum against EGFP followed by protein A complexed with 10 nm gold. Xp24δ1-GFP was found in structures that resemble ER (left panel) and Golgi (right panel) compartments. Bars equal 0,1µm.

Immunoelectron microscopy analysis of melanotrope cells expressing Xp24δ1-GFP

Since overexpression of mammalian p24δ1 causes the expansion of smooth ER membranes in transfected mammalian cells in culture (Blum et al., 1999; Rojo et al., 1997), we wondered what the effect of expressing Xp24δ1-GFP at high levels was on the morphology of the transgenic melanotrope cells. Furthermore, to examine to which early secretory pathway membranes Xp24δ1-GFP would localize, we performed immunoelectron microscopy using an antibody directed against GFP. Xp24δ1-GFP was found in structures that resemble the ER and the Golgi, and this finding is in line with previous results showing that the mammalian p24δ1 protein continuously cycles between the ER and the Golgi (Nickel et al., 1997) (Fig. 3). We did not observe any change in ER and Golgi morphology between wild-type and transgenic cells, suggesting that the overexpression of Xp24δ1-GFP was to such extent that did not induce any morphological changes in the early secretory pathway, comparable to what was found when Xp24δ2-GFP was overexpressed in the Xenopus melanotrope cells (Bouw et al., 2003) (data not shown).

Background adaptation of Xenopus transgenic for Xp24δ1-GFP

To investigate whether the overexpression of Xp24δ1-GFP and the subsequent slight upregulation of other p24 members (Xp24α3 and -γ3) would have any consequences for the process of background adaptation, transgenic animals were placed on a black background for at least four months. As expected, wild-type Xenopus were black and contained many completely dispersed pigment-filled granules in the dermal melanophores of their webs. The #252 transgenic animals were similar to wild-type animals with respect to the number and pigment content of their melanophores (Fig. 4).

Figure 4: Background adaptation of Xenopus transgenic for Xp24δ1-GFP.

Wild-type (wt) and #252 transgenic animals (tr) were placed on a black background for 4 months. Shown below are the webs (lower panel) and pigment-containing dermal melanophores in the webs (upper panel); bars equal 1 mm and 250 µm for the lower- and upper panel, respectively.

 

Steady-state and newly synthesized levels of Xp24δ, POMC and (pro)PC2 in intermediate pituitary melanotrope cells transgenic for Xp24δ1-GFP

Similar to what was found for Xenopus that express Xp24δ2-GFP (Bouw et al., 2004), the expression of the Xp24δ1-GFP fusion protein was dependent on the background colour (Fig. 5A). The process of background adaptation is controlled by α-MSH, a cleavage product of POMC. Since the animals transgenic for Xp24δ1-GFP were able to normally adapt to their background, we next wondered whether the steady-state and newly synthesized protein levels of POMC in wild-type and transgenic melanotrope cells were similar. Interestingly, western blot analysis showed that the steady-state level of POMC in #252 transgenic cells was slightly induced (2,5x ± 0,7; n=8) as compared to wild-type cells, whereas the amounts of both the proenzyme and mature forms of the POMC cleavage enzyme PC2 were comparable between transgenic and wild-type cells (1,1x ± 0,4; n=8, Fig. 5B). In both wild-type and transgenic animals, the steady-state levels of POMC were dependent on the background colour to the same extent and overexpression of Xp24δ1-GFP did thus not change this physiological manipulation of gene expression (data not shown). To investigate the dynamics of POMC biosynthesis and processing, we performed pulse-chase experiments with melanotrope cells from wild-type and transgenic animals. For the analysis of the degree of prohormone cleavage to 18-kDa POMC, we used apomorphine to block secretion, since released 18-kDa POMC may be degraded in the medium and this would then interfere with our analysis. The transgenic melanotrope cells showed no affected POMC biosynthesis and subsequent processing to 18-kDa POMC, suggesting that these processes are not affected by the overexpression of Xp24δ1-GFP in the melanotrope cells (Fig. 6).

Figure 5: Steady-state protein levels of Xp24δ, POMC and PC2 in black- and white-adapted wild-type and transgenic Xenopus.

(A) Western blot analysis of p24δ protein expression in the neurointermediate lobe (NIL) of black-adapted (BA) and white-adapted (WA) wild-type (wt) and transgenic (tr) Xenopus using a p24δ1/-δ2-specific antibody mix. The lower panel shows direct fluorescent signals in the NILs. (B) Western blot analysis of NIL proteins of wild-type and transgenic Xenopus adapted to a black (BA) or white (WA) background using anti-POMC and anti-PC2 antibodies.

Figure 6: Newly synthesized proteins produced in wild-type and transgenic Xenopus intermediate pituitary cells.

Neurointermediate lobes (NILs) of black-adapted wild-type (wt) and #252 transgenic (tr) animals were pulse labelled for 30 min and chased for 150 min. Newly synthesized proteins extracted from the lobes were resolved by SDS-PAGE and visualized by fluorography.

Profile of POMC-derived peptides in intermediate pituitary cells transgenic for p24δ1-GFP

Since no effect on the level of synthesis of POMC and processing to 18-kDa POMC was observed in the transgenic animals, we next wondered whether the processing of POMC into smaller bioactive peptides was normal. For this, we performed HPLC analysis of melanotrope cell lysates and -media of wild-type- and transgenic animals. The peptide profiles showed that the amounts of peptides produced and secreted into the medium were not affected in the transgenic animals (data not shown). Together with the unaffected biosynthesis of POMC this suggests that the efficiency of processing is similar in wild-type melanotrope cells and cells that express Xp24δ1-GFP.

 

 

DISCUSSION

The p24 proteins are major type I transmembrane constituents of early secretory pathway membranes and represent about 30% of the total membrane protein content of COP I- and COP II-coated vesicles (Rojo et al., 1997). Their structural characteristics, such as an N-terminal putative cargo-binding loop structure and the ability to bind to both COP I- and COP II coat proteins through motifs in the short cytoplasmic tail, suggest that they play an important role in cargo-selective transport through the early secretory pathway. Several approaches have been employed to unravel the molecular mechanisms behind the action of p24 proteins. However, the complex nature of these proteins has hampered functional analysis. In the past, we overexpressed a GFP-tagged Xp24δ2 protein specifically in the melanotrope cell using Xenopus transgenesis and this manipulation caused displacement of the endogenous p24 complex. Overexpression of this p24 family member affected the maturation and processing of POMC (the major cargo molecule in the melanotrope cell) and as a result, the transgenic animals were not able to fully adapt to a black background (Bouw et al., 2004). Since Xp24δ2 is one of the p24 members that are coordinately expressed with POMC (Xp24α3, -β1, -γ3 and -δ2; Kuiper et al., 2000; Rötter et al., 2002), we next wondered what would happen when the melanotrope cell has to cope with high amounts of one of the p24 members that is not upregulated upon black-background adaptation (Xp24δ1 or -γ1/2; Rötter et al., 2002). For this, we chose Xp24δ1, the p24δ subfamily member that is closely related to Xp24δ2 with which it shares a high degree of similarity at the amino acid level, especially in the coiled-coil region, transmembrane domain and the short cytoplasmic tail where they are virtually identical (Kuiper et al., 2000; Rötter et al., 2002). Since the expression level of Xp24δ1 was not dependent on background colour, we hypothesized that overexpression of this protein would interfere with the endogenous p24 content and may affect POMC transport and subsequent processing even more than by overexpressing Xp24δ2. We overexpressed a GFP-tagged form of Xp24δ1 and examined the effects of this manipulation on the functioning of the melanotrope cell. Interestingly, high levels of Xp24δ1-GFP did not displace endogenous Xp24δ1 and -δ2, but rather slightly induced their expression. This is in contrast to what has been observed for transgenic melanotrope cells overexpressing Xp24δ2-GFP where a clear downregulation of endogenous Xp24δ1 and -δ2 was found (Bouw et al., 2004). Remarkably, while Xp24δ2 overexpression also downregulated Xp24α3, -β1 and -γ3, the levels of endogenous Xp24α and -γ were significantly increased (~4x) in cells that overexpressed Xp24δ1. This ~four-fold increase was similar to the increase in the level of expression of Xp24δ1-GFP as compared to endogenous Xp24δ. In yeast and mammalian cells, the presence of α-γ dimers has been proposed (Belden and Barlowe, 1996; Ciufo and Boyd, 2000; Gommel et al., 1999). Furthermore, crosslinking studies have shown that dimers of Xp24α3-p24γ3 occur in the ER-Golgi intermediate compartment of Xenopus melanotrope cells (Kuiper, R.P., Janssen, K.P.C., Bouw, G., Rötter, J., van Herp, F., Martens, G.J.M., unpublished observations). However, the available antibodies do not discriminate between the various members of the subfamilies, and two forms of Xp24α and two -γ forms are recognized. Therefore, Xp24δ1-GFP might be part of a complex containing Xp24α2 and -γ2. In this scenario, the p24δ family would be involved in the formation of two independent functional p24 complexes, whereby the p24 complex containing Xp24δ2 provides cargo specificity and is involved in POMC biosynthesis. Overexpression of Xp24δ2-GFP resulted in a distorted p24 system in the melanotrope cell, ultimately leading to inefficient processing of POMC and the subsequent impaired ability of the animal to adapt to a black background. In transgenic animals overexpressing Xp24δ1-GFP, the fact that a distortion of the endogenous p24 complex apparently does not occur could account for the observed differential effect. Alternatively, the newly synthesized level of Xp24δ1-GFP in the #252 animal could have been too low to compete away the endogenous p24 members. For Xp24δ2, we observed that the degree of displacement was dependent on the level of newly synthesized Xp24δ2-GFP fusion protein. Transgenic cells expressing lower levels of the Xp24δ2 transgene product did not show an affected p24 system and as a result did not show any phenotypic effects (affected POMC-peptide profile, reduced number and size of melanophores; Bouw et al., 2004). However, in the #252 cells, the overexpression of Xp24δ1-GFP caused an upregulation of endogenous p24 members instead of reduced levels. In addition, preliminary experiments show that even in transgenic animals that express high levels of newly synthesized wild-type Xp24δ1 (untagged), Xp24α3 is not competed away (G. Bouw, J. Strating, data not shown). Therefore, the underlying mechanism that determines the p24 content in the melanotrope cells transgenic for Xp24δ1-GFP seems different from that of melanotrope cells overexpressing Xp24δ2-GFP. It is thought that the coiled-coil domains determine the p24 partners in the final complex (Ciufo and Boyd, 2000). The change in the loop or coiled-coil structure of Xp24δ1 and -δ2 may therefore explain the differences in the ability of Xp24δ1 and -δ2 fusion proteins to displace the endogenous p24 subfamily members.

Since in the ER specific inclusion of proteins into distinct transport vesicles determines the final destination of a certain cargo molecule (Campbell and Schekman, 1997; Schekman and Orci, 1996), we hypothesize that, based on the results described here, the p24 proteins could provide the appropriate signals to direct components that are necessary for secretory protein processing to their proper secretory pathway subcompartments. Individual functional p24 complexes composed of either Xp24αx, -βx, -γx, -δ1 or Xp24α3, -β1, -γ3, -δ2 target such components to separate and specific subcompartments of the Golgi apparatus, thereby providing the proper machinery for efficient and correct protein maturation and processing.

 

 

ACKNOWLEDGEMENTS

We would like to thank Ron Engels for animal care, Tony Coenen and Huib Croes for technical assistance with confocal- and electron microscopy, respectively. We also thank Drs Irene Schulz, Tommy Nilsson, Wim Van de Ven, Jack Franssen and Shige Tanaka for providing antibodies. This work was supported by grant 811.38.002 from the Netherlands Organization for Scientific Research - Earth and Life Sciences (NWO-ALW).

 

 

REFERENCES

Barlowe, C. (2000). Traffic COPs of the early secretory pathway. Traffic 1, 371-7.

Belden, W. J. and Barlowe, C. (1996). Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J Biol Chem 271, 26939-26946.

Belden, W. J. and Barlowe, C. (2001). Deletion of yeast p24 genes activates the unfolded protein response. Molecular Biology of the Cell 12, 957-69.

Berghs, C. A., Tanaka, S., Van Strien, F. J., Kurabuchi, S. and Roubos, E. W. (1997). The secretory granule and pro-opiomelanocortin processing in Xenopus melanotrope cells during background adaptation. Journal of Histochemistry and Cytochemistry 45, 1673-82.

Blum, R., Feick, P., Puype, M., Vandekerckhove, J., Klengel, R., Nastainczyk, W. and Schuldz, I. (1996). Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal membranes, are members of a protein family involved in vesicular trafficking. J Biol Chem 271, 17183-17189.

Blum, R., Pfeiffer, F., Feick, P., Nastainczyk, W., Kohler, B., Schäfer, K. H. and Schulz, I. (1999). Intracellular localization and in vivo trafficking of p24A and p23. Journal of Cell Science 112, 537-548.

Bouw, G., Van Huizen, R., Jansen, E. J. R. and Martens, G. J. M. (2004). A cell-specific transgenic approach in Xenopus reveals the importance of a functional p24 system for a secretory cell. Molecular Biology of the Cell, in press

Braks, J. A. and Martens, G. J. M. (1994). 7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway. Cell 78, 263-273.

Campbell, J. L. and Schekman, R. (1997). Selective packaging of cargo molecules into endoplasmic reticulum- derived COPII vesicles. Proc Natl Acad Sci U S A 94, 837-842.

Ciufo, L. F. and Boyd, A. (2000). Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway. Journal of Biological Chemistry 275, 8382-8.

Creemers, J. W., Usac, E. F., Bright, N. A., Van de Loo, J. W., Jansen, E., Van de Ven, W. J. M. and Hutton, J. C. (1996). Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2. J Biol Chem 271, 25284-25291.

Cuppen, E., Wijers, M., Schepens, J., Fransen, J., Wieringa, B. and Hendriks, W. (1999). A FERM domain governs apical confinement of PTP-BL in epithelial cells. Journal of Cell Science 112 ( Pt 19), 3299-308.

De Rijk, E. P., Jenks, B. G. and Wendelaar Bonga, S. E. (1990). Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation. General and Comparative Endocrinology 79, 74-82.

Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J. J., Solari, R. C. and Owen, M. J. (2000). The p24 family member p23 is required for early embryonic development. Current Biology 10, 55-8.

Dominguez, M., Dejgaard, K., Füllekrug, J., Dahan, S., Fazel, A., Paccaud, J. P., Thomas, D. Y., Bergeron, J. J. and Nilsson, T. (1998). gp25l/emp24/p24 protein family members of the cis-Golgi network bind both Cop I and II coatomer. Journal of Cell Biology 140, 751-65.

Emery, G., Rojo, M. and Gruenberg, J. (2000). Coupled transport of p24 family members. Journal of Cell Science 113 ( Pt 13), 2507-16.

Fransen, J. A., Ginsel, L. A., Hauri, H. P., Sterchi, E. and Blok, J. (1985). Immuno-electronmicroscopical localization of a microvillus membrane disaccharidase in the human small-intestinal epithelium with monoclonal antibodies. European Journal of Cell Biology 38, 6-15.

Füllekrug, J., Suganuma, T., Tang, B. L., Hong, W., Storrie, B. and Nilsson, T. (1999). Localization and Recycling of gp27 (hp24gamma3): Complex Formation with Other p24 Family Members. Molecular Biology of the Cell 10, 1939-1955.

Gommel, D., Orci, L., Emig, E. M., Hannah, M. J., Ravazzola, M., Nickel, W., Helms, J. B., Wieland, F. T. and Sohn, K. (1999). p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway. FEBS Letters 447, 179-85.

Holthuis, J. C., van Riel, M. C. and Martens, G. J. M. (1995). Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary. Biochem J 312, 205-213.

Jansen, E. J. R., Holling, T. M., van Herp, F. and Martens, G. J. M. (2002). Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis. FEBS Letters 516, 201-7.

Jenne, N., Frey, K., Brugger, B. and Wieland, F. T. (2002). Oligomeric state and stoichiometry of p24 proteins in the early secretory pathway. Journal of Biological Chemistry 277, 46504-11.

Kaiser, C. (2000). Thinking about p24 proteins and how transport vesicles select their cargo. Proc Natl Acad Sci U S A 97, 3783-3785.

Kroll, K. L. and Amaya, E. (1996). Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173-83.

Kuiper, R. P., Bouw, G., Janssen, K. P. C., Rötter, J., van Herp, F. and Martens, G. J. M. (2001). Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell. Biochemical Journal 360, 421-9.

Kuiper, R. P., Waterham, H. R., Rötter, J., Bouw, G. and Martens, G. J. M. (2000). Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell. Molecular Biology of the Cell 11, 131-40.

Martens, G. J. M., Jenks, B. G. and Overbeeke, A. P. (1982). Biosynthesis of pairs of peptides related to melanotropin, corticotropin and endorphin in the pars intermedia of the amphibian pituitary gland. European Journal of Biochemistry 122, 1-10.

Marzioch, M., Henthorn, D. C., Herrmann, J. M., Wilson, R., Thomas, D. Y., Bergeron, J. J., Solari, R. C. and Rowley, A. (1999). Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex. Molecular Biology of the Cell 10, 1923-1938.

Nickel, W., Sohn, K., Bunning, C. and Wieland, F. T. (1997). p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway. Proc Natl Acad Sci U S A 94, 11393-8.

Nickel, W. and Wieland, F. T. (1998). Biosynthetic protein transport through the early secretory pathway. Histochemistry and Cell Biology 109, 477-86.

Rojo, M., Emery, G., Marjomaki, V., McDowall, A. W., Parton, R. G. and Gruenberg, J. (2000). The transmembrane protein p23 contributes to the organization of the Golgi apparatus. Journal of Cell Science 113, 1043-57.

Rojo, M., Pepperkok, R., Emery, G., Kellner, R., Stang, E., Parton, R. G. and Gruenberg, J. (1997). Involvement of the transmembrane protein p23 in biosynthetic protein transport. Journal of Cell Biology 139, 1119-35.

Rötter, J., Kuiper, R. P., Bouw, G. and Martens, G. J. M. (2002). Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors. Journal of Cell Science 115, 1049-58.

Roubos, E. W. (1997). Background adaptation by Xenopus laevis: a model for studying neuronal information processing in the pituitary pars intermedia. Comparative Biochemistry and Physiology. Part A, Physiology 118, 533-50.

Schekman, R. and Orci, L. (1996). Coat proteins and vesicle budding. Science 271, 1526-33.

Schimmöller, F., Singer Krüger, B., Schröder, S., Krüger, U., Barlowe, C. and Riezman, H. (1995). The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO Journal 14, 1329-39.

Schweizer, A., Fransen, J. A., Bachi, T., Ginsel, L. and Hauri, H. P. (1988). Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. Journal of Cell Biology 107, 1643-53.

Sohn, K., Orci, L., Ravazzola, M., Amherdt, M., Bremser, M., Lottspeich, F., Fiedler, K., Helms, J. B. and Wieland, F. T. (1996). A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding. Journal of Cell Biology 135, 1239-48.

Sparrow, D. B., Latinkic, B. and Mohun, T. J. (2000). A simplified method of generating transgenic Xenopus. Nucleic Acids Research 28, E12.

Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P. and Rothman, J. E. (1995). An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding [published erratum appears in Proc Natl Acad Sci U S A 1995 Nov 7;92(23):10816]. Proc Natl Acad Sci U S A 92, 8011-5.

 

 

 

Chapter 7: General Discussion

 

GENERAL DISCUSSION

Following quality control, secretory cargo molecules that are destined for the exterior of the cell exit from the endoplasmic reticulum (ER) and are selectively packaged into transport vesicles for their subsequent release from the cell. Many studies have been performed to understand the molecular actions that underlie these cellular processes. One of the findings was a major family of so-called p24 proteins that is abundantly present in COP I- and COP II-coated transport vesicles originating from the ER and the Golgi (Stamnes et al., 1995). This family of type-I transmembrane proteins can be divided into four main subfamilies, designated p24α, -β, -γ and -δ, that share several structural characteristics: an N-terminal putative cargo binding domain with two conserved cysteine residues that are possibly involved in the formation of a loop structure, a coiled-coil region involved in multimerization, a transmembrane domain and a short cytoplasmic tail containing trafficking motifs that can bind coat proteins (Belden and Barlowe, 1996; Dominguez et al., 1998; Schimmöller et al., 1995; Stamnes et al., 1995). The coat binding allows p24 proteins to shuttle from the ER to the Golgi and back (for review see Kaiser, 2000). Sequences similar to the N-terminal p24 loop domain have recently also been identified in classes of proteins that possess a novel β-strand-rich protein module, the GOLgi Dynamics (GOLD) domain. This domain is predicted to be involved in specific protein-protein interactions and is present in many eukaryotic proteins that appear to have a role in Golgi dynamics (Anantharaman and Aravind, 2002). Several functions for the p24 proteins in ER-to-Golgi transport have been implicated, including a function as cargo receptor, membrane organizer, regulator of vesicle budding, as well as in ER quality control, or excluding ER resident proteins from the vesicular lumen (Belden and Barlowe, 2001a; Bremser et al., 1999; Denzel et al., 2000; Elrod Erickson and Kaiser, 1996; Kaiser, 2000; Lavoie et al., 1999; Muñiz et al., 2000; Rojo et al., 1997; Schimmöller et al., 1995; Springer et al., 2000; Wen and Greenwald, 1999) (for details concerning the proposed functions of p24, see the introductory chapter of this thesis).

In the first part of our research, we identified and subsequently characterized the members of the p24 family in a well-defined secretory cell, the intermediate pituitary melanotrope cell of the amphibian Xenopus laevis (chapter 2). This cell is dedicated to produce a single major soluble cargo protein, the prohormone proopiomelanocortin (POMC). The expression level of POMC in the melanotrope cells can be physiologically regulated by placing the animal on a white- or a black background (Roubos, 1997). On a black background these cells are highly active, producing vast amounts of POMC, whereas on a white background they are virtually inactive. By analyzing the subcellular localization and the expression levels of the p24 members in the melanotrope cells of black- and white-adapted Xenopus, we could show the highly dynamic behaviour of these proteins (chapter 3). Furthermore, we combined the unique properties of the melanotrope cell with the recently developed technique of stable Xenopus transgenesis (Kroll and Amaya, 1996; Sparrow et al., 2000) to overexpress green fluorescent protein (GFP)-tagged versions of two Xenopus p24 proteins specifically in this cell type (chapter 4). With this in vivo approach, we were able to interfere with the endogenous p24 system in the melanotrope cells (chapters 5 and 6). The results obtained with this approach have provided more insight into the complex nature and functioning of the p24 proteins.

 

The p24 protein family in the Xenopus melanotrope cell

In order to efficiently perform its task, the highly specialized melanotrope cell produces a number of proteins that are involved in POMC biosynthesis and processing, and these proteins are coordinately expressed with the prohormone. A differential screening approach to identify genes encoding such proteins resulted in the isolation of a member of the Xenopus p24 family, Xp24δ2 (Holthuis et al., 1995; Kuiper et al., 2000). Subsequent screening of several Xenopus cDNA libraries and the use of database searches revealed that seven additional members (Xp24α2, -α3, -β1, -γ1, -γ2, -γ3 and -δ1) are present in Xenopus (chapter 2). Remarkably, in all other species examined, the p24δ family is comprised only of p24δ1, whereas Xenopus contains the additional Xp24δ2 member. Since Xenopus has a duplicated genome (Bisbee et al., 1977), the Xp24δ2 gene could have diverged from the primordial Xp24δ1 gene and now fulfills a specialized function in neuroendocrine cells, including the melanotrope cell. In human, the presence of a second p24δ protein has been suggested (Blum et al., 1996), but this sequence appeared to be derived from a pseudogene that presumably originated from a duplication event (Horer et al., 1999).

Expression studies showed that two of the eight Xenopus p24 family members (Xp24α2 and -γ1) were not expressed in the Xenopus melanotrope cell suggesting that p24 proteins can display tissue-specific expression patterns. Interestingly, upon induction of the melanotrope cells by placing the animals on a black background, four of the p24 members identified in the melanotrope cell (Xp24α3, -β1, γ3 and -δ2) were coordinately expressed with POMC, whereas others (Xp24γ2 and -δ1) were not or only slightly induced (chapter 2). This suggests that, based on the characteristics of p24 proteins, the coexpressed members are somehow involved in the transport of POMC through the early secretory pathway and could serve as cargo receptors. In rat, p24δ1 has been found to be upregulated during development of the nephrogenic cortex of the newborn kidney and as the nephron matures, the levels of p24δ1 declined. The p24δ1 protein may therefore direct intracellular trafficking or secretion of proteins specifically responsible for nephrogenesis and is therefore upregulated during this stage (Baker and Gomez, 2000). Furthermore, an increase in the relative amounts of certain p24 members in mammary glands from lactating rabbits confirms their presumed function in cargo-selective processes (Dr. E. Chanat, personal communication).


Dynamic behavior of p24 proteins in the early secretory pathway of Xenopus melanotrope cells

It has been shown previously that p24 proteins are able to bind to both COP-I and COP-II coat complexes, and are thought to shuttle between the ER and the Golgi (Dominguez et al., 1998). Sequence alignment of the Xenopus p24 proteins however revealed that while all members have the double phenylalanine (FF) motif used for exit from the ER, only a few members have the K(X)KXX-like motif for retrieval in COP-I coated vesicles from the Golgi back to the ER (Rötter et al., 2002)). In yeast cells, it has been shown that in addition to these classical trafficking motifs the C-terminal residues as well as sequences in the transmembrane domain determine the localization of p24 proteins to the various subcompartments of the early secretory pathway (Nakamura et al., 1998). In general, the steady-state localization of p24 proteins has been found to be in the intermediate- and in cis-Golgi compartments (Blum et al., 1999; Dominguez et al., 1998; Füllekrug et al., 1999; Lin et al., 1999; Rojo et al., 1997; Sohn et al., 1996). Some p24 proteins (p24α2 and -β1) are capable of binding to Golgi Reassembly Stacking Proteins (GRASPs), GRASP55 and GRASP65, which aids their retention (and thus steady-state localization) in the Golgi apparatus (Barr et al., 2001). The steady-state localizations of p24 proteins in Xenopus melanotrope cells were found to be dependent on the biosynthetic activity of the cells. In melanotrope cells from white-adapted animals (biosynthetically virtually inactive cells), at steady-state all p24 members were found in the cis-Golgi compartment. Upon activation of the cells, the p24 proteins were recruited to earlier compartments, such as the ER-Golgi intermediate compartment (ERGIC) and ER subdomains. In the activated state, the ER contains high amounts of newly synthesized POMC molecules that have to be transported and processed. For these processes, an efficient ER-to-Golgi transport machinery is required. Therefore, the p24 proteins seem to be recruited to play an important role in the increased vesicular transport processes in the active melanotrope cells of black-adapted animals (chapter 3).

 

Multimerization of p24 proteins

In yeast and mammalian cells, p24 proteins are able to form functional heterotetrameric complexes containing one representative of each subfamily, whereby the composition of the complex may differ in various cell types and certain family members are excluded from the complex (Belden and Barlowe, 2001a; Ciufo and Boyd, 2000; Dominguez et al., 1998; Emery et al., 2000; Füllekrug et al., 1999; Marzioch et al., 1999). The stability of the p24 members appears to be compromised when cells are deficient in the expression of a single p24 protein (Denzel et al., 2000; Marzioch et al., 1999). The existence of p24α-γ and p24β-δ dimers has been proposed and based on these findings, p24 multimers are thought to be formed through the assemblage of heterodimers (Belden and Barlowe, 1996; Ciufo and Boyd, 2000; Emery et al., 2000; Gommel et al., 1999). Furthermore, the steady-state localization and trafficking of p24 proteins depends largely on their assembly into functional complexes (Emery et al., 2000; Füllekrug et al., 1999). Recent evidence suggests a complex and dynamic p24 system of mostly monomers and homo-/hetero-dimers, and that the degree of oligomerization constantly alters and largely depends on the subcellular localizations of the p24 subfamily members (Jenne et al., 2002). In Xenopus melanotrope cells, the coordinate expression of Xp24α3, -β1, -γ3 and -δ2 suggests that in these cells the p24 complex is composed of these p24 family members. Since not all members of the p24 family have the COP I retrieval motif, these members largely depend on other mechanisms to be transported back to the ER and thus p24 complex formation could be a mechanism for this. Our attempts to cross-link and immunoprecipitate p24 protein complexes using anti-p24 antibody-coated magnetic beads did not provide a clue as to which p24 complexes exist in the Xenopus melanotrope cell. Similar to what has been found in yeast and mammalian cells (Belden and Barlowe, 1996; Ciufo and Boyd, 2000; Emery et al., 2000; Gommel et al., 1999), localization studies using subcellular fractionation gradients suggested that α-γ and β-δ dimers exist in the intermediate compartment of Xenopus melanotrope cells, since the migration of these pairs appeared to be coupled (Kuiper, R.P., Janssen, K.P.C., Bouw, G., Rötter, J., Van Herp, F., Martens, G.J.M., unpublished results). However, the continuous assembly and disassembly of protein complexes makes it difficult to pinpoint the state of the multimers in the various compartments of the early secretory pathway of Xenopus melanotrope cells and our studies did not provide clear insight into the composition of the functional p24 complex existing in this cell. Additional research should be performed using subcellular fractionation, protein separation analysis of the subcompartments and subsequent proteomic analysis in order to identify the molecular build-up of the functional p24 machinery in the various compartments.

 

Functional studies on p24 proteins

Despite extensive studies with a variety of cell biological and genetic approaches in a number of species (e.g. mutant yeasts and worms, knock-out mice, transfected mammalian cells in culture), defining a function for p24 has proven to be difficult. For instance, deletion of all p24 proteins resulted in viable yeast (Marzioch et al., 1999; Springer et al., 2000), whereas genetic ablation of a single p24 family member caused early lethality in mice (Denzel et al., 2000). In order to establish a role for p24 proteins in the early secretory pathway, we initially used transfection studies by overexpressing various combinations of tagged p24 proteins and mutants thereof in mouse anterior pituitary-derived AtT20 cells that produce POMC endogenously. The attempts to determine the effect of this overexpression on POMC transport were not successful, probably because of difficulties to produce an appropriate balance in expression of the various transfected p24 members, which is required for proper p24 functioning (Dominguez et al., 1998; Emery et al., 2000; Füllekrug et al., 1999). Furthermore, since others have shown that in yeast p24β and p24δ could be directly cross-linked to Gas1p cargo molecules in ER-derived vesicles (Muñiz et al., 2000), we wondered whether we could show a specific interaction of the putative cargo molecule POMC with p24 proteins in the melanotrope cells. We used the mammalian two-hybrid system to examine a possible interaction between p24 members and POMC. In this approach, the N-terminal domains of the various Xenopus p24 proteins were expressed together with POMC in COS-1 cells to examine whether the prohormone would bind specifically to one of the members using a luciferase readout system. The system is designed in such a way that the interaction of two proteins of interest has to take place in the cytoplasm whereby the transcription of a reporter gene is initiated once the protein complex is formed. This means that a possible interaction between p24 and a cargo is studied in an environment different from the physiological one, namely the ER-lumen. Furthermore, the absence of the appropriate microenvironment may have hampered the proper folding of the p24 fragment, affecting the binding of cargo molecules. It is therefore not surprising that during these studies we could not detect any specific interaction, also due to high background values (Bouw, G. and Van Roosmalen, M., unpublished results). To further investigate the possibility of a specific binding of POMC molecules to p24 proteins, we used anti-p24 antibody-coated magnetic beads to pull down any interacting proteins in melanotrope cell lysates under native conditions. However, because of nonspecific binding of POMC to even uncoated beads, we were not able to draw conclusions from these experiments with respect to the specific binding of p24 proteins to any soluble cargo (Bouw, G., unpublished observations).

 

Stable Xenopus transgenesis as a tool to study in vivo the role of a protein of unknown function

In order to study close to the in vivo situation the role of a protein of unknown function, the need for a well-characterized model system is obvious. We therefore set up the technique of stable Xenopus transgenesis in our lab (chapter 4). This technique has been developed by Kroll and Amaya (Kroll and Amaya, 1996) and simplified by the group of Mohun (Sparrow et al., 2000). In this method, sperm nuclei are incubated with the transgene DNA of interest and this mixture is then microinjected into unfertilized Xenopus eggs. If desired, expression of the transgene can be directed to the appropriate tissue and at the appropriate time by using specific promoters. Possible applications of stable Xenopus transgenesis include (a) studying protein function by ubiquitous or spatio-temporally restricted (over)expression of wild-type or (dominant-negative) mutant proteins or underexpression via expression of antisense RNA, (b) mapping transcription/translation regulatory elements in Xenopus and non-Xenopus genes by using reporter genes such as GFP (reviewed in Dirks et al., 2003).

 

Xenopus transgenesis and commercialization

We recently initiated an exploration of the possibility to commercialize the Xenopus transgenesis technique. Stimulated by the current revolution in Genomics, Genetics and Biotechnology, many disease-associated genes have been discovered. The ultimate goal is to use this information to effectively develop new diagnostics and drugs. This process is, however, heavily hampered by the fact that the functions of many of the genes concerned are not known and knowledge of the molecular regulatory mechanisms underlying the disease-associated pathways is lacking. It is recognised and well accepted that elucidation of the roles of the genes and the underlying mechanisms is necessary to be able to specifically interfere with the disease. Therefore, we set out to examine the possibility to start a company that would exploit the benefits of the stable Xenopus transgenesis technology platform by applying for a BioPartner First Stage Grant to financially support this venture. The project called “Xenogene Solutions – Discover the function of your Central Nervous System (CNS)-disease associated gene” was granted an initial phase. The goal was to start as a contract research company generating transgenic Xenopus embryos, tadpoles and frogs to perform Functional Neurogenomics studies on these in vivo models in order to provide pharmaceutical and biotechnology companies with functional data on gene products of unknown function. The in vivo models will significantly facilitate the elucidation of the function and underlying pathways of genes of interest. This technology will reduce failure rates in the process of target validation and therefore decrease overall costs of drug discovery. The generation of the transgenic Xenopus may also result in animal disease models that can be used to screen compound libraries. The characteristics of the Xenopus model system may greatly enhance the feasibility to become a player in the field of drug discovery: the possibility to generate in a day in a cost-effective and efficient way hundreds of transgenic Xenopus embryos allows to combine the traditional advantages of Xenopus - large embryos, ease of microinjection, external embryonic development, a reliable fate map, ease of dissection/micromanipulation - with the ability to express a gene any time and any place. Stable Xenopus transgenesis could be used in combination with selected neuronal and endocrine gene promoters to specifically direct transgene expression, in particular to the intermediate pituitary melanotrope cells, the cell of which our lab studied the functioning extensively in the past. The generated transgenic embryos, tadpoles and frogs could then be analysed with a variety of biochemical, physiological, morphological, cell biological and molecular biological techniques that are established in the lab. In the initial phase of the BioPartner First Stage Grant, we tried to investigate the chances of founding Xenogene Solutions B.V., before applying for a second phase of the grant. We performed a thorough market analysis and survey to investigate to which customer we should offer our product that should be of additional value. These efforts resulted in the positioning of Xenogene Solutions in the field of target validation and small-scale target identification, rather than large-scale target identification, target optimization and drug screening approaches. Because of the risks involved, large pharmaceutical companies shifted their activities more towards clinical phases of the drug discovery process than to functional research to identify targets and validate them. Knowing this, we approached several potential future customers and explained to them in which way our technology platform could be of additional value to their drug development process. Four of these companies have acknowledged the value of the Xenopus model as a functional research platform and sent us letters of intent. With one of them, we are now in the process of investigating future cooperations. This collaboration shows that biotech companies indeed consider the transgenic Xenopus approach of additional value to them and wish to include this model system in their future research strategies.

By thorough investigation of the feasibility to start a company, we proved that there is enough foundation for Xenogene Solutions to become a key player in the field of drug discovery. In addition, we were able to make the translation from technology platform to a well-defined product: information about the functioning of gene products in a physiological context. In addition to the commercial activities performed, we continued to use the technique in the lab to investigate the role of several interesting proteins.

 

Functional characterization of the p24δ proteins in transgenic Xenopus melanotrope cells

Using stable Xenopus transgenesis, we set out to explore the role of p24 proteins in cargo transport by overexpressing GFP-tagged Xp24δ1 and Xp24δ2 specifically in the Xenopus intermediate pituitary melanotrope cell (chapters 4-6). The reason to select the two members of the Xp24δ subfamily was their differential expression in the Xenopus melanotrope cell. The Xp24δ2 protein, but not the Xenopus orthologue of the mammalian p24δ1 protein (Xp24δ1), was found to be coordinately expressed with POMC. Using GFP fusion proteins, we could easily identify transgenic animals by direct visualization of the embryo through a fluorescence microscope. The melanotrope cell-specific expression of the fusion protein was accomplished by using a Xenopus POMC gene promoter fragment (Jansen et al., 2002). The use of a cell-specific promoter has several advantages. First, the effect observed in the transgenic cells is a primary effect of the overexpression of the transgene product, and is not influenced by the expression of the exogenous proteins in neighbouring cells that for instance control the target cells (in our case, the hypothalamic neurons innervating the melanotrope cells). Second, one increases the chances of being able to study a potentially toxic protein, since non-functional melanotrope cells are probably not lethal for the animal. Third, any phenotype at the level of the organism can be attributed to transgene expression in the specific cell type. In adult animals, using both immunocytochemistry and western blotting, we were able to establish that both Xp24δ1-GFP and Xp24δ2-GFP were expressed exclusively in the intermediate pituitary melanotrope cells and not in the anterior lobe of the pituitary or any other parts of the brain. Thus, any effect observed in the transgenic melanotrope cells results directly from the expression of a transgene product in these cells. We generated Xenopus transgenic for the pPOMC-Xp24δ12-GFP transgene in several rounds of transgenesis expressing different levels of the fusion protein. Southern blot analysis revealed various numbers and sites of concatemeric integration of the transgene. Interestingly, the site of integration within the genomic DNA rather than the number of integration sites and amount of copies of the transgene determined the level of expression of the fusion protein. That is, it appeared that a lower number of copies meant less reason for silencing and thus more transgene expression. The silencing could well involve a methylation event.

Analysis of the transgenic animals expressing p24 fusion proteins revealed several interesting aspects. Microscopy analysis confirmed that Xp24δ2-GFP and Xp24δ1-GFP were targeted specifically to the melanotrope cells in the pituitary and partly colocalized intracellularly with POMC. The fusion protein localized to structures that resembled ER and Golgi, indicating that the C-terminal fusion with GFP did not hamper proper trafficking of the p24 protein between the ER and the Golgi. To further characterize the intracellular compartments to which the fusion proteins are localized, additional studies using specific markers for these compartments are needed. Upon overexpression of Xp24δ2-GFP, at the steady-state protein level, endogenous Xp24δ2 and Xp24δ1 were competed away. The degree of displacement was correlated with the level of newly synthesized fusion protein and the resulting amount of steady-state overexpressed transgene product. We generated several F1 lines of transgenic animals producing p24 fusion proteins specifically in the melanotrope cells with various levels of transgene expression. The F1 animals were, apart from the number of integrated transgenes, genetically identical. These lines were used to functionally characterize p24δ proteins in the Xenopus melanotrope cell.

In the Xenopus melanotrope cell, the early secretory pathway and processing compartments are highly specialized and committed to the biosynthesis of POMC, since ~80% of all newly synthesized proteins concerns this prohormone. Transgenic intervention in this specialized secretory cell may thus well result in the distortion of cell physiological processes and thus the POMC biosynthetic machinery. Overexpression of Xp24δ2-GFP resulted in the distortion of the endogenous p24 complex at steady-state protein level. It therefore appears that the number of ER/Golgi subcompartments that can harbour p24 proteins is limited and that the relative amounts of the various newly synthesized p24 family members expressed in a cell determine the final composition of the p24 machinery in the early secretory pathway. As already mentioned, the degree of competition was linked to the level of overexpression of newly synthesized Xp24δ2-GFP (chapters 4 and 5). Taken into account that more than 30% of the total protein content of membranes of the early secretory pathway is comprised of p24 proteins (Rojo et al., 1997), it was not surprising to see that high levels of expression of the transgene product were needed to achieve such a displacement effect. Interestingly, in cells overexpressing Xp24δ1-GFP, no competition with endogenous p24 proteins was observed. In contrast, steady-state protein levels of Xp24α3 and -γ3 were increased. An explanation for these observations could be that the level of Xp24δ1-GFP expression was too low to compete with other p24 proteins. However, preliminary experiments have shown that even in transgenic animals that express high levels of newly synthesized exogenous wild-type Xp24δ1 (untagged), Xp24α3 was not competed away, as observed with the overexpression of the Xp24δ2-GFP fusion protein. Interestingly, endogenous Xp24δ2 was competed away to some extent by the high transgene-derived Xp24δ1 levels (chapter 6). Therefore, the underlying mechanism that determines the p24 content in the melanotrope cells transgenic for Xp24δ1-GFP seems different from that in melanotrope cells overexpressing Xp24δ2-GFP. The amino acid sequence differences present in the loop domains of Xp24δ1 and -δ2 might explain this phenomenon. The highly distorted p24 system in the melanotrope cells transgenic for Xp24δ2-GFP or the upregulation of Xp24α and -γ subfamily members by the overexpression of Xp24δ1-GFP did not lead to a change in cell morphology and the various subcellular compartments seemed intact. In contrast, in transfected mammalian cells overexpression of p24δ1 induced expansion of smooth ER membranes and changed the morphology of cis-Golgi and Golgi membranes (Rojo et al., 2000). Apparently, the levels of Xp24δ2-GFP and Xp24δ1-GFP transgene expression may have been relatively less than the amount of exogenous p24 produced in the transfected cells and thus to an extent that did not destroy the early secretory pathway in the transgenic Xenopus melanotrope cells.

In the following part, possible roles of p24 in the Xenopus melanotrope cell will be discussed on the basis of the results of our transgenic studies.

 

Xp24δ2 as cargo receptor for POMC

Several groups have implied a role for p24 proteins as cargo receptors (Kaiser, 2000; Muñiz et al., 2000; Schimmöller et al., 1995). Since the structural characteristics are in line with such a function in the early secretory pathway (N-terminal globular domain, transmembrane domain and cytoplasmic tail containing COP I/II binding motifs) and Xp24δ2 expression is upregulated coordinately with the major cargo protein POMC, the most obvious role for Xp24δ2 in the melanotrope cell would be in the cargo-specific transport of POMC. However, interference with the endogenous p24 system in the transgenic melanotrope cell by overexpression of Xp24δ2-GFP, resulting in a p24 system comprised of almost only Xp24δ2-GFP, did not result in impaired POMC transport and subsequent processing to 18-kDa POMC. Furthermore, experiments using radioactively labelled sulphate showed that in both transgenic and wild-type melanotrope cells, the rate of POMC sulfation in the Golgi apparatus was the same (our unpublished results). These results indicate that the rate of transport of POMC from the ER to the Golgi is unaffected in cells overexpressing Xp24δ2-GFP. However, we found that in transgenic cells that express Xp24δ2-GFP the amounts of POMC-derived peptides were lower in the cell lysates (chapter 5). These results point to the inefficient processing of POMC rather than to an impaired POMC cargo transport. We suspected that if Xp24δ2 proteins would act as specific cargo receptors for POMC, overexpression of Xp24δ1 would interfere with the transport of POMC. However, we did not observe any effect on POMC biosynthesis in the melanotrope cells transgenic for Xp24δ1-GFP. Therefore, it is improbable that p24 proteins physically interact with POMC, thereby directing cargo molecules into transport vesicles. Together, these results indicate that a role for Xp24δ2 in cargo-specific transport of POMC is implausible.

Since the amount of POMC-derived peptides was clearly affected and the 18-kDa product was not properly processed in the transgenic cells, Xp24δ2 could be involved in the cargo-specific transport of proteins that are involved in the processing of POMC. Alternatively, the distorted p24 system might fail to target components of the POMC processing machinery to the correct secretory pathway subcompartments. To establish these roles for p24 proteins, cross-linking studies should be performed to show cargo-specific binding of p24 members to processing enzymes and other factors that are involved in POMC maturation and processing. Alternatively, the transgenic expression mutants that lack the ability to bind cargo could reveal the importance of the putative cargo-binding loop domain in the specific transport of cargo molecules. Furthermore, live-imaging experiments using tagged versions of POMC and p24 proteins could shed light on the targeting of POMC-containing cargo vesicles and the involvement of certain p24 proteins in this process.

 

Xp24δ2 as player in the quality control system

One of the proposed functions of p24 proteins is their involvement in quality control. In C. elegans, mutations in p24β1 led to the trafficking of a mutant protein to the plasma membrane that would otherwise accumulate within the cell (Wen and Greenwald, 1999). This finding made the authors conclude that p24 is involved in the ER quality control machinery. Our data could be consistent with such a function, since in the transgenic melanotrope cells overexpressing Xp24δ2-GFP, an aberrant form of 18-kDa POMC was produced (18-kDa POMC*). This could be the result of the distorted p24 system in these cells, allowing misfolded POMC molecules to leave the ER and reach the processing site later in the secretory pathway, resulting in abnormal POMC processing products. In addition, an affected quality control machinery could result in less properly folded POMC molecules that are inefficiently processed to less POMC-derived peptides. Interestingly, overexpression of Xp24δ1-GFP did not lead to the production of the aberrant 18-kDa POMC* product, which supports the hypothesis that there is a discrepancy in the mode of action of Xp24δ1 and -δ2. The nature of the aberrant 18-kDa POMC* processing product remains to be elucidated. Until now, the characterization of this product using 2D electrophoresis and mass-spectrometry was unsuccessful (Bouw, G., Janssen, K.P.C., Van Herp, F., unpublished observations). Based on the production of an aberrant 18-kDa POMC* cleavage product and less POMC-derived peptides in the Xp24δ2-GFP transgenic melanotrope cells, it is possible that p24 proteins are involved in quality control mechanisms. However, this could as well be a secondary effect, namely as a consequence of the affected targeting processes in the early secretory pathway (see below). For instance, improper targeting and retrieval of the KDEL receptor could allow BiP to escape the ER, resulting in an affected quality control machinery. In the cells transgenic for Xp24δ2-GFP the amount of BiP had indeed decreased (chapter 5, appendix)

 

The role of p24 proteins in the Xenopus melanotrope cell: targeting the POMC processing machinery to subcompartments of the secretory pathway?

The results presented allow us to propose a model for the function of p24 proteins in the early secretory pathway that is different from our initial working hypothesis in which the p24 family in the Xenopus melanotrope cell would act as cargo receptors for POMC. Since the expression of the p24δ transgenes was restricted to the melanotrope cell, the effects of the overexpression observed in these cells can be directly linked to a role of p24δ in POMC biosynthesis. Interestingly, overexpression of Xp24δ1 and -δ2 specifically in the melanotrope cell resulted in different phenotypes. Because the Xp24δ members show a high degree of amino acid sequence identity in predominantly the C-terminal and middle parts of the protein (including the coiled-coil region) (chapter 2), we hypothesize that the differential effects were due to the amino acid sequence differences in the N-terminal loop domains. As already mentioned, overexpression of both Xp24δ1 and -δ2 did not disturb the transport of POMC molecules from the ER to the Golgi apparatus, indicating that p24 proteins are not absolutely necessary for cargo transport by itself. Since in Xp24δ2-overexpressing cells the maturation and processing of POMC is inefficient and inaccurate, p24 may be involved in POMC biosynthetic steps other than prohormone transport. In wild-type cells, Xp24δ1 and -δ2 are part of separate functional p24 complexes that might play a role in separate events (Fig. 1). Overexpression of Xp24δ1-GFP did not have an effect on the endogenous levels of the components of the p24 complex that is upregulated with POMC in the melanotrope cell. Instead, the levels of the Xp24α and -γ subfamily members were upregulated, possibly because overexpressed Xp24δ1 needed binding partners since otherwise the Xp24δ1-containing complex would not be functional (Fig. 1A).

Figure 1: Schematic representation of the composition of the p24 machinery in the melanotrope cells of wild-type and transgenic Xenopus, and the effect of the transgenic manipulation on the targeting of ‘machinery protein’ and the processing of proopiomelanocortin (POMC) in the melanotrope cells.

(A) Wild-type melanotrope cells contain ~10-15 times more Xp24δ2 proteins than Xp24δ1. This is reflected by the number of the two functional p24 complexes, containing either Xp24δ1 or -δ2 that are present in these cells (I). In melanotrope cells transgenic for Xp24δ2-GFP, the endogenous p24 system is severely distorted, resulting in a p24 machinery that is virtually completely comprised of the Xp24δ2-GFP protein (II). Transgenic overexpression of Xp24δ1-GFP did not lead to a displacement of endogenous p24 members. In these cells, more Xp24δ1-containing p24 complexes are present than in wild-type cells (III). (B) In wild-type melanotrope cells, POMC travels from the ER in large transport carriers to the Golgi. The p24- and ‘machinery proteins’-containing transport vesicles, including proteins that are needed for a proper lumenal environment, are incorporated into the correct outgoing POMC-containing transport carrier, delivering components for efficient and accurate POMC processing to 18-kDa POMC and POMC-derived peptides (left diagram). Transgenic cells that overexpress Xp24δ2-GFP are equipped with a severely distorted p24 system that causes mistargeting of ‘machinery proteins’. As a result, POMC is inefficiently and inaccurately processed leading to the production of 18-kDa POMC* and less POMC-derived peptides (middle diagram). Overexpression of Xp24δ1-GFP in the melanotrope cell does not interfere with the processing of POMC (right diagram).

At present it is not clear which signaling molecules are involved in the upregulation. The contents of transport vesicles that are defined by the Xp24δ1, -αx, -βx and -γx complex have to be investigated, as well as the natures of the p24 subfamily members in this complex. In cells that overexpress Xp24δ2-GFP, the endogenous p24 complex is severely disturbed and the resulting effect on the production of POMC-derived peptides is likely caused by inefficient or improper cleavage of the prohormone. This effect may have been caused by a lower rate of transport of newly synthesized POMC and cleavage enzymes through the secretory pathway, resulting in a lower rate of precursor protein processing and less newly synthesized precursor-derived peptides. However, the production of the aberrant 18-kDa POMC* product support an alternative role for p24, namely that the distorted p24 system may have exerted a more direct effect on the POMC processing event itself, e.g. because it failed to provide the proper processing conditions in the various secretory pathway subcompartments. Collectively, we conclude from our transgenic studies that p24 proteins may not be involved in transport vesicle budding or secretory cargo transport by itself, but rather are responsible for supplying the appropriate secretory pathway components to the correct subcompartments to allow proper secretory protein processing (Fig. 1B). In this connection, the results from recent morphological studies by Mironov et al (2003) are intriguing. They have shown that in mammalian cells secretory cargo (in their case large supramolecular procollagen molecules) exits the ER by bulk flow in large carriers via distinct, specialized ER exit sites. The transport of these secretory cargo-containing carriers from the ER to the Golgi was COP II-dependent but did not involve the budding and fusion of COP II-coated vesicles. Instead, the key role for COP II would be to recruit to the carriers components (so-called ‘machinery proteins’) that are needed for docking/fusion and creating the proper lumenal environment of the secretory cargo carrier. At each carrier departure, a complement set of ‘machinery proteins’ would be incorporated into the outgoing secretory cargo-containing carrier (Mironov et al., 2003). Since in the transgenic Xenopus melanotrope cells with a disrupted p24 system the synthesis and subsequent transport of POMC to the Golgi is not affected per se, we hypothesize that, independently of COP II budding events, such large transport carriers still transport POMC to the Golgi. In this setting, the p24 proteins would recruit ‘machinery proteins’ into specialized ER exit sites and subsequently incorporate them into transport carriers containing POMC. The recruited proteins are needed to create the proper environment for efficient and accurate POMC processing. In the early secretory pathway of the transgenic cells that overexpress Xp24δ2-GFP and hence have a distorted p24 system, the supply of the proper ‘machinery proteins’ to the outgoing POMC-containing transport carriers is disturbed, leading to inefficient and inaccurate processing of POMC that results in the production of 18-kDa POMC* and less POMC-derived peptides (Fig. 1B).

Recently, it was shown that p24β1 and -α2 could bind to the Golgi matrix proteins GRASP55 and GRASP65. Yeast-two-hybrid experiments resulted in the identification of a GRASP-binding domain in p24β1. Furthermore, multimerization of the p24 proteins into a functional complex (p24α2, -β1, -δ1 and -γ3) was necessary for the interaction with GRASPs. Interestingly, a p24 member that is not in the presumed mammalian p24 complex (p24γ4) could also bind to GRASPs (Barr et al., 2001). Upon black-background adaptation, certain members of the Xenopus p24 family (Xp24α3, -β1, -γ3 and -δ2) are coordinately expressed with POMC. Since it has been shown that certain members of the GRASP family can define a subdomain in the intermediate compartment (Marra et al., 2001), binding of Xp24β1 to a specific GRASP could target vesicles that bud off the ER membrane to a predefined subcompartment of the cis-Golgi (Fig 1B). In addition, it has been suggested that the functional p24 system consisted of monomers and dimers (Jenne et al., 2002). In this scenario, members of the Xp24α and -β subfamilies could be part of p24 dimers that are responsible for the recruitment of a specific set of ‘machinery proteins’ that are incorporated into distinct secretory cargo-containing transport carriers. Exclusion of one of the GRASP-binding p24 proteins from a functional dimer could result in the misdirection to the wrong cargo-containing carrier (Fig. 1B). Overexpression of Xp24δ1 did not result in the distortion of the p24 complex and thus the supply of machinery cargo to POMC-containing subcompartments was not affected, resulting in a normal amount and profile of POMC-derived peptides. In contrast, high levels of Xp24δ2-GFP competed away endogenous p24 proteins and as a result, p24 proteins that were able to bind to GRASPs were no longer available, leading to the mistargeting of the ‘machinery proteins’. This defective targeting mechanism resulted in the affected profile of POMC-derived peptides and eventually to a reduced capacity of the animal transgenic for Xp24δ2-GFP to fully adapt to a black background.

 

Future prospects

During the last decade, considerable progress has been made in characterizing p24 proteins in various species and their role in the early secretory pathway. However, the complex nature of this family of putative cargo receptors has hampered functional analysis of these proteins. A number of questions have to be answered in order to fully understand p24 proteins and their molecular action in the secretory pathway. Therefore, a well-defined model system such as the Xenopus melanotrope cell may enhance the current understanding of the role of p24 proteins. To study p24 action in the Xenopus melanotrope cell, further experiments with transgenic Xenopus expressing tagged p24 proteins is the most straightforward line of research in the near future. It would be very interesting to investigate the overexpression of other p24 family members (Xp24α2,3, -β1, and -γ1,2,3) and the effect of this on the transport and processing of POMC. In order to perform functional studies using stable Xenopus transgenesis, an F1 generation of transgenic animals with similar genetic background and expression levels is desirable. Furthermore, analyses using biochemical techniques require significant amounts of biological material, which is easily obtained from an F1 population. In the recent past, we have generated transgenic F0 and subsequent F1 animals expressing Xp24α3-GFP or Xp24γ2-GFP, of which the first but not the latter is upregulated in the melanotrope cells of black-adapted animals (Rötter et al., 2002). Until now, we were not able to obtain F0 transgenic animals that express high levels of Xp24α3-GFP in melanotrope cells. As a result, in the F0 animals, no effect of the overexpression of the fusion protein on POMC biosynthesis and transport could be observed. However, several attempts to produce F1 offspring that express high levels of Xp24α3-GFP have resulted in one high-expressing F1 line that is now ready for analysis. Since p24α2 has been suggested to play a role in cargo exit site formation in mammalian cells (Lavoie et al., 1999), overexpression of Xp24α3-GFP in Xenopus may disrupt these specialized regions in the ER and interfere with proper selection of ‘machinery proteins’ into transport vesicles.

Several groups have investigated the trafficking of p24 proteins in the early secretory pathway by mutating transport motifs in their transmembrane domain or cytoplasmic tail. Such motifs bind to COP II- or COP I-coats for either export out of the ER or retrieval from the Golgi, respectively. Transfection experiments and yeast-two-hybrid experiments have identified a number of transport motifs that are crucial for p24 trafficking (Belden and Barlowe, 2001b; Dominguez et al., 1998; Emery et al., 2000; Fiedler and Rothman, 1997; Nakamura et al., 1998; Nickel et al., 1997). We have substituted the double phenylalanine motif in the C-tail of Xp24δ2 for two alanines giving the Xp24δ2FF/AA mutant. The double phenylalanine motif regulates ER exit of p24 proteins by binding of COP II to this motif (Dominguez et al., 1998; Nakamura et al., 1998). In addition, we constructed an Xp24δ2KK/SS mutant lacking the double lysine motif that is involved in COP I-mediated retrieval of the protein. COS-1 cells transfected with the FF/AA mutant construct showed intense ER staining, whereas the Xp24δ2KK/SS mutant was expressed at the plasma membrane, indicating that interference with the trafficking motifs alters the subcellular localization of Xp24δ2 (Bouw, G. and Rötter, J., unpublished observations). These mutants can be expressed in transgenic Xenopus melanotrope cells to investigate the role of the Xp24δ2 protein in the functioning of the p24 system, and its effect on POMC transport and processing. For instance, mutating the ER exit motif (FF) may result in the accumulation of Xp24δ2 proteins in the ER. If this p24 protein would be involved in the transport of ‘machinery proteins’ from the ER to the Golgi and acts as specific receptor for this type of proteins, these proteins would then accumulate within the ER and as a result may be degraded. Assuming that Xp24δ2 plays a role in anterograde transport of properly folded POMC as well as retrograde retrieval of ‘machinery proteins’, mutating the COP-I (KKXX) retrieval signal would direct the Xp24δ2 protein to the plasma membrane and as a result hamper the proper retrieval of ‘machinery proteins’ back to the ER. In addition, since Xp24δ2 cannot be retrieved from cis-Golgi compartments, this could lead to a shortage of putative cargo receptors. This would eventually result in an affected exit of ‘machinery cargo’ molecules from the ER. However, considering our current hypothesis, the targeting and inclusion of ‘machinery proteins’ into the proper secretory cargo-containing carriers would not be affected by mutating the COP I retrieval signal (because it is COP II-dependent), but a lack of p24 retrieval to the ER could deplete the necessary p24 proteins in this compartment and as a result, ‘machinery cargo’ would then not be properly included into POMC-containing carriers anymore.

To examine a role for p24 in cargo-specific transport of POMC, a role that according to our transgenic results is unlikely, the most obvious question is whether p24 proteins can bind to putative cargo proteins. To show cargo binding, swapping the N-terminal putative cargo domains of the two p24δ members or mutating the conserved cysteine residues could reveal the individual specificity for certain cargo molecules. In yeast, such studies have already been performed and provided some evidence for such a role for p24 as specific cargo receptor by successfully crosslinking p24 proteins to Gas1p cargo molecules (Muñiz et al., 2000).

To support our current hypothesis that p24 proteins act in the targeting of ‘machinery proteins’ to proper secretory pathway subcompartments, it would be interesting to mutate the GRASP-binding sites in several Xenopus p24 members to identify their involvement in subcompartmentalization and targeting of subsets of proteins to their proper Golgi environment. The initial and most obvious experiment would be to mutate the GRASP-binding site in Xp24β1 (C-terminal residues RRVV) since this motif represents the best-characterized site (Barr et al., 2001). Mutations of the sites in different members of the Xp24α subfamily or in Xp24β1 could reveal the specificity of these members to target selected ‘machinery proteins’ to specific subcompartments.

One of the poorly understood issues in p24 research concerns p24 complex formation and the implication of these complexes for p24 function. Multimeric complexes containing one representative of each p24 subfamily have been shown in yeast and mammalian cells, but the importance of such complexes is still unknown (Belden and Barlowe, 1996; Füllekrug et al., 1999; Marzioch et al., 1999). Furthermore, others have stated that p24 proteins exist as monomers or dimers cycling differentially in the early secretory pathway (Jenne et al., 2002). Mutating the coiled-coil domains of individual p24 members and transgenically expressing these mutants in the melanotrope cell could provide an approach to study p24 complex formation and its contribution to p24 function. Subsequent immunoprecipitation or cell fractionation studies may provide more insight into this complex issue.

Knocking out p24 genes in yeast had only a minor effect on the transport of cargo (Belden and Barlowe, 1996; Marzioch et al., 1999; Schimmöller et al., 1995). In addition, deletion of all eight yeast p24 genes did not prevent yeast from growing and cargo transport was normal (Springer et al., 2000). In contrast, genetic deletion of one p24 member (p24δ1) in mouse caused early embryonic death (Denzel et al., 2000). This clearly demonstrates that in higher eukaryotes the p24 proteins have a crucial role in embryonic development (presumably allowing cell-cell communication) and interference with p24 function causes lethality. The technique of Xenopus transgenesis provides an elegant system to genetically interfere with p24 function by using tissue-specific promoters to drive transgene expression. In the last few years, the technique of RNA interference has been developed to silence gene expression (Dykxhoorn et al., 2003; Sharp, 1999) and can be used to block the expression of target genes in Xenopus by microinjecting double-stranded RNA (Nakano et al., 2000; Zhou et al., 2002). We are now combining RNA interference with the technique of stable Xenopus transgenesis to stably and tissue-specifically knock down genes by the expression of small interfering RNAs. With this technique we might be able to specifically knock down individual p24 family members in the melanotrope cell and investigate the effect of this manipulation on p24 complex formation, and POMC cargo transport and subsequent processing. However, thus far, our attempts to accomplish transgene-driven RNA interference in Xenopus have not been successful (Dirks et al., 2003). It is likely that the cell-specific transgenic overexpression experiments with dominant negative and mutant forms of p24 family members that are now in progress will be more successful.

In conclusion, the research described in this thesis has given us more insight into the p24 family and the dynamic behavior of these proteins in the Xenopus melanotrope cell. Furthermore, we provided the first proof of principle for the Xenopus transgenesis platform and have shown that functional studies can be performed in a physiological context, ultimately leading to a further understanding of the functioning of complex protein families. The p24 family was the subject of this proof of principle. Using cell-specific transgene expression, we were able to establish that in the secretory pathway an intact p24 system is not essential for transport vesicle budding or secretory cargo transport per se, but rather for directing ‘machinery proteins’ to specific secretory cargo-containing transport carriers to allow proper secretory protein processing.

 

 

REFERENCES

Anantharaman, V. and Aravind, L. (2002). The GOLD domain, a novel protein module involved in Golgi function and secretion. Genome Biol 3, research0023.

Baker, L. A. and Gomez, R. A. (2000). Tmp21-I, a vesicular trafficking protein, is differentially expressed during induction of the ureter and metanephros. Journal of Urology 164, 562-6.

Barr, F. A., Preisinger, C., Kopajtich, R. and Korner, R. (2001). Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus. Journal of Cell Biology 155, 885-91.

Belden, W. J. and Barlowe, C. (1996). Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J Biol Chem 271, 26939-26946.

Belden, W. J. and Barlowe, C. (2001a). Deletion of yeast p24 genes activates the unfolded protein response. Molecular Biology of the Cell 12, 957-69.

Belden, W. J. and Barlowe, C. (2001b). Distinct roles for the cytoplasmic tail sequences of Emp24p and Erv25p in transport between the endoplasmic reticulum and Golgi complex. Journal of Biological Chemistry 276, 43040-8.

Bisbee, C.A., Baker, M.A., Wilson, A.C., Haji-Azimi, I. and Fischberg, M. (1977). Albumin phylogeny for clawed frogs (Xenopus). Science 195, 785-787.

Blum, R., Feick, P., Puype, M., Vandekerckhove, J., Klengel, R., Nastainczyk, W. and Schuldz, I. (1996). Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal membranes, are members of a protein family involved in vesicular trafficking. J Biol Chem 271, 17183-17189.

Blum, R., Pfeiffer, F., Feick, P., Nastainczyk, W., Kohler, B., Schafer, K. H. and Schulz, I. (1999). Intracellular localization and in vivo trafficking of p24A and p23. Journal of Cell Science 112, 537-548.

Bremser, M., Nickel, W., Schweikert, M., Ravazzola, M., Amherdt, M., Hughes, C. A., Sollner, T. H., Rothman, J. E. and Wieland, F. T. (1999). Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors. Cell 96, 495-506.

Ciufo, L. F. and Boyd, A. (2000). Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway. Journal of Biological Chemistry 275, 8382-8.

Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J. J., Solari, R. C. and Owen, M. J. (2000). The p24 family member p23 is required for early embryonic development. Current Biology 10, 55-8.

Dirks, P. H., Bouw, G., Van Huizen, R., Jansen, E. J. R. and Martens, G. J. M. (2003). Functional Genomics in Xenopus laevis: towards transgene-driven RNA interference and cell-specific transgene expression. Current Genomics, in press.

Dominguez, M., Dejgaard, K., Fullekrug, J., Dahan, S., Fazel, A., Paccaud, J. P., Thomas, D. Y., Bergeron, J. J. and Nilsson, T. (1998). gp25l/emp24/p24 protein family members of the cis-Golgi network bind both Cop I and II coatomer. Journal of Cell Biology 140, 751-65.

Dykxhoorn, D. M., Novina, C. D. and Sharp, P. A. (2003). Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4, 457-67.

Elrod Erickson, M. J. and Kaiser, C. A. (1996). Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations. Molecular Biology of the Cell 7, 1043-1058.

Emery, G., Rojo, M. and Gruenberg, J. (2000). Coupled transport of p24 family members. Journal of Cell Science 113 ( Pt 13), 2507-16.

Fiedler, K. and Rothman, J. E. (1997). Sorting determinants in the transmembrane domain of p24 proteins. J Biol Chem 272, 24739-24742.

Füllekrug, J., Suganuma, T., Tang, B. L., Hong, W., Storrie, B. and Nilsson, T. (1999). Localization and Recycling of gp27 (hp24gamma3): Complex Formation with Other p24 Family Members. Molecular Biology of the Cell 10, 1939-1955.

Gommel, D., Orci, L., Emig, E. M., Hannah, M. J., Ravazzola, M., Nickel, W., Helms, J. B., Wieland, F. T. and Sohn, K. (1999). p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway. FEBS Letters 447, 179-85.

Holthuis, J. C., van Riel, M. C. and Martens, G. J. M. (1995). Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary. Biochem J 312, 205-213.

Horer, J., Blum, R., Feick, P., Nastainczyk, W. and Schulz, I. (1999). A comparative study of rat and human Tmp21 (p23) reveals the pseudogene- like features of human Tmp21-II. DNA Sequence 10, 121-6.

Jansen, E. J. R., Holling, T. M., van Herp, F. and Martens, G. J. M. (2002). Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis. FEBS Letters 516, 201-7.

Jenne, N., Frey, K., Brugger, B. and Wieland, F. T. (2002). Oligomeric state and stoichiometry of p24 proteins in the early secretory pathway. Journal of Biological Chemistry 277, 46504-11.

Kaiser, C. (2000). Thinking about p24 proteins and how transport vesicles select their cargo. Proc Natl Acad Sci U S A 97, 3783-3785.

Kroll, K. L. and Amaya, E. (1996). Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173-83.

Kuiper, R. P., Waterham, H. R., Rötter, J., Bouw, G. and Martens, G. J. M. (2000). Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell. Molecular Biology of the Cell 11, 131-40.

Lavoie, C., Paiement, J., Dominguez, M., Roy, L., Dahan, S., Gushue, J. N. and Bergeron, J. J. (1999). Roles for alpha(2)p24 and COPI in Endoplasmic Reticulum Cargo Exit Site Formation. Journal of Cell Biology 146, 285-300.

Lin, C. C., Love, H. D., Gushue, J. N., Bergeron, J. J. and Ostermann, J. (1999). ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro. Journal of Cell Biology 147, 1457-72.

Marra, P., Maffucci, T., Daniele, T., Tullio, G. D., Ikehara, Y., Chan, E. K., Luini, A., Beznoussenko, G., Mironov, A. and De Matteis, M. A. (2001). The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment. Nat Cell Biol 3, 1101-13.

Marzioch, M., Henthorn, D. C., Herrmann, J. M., Wilson, R., Thomas, D. Y., Bergeron, J. J., Solari, R. C. and Rowley, A. (1999). Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex. Molecular Biology of the Cell 10, 1923-1938.

Mironov, A. A., Mironove Jr., A. A., Beznoussenko, G. V., Trucco, A., Lupetti, P., Smith, J. D., Geerts, W. J. C., Koster, A. J., Burger, K. N. J., Martone, M. E., Deerinck, T. J., Ellisman, M. H. and Luini, A. (2003). ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains. Developmental Cell 5, 583-594.

Muñiz, M., Nuoffer, C., Hauri, H. P. and Riezman, H. (2000). The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles. Journal of Cell Biology 148, 925-30.

Nakamura, N., Yamazaki, S., Sato, K., Nakano, A., Sakaguchi, M. and Mihara, K. (1998). Identification of potential regulatory elements for the transport of emp24p. Molecular Biology of the Cell 9, 3493-503.

Nakano, H., Amemiya, S., Shiokawa, K. and Taira, M. (2000). RNA interference for the organizer-specific gene Xlim-1 in Xenopus embryos. Biochemical and Biophysical Research Communications 274, 434-9.

Nickel, W., Sohn, K., Bunning, C. and Wieland, F. T. (1997). p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway. Proc Natl Acad Sci U S A 94, 11393-8.

Rojo, M., Emery, G., Marjomaki, V., McDowall, A. W., Parton, R. G. and Gruenberg, J. (2000). The transmembrane protein p23 contributes to the organization of the Golgi apparatus. Journal of Cell Science 113, 1043-57.

Rojo, M., Pepperkok, R., Emery, G., Kellner, R., Stang, E., Parton, R. G. and Gruenberg, J. (1997). Involvement of the transmembrane protein p23 in biosynthetic protein transport. Journal of Cell Biology 139, 1119-35.

Rötter, J., Kuiper, R. P., Bouw, G. and Martens, G. J. M. (2002). Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors. Journal of Cell Science 115, 1049-58.

Roubos, E. W. (1997). Background adaptation by Xenopus laevis: a model for studying neuronal information processing in the pituitary pars intermedia. Comparative Biochemistry and Physiology. Part A, Physiology 118, 533-50.

Schimmöller, F., Singer Krüger, B., Schröder, S., Krüger, U., Barlowe, C. and Riezman, H. (1995). The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO Journal 14, 1329-39.

Sharp, P. A. (1999). RNAi and double-strand RNA. Genes and Development 13, 139-41.

Sohn, K., Orci, L., Ravazzola, M., Amherdt, M., Bremser, M., Lottspeich, F., Fiedler, K., Helms, J. B. and Wieland, F. T. (1996). A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding. Journal of Cell Biology 135, 1239-48.

Sparrow, D. B., Latinkic, B. and Mohun, T. J. (2000). A simplified method of generating transgenic Xenopus. Nucleic Acids Research 28, E12.

Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S. and Schekman, R. (2000). The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 97, 4034-9.

Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P. and Rothman, J. E. (1995). An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding [published erratum appears in Proc Natl Acad Sci U S A 1995 Nov 7;92(23):10816]. Proc Natl Acad Sci U S A 92, 8011-5.

Wen, C. and Greenwald, I. (1999). p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans. Journal of Cell Biology 145, 1165-75.

Zhou, Y., Ching, Y. P., Kok, K. H., Kung, H. F. and Jin, D. Y. (2002). Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA. Nucleic Acids Research 30, 1664-9.

 
 
 
Summary / Samenvatting

 

SUMMARY

Like all of us in everyday life, body cells have to communicate with each other and with their direct environment in order to establish and preserve cell-cell interactions and, at a higher level, maintain tissue integrity. In addition, long-distance signaling allows the body to have centralized organs that regulate peripheral processes. For these processes, cells secrete proteins to the extracellular environment using a well-defined secretory machinery that is composed of specialized membrane-bounded compartments. Each of these distinct compartments is involved in a specific and crucial step in the biosynthesis and processing of secretory proteins. In the early secretory pathway, proteins are synthesized in the endoplasmic reticulum (ER) and subsequently transported to the Golgi apparatus. So-called transport vesicles allow the cargo transport from the ER to the Golgi. These vesicles are either coated with coat protein (COP) I or COP II and are involved in retrograde (Golgi-to-ER) or anterograde (ER-to-Golgi) transport, respectively. Once proteins are synthesized in the ER and processed or folded properly, they enter COP II-coated vesicles, which fuse to form vesicular tubular clusters (VTCs), and finally form the intermediate ER-Golgi compartment (ERGIC) that is transported to the Golgi. During the subsequent transport steps, resident components of the ER are recycled from the ERGIC and the Golgi by COP I-coated vesicles. In the transport vesicles, the p24 family of type I transmembrane proteins is highly abundant (~30% of the total membrane protein content). According to their amino acid sequences, the p24 proteins can be subdivided into four main subfamilies that are structurally related (α, β, γ and δ subfamilies). All p24 proteins have a lumenal portion (with a loop domain containing two conserved cysteine residues and a coiled-coil region), a transmembrane domain and a short cytoplasmic region (containing several transport motifs binding COP I or COP II). The N-terminal coiled-coil regions enable p24 proteins to form multimeric complexes. Although the function of the p24 proteins in the early secretory pathway is still unknown, in yeast they have been suggested to play a role in efficient cargo-selective transport of a subset of proteins out of the ER. Furthermore, they have been implicated in transport vesicle biogenesis, cargo exclusion, membrane architecture and ER quality control.

The goal of the research described in this thesis was to shed light on the function of p24 proteins in the early secretory pathway and especially in the transport of cargo from the ER to the Golgi in a highly specialized secretory cell, the intermediate pituitary melanotrope cell of the amphibian Xenopus laevis. This cell type is unique because it is involved in the process of background adaptation of the animal and is therefore dedicated to produce vast amounts of a single secretory cargo protein, the prohormone proopiomelanocortin (POMC). POMC is proteolytically cleaved to α-melanophore stimulating hormone (α-MSH) that, once released into the blood, regulates blackening of the skin by causing the dispersion of melanin in the skin melanophores. While adapting to a black background, the melanotrope cells become highly active, whereas placing the animal on a white background causes these cells to be virtually inactive. Thus, by placing the animal on a white- or a black background, the biosynthetic and secretory activities of the melanotrope cells can be physiologically manipulated.

Extensive screening of Xenopus cDNA libraries revealed all p24 proteins (Xp24α3, -β1, -γ2,3, -δ1,2) expressed in the Xenopus melanotrope cell. In addition, we found two members (Xp24α2 and -γ1) that are not expressed in the melanotrope cell (chapter 2). We then characterized the p24 family in active and inactive Xenopus melanotrope cells and found that a subset of the eight Xenopus p24 members (Xp24α3, -β1, γ3 and δ2) was coexpressed with POMC upon black-background adaptation, indicating that these members could be involved in the cargo-specific transport of POMC. Xp24δ1 and -γ2 were not upregulated and might therefore play a role in processes other than POMC cargo transport. In order to investigate the dynamic behavior of the p24 proteins, we examined their subcellular localization upon manipulation of the activity of the melanotrope cell (chapter 3). We found that when the animal is adapted to a black background, p24 proteins are recruited from early Golgi structures to specialized ER exit sites and the intermediate compartment in the melanotrope cell, suggesting that following activation of POMC biosynthesis, high amounts of cargo proteins apparently increase the demand for p24 proteins to direct their transport out of the ER and through the secretory pathway.

Many studies using a variety of model organisms and experimental approaches have not provided a clear answer for the precise role of p24 proteins in the early secretory pathway. Therefore, for our studies in the melanotrope cell, we exploited the technique of stable Xenopus transgenesis as a tool to gain more insight into the function of p24 proteins. We used this technique to manipulate the expression of two highly similar members of the Xenopus p24δ family (Xp24δ1 and -δ2). In the recent past, Xenopus transgenesis has been developed and simplified in such a way that we are able to obtain transgenic animals by injecting sperm nuclei mixed with the DNA that codes for the desired transgene product into unfertilized Xenopus eggs. With stable Xenopus transgenesis we are able to express a transgene in any place of the animal and at any time by using tissue-specific promoters to drive transgene expression. We generated a number of transgenic animals and subsequently used transgenic testes of males to obtain different F1 generations of transgenic Xenopus expressing a number of p24 proteins and tagged versions of p24 proteins at various levels. Microscopy analyses on transgenic melanotrope cells that expressed a green fluorescent protein (GFP)-tagged version of Xp24δ1 or -δ2 revealed that by using a Xenopus POMC gene promoter fragment, we could successfully drive transgene expression specifically to the melanotrope cell and in structures that resemble ER and Golgi (chapters 4 to 6). Furthermore, we analyzed the transgenic animals with a variety of biochemical, physiological and cell biological techniques (chapter 4).

In chapter 5, we investigated the effect of overexpressing Xp24δ2-GFP on the functioning of the melanotrope cell. The transgene product effectively displaced the endogenous p24 proteins (Xp24α3, -β1, γ3 and -δ2), resulting in a p24 system that is comprised only of the transgene Xp24δ2 protein. Despite the severely distorted p24 system, the subcellular structures and the rates of POMC synthesis and transport were normal in the transgenic cells. However, the number and pigment content of skin melanophores were reduced, which was likely the result of the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. As a consequence, the transgenic animals were not able to fully adapt to a black background. These results point towards a role for p24 in directing or supplying secretory pathway components to specific subcompartments that then harbor the proper environment for efficient and correct secretory protein processing. In chapter 6, we examined the overexpression of Xp24δ1, the Xenopus p24δ subfamily member that is highly similar to Xp24δ2, except for the N-terminal loop. Since these p24δ subfamily members are closely related, we wondered what would happen when we would overexpress Xp24δ1 in the melanotrope cell. Interestingly, overexpression of Xp24δ1-GFP did not compete away the major endogenous p24 complex but rather increased the steady state levels of the two p24 subfamily members examined (Xp24α3 and -γ3). Furthermore, in these cells we did not observe the phenotypic effects that were caused by the overexpression of Xp24δ2-GFP, strengthening the conclusion that an intact p24 system is crucial for proper POMC processing.

Together, the transgenic results suggest that the N-terminal domains of p24 proteins are involved in selective complex formation and support a role for p24 in targeting components of the POMC processing machinery to their proper destination in the secretory pathway of the melanotrope cell, rather than acting as specific cargo receptors for POMC. To understand the exact molecular mechanisms behind the mode of action of p24 in the early secretory pathway, we need to generate and analyze more transgenic animals expressing other GFP-tagged p24 members and mutants thereof specifically in the melanotrope cells.

In conclusion, we successfully used the technique of stable Xenopus transgenesis as a tool to study the function of p24 proteins and the results offer new insights into the role of p24 proteins in the early secretory pathway of the melanotrope cell. We thus provide a proof of principle that the Xenopus transgenesis technique is a valuable and promising tool to perform functional studies on proteins of unknown function in a physiological context and close to the in vivo situation.

 

SAMENVATTING

Lichaams cellen moeten net als wij in ons dagelijks leven communiceren met elkaar en hun directe omgeving om cel-cel interacties tot stand te brengen, ze te onderhouden en te zorgen voor de integriteit van een weefsel of orgaan. Signaaloverdracht over grotere afstanden schept een mogelijkheid voor centrale organen om perifere processen te reguleren. Voor deze processen worden eiwitten uitgescheiden in de extracellulaire omgeving door middel van een goed gedefinieerd secretie systeem, dat bestaat uit verschillende gespecialiseerde membraan omgeven compartimenten. Elk van deze compartimenten zijn betrokken bij specifieke en cruciale stappen in de biosynthese en modificatie van secretie eiwitten. In de vroege secretie route worden eiwitten gemaakt in het endoplasmatisch reticulum (ER) en vervolgens getransporteerd naar het Golgi aparaat. Dit transport wordt mogelijk gemaakt door zogenaamde transport blaasjes die omgeven zijn door een membraan en de mantel eiwitten COP I of COP II voor respectievelijk het voorwaarts transport van het ER naar het Golgi of transport terug. Wanneer eiwtten eenmaal gemaakt zijn in het ER worden ze gemodificeerd en correct gevouwen voordat ze in transport blaasjes verzameld worden. Deze transport blaasjes snoeren af van het ER en fuseren vervolgens tot vesiculaire tubulaire clusters (VTC’s), welke het intermediaire ER-Golgi compartiment (ERGIC) gaan vormen. Dit compartiment wordt getransporteerd naar het Golgi. Tijdens deze stappen worden eiwitten die in het ER thuishoren van het ERGIC teruggesluisd naar het ER door transport blaasjes omgeven door COP I mantel eiwitten. Van de eiwitten die zich in het membraan van de transport blaasjes bevinden behoort ongeveer 30% tot de p24 family van type I transmembraan eiwitten. Volgens een classificatie naar aminozuur volgorde kunnen deze eiwitten ingedeeld worden in vier subfamilies die structureel gerelateerd zijn (α, β, γ en δ subfamilies). Alle p24 eiwitten hebben een lumenaal deel (met twee geconserveerde cysteine residuen die een loop structuur kunnen vormen, en een coiled-coil regio), een transmembraan regio en een korte cytoplasmatische staart (met daarin motieven welke herkend kunnen worden door COP I en COP II mantel eiwitten). De coiled-coil structuren in p24 maken het mogelijk om multimere complexen te vormen met elkaar. Alhoewel de functie van de p24 eiwitten nog steeds niet helemaal duidelijk is, heeft men in gist gevonden dat ze een rol spelen in efficient en selectieve export van een deel van de cellulaire eiwitten uit het ER. Verder denkt men dat ze een rol spelen in het genereren van transport blaasjes, het uitsluiten van cargo, membraan architectuur en ER kwaliteits controle.

Het doel van het onderzoek, beschreven in dit proefschrift, was om de functie van p24 eiwitten in de vroege secretieroute te ontrafelen, met name het transport van cargo van het ER naar het Golgi in een gespecialiseerde secreterende cel, de hypofyse middenkwab melanotrope cel van de klauwpad (Xenopus laevis). Dit celtype is uniek omdat het betrokken is bij de zwart-wit adaptatie van het beest en is daarom bestemd om grote hoeveelheden van een enkel cargo eiwit te maken, het prohormoon proopiomelanocortine (POMC). POMC wordt nadat het gemaakt is door endoproteasen in het ER gekliefd tot α-MSH dat, zodra het in het bloed gesecreteerd wordt, ervoor zorgt dat door middel van dispersie van melanine in de melanoforen in de huid het dier zwart wordt. Tijdens de adaptatie op een zwarte achtergrond zal de melanotrope cel zeer aktief worden, terwijl adaptatie op een witte achtergrond de cellen inaktiveert. Samengevat kunnen we dus de biosynthetische aktiviteit van deze gespecialiseerde cellen reguleren op een fyiologische manier, namelijk door de beesten of op een witte- of op een zwarte achtergrond te plaatsen.

Intensieve screening van een Xenopus cDNA bibliotheek leverde sequencie informatie op van alle p24 eiwitten (Xp24α3, -β1, -γ2,3, -δ1,2) die in de melanotrope cel tot expressie komen. Daarnaast vonden we twee familieleden (Xp24α2 and -γ1) die niet in de melanotrope cel voorkomen (hoofdstuk 2).

De volgende stap was om in aktieve en inaktieve cellen de p24 familie te charakteriseren en we vonden dat een aantal van de in totaal acht Xenopus p24 eiwitten in de melanotrope cel (Xp24α3, -β1, γ3 and δ2) opgereguleerd waren met POMC na adaptatie op een zwarte achtergrond. Dit betekent dat deze p24 leden te maken kunnen hebben met het transport van POMC cargo eiwitten, terwijl de leden die niet opgereguleerd zijn (Xp24δ1 and -γ2) in andere transport processen betrokken zouden kunnen zijn. Om het dynamische gedrag van de p24 eiwitten te onderzoeken hebben we de subcellulaire localizatie van deze eiwitten onderzocht in aktieve en inactive melanotrope cellen (hoofdstuk 3). We vonden dat wanneer het dier op een zwarte achtergrond geadapteerd werd, de p24 eiwitten gerecruteerd werden vanuit vroeg-Golgi compartimenten naar gespecialiseerde ER exit sites en naar het intermediaire compartiment in de melanotrope cel. Dit suggereert dat, na aktivatie van POMC biosynthese, een grote hoeveelheid cargo eiwitten de vraag naar p24 eiwitten verhoogt om de export uit het ER en hun transport door de secretieroute te dirigeren.

Vele studies, gebruikmakend van een aantal model organismen en experimentele benaderingen, hebben niet geresulteerd in de opheldering van een precieze rol van p24 eiwitten in de vroege secretie route. Voor onze studies in de melanotrope cel hebben we daarom de stabiele Xenopus transgenese techniek gebruikt en uitgewerkt om meer inzicht te krijgen in de functie van p24 eiwitten. We gebruikten deze techniek om de epxressie van twee zeer gelijkende leden van de Xenopus p24δ familie (Xp24δ1 and -δ2) te manipuleren. Recentelijk is de Xenopus transgenese techniek zodanig ontwikkeld en gesimplificeerd dat we transgene dieren verkrijgen door spermakernen te mengen met het DNA dat codeert voor het transgen product en dit mengsel te injecteren in onbevruchte Xenopus eitjes. Met de Xenopus transgenese zijn we in staat om een transgen tot expressie te brengen in elke gewenste plaats in het dier en op elk gewenst tijdstip. We hebben een aantal transgene dieren gegenereerd en hebben de testes gebruikt van transgene mannetjes om generaties F1 transgene nakomelingen te maken die diverse p24 eiwitten en getagde versies daarvan tot expressie brengen met verschillende nivo’s van expressie. Microscopische analyse van transgene melanotrope cellen die een met GFP-getagde versie van Xp24δ1 of -δ2 tot expressie brachten liet zien dat met behulp van een POMC gen promoter fragment de expressie van een transgen gestuurd kon worden specifiek naar de melanotrope cell en in structuren die leken op ER en Golgi (hoofdstukken 4-6). Deze transgene beesten hebben we geanalyseerd met een diversiteit aan biochemische, fysiologische en celbiologische technieken (hoofdstuk 4).

In hoofdstuk 5 hebben we het effect onderzocht van de overexpressie van Xp24δ2-GFP op het functioneren van de melanotrope cel. Het transgen product kon effectief competeren met de endogene p24 eiwitten (Xp24α3, -β1, γ3 en -δ2), resulterende in een p24 systeem dat louter uit het transgene Xp24δ2 eiwit bestond. Ondanks het ernstig verstoorde p24 systeem waren de subcellulaire structuren en de snelheid van POMC synthese en transport normaal in deze transgene cellen. Het aantal melanophore en hun pigment inhoud waren echter verlaagd, waarschijnlijk door een aangetast profiel van POMC-afgeleid bioaktieve peptiden in de transgene melanotrope cellen. Een consequentie van dit alles was dat de transgene dieren niet meer in staat waren om volledig te adapteren aan een zwarte achtergrond. Deze resultaten wijzen in de richting van een rol voor p24 eiwitten in het dirigeren of verstrekken van componenten van de secretieroute naar specifieke subcompartimenten die dan de juiste omgeving krijgen voor efficient en correcte processing van secretie eiwitten.

In hoofdstuk 6 hebben we de overexpressie bestudeerd van Xp24δ1, een Xenopus p24δ subfamilielid dat zeer veel lijkt op Xp24δ2, behalve voor de N-terminale loop structuur. Omdat deze twee familieleden sterk op elkaar lijken vroegen we ons af wat het effect zou zijn wanneer we Xp24δ1 tot overexpressie zouden brengen in de melanotrope cel. Overexpressie van Xp24δ1-GFP had niet tot gevolg dat het endogene p24 complex weg gecompeteerd werd, maar verhoogde juist de steady-state expressie nivo’s van twee p24 subfamilieleden (Xp24α3 en -γ3). Ook konden we in deze cellen niet de fenotypische effecten zien die werden veroorzaakt door de overexpressie van Xp24δ2-GFP. Dit versterkt de conclusie dat een intakt p24 systeem cruciaal is voor het juist processen van POMC.

Samengevat suggereren de transgene resultaten dat de N-terminale domeinen van p24 eiwitten betrokken zijn bij het selectief formeren van complexen en onderbouwen een rol voor p24 eiwitten in het sturen van componenten van de POMC-processing machinerie naar de juiste subcompartimenten van de secretieroute in de melanotrope cel. De p24 eiwitten fungeren dus niet als specifieke cargo-receptoren voor POMC. Om de exacte onderliggende moleculaire mechanismen van de akties van p24 eiwitten te begrijpen moeten we meer transgene dieren genereren en analyzeren, die GFP-getagde p24 leden en mutanten daarvan tot expressie brengen in de melantrope cel.

In conclusie kunnen we zeggen dat we succesvol de techniek van stabiele Xenopus transgenese hebben gebruikt om de functie van p24 eiwitten op te helderen. De resultaten beschreven in dit proefschrift geven nieuwe inzichten in de rol van p24 eiwitten in de vroege secretieroute van de melanotrope cel. We bewijzen hiermee dat de Xenopus transgenese techniek een waardevol en veelbelovend stuk gereedschap is om eiwitten met een onbekende functie te onderoeken in een fysiologische omgeving en dichtbij de in vivo situatie.

 

Curriculum Vitae

Gerrit Bouw werd geboren op 10 april 1973 in Dordrecht en groeide op in Hardinxveld-Giessendam. In 1992 behaalde hij het VWO diploma aan de Christelijke scholengemeenschap De Lage Waard te Papendrecht. In datzelfde jaar startte hij met de studie Medische Biologie aan de Universiteit Utrecht. Deze studie duurde iets meer dan vijf jaar, waarin hij twee afstudeerstages voltooide. Tijdens de tweede afstudeerstage bij de vakgroep Celbiologie onder leiding van dr. Willem Stoorvogel werd zijn interesse gewekt in celbiologie, en met name het proces van vesiculair transport en de daarbij betrokken eiwitten. Op 22 december 1997 werd de studie afgesloten met het behalen van het doctoraal diploma. Op 1 januari 1998 begon Gerrit Bouw als assistent in opleiding (AIO) bij de vakgroep Moleculaire Dierfysiologie aan de Katholieke Universiteit Nijmegen (nu Radboud Universiteit Nijmegen) onder leiding van prof. dr. Gerard J.M. Martens. Het onderzoek naar de functie van p24 eiwitten in de vroege secretieroute van de Xenopus hypofyse middenkwab melanotrope cell heeft geleid tot de in dit proefschrift beschreven resultaten en conclusies. Tijdens het promotieonderzoek startte Gerrit Bouw, in samenwerking met Multigen B.V., Vengen B.V. en prof. dr. Gerard J.M. Martens een project voor 6 maanden om de Xenopus transgenese techniek te commercialiseren. Dit project werd gefinancierd door een aan hem toegekende BioPartner First Stage Grant. Per 3 november 2003 is hij werkzaam als Verkoop Specialist Microscopie in de Life Sciences bij de firma Paes Nederland, Olympus.