The Pallidin (Pldn) Gene and the Role of SNARE Proteins in Melanosome Biogenesis

Authors


Dr Esteban C. Dell'Angelica Department of Human Genetics, UCLA School of Medicine, Gonda Center, Room 6357B, Los Angeles, CA 90095. E-mail: Edellangelica@mednet.ucla.edu

Abstract

This review focuses on the product of the pallidin (Pldn) gene, one of a number of genes that in mice are associated with pigmentation defects and platelet dense granule deficiency. A similar combination of defects is also observed in patients suffering from Hermansky–Pudlak (HPS) and Chediak–Higashi (CHS) syndromes. Pldn encodes a novel, ∼20-kDa protein that is expressed ubiquitously in mammalian tissues. The pallidin protein was found to bind to syntaxin 13, a member of the syntaxin family of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). As SNARE proteins mediate fusion of intracellular membranes, pallidin may play a role in membrane fusion events required for melanosome biogenesis.

Abbreviations:
CHS

Chediak–Higashi syndrome

EEA1

early endosome antigen 1

EST

expressed sequence tag

HPS

Hermansky–Pudlak syndrome

SNAP

synaptosome-associated protein

SNARE

soluble N-ethylmaleimide-sensitive factor attachment protein receptor

SPD

storage pool deficiency

TRP

tyrosinase-related protein

VAMP

vesicle-associated membrane protein

INTRODUCTION

In mice, at least 15 distinct gene loci associated with pigmentation defects also are associated with platelet dense granule deficiency (Table 1; reviewed in [1]). This combination of deficiency in platelet dense granules [also known as storage pool deficiency (SPD)] and pigmentation dilution is likewise observed in two human genetic disorders: Hermansky–Pudlak syndrome (HPS) and Chediak–Higashi syndrome (CHS). HPS is characterized by a triad of oculocutaneous albinism, prolonged bleeding and mild ceroid lipofuscinosis, and comprises at least three related disorders, referred to as HPS-1, -2 and -3, that are caused by mutations in distinct genes (2). CHS manifestations include, in addition to reduced pigmentation and SPD, immunodeficiency associated with defective natural killer cell function and an accelerated lymphoproliferative phase that can be fatal (3).

Table 1.  . Gene loci associated with reduced pigmentation and platelet dense granule deficiency in mice Thumbnail image of

Although the molecular basis for the combined deficiency of pigmentation and platelet dense granules remains poorly understood, the current hypothesis is that each affected gene encodes a component of a previously unknown cellular machinery required for the biogenesis of both melanosomes and platelet dense granules. The ongoing identification of the affected genes and characterization of the corresponding encoded proteins are beginning to shed some light on this issue (4[5]–6).

In this review, we focus on one of the mouse genes associated with pigmentation and platelet dense granule deficiencies, pallidin (Pldn), formerly known as pa or p2. We also discuss evidence suggesting a role for the Pldn gene product in membrane fusion events mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs).

THE PALLID MOUSE

The recessive pallid mutation was found in the wild in 1926 and tentatively named pink-eyed 2 (p2) because of the similarity between eye colours of pallid and pink-eyed (p) mutant mice (7). Coat colour of pallid mice was lighter than that of pink-eyed mutants and darker than that of tyrosinase-negative, albino (Tyrc) mice. Therefore, pigments are severely reduced but not absent from pallid mice.

Homozygous pallid mice manifest several defects in addition to hypopigmentation. Long-term survival is reduced as compared with wild-type animals (8). Importantly, pallid mice display platelet dense granule deficiency, which results in prolonged bleeding times (>15 min) because of defective platelet aggregation (1). It has been suggested (9) that platelet dense granules are not absent from platelets or megakaryocytes derived from pallid mice, but that these granules fail to accumulate their normal content (e.g. adenosine diphosphate, serotonin). Adult pallid mice slowly develop emphysematous lung lesions consisting of air space enlargement and destruction of alveolar septa (10). Lung lesion development in pallid mice was correlated with decreased serum levels of α1-antitrypsin, which could potentially cause lung damage because of increased elastase activity. However, mRNA levels of α1-antitrypsin in liver from pallid mice were similar to those of wild-type controls, suggesting a possible defect in α1-antitrypsin processing or trafficking (10). Finally, some homozygous pallid mice display defects in otolith formation, which often results in head tilting and/or reduced ability to swim (1). The combination of hypopigmentation, platelet dense granule deficiency and otolith defect has been described for two additional mouse mutants, named muted (mu) and mocha (Apd3d mh). Intriguingly, the otolith and behavioural defects of pallid mice could be duplicated in wild-type mice that were deprived of manganese during embryonic development and, conversely, could be prevented by supplementing the diet of pregnant pallid mice with high amounts of manganese (11). These observations suggest that the Pldn gene product may be required for uptake, storage and/or intracellular utilization of manganese ions during otolith formation, although the molecular bases for this function remain unclear.

THE Pldn GENE

The pallid mutant allele, originally found in wild Mus domesticus, was transferred by repeated backcrossing into the genetically defined, M. musculus C57BL/6J inbred strain. This allele transfer between two mouse species complicated somewhat the identification of Pldn as the transfer turned out to involve not only this gene but also neighbouring genes. For instance, the gene encoding erythrocyte protein band 4.2 (Epb4.2) was proposed to be identical to Pldn on the basis of genetic colocalization of Epb4.2 and Pldn on mouse chromosome 2, 68 cM, and of expression in pallid mice tissues of a truncated Epb4.2 mRNA form (12). However, it was subsequently demonstrated that the Epb4.2 and Pldn loci could be segregated by recombination upon interspecific crossing, and that the truncated Epb4.2 mRNA species found in pallid mice was also expressed in wild-type M. domesticus and encoded a protein of normal size and primary structure (13). Therefore, the truncated Epb4.2 mRNA species expressed in pallid mice most likely represents an allelic variation with no consequences to the encoded protein. Similarly, silent allelic variations between C57BL/6J-pallid and C57BL/6J mice were found in mRNA species derived from the mouse Vps39/Vamp6 gene (GenBank accession codes AF281050 and AF281051), which also maps to the chromosomal region that contains Pldn (E.C. Dell'Angelica, unpublished results).

More recently, the Pldn gene was identified through Northern blot analysis of transcripts from genes that map to the region containing the Pldn locus (14). A single mRNA of ˜2.5 kb was found at reduced levels in tissues from pallid mice as compared with tissues from wild-type mice. In wild-type mice, this mRNA species encodes a protein of 172 amino acid residues, whereas in pallid mice there is a nonsense mutation at codon 69 that might affect mRNA stability because of nonsense-mediated mRNA decay (14).

The human orthologue of the Pldn gene, PLDN, maps to chromosome 15q21.1 and, like the mouse gene, consists of five exons (Fig. 1). Although the pallid mutant mouse has long been considered a mouse model of HPS, so far no HPS patients bearing mutations in PLDN have been described (2).

Figure 1.

. Genome organization and alternative splicing of the human PLDN gene. The different exonic segments are identified using shades of grey. The first four bases of exon 3, which in one mRNA species are missing because of the use of a cryptic splicing acceptor site, are represented out of scale by a black, vertical solid line. Numbers in parenthesis indicate segment sizes (in base pairs). Triangles and squares in each alternatively spliced mRNA form indicate the approximate positions of initiation and termination codons, respectively.

EXPRESSION PATTERN AND ALTERNATIVE SPLICING

The mouse pallidin mRNA was detected in a wide variety of tissues, with the highest expression levels observed in brain, heart, kidney and liver (14). In addition to the originally described mRNA species, an alternatively spliced variant lacking exon 2 was detected in tissues from both pallid (14) and wild-type (E.C. Dell'Angelica, unpublished results) mice. Skipping exon 2 causes a frameshift that leads to a premature termination codon. Another alternatively spliced form, resulting from the use of a cryptic splice acceptor site at the beginning of exon 3, generates a mRNA species with a deletion of four bases (e.g. mouse EST clone IMAGE: 3256910, GenBank accession code BC016554). Again, this mRNA species encodes a truncated polypeptide because of early termination caused by frameshift. The physiological significance of these two alternatively spliced forms is unknown.

The human pallidin mRNA is ∼2 kb in length and encodes a polypeptide of 172 amino acids that shares 86% identical residues with the mouse protein. Two additional alternatively spliced forms can be detected (Fig. 1). One of them is identical to a form occurring in mice and involves a cryptic splice acceptor site at the beginning of exon 3 that generates a four-base deletion and early termination of translation (e.g. human EST clone IMAGE: 5422585, GenBank accession code BM008571). The other form results from skipping of exons 2 and 3 (e.g. human EST clone IMAGE: 3353601, GenBank accession code BE258923) and is also predicted to code for a truncated polypeptide because of an early termination codon. As in the mouse, the physiological relevance of these alternatively spliced forms of PLDN is obscure.

THE PALLIDIN PROTEIN

The major pallidin transcript is predicted to encode a protein of 19.7 kDa (14). Using an antibody raised against residues 129–144, pallidin was detected as a single polypeptide that had an electrophoretic mobility corresponding to a ˜25-kDa protein and was present in cell extracts derived from wild-type animals but absent from pallid mice cell extracts (14). Because the segment used to raise antibodies to pallidin is absent from the truncated polypeptides encoded by the alternatively spliced mRNA species described above, it remains to be determined whether or not these putative truncated forms of pallidin are expressed in normal cells.

The primary structure of pallidin displays no homology to that of any known protein, and no recognizable homologues are found in lower eukaryotes such as Saccharomyces cerevisiae, Drosophila melanogaster or Caenorhabditis elegans. Secondary structure predictions (not shown) suggest that pallidin has very high α-helical content and that both the central and carboxyl terminal regions have a significant propensity to form coiled-coil structures. These two regions display the highest degrees of conservation among the pallidin proteins from different species (not shown), thus suggesting a structural or functional role for these coiled-coil domains. As coiled-coil domains often mediate protein–protein interaction (15) the central and carboxyl terminal regions of pallidin could be involved in either oligomerization or binding to other cellular proteins.

Importantly, yeast two-hybrid experiments resulted in the identification of syntaxin 13 as a potential binding partner of pallidin (14). Syntaxin 13 is a member of the syntaxin family of SNARE proteins that mediate intracellular membrane fusion through the formation of heteromeric coiled-coil structures (16). Interaction between pallidin and syntaxin 13 was also detected by coimmunoprecipitation of the endogenous proteins from normal, rat kidney cells (14). Interestingly, steady-state protein levels of syntaxin 13 appeared to be reduced in cell extracts from pallid mouse kidney, suggesting that the pallidin protein might be important for in vivo stability of syntaxin 13 (14). These observations suggest that pallidin may play a role in SNARE-dependent membrane fusion events. Whether pallidin interacts only with syntaxin 13 or also with other SNAREs remains to be determined.

POTENTIAL ROLES OF SYNTAXIN 13 AND OTHER SNARE PROTEINS IN MELANOSOME BIOGENESIS

The SNAREs are membrane-associated proteins that share a conserved coiled-coil-forming domain and play a fundamental role in the fusion of intracellular organelles (for recent reviews, see [17, 18]). By sequence homology, they can be divided into three protein families: the syntaxins, the vesicle-associated membrane proteins (VAMPs) and the synaptosome-associated proteins (SNAPs). Both the syntaxins and the VAMPs are integral membrane proteins having a coiled-coil domain and a transmembrane domain close to their C-termini. The SNAPs, on the other hand, have two coiled-coil domains and are peripherally associated to membranes through palmitoyl moieties attached to cysteine residues within the central region. Assembly of SNARE coiled-coil domains into highly stable complexes is thought to provide a driving force that allows fusion of lipid bilayers (17, 18). Typically, a SNARE core complex consists of one coiled-coil-forming domain from a syntaxin, one from a VAMP, and two from a SNAP.

In addition to the role of SNAREs in driving membrane fusion, it has long been proposed that these proteins are key to ensure the specificity of intracellular membrane trafficking. According to the SNARE hypothesis (19), the specificity of recognition between SNAREs associated with the membranes of the transport vesicles (v-SNAREs) and of the target compartments (t-SNAREs) would be sufficient to allow transport vesicles to fuse only with their correct target organelle. In support of this notion, every eukaryotic cell expresses a number of syntaxins, VAMPs and SNAPs that localize to specific subcellular compartments. The SNARE hypothesis, however, still remains a matter of controversy (17, 18). Other proteins proposed to contribute to the specificity of intracellular membrane transport include the Rab GTPases, which also localize specifically to different compartments and can recruit cytosolic factors to mediate membrane tethering and docking. Interestingly, some studies have suggested that Rabs and SNAREs can cooperate to determine the specificity of membrane fusion (17). For instance, there is evidence for physical association between Rab5 effectors and components of the SNARE fusion machinery on endosomes (see below).

Syntaxin 13 was localized by immunoelectron microscopy to tubular extensions of early endosomes and recycling endosomes containing the transferrin receptor, as well as to endosomal vacuoles coated with the scaffolding protein, clathrin (20). Co-localization of syntaxin 13 with transferrin receptor at the electron microscopic level suggested a role for this SNARE in receptor recycling from endosomes to the plasma membrane. In support of this hypothesis, anti-syntaxin 13 antibodies inhibited the recycling of internalized transferrin to the plasma membrane in streptolysin-O-permeabilized PC12 cells (20).

Another study concluded that syntaxin 13 is required for homotypic endosome–endosome fusion (21). Thus, antibodies to syntaxin 13, and a recombinant protein bearing the syntaxin 13 cytoplasmic domain, both inhibited in a dose-dependent manner the fusion of early endosomes in vitro. In addition, the cytoplasmic domain of syntaxin 13 was found to interact directly with the carboxyl terminal region of Early Endosome Antigen 1 (EEA1), an effector of the Rab5 GTPase that functions in tethering of early endosomes prior to fusion. Further, syntaxin 13 transiently associated to large oligomeric complexes containing EEA1, Rabaptin-5 (another Rab5 effector) and N-ethylmaleimide-sensitive factor (a soluble ATPase involved in SNARE-dependent membrane fusion). These oligomeric complexes are thought to represent a functional link between tethering factors and the SNARE fusion machinery.

How do the functions proposed for syntaxin 13 relate to the biogenesis of melanosomes? A current model for melanosome biogenesis involves the existence of an early endosomal compartment, the so-called `coated endosome' (6). A characteristic feature of the coated endosome is the presence of large clathrin-containing lattices decorating the organelle's cytoplasmic side (22). In the coated endosome, the premelanosomal protein, Pmel17, is sorted into intraluminal membrane vesicles. Subsequently, these intraluminal vesicles containing Pmel17 form striations, characteristic of stage II premelanosomes, in which insoluble melanin pigments accumulate upon synthesis (6). Because of its endosomal localization, syntaxin 13 (and, by extension, its binding partner pallidin) may be involved in pmel17 sorting, for instance by driving homotypic early endosome fusions to generate the coated endosome or by mediating removal of recycling receptors (e.g. transferrin receptor). In line with this notion, it is worth noting that the clathrin-coated endosomal structures to which a subset of syntaxin 13 was localized in Chinese hamster ovary cells (20) are reminiscent of the coated endosomes involved in melanosome biogenesis (22).

Other melanosomal membrane proteins, such as tyrosinase and tyrosinase-related proteins (TRP)-1 and -2, are thought to traffic from the trans-Golgi network to the premelanosome following a route that bypasses the coated endosome (6). In this context, it is conceivable that syntaxin 13 could function as a premelanosomal t-SNARE, thus mediating the fusion of vesicles carrying these melanosomal proteins.

Other SNAREs besides syntaxin 13 are likely to be involved in the multistep process of melanosome biogenesis. In this regard, a recent study has found that purified melanosome preparations contained syntaxin 4, VAM-2, SNAP-23 and SNAP-25 (23). The function of these SNARE proteins in melanosome biogenesis, however, remains to be ascertained.

In summary, initial characterization of the pallidin protein has provided a first link between the SNARE-dependent fusion machinery and melanosome biogenesis. Further experimental work, however, will be required to establish the exact function of pallidin in this process.

Acknowledgements

We thank David E. Krantz, Marta Starcevic and Ramin Nazarian for critical reading of the manuscript. Work in Dell'Angelica's laboratory is supported in part by grant HL68117 from the National Institutes of Health.

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