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.     

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.     

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.     

BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.     

BLOC-2 subunit HPS6 deficiency affects the tubulation and secretion of von Willebrand factor from mouse endothelial cells

Hermansky-Pudlak syndrome(HPS) is a recessive disorder with bleeding diathesis, which has been linked to platelet granule defects. Both platelet granules and endothelial Weibel-Palade bodies(WPBs)are members of lysosome-related organelles(LROs) whose formation is regulated by HPS protein associated complexes such as BLOC(biogenesis of lysosome-related organelles complex)-1,-2,-3, AP-3(adaptor protein complex-3) and HOPS(homotypic fusion and protein sorting complex). Von Willebrand factor(VWF) is critical to hemostasis, which is stored in a highly-multimerized form as tubules in the WPBs. In this study, we found the defective, but varying, release of VWF into plasma after desmopressin(DDAVP) stimulation in HPS1(BLOC-3 subunit), HPS6(BLOC-2 subunit), and HPS9(BLOC-1 subunit)deficient mice. In particular, VWF tubulation, a critical step in VWF maturation, was impaired in HPS6 deficient WPBs. This likely reflects a defective endothelium, contributing to the bleeding tendency in HPS mice or patients. The differentially defective regulated release of VWF in these HPS mouse models suggests the need for precise HPS genotyping before DDAVP administration to HPS patients.     

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.     

The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5)

Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.     

The pink gene encodes the Drosophila orthologue of the human Hermansky-Pudlak syndrome 5 (HPS5) gene.

Hermansky-Pudlak syndrome (HPS) consists of a set of human autosomal recessive disorders, with symptoms resulting from defects in genes required for protein trafficking in lysosome-related organelles such as melanosomes and platelet dense granules. A number of human HPS genes and rodent orthologues have been identified whose protein products are key components of 1 of 4 different protein complexes (AP-3 or BLOC-1, -2, and -3) that are key participants in the process. Drosophila melanogaster has been a key model organism in demonstrating the in vivo significance of many genes involved in protein trafficking pathways; for example, mutations in the "granule group" genes lead to changes in eye colour arising from improper protein trafficking to pigment granules in the developing eye. An examination of the chromosomal positioning of Drosophila HPS gene orthologues suggested that CG9770, the Drosophila HPS5 orthologue, might correspond to the pink locus. Here we confirm this gene assignment, making pink the first eye colour gene in flies to be identified as a BLOC complex gene.     

Instability of BLOC‐2 and BLOC‐3 in Chinese patients with Hermansky‐Pudlak syndrome

Hermansky‐Pudlak syndrome (HPS) is a rare recessive disorder characterized by oculocutaneous albinism (OCA) or ocular albinism (OA), bleeding tendency, and other symptoms due to multiple defects in tissue‐specific lysosome‐related organelles. Ten HPS subtypes have been characterized with mutations in HPS1 to HPS10, which encode the subunits of BLOC‐1, ‐2, ‐3, and AP‐3. Using next‐generation sequencing (NGS), we have screened 100 hypopigmentation genes in OCA or OA patients and identified four HPS‐1, one HPS‐3, one HPS‐4, one HPS‐5, and three HPS‐6. The HPS‐4 case is the first report in the Chinese population. Among these 20 mutational alleles, 16 were previously unreported alleles (6 in HPS1, 1 in HPS3, 2 in HPS4, 2 in HPS5, and 5 in HPS6). BLOC‐2 and BLOC‐3 were destabilized due to the mutation of these HPS genes which are so far the only reported causative genes in Chinese HPS patients, in which HPS‐1 and HPS‐6 are the most common subtypes. The mutational spectrum of Chinese HPS is population specific.     

Novel variants in the BLOC1S3 gene in patients presenting a mild form of Hermansky–Pudlak syndrome

Hermansky–Pudlak syndrome (HPS) associates oculocutaneous albinism and systemic affections including platelet dense granules anomalies leading to bleeding diathesis and, depending on the form, pulmonary fibrosis, immunodeficiency, and/or granulomatous colitis. So far, 11 forms of autosomal recessive HPS caused by pathogenic variants in 11 different genes have been reported. We describe three HPS‐8 consanguineous families with different homozygous pathogenic variants in BLOC1S3 (NM_212550.3), one of which is novel. These comprise two deletions leading to a reading frameshift (c.385_403del, c.338_341del) and one in frame deletion (c.444_467del). All patients have moderate oculocutaneous albinism and bleeding diathesis, but other HPS symptoms are not described. One patient diagnosed with HPS‐8 suffered from lymphocyte‐predominant Hodgkin lymphoma. The mild severity of HPS‐8 is consistent with other HPS forms caused by variants in BLOC‐1 complex coding genes (HPS‐7, DTNBP1; HPS‐9, BLOC1S6, HPS‐11, BLOC1S5).     

BLOC-3, a protein complex containing the Hermansky-Pudlak syndrome gene products HPS1 and HPS4

The Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. HPS results from mutations in either one of six human genes named HPS1 to HPS6, most of which encode proteins of unknown function. Here we report that the human HPS1 and HPS4 proteins are part of a complex named BLOC-3 (for biogenesis of lysosome-related organelles complex 3). Co-immunoprecipitation experiments demonstrated that epitope-tagged and endogenous HPS1 and HPS4 proteins assemble with each other in vivo. The HPS1.HPS4 complex is predominantly cytosolic, with a small amount being peripherally associated with membranes. Size exclusion chromatography and sedimentation velocity analyses of the cytosolic fraction indicate that HPS1 and HPS4 form a moderately asymmetric protein complex with a molecular mass of approximately 175 kDa. HPS4-deficient fibroblasts from light ear mice display normal distribution and trafficking of the lysosomal membrane protein, Lamp-2, in contrast to fibroblasts from AP-3-deficient pearl mice (HPS2), which exhibit increased trafficking of this lysosomal protein via the plasma membrane. Similarly, light ear fibroblasts display an apparently normal accumulation of Zn2+ in intracellular vesicles, unlike pearl fibroblasts, which exhibit a decreased intracellular Zn2+ storage. Taken together, these observations demonstrate that the HPS1 and HPS4 proteins are components of a cytosolic complex that is involved in the biogenesis of lysosomal-related organelles by a mechanism distinct from that operated by AP-3 complex.     

Melanoregulin, Product of the dsu Locus, Links the BLOC-Pathway and Oa1 in Organelle Biogenesis

Humans with Hermansky-Pudlak Syndrome (HPS) or ocular albinism (OA1) display abnormal aspects of organelle biogenesis. The multigenic disorder HPS displays broad defects in biogenesis of lysosome-related organelles including melanosomes, platelet dense granules, and lysosomes. A phenotype of ocular pigmentation in OA1 is a smaller number of macromelanosomes, in contrast to HPS, where in many cases the melanosomes are smaller than normal. In these studies we define the role of the Mreg^dsu gene, which suppresses the coat color dilution of Myo5a, melanophilin, and Rab27a mutant mice in maintaining melanosome size and distribution. We show that the product of the Mreg^dsu locus, melanoregulin (MREG), interacts both with members of the HPS BLOC-2 complex and with Oa1 in regulating melanosome size. Loss of MREG function facilitates increase in the size of micromelanosomes in the choroid of the HPS BLOC-2 mutants ruby, ruby2, and cocoa, while a transgenic mouse overexpressing melanoregulin corrects the size of retinal pigment epithelium (RPE) macromelanosomes in Oa1^ko/ko mice. Collectively, these results suggest that MREG levels regulate pigment incorporation into melanosomes. Immunohistochemical analysis localizes melanoregulin not to melanosomes, but to small vesicles in the cytoplasm of the RPE, consistent with a role for this protein in regulating membrane interactions during melanosome biogenesis. These results provide the first link between the BLOC pathway and Oa1 in melanosome biogenesis, thus supporting the hypothesis that intracellular G-protein coupled receptors may be involved in the biogenesis of other organelles. Furthermore these studies provide the foundation for therapeutic approaches to correct the pigment defects in the RPE of HPS and OA1.     

NGS‐based targeted resequencing identified rare subtypes of albinism: Providing accurate molecular diagnosis for Japanese patients with albinism

Albinism, which is commonly inherited as an autosomal recessive trait, is characterized by a reduction or absence of melanin in the eyes, skin, and hair. To date, more than 20 causal genes for albinism have been identified; thus, the accurate diagnosis of albinism requires next‐generation sequencing (NGS). In this study, we analyzed 46 patients who tested negative for oculocutaneous albinism (OCA)1–4 and Hermansky‐Pudlak syndrome (HPS)1 based on conventional analysis, in addition to 28 new Japanese patients, using NGS‐based targeted resequencing. We identified a genetic background for albinism in 18 of the 46 patients (39%), who were previously tested negative according to the conventional analysis. In addition, we unveiled a genetic predisposition toward albinism in 23 of the 28 new patients (82%). We identified six patients with rare subtypes of albinism, including HPS3, HPS4, and HPS6, and found 12 novel pathological mutations in albinism‐related genes. Furthermore, most patients who were not diagnosed with albinism by the NGS analysis showed mild manifestations of albinism without apparent eye symptoms and harbored only one heterozygous mutation, occasionally in combination with skin‐color associated gene variants.     

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.     

The Hermansky-Pudlak syndrome 3 (cocoa) protein is a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2)

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease affecting vesicle trafficking among lysosome-related organelles. The Hps3, Hps5, and Hps6 genes are mutated in the cocoa, ruby-eye-2, and ruby-eye mouse pigment mutants, respectively, and their human orthologs are mutated in HPS3, HPS5, and HPS6 patients. These three genes encode novel proteins of unknown function. The phenotypes of Hps5/Hps5,Hps6/Hps6 and Hps3/Hps3,Hps6/Hps6 double mutant mice mimic, in coat and eye colors, in melanosome ultrastructure, and in levels of platelet dense granule serotonin, the corresponding phenotypes of single mutants. These facts suggest that the proteins encoded by these genes act within the same pathway or protein complex in vivo to regulate vesicle trafficking. Further, the Hps5 protein is destabilized within tissues of Hps3 and Hps6 mutants, as is the Hps6 protein within tissues of Hps3 and Hps5 mutants. Also, proteins encoded by these genes co-immunoprecipitate and occur in a complex of 350 kDa as determined by sucrose gradient and gel filtration analyses. Together, these results indicate that the Hps3, Hps5, and Hps6 proteins regulate vesicle trafficking to lysosome-related organelles at the physiological level as components of the BLOC-2 (biogenesis of lysosome-related organelles complex-2) protein complex and suggest that the pathogenesis and future therapies of HPS3, HPS5, and HPS6 patients are likely to be similar. Interaction of the Hps5 and Hps6 proteins within BLOC-2 is abolished by the three-amino acid deletion in the Hps6(ru) mutant allele, indicating that these three amino acids are important for normal BLOC-2 complex formation.     

BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.     

Characterization of melanosomes and melanin in Japanese patients with Hermansky–Pudlak syndrome types 1, 4, 6, and 9

Hermansky–Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism (OCA), a bleeding tendency, and ceroid deposition. Most of the causative genes for HPS encode subunits of the biogenesis of lysosome‐related organelles complex (BLOC). In this study, we identified one patient each with HPS4, HPS6, and HPS9 by whole‐exome sequencing. Next, we analyzed hair samples from the three patients and representative patients with HPS1 and controls using electron microscopy and chemical methods. All HPS patients had fewer, smaller, and more immature melanosomes than healthy controls. Further, all patients showed reduced total melanin content and increased levels of benzothiazine‐type pheomelanin. The results of this study demonstrate the impact of the dysfunctions of BLOCs on the maturation of melanosomes and melanin levels and composition through analysis of their hair samples.     

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.     

BLOC-1 complex deficiency alters the targeting of adaptor protein complex-3 cargoes

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1-deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.