Melanoregulin is stably targeted to the melanosome membrane by palmitoylation

In mammals, pigments are made by melanocytes within a specialized organelle, the melanosome. Mature, pigment-laden melanosomes are then transferred to keratinocytes to drive the visible pigmentation of the animal’s hair and skin. The dilute suppressor (dsu) locus encodes an extragenic suppressor of the pigmentation defect exhibited by mice lacking myosin Va (i.e. dilute mice). We recently showed that melanoregulin, the product of the dsu locus, functions as a negative regulator of a shedding mechanism that drives the intercellular transfer of melanosomes from the melanocyte to the keratinocyte. Here we address melanoregulin’s localization within the melanocyte, as well as the molecular basis for its localization. First, we confirm and extend recently published results using exogenous, GFP-tagged melanoregulin by showing that endogenous melanoregulin also targets extensively to melanosomes. Second, using site-directed mutagenesis, metabolic labeling with H³-palmitate, and an inhibitor of palmitoylation in vivo, we show that the targeting of melanoregulin to the limiting membranes of melanosomes in melanocytes and lysosomes in CV1 cells depends critically on the palmitoylation of one or more of six closely-spaced cysteine residues located near melanoregulin’s N-terminus. Finally, using Fluorescence Recovery after Photobleaching (FRAP), we show that melanoregulin-GFP exhibits little if any tendency to cycle in and out of the melanosome membrane. We conclude that multiple palmitoylation serves to stably anchor melanoregulin in the melanosome membrane.     

The Hermansky-Pudlak syndrome 1 (HPS1) and HPS4 proteins are components of two complexes,BLOC-3 and BLOC-4, involved in the biogenesis of lysosome-related organelles

Hermansky-Pudlak syndrome (HPS) is a genetic disease of lysosome, melanosome, and granule biogenesis. Mutations of six different loci have been associated with HPS in humans, the most frequent of which are mutations of the HPS1 and HPS4 genes. Here, we show that the HPS1 and HPS4 proteins are components of two novel protein complexes involved in biogenesis of melanosome and lysosome-related organelles: biogenesis of lysosome-related organelles complex-(BLOC) 3 and BLOC-4. The phenotypes of Hps1-mutant (pale-ear; ep) and Hps4-mutant (light-ear; le) mice and humans are very similar, and cells from ep and le mice exhibit similar abnormalities of melanosome morphology. HPS1 protein is absent from ep-mutant cells, and HPS4 from le-mutant cells, but le-mutant cells also lack HPS1 protein. HPS4 protein seems to be necessary for stabilization of HPS1, and the HPS1 and HPS4 proteins co-immunoprecipitate, indicating that they are in a complex. HPS1 and HPS4 do not interact directly in a yeast two-hybrid system, although HPS4 interacts with itself. In a partially purified vesicular/organellar fraction, HPS1 and HPS4 are both components of a complex with a molecular mass of approximately 500 kDa, termed BLOC-3. Within BLOC-3, HPS1 and HPS4 are components of a discrete approximately 200-kDa module termed BLOC-4. In the cytosol, HPS1 (but not HPS4) is part of yet another complex, termed BLOC-5. We propose that the BLOC-3 and BLOC-4 HPS1.HPS4 complexes play a central role in trafficking cargo proteins to newly formed cytoplasmic organelles.     

The ocular albinism type 1 (OA1) protein and the evidence for an intracellular signal transduction system involved in melanosome biogenesis

Ocular albinism type 1 is an X-linked disorder characterized by severe reduction of visual acuity, retinal hypopigmentation, foveal hypoplasia, optic misrouting and the presence of giant melanosomes (macromelanosomes) in skin melanocytes and retinal pigment epithelium. The protein product of the OA1 gene is a pigment cell specific membrane glycoprotein, displaying structural and functional features of G protein-coupled receptors (GPCRs). However, in contrast to all other previously characterized GPCRs, OA1 is not localized to the plasma membrane, but is targeted to intracellular organelles, namely late endosomes/lysosomes and melanosomes. These unique characteristics suggest that OA1 represents the first example described so far of an exclusively intracellular GPCR and regulates melanosome biogenesis by transducing signals from the organelle lumen to the cytosol. These findings support previous hypotheses that GPCR-mediated signaling might also operate at the internal membranes in mammalian cells.     

Assembly of the Biogenesis of Lysosome-related Organelles Complex-3 (BLOC-3) and Its Interaction with Rab9

The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs.     

BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

Hermansky–Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2–deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2–deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.     

BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1-deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.     

Specific interaction of Gαi3 with the Oa1 G-protein coupled receptor controls the size and density of melanosomes in retinal pigment epithelium

Background

Ocular albinism type 1, an X-linked disease characterized by the presence of enlarged melanosomes in the retinal pigment epithelium (RPE) and abnormal crossing of axons at the optic chiasm, is caused by mutations in the OA1 gene. The protein product of this gene is a G-protein-coupled receptor (GPCR) localized in RPE melanosomes. The Oa1-/- mouse model of ocular albinism reproduces the human disease. Oa1 has been shown to immunoprecipitate with the Gαi subunit of heterotrimeric G proteins from human skin melanocytes. However, the Gαi subfamily has three highly homologous members, Gαi1, Gαi2 and Gαi3 and it is possible that one or more of them partners with Oa1. We had previously shown by in-vivo studies that Gαi3-/- and Oa1-/- mice have similar RPE phenotype and decussation patterns. In this paper we analyze the specificity of the Oa1-Gαi interaction.

Methodology

By using the genetic mouse models Gαi1-/-, Gαi2-/-, Gαi3-/- and the double knockout Gαi1-/-, Gαi3-/- that lack functional Gαi1, Gαi2, Gαi3, or both Gαi1 and Gαi3 proteins, respectively, we show that Gαi3 is critical for the maintenance of a normal melanosomal phenotype and that its absence is associated with changes in melanosomal size and density. GST-pull-down and immunoprecipitation assays conclusively demonstrate that Gαi3 is the only Gαi that binds to Oa1. Western blots show that Gαi3 expression is barely detectable in the Oa1-/- RPE, strongly supporting a previously unsuspected role for Gαi3 in melanosomal biogenesis.

Conclusion

Our results identify the Oa1 transducer Gαi3 as the first downstream component in the Oa1 signaling pathway.     

Melanosome maturation defect in Rab38-deficient retinal pigment epithelium results in instability of immature melanosomes during transient melanogenesis

Pathways of melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce melanosomes. Here, we show that progression from pmel17-positive premelanosomes to tyrosinase-positive mature melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the "chocolate" (cht) mouse causes dramatically reduced numbers of melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced melanosome numbers, but a continuing low level of melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for melanosome biogenesis. In cht RPE, premelanosomes form but delivery of tyrosinase is impaired. Premelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing melanosome from the activity of lumenal acid hydrolases.     

A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8)

Hermansky-Pudlak syndrome (HPS) is genetically heterogeneous, and mutations in seven genes have been reported to cause HPS. Autozygosity mapping studies were undertaken in a large consanguineous family with HPS. Affected individuals displayed features of incomplete oculocutaneous albinism and platelet dysfunction. Skin biopsy demonstrated abnormal aggregates of melanosomes within basal epidermal keratinocytes. A homozygous germline frameshift mutation in BLOC1S3 (p.Gln150ArgfsX75) was identified in all affected individuals. BLOC1S3 mutations have not been previously described in patients with HPS, but BLOC1S3 encodes a subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Mutations in other BLOC-1 subunits have been associated with an HPS phenotype in humans and/or mouse, and a nonsense mutation in the murine orthologue of BLOC1S3 causes the reduced pigmentation (rp) model of HPS. Interestingly, eye pigment formation is reported to be normal in rp, but we found visual defects (nystagmus, iris transilluminancy, foveal hypoplasia, reduced visual acuity, and evidence of optic pathway misrouting) in affected individuals. These findings define a novel form of human HPS (HPS8) and extend genotype-phenotype correlations in HPS.     

Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells

In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.     

Ocular albinism type 1: more than meets the eye

Ocular albinism type 1 (OA1) is an X-linked recessive disorder characterized by a severe reduction of visual acuity, and hypopigmentation of the retina that leads to nystagmus, strabismus, and photophobia/photodysphoria. Microscopic examination of both retinal pigment epithelium and skin melanocytes in OA1 reveals the presence of macrome-lanosomes, suggesting that the OA1 gene product plays a role in melanosome biogenesis. Studies of mutations identified from OA1 patients and an Oa1 knock-out mouse model further implicate OA1 protein function in the late stage of melanosome development. Because its effects are primarily limited to the eye, OA1 represents an ideal model system to study the relationship between pigmentation and visual development. Based upon sequence homology and biochemical studies, OA1 may represent a novel intracellular G-protein coupled receptor. Understanding the function of OA1 will contribute greatly to our understanding of melanosome biogenesis and the role of pigmentation in visual development.     

Functional interactions between OCA2 and the protein complexes BLOC-1, BLOC-2, and AP-3 inferred from epistatic analyses of mouse coat pigmentation

The biogenesis of melanosomes is a multistage process that requires the function of cell-type-specific and ubiquitously expressed proteins. OCA2, the product of the gene defective in oculocutaneous albinism type 2, is a melanosomal membrane protein with restricted expression pattern and a potential role in the trafficking of other proteins to melanosomes. The ubiquitous protein complexes AP-3, BLOC-1, and BLOC-2, which contain as subunits the products of genes defective in various types of Hermansky-Pudlak syndrome, have been likewise implicated in trafficking to melanosomes. We have tested for genetic interactions between mutant alleles causing deficiency in OCA2 (pink-eyed dilution unstable), AP-3 (pearl), BLOC-1 (pallid), and BLOC-2 (cocoa) in C57BL/6J mice. The pallid allele was epistatic to pink-eyed dilution, and the latter behaved as a semi-dominant phenotypic enhancer of cocoa and, to a lesser extent, of pearl. These observations suggest functional links between OCA2 and these three protein complexes involved in melanosome biogenesis.     

Identification of snapin and three novel proteins (BLOS1, BLOS2, and BLOS3/reduced pigmentation) as subunits of biogenesis of lysosome-related organelles complex-1 (BLOC-1)

Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a ubiquitously expressed multisubunit protein complex required for the normal biogenesis of specialized organelles of the endosomal-lysosomal system, such as melanosomes and platelet dense granules. The complex is known to contain the coiled-coil-forming proteins, Pallidin, Muted, Cappuccino, and Dysbindin. The genes encoding these proteins are defective in inbred mouse strains that serve as models of Hermansky-Pudlak syndrome (HPS), a genetic disorder characterized by hypopigmentation and platelet storage pool deficiency. In addition, mutation of human Dysbindin causes HPS type 7. Here, we report the identification of another four subunits of the complex. One is Snapin, a coiled-coil-forming protein previously characterized as a binding partner of synaptosomal-associated proteins 25 and 23 and implicated in the regulation of membrane fusion events. The other three are previously uncharacterized proteins, which we named BLOC subunits 1, 2, and 3 (BLOS1, -2, and -3). Using specific antibodies to detect endogenous proteins from human and mouse cells, we found that Snapin, BLOS1, BLOS2, and BLOS3 co-immunoprecipitate, and co-fractionate upon size exclusion chromatography, with previously known BLOC-1 subunits. Furthermore, steady-state levels of the four proteins are significantly reduced in cells from pallid mice, which carry a mutation in Pallidin and display secondary loss of other BLOC-1 subunits. Yeast two-hybrid analyses suggest a network of binary interactions involving all of the previously known and newly identified subunits. Interestingly, the HPS mouse model strain, reduced pigmentation, carries a nonsense mutation in the gene encoding BLOS3. As judged from size exclusion chromatographic analyses, the reduced pigmentation mutation affects BLOC-1 assembly less severely than the pallid mutation. Mutations in the human genes encoding Snapin and the BLOS proteins could underlie novel forms of HPS.     

Biogenesis of melanosomes - the chessboard of pigmentation

Melanosomes are lysosome-related organelles in retinal pigment epithelial cells and epidermal melanocytes in which melanin pigments are synthesized and stored. Melanosomes are generated by multistep processes in which an immature unpigmented organelle forms and then subsequently matures. Such maturation requires inter-organellar transport of protein cargos required for pigment synthesis but also recruitment of effector proteins necessary for the correct transport of melanosomes to the cell periphery. Several studies have started to unravel the main pathways and mechanisms exploited by melanosomal proteins involved in melanosome structure and melanin synthesis. A major unexpected finding seen early in melanosome biogenesis showed the similarities between the fibrillar sheets of premelanosomes and amyloid fibrils. Late steps of melanosome formation are dependent on pathways regulated by proteins encoded by genes mutated in genetic diseases such as the Hermansky-Pudlak Syndrom (HPS) and different types of albinism. Altogether the findings from the past recent years have started to unravel how specialized cells integrate unique and ubiquitous molecular mechanisms in subverting the endosomal system to generate cell-type specific structures and their associated functions. Further dissection of the melanosomal system will likely shed light not only on the biogenesis of lysosome-related organelles but also on general aspects of vesicular transport in the endosomal system.     

Signaling pathways in melanosome biogenesis and pathology

Melanosomes are the specialized intracellular organelles of pigment cells devoted to the synthesis, storage and transport of melanin pigments, which are responsible for most visible pigmentation in mammals and other vertebrates. As a direct consequence, any genetic mutation resulting in alteration of melanosomal function, either because affecting pigment cell survival, migration and differentiation, or because interfering with melanosome biogenesis, transport and transfer to keratinocytes, is immediately translated into color variations of skin, fur, hair or eyes. Thus, over 100 genes and proteins have been identified as pigmentary determinants in mammals, providing us with a deep understanding of this biological system, which functions by using mechanisms and processes that have parallels in other tissues and organs. In particular, many genes implicated in melanosome biogenesis have been characterized, so that melanosomes represent an incredible source of information and a model for organelles belonging to the secretory pathway. Furthermore, the function of melanosomes can be associated with common physiological phenotypes, such as variation of pigmentation among individuals, and with rare pathological conditions, such as albinism, characterized by severe visual defects. Among the most relevant mechanisms operating in melanosome biogenesis are the signal transduction pathways mediated by two peculiar G protein-coupled receptors: the melanocortin-1 receptor (MC1R), involved in the fair skin/red hair phenotype and skin cancer; and OA1 (GPR143), whose loss-of-function results in X-linked ocular albinism. This review will focus on the most recent novelties regarding the functioning of these two receptors, by highlighting emerging signaling mechanisms and general implications for cell biology and pathology.     

Intracellular distribution and late endosomal effects of the ocular albinism type 1 gene product: consequences of disease-causing mutations and implications for melanosome biogenesis

To investigate the function of ocular albinism type 1 ( OA1 ), the gene responsible for X-linked ocular albinism, we employed a construct containing murine Oa1 fused to green fluorescent protein (GFP) in a heterologous COS cell expression system. The cellular distribution of wild-type (WT) Oa1 protein and Oa1 proteins reflecting mutations causing X-linked ocular albinism were examined. Comparison with different organelle markers revealed that Oa1-GFP localized to the late endolysosomal compartments. Some Oa1 mutant proteins failed to exit the endoplasmic reticulum (ER) (Class I mutants), while other mutants partially (Class II mutants) or fully (Class III mutants) exited the ER and trafficked to endolysosomal compartments. We observed that expression of WT Oa1-GFP in COS cells caused an apparent enlargement of late endosomes and a redistribution of the mannose-6-phosphate receptor (M6PR). None of the mutants displayed the full range of effects on the redistribution of M6PR exhibited by WT Oa1. The effects of Oa1 on late endosome structure and content are thus likely to reflect an important biological property of Oa1. We propose that OA1 is involved in reorganizing the endolysosomal compartment as a necessary step in ocular melanosome biogenesis.     

Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II �� in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting. Disease is triggered by defects in any of 15 mouse gene products, which are part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular associations of these complexes, we used in vivo cross-linking followed by purification of cross-linked AP-3 complexes and mass spectrometric identification of associated proteins. AP-3 was co-isolated with BLOC-1, BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits; clathrin; and phosphatidylinositol-4-kinase type II α (PI4KIIα). We previously reported that this membrane-anchored enzyme is a regulator of AP-3 recruitment to membranes and a cargo of AP-3 (Craige, B., Salazar, G., and Faundez, V. (2008) Mol. Biol. Cell 19,1415 -1426). Using cells deficient in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1, but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIα inclusion into AP-3 complexes. BLOC-1, PI4KIIα, and AP-3 belong to a tripartite complex, and down-regulation of either PI4KIIα, BLOC-1, or AP-3 complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1 complex modulates the association of PI4KIIα with AP-3. These results suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to specify sorting of PI4KIIα along the endocytic route.Membranous organelles along the exocytic and endocytic pathways are each defined by unique lipid and protein composition. Vesicle carriers communicate and maintain the composition of these organelles (2). Consequently defining the machineries that specify vesicle formation, composition, and delivery are central to understanding membrane protein traffic. Generally vesicle biogenesis uses multiprotein cytosolic machineries to select membrane components for inclusion in nascent vesicles (2, 3). Heterotetrameric adaptor complexes (AP-1 to AP-4) are critical to generate vesicles of specific composition from the different organelles constituting the exocytic and endocytic routes (2-4).The best understood vesicle formation machinery in mammalian cells is the one organized around the adaptor complex AP-2 (5). This complex generates vesicles from the plasma membrane using clathrin. Our present detailed understanding of AP-2 vesicle biogenesis mechanisms and interactions emerged from a combination of organellar and in vitro binding proteomics analyses together with the study of binary interactions in cell-free systems (5-9). In contrast, the vesicle biogenesis pathways controlled by AP-3 are far less understood. AP-3 functions to produce vesicles that traffic selected membrane proteins from endosomes to lysosomes, lysosome-related organelles, or synaptic vesicles (10-13). AP-3 is one of the protein complexes affected in the Hermansky-Pudlak syndrome (HPS;³ Online Mendelian Inheritance in Man (OMIM) 203300). So far, mutations in any of 15 mouse or eight human genes trigger a common syndrome. This syndrome encompasses defects that include pigment dilution, platelet dysfunction, pulmonary fibrosis, and occasionally neurological phenotypes (14, 15). All forms of HPS show defective vesicular biogenesis or trafficking that affects lysosomes, lysosome-related organelles (for example melanosomes and platelet dense granules), and, in some of them, synaptic vesicles (11-13). Most of the 15 HPS loci encode polypeptides that assemble into five distinct molecular complexes: the adaptor complex AP-3, HOPS, and the BLOC complexes 1, 2, and 3 (14). Recently binary interactions between AP-3 and BLOC-1 or BLOC-1 and BLOC-2 suggested that arrangements of these complexes could regulate membrane protein targeting (16). Despite the abundance of genetic deficiencies leading to HPS and genetic evidence that HPS complexes may act on the same pathway in defined cell types (17), we have only a partial picture of protein interactions organizing these complexes and how they might control membrane protein targeting.In this study, we took advantage of cell-permeant and reversible cross-linking of HPS complexes followed by their immunoaffinity purification to identify novel molecular interactions. Cross-linked AP-3 co-purified with BLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KIIα. We previously identified PI4KIIα as a cargo and regulator of AP-3 recruitment to endosomes (1, 18). Using mutant cells deficient in either individual HPS complexes or a combination of them, we found that BLOC-1 facilitates the interaction of AP-3 and PI4KIIα. Our studies demonstrate that subunits of four of the five HPS complexes co-isolate with AP-3. Moreover BLOC-1, PI4KIIα, and AP-3 form a tripartite complex as demonstrated by sequential co-immunoprecipitations as well as by similar LAMP1 distribution phenotypes induced by down-regulation of components of this tripartite complex. Our findings indicate that BLOC-1 complex modulates the recognition of PI4KIIα by AP-3. These data suggest that AP-3, either in concert or sequentially with BLOC-1, participates in the sorting of common membrane proteins along the endocytic route.     

Characterization of BLOC-2, a complex containing the Hermansky-Pudlak syndrome proteins HPS3, HPS5 and HPS6

Hermansky–Pudlak syndrome (HPS) defines a group of at least seven autosomal recessive disorders characterized by albinism and prolonged bleeding due to defects in the lysosome-related organelles, melanosomes and platelet-dense granules, respectively. Most HPS genes, including HPS3, HPS5 and HPS6 , encode ubiquitously expressed novel proteins of unknown function. Here, we report the biochemical characterization of a stable protein complex named Biogenesis of Lysosome-related Organelles Complex-2 (BLOC-2), which contains the HPS3, HPS5 and HPS6 proteins as subunits. The endogenous HPS3, HPS5 and HPS6 proteins from human HeLa cells coimmunoprecipitated with each other from crude extracts as well as from fractions resulting from size-exclusion chromatography and density gradient centrifugation. The native molecular mass of BLOC-2 was estimated to be 340 ± 64 kDa. As inferred from the biochemical properties of the HPS6 subunit, BLOC-2 exists in a soluble pool and associates to membranes as a peripheral membrane protein. Fibroblasts deficient in the BLOC-2 subunits HPS3 or HPS6 displayed normal basal secretion of the lysosomal enzyme β-hexosaminidase. Our results suggest a common biological basis underlying the pathogenesis of HPS-3, -5 and -6 disease.     

The Ocular Albinism Type 1 Gene Product is an N‐Glycoprotein but Glycosylation is not Required for its Subcellular Distribution

The ocular albinism type 1 (OA1) gene product is a membrane glycoprotein that may play a role in controlling melanosome growth and maturation. A number of mutations in the OA1 gene lead to ocular albinism due at least in part to retention of the aberrant protein in the endoplasmic reticulum. To examine whether N‐glycosylation plays a role in the post‐translational trafficking of the Oa1 protein, we constructed a series of mutant mouse Oa1 cDNAs encoding an Oa1‐green fluorescent protein fusion in which some or all of the potential glycosylation sites were eliminated by site‐directed mutagenesis. Biochemical studies in transfected cells treated with tunicamycin and peptide:N‐glycosidase F suggest that asparagine at amino acid 106 is essential for N‐glycosylation of the protein. Mutation at amino acid 106 that eliminated glycosylation did not affect the endo/lysosomal distribution of the Oa1 protein in either COS cells or cultured murine melanocytes.