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The function(s) of the Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1) during brain development is to date largely unknown. Here, we investigated how its absence alters the trajectory of postnatal brain development using as model the pallid mouse. Most of the defects observed early postnatally in the mutant mice were more prominent in males than in females and in the hippocampus. Male mutant mice, but not females, had smaller brains as compared to sex-matching wild types at postnatal day 1 (P1), this deficit was largely recovered by P14 and P45. An abnormal cytoarchitecture of the pyramidal cell layer of the hippocampus was observed in P1 pallid male, but not female, or juvenile mice (P45), along with severely decreased expression levels of the radial glial marker Glutamate-Aspartate Transporter. Transcriptomic analyses showed that the overall response to the lack of functional BLOC-1 was more pronounced in hippocampi at P1 than at P45 or in the cerebral cortex. These observations suggest that absence of BLOC-1 renders males more susceptible to perinatal brain maldevelopment and although most abnormalities appear to have been resolved in juvenile animals, still permanent defects may be present, resulting in faulty neuronal circuits, and contribute to previously reported cognitive and behavioral phenotypes in adult BLOC-1-deficient mice., (© 2020 Wiley Periodicals, Inc.)

Purpose: We investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications., Methods: From 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide., Results: The molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality., Conclusion: In this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.

Protein coats are supramolecular complexes that assemble on the cytosolic face of membranes to promote cargo sorting and transport carrier formation in the endomembrane system of eukaryotic cells. Several types of protein coats have been described, including COPI, COPII, AP-1, AP-2, AP-3, AP-4, AP-5, and retromer, which operate at different stages of the endomembrane system. Defects in these coats impair specific transport pathways, compromising the function and viability of the cells. In humans, mutations in subunits of these coats cause various congenital diseases that are collectively referred to as coatopathies. In this article, we review the fundamental properties of protein coats and the diseases that result from mutation of their constituent subunits.

Mice lacking a functional Biogenesis of Lysosome-related Organelles Complex 1 (BLOC-1), such as those of the pallid line, display cognitive and behavioural impairments reminiscent of those presented by individuals with intellectual and developmental disabilities. Although disturbances in the sleep/wake cycle are commonly lamented by these individuals, the underlying mechanisms, including the possible role of the circadian timing system, are still unknown. In this paper, we have explored sleep/circadian malfunctions and underlying mechanisms in BLOC-1-deficient pallid mice. These mutants exhibited less sleep behaviour in the beginning of the resting phase than wild-type mice with a more broken sleeping pattern in normal light-dark conditions. Furthermore, the strength of the activity rhythms in the mutants were reduced with significantly more fragmentation and lower precision than in age-matched controls. These symptoms were accompanied by an abnormal preference for the open arm in the elevated plus maze in the day and poor performance in the novel object recognition at night. At the level of the central circadian clock (the suprachiasmatic nucleus, SCN), loss of BLOC-1 caused subtle morphological changes including a larger SCN and increased expression of the relative levels of the clock gene Per2 product during the day but did not affect the neuronal activity rhythms. In the hippocampus, the pallid mice presented with anomalies in the cytoarchitecture of the Dentate Gyrus granule cells, but not in CA1 pyramidal neurones, along with altered PER2 protein levels as well as reduced pCREB/tCREB ratio during the day. Our findings suggest that lack of BLOC-1 in mice disrupts the sleep/wake cycle and performance in behavioural tests associated with specific alterations in cytoarchitecture and protein expression.

Selective immunoprecipitation of proteins is a useful tool for characterizing proteins and protein-protein interactions. Clear step-by-step protocols are provided for preparing lysates of cells and yeast under a variety of conditions, for binding the antibody to a solid matrix, and for performing the actual immunoprecipitation. An additional method is provided for increasing the specificity of the technique by reprecipitating the antigen with the same or a different antibody. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.

Dysbindin (also known as dysbindin-1 or dystrobrevin-binding protein 1) was identified 10 years ago as a ubiquitously expressed protein of unknown function. In the following years, the protein and its encoding gene, DTNBP1, have become the focus of intensive research owing to genetic and histopathological evidence suggesting a potential role in the pathogenesis of schizophrenia. In this review, we discuss published results demonstrating that dysbindin function is required for normal physiology of the mammalian central nervous system. In tissues other than brain and in non-neuronal cell types, the protein has been characterized as a stable component of a multi-subunit complex, named BLOC-1 (biogenesis of lysosome-related organelles complex-1), which has been implicated in intracellular protein trafficking and the biogenesis of specialized organelles of the endosomal-lysosomal system. In the brain, however, dysbindin has been proposed to associate into multiple complexes with alternative binding partners, and to play a surprisingly wide variety of functions including transcriptional regulation, neurite and dendritic spine formation, synaptic vesicle biogenesis and exocytosis, and trafficking of glutamate and dopamine receptors. This puzzling array of molecular and functional properties ascribed to the dysbindin protein from brain underscores the need of further research aimed at ascertaining its biological significance in health and disease.

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., (© 2010 John Wiley & Sons A/S.)

Biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3 are three multi-subunit protein complexes that are deficient in various forms of Hermansky-Pudlak syndrome, a human disease characterized by abnormal formation of lysosome-related organelles. Contrasting views have arisen on the evolutionary origin of these protein complexes. One view is that the BLOCs represent a recent evolutionary 'acquisition' unique to metazoans. However, the yeast proteins Mon1, Ccz1 and She3 have been reported to display homology to the HPS1 and HPS4 subunits of BLOC-3 and the BLOS2 subunit of BLOC-1, respectively. In this work, we have systematically searched for orthologs of BLOC subunits in the annotated genomes of over 160 species of eukaryotes, including metazoans and fungi in the Opisthokonta group as well as highly divergent organisms. We have found orthologs of six of the eight BLOC-1 subunits, two of the three BLOC-2 subunits, and the two BLOC-3 subunits, in some non-opisthokonts such as Dictyostelium discoideum, suggesting an early evolutionary origin for these complexes. On the other hand, we have obtained no evidence in support of the notion that yeast She3 would be an ortholog of BLOS2, and found that yeast Mon1 and Ccz1, despite displaying restricted homology to portions of HPS1 and HPS4, are unlikely to represent the orthologs of these BLOC-3 subunits. Potential orthologs of Mon1 and Ccz1 were found in humans and several other eukaryotes.

Biogenesis of lysosome-related organelles complex 1 (BLOC-1) is a protein complex formed by the products of eight distinct genes. Loss-of-function mutations in two of these genes, DTNBP1 and BLOC1S3, cause Hermansky-Pudlak syndrome, a human disorder characterized by defective biogenesis of lysosome-related organelles. In addition, haplotype variants within the same two genes have been postulated to increase the risk of developing schizophrenia. However, the molecular function of BLOC-1 remains unknown. Here, we have generated a fly model of BLOC-1 deficiency. Mutant flies lacking the conserved Blos1 subunit displayed eye pigmentation defects due to abnormal pigment granules, which are lysosome-related organelles, as well as abnormal glutamatergic transmission and behavior. Epistatic analyses revealed that BLOC-1 function in pigment granule biogenesis requires the activities of BLOC-2 and a putative Rab guanine-nucleotide-exchange factor named Claret. The eye pigmentation phenotype was modified by misexpression of proteins involved in intracellular protein trafficking; in particular, the phenotype was partially ameliorated by Rab11 and strongly enhanced by the clathrin-disassembly factor, Auxilin. These observations validate Drosophila melanogaster as a powerful model for the study of BLOC-1 function and its interactions with modifier genes.

The adaptor protein (AP)-3 complex defines a pathway for the intracellular trafficking of membrane-associated proteins in most eukaryotic cells. Ten years ago, genetic defects in AP-3 were linked to a human Mendelian disease, named Hermansky-Pudlak syndrome, characterized by abnormal biogenesis and function of lysosome-related organelles such as melanosomes and platelet dense granules. During recent years, research on this trafficking pathway has significantly expanded its horizons to include evolutionarily divergent eukaryotic models and to embrace functional genomics and proteomics approaches. These studies have brought into focus ideas about the specific roles of this pathway in protein trafficking and organelle biogenesis within the endosomal-lysosomal system.

Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steady-state lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes.

Hermansky-Pudlak syndrome (HPS) comprises a constellation of human autosomal recessive disorders characterized by albinism and platelet storage pool deficiency. At least eight types of HPS have been defined based on the identity of the mutated gene. These genes encode components of four ubiquitously expressed protein complexes, named Adaptor Protein (AP)-3 and Biogenesis of Lysosome-related Organelles Complex (BLOC)-1 through -3. In patients of Puerto Rican origin, the molecular diagnosis can be based on analysis of two founder mutations. On the other hand, identification of the HPS type in other patients relies on the sequencing of all candidate genes. In this work, we have developed a biochemical assay to minimize the number of candidate genes to be sequenced per patient. The assay consists of immunoblotting analysis of extracts prepared from skin fibroblasts, using antibodies to one subunit per protein complex. The assay allowed us to determine which complex was defective in each of a group of HPS patients with unknown genetic lesions, thus subsequent sequencing was limited to genes encoding the corresponding subunits. Because no mutations within the two genes encoding BLOC-3 subunits could be found in two patients displaying reduced BLOC-3 levels, the possible existence of additional subunits was considered. Through size-exclusion chromatography and sedimentation velocity analysis, the native molecular mass of BLOC-3 was estimated to be 140+/-30 kDa, a value most consistent with the idea that BLOC-3 is a HPS1HPS4 heterodimer (approximately 156 kDa) albeit not inconsistent with the putative existence of a relatively small third subunit.

Zinc accumulation in the lumen of cytoplasmic vesicles is one of the mechanisms by which cells can store significant amounts of this essential but potentially toxic biometal. Previous studies had demonstrated reduced vesicular zinc levels in fibroblasts from mutant mice deficient in adaptor protein 3 (AP-3), a complex involved in protein trafficking to late endosomes and lysosomes. We have observed a similar phenotype in the human fibroblastoid cell line, M1, upon small interference RNA-mediated AP-3 knockdown. A survey of the expression and localization of zinc transporter (ZnT) family members identified ZnT2, ZnT3, and ZnT4 as likely mediators of vesicular zinc accumulation in M1 cells. Expression of green fluorescence protein (GFP)-tagged ZnT2 and ZnT3 promoted accumulation of vesicular zinc as visualized using the indicator zinquin. Moreover, GFP-ZnT2 overexpression elicited a significant accumulation of zinc within mature lysosomes, which in untransfected M1 cells contained little or no chelatable zinc, and restored the zinc storage capability of AP-3-deficient cells. These results suggest that ZnT2 can facilitate vesicular zinc accumulation independently of AP-3 function, and validate the M1 fibroblastoid line as a human cell culture system amenable to the study of vesicular zinc regulation using techniques compatible with functional genomic approaches.

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.

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 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.

Dysbindin was identified as a dystrobrevin-binding protein potentially involved in the pathogenesis of muscular dystrophy. Subsequently, genetic studies have implicated variants of the human dysbindin-encoding gene, DTNBP1, in the pathogeneses of Hermansky-Pudlak syndrome and schizophrenia. The protein is a stable component of a multisubunit complex termed BLOC-1 (biogenesis of lysosome-related organelles complex-1). In the present study, the significance of the dystrobrevin-dysbindin interaction for BLOC-1 function was examined. Yeast two-hybrid analyses, and binding assays using recombinant proteins, demonstrated direct interaction involving coiled-coil-forming regions in both dysbindin and the dystrobrevins. However, recombinant proteins bearing the coiled-coil-forming regions of the dystrobrevins failed to bind endogenous BLOC-1 from HeLa cells or mouse brain or muscle, under conditions in which they bound the Dp71 isoform of dystrophin. Immunoprecipitation of endogenous dysbindin from brain or muscle resulted in robust co-immunoprecipitation of the pallidin subunit of BLOC-1 but no specific co-immunoprecipitation of dystrobrevin isoforms. Within BLOC-1, dysbindin is engaged in interactions with three other subunits, named pallidin, snapin and muted. We herein provide evidence that the same 69-residue region of dysbindin that is sufficient for dystrobrevin binding in vitro also contains the binding sites for pallidin and snapin, and at least part of the muted-binding interface. Functional, histological and immunohistochemical analyses failed to detect any sign of muscle pathology in BLOC-1-deficient, homozygous pallid mice. Taken together, these results suggest that dysbindin assembled into BLOC-1 is not a physiological binding partner of the dystrobrevins, likely due to engagement of its dystrobrevin-binding region in interactions with other subunits.

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in vitro-translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means (e.g., with and without detergent, using glass beads, etc.). Flow charts and figures give the user a clear-cut explanation of the options for employing the technology.

Late endosomes and lysosomes of mammalian cells in interphase tend to concentrate in the perinuclear region that harbors the microtubule-organizing center. We have previously reported abnormal distribution of these organelles - as judged by reduced percentages of cells displaying pronounced perinuclear accumulation - in mutant fibroblasts lacking BLOC-3 (for ;biogenesis of lysosome-related organelles complex 3'). BLOC-3 is a protein complex that contains the products of the genes mutated in Hermansky-Pudlak syndrome types 1 and 4. Here, we developed a method based on image analysis to estimate the extent of organelle clustering in the perinuclear region of cultured cells. Using this method, we corroborated that the perinuclear clustering of late endocytic organelles containing Lamp1 (for ;lysosome-associated membrane protein 1') is reduced in BLOC-3-deficient murine fibroblasts, and found that it is apparently normal in fibroblasts deficient in BLOC-1 or BLOC-2, which are another two protein complexes associated with Hermansky-Pudlak syndrome. Wild-type and mutant fibroblasts were transfected to express human LAMP1 fused at its cytoplasmic tail to green fluorescence protein (GFP). At low expression levels, LAMP1-GFP was targeted correctly to late endocytic organelles in both wild-type and mutant cells. High levels of LAMP1-GFP overexpression elicited aberrant aggregation of late endocytic organelles, a phenomenon that probably involved formation of anti-parallel dimers of LAMP1-GFP as it was not observed in cells expressing comparable levels of a non-dimerizing mutant variant, LAMP1-mGFP. To test whether BLOC-3 plays a role in the movement of late endocytic organelles, time-lapse fluorescence microscopy experiments were performed using live cells expressing low levels of LAMP1-GFP or LAMP1-mGFP. Although active movement of late endocytic organelles was observed in both wild-type and mutant fibroblasts, quantitative analyses revealed a relatively lower frequency of microtubule-dependent movement events, either towards or away from the perinuclear region, within BLOC-3-deficient cells. By contrast, neither the duration nor the speed of these microtubule-dependent events seemed to be affected by the lack of BLOC-3 function. These results suggest that BLOC-3 function is required, directly or indirectly, for optimal attachment of late endocytic organelles to microtubule-dependent motors.

Hermansky-Pudlak syndrome (HPS) defines a group of at least seven autosomal recessive disorders characterized by albinism and prolonged bleeding. These manifestations arise from defects in the biogenesis of lysosome-related organelles, including melanosomes and platelet dense granules. Most genes associated with HPS in humans and rodent models of the disease encode components of multisubunit protein complexes that are expressed ubiquitously and play roles in intracellular protein trafficking and/or organelle distribution. A small GTPase of the Rab family, Rab38, is also implicated in the pathogenesis of the disease. This article reviews recent progress toward elucidating the cellular functions of these proteins.

Melanocytes and cells of the immune system share an unusual secretory mechanism which uses the lysosome as a regulated secretory organelle. Recently, a number of the proteins required for these 'secretory lysosomes' to undergo exocytosis have been identified. These include Rab27a, Lyst, Rab geranyl geranyl transferase and the adapter protein complex AP-3. Patients lacking any of these proteins are characterized by the rare combination of albinism and immunodeficiency, revealing roles for these proteins in both melanocyte and immune cell secretion. In order to ask how far the link between albinism and immunodeficiency extends we have examined cytotoxic T-lymphocyte (CTL) secretion from two BLOC-3-deficient patients and seven different mouse models of Hermansky-Pudlak syndrome, all of which display defects in pigmentation and platelet function. We find that CTL function is normal in HPS patients and pale-ear mice deficient in BLOC-3, pallid, muted and sandy mice deficient in BLOC-1, ruby-eye mice deficient in BLOC-2 and buff mice deficient in Vps33a. Similarly, the unconventional myosins, Va, VIIa and XV, which can act as effectors for Rab27a in some cell types, are not required in CTL. These results reveal differences in the protein machinery required for biogenesis and/or secretion of lysosome-related organelles in CTL and melanocytes.

Hermansky-Pudlak syndrome defines a group of genetic disorders characterized by defects in organelles of the endosomal-lysosomal system, most notably melanosomes and platelet-dense granules. About a dozen genes have been implicated in the pathogenesis of the disease in humans and mice. Most of these genes encode novel polypeptides that are not conserved in unicellular eukaryotes. Recent studies have revealed that these polypeptides are stable components of at least three distinct, ubiquitously expressed protein complexes, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3. These findings provide a framework for studies on the function of these proteins and the pathogenesis of Hermansky-Pudlak syndrome.

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.

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 beta-hexosaminidase. Our results suggest a common biological basis underlying the pathogenesis of HPS-3, -5 and -6 disease.

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.

Melanosomes are specialized intracellular organelles in which melanin pigments are synthesized and stored. The ontogenesis of these morphologically unique organelles, as well as their relationship to "conventional" organelles of the secretory and endocytic pathways, has for decades been a matter of study - and controversy. Recent work by the groups of Michael Marks and Graça Raposo has uncovered the molecular mechanism that results in the formation of the lumenal striations characteristic of melanosome precursor organelles.

Hermansky-Pudlak syndrome (HPS; MIM 203300) is a genetically heterogeneous disorder characterized by oculocutaneous albinism, prolonged bleeding and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules. In mice, at least 16 loci are associated with HPS, including sandy (sdy; ref. 7). Here we show that the sdy mutant mouse expresses no dysbindin protein owing to a deletion in the gene Dtnbp1 (encoding dysbindin) and that mutation of the human ortholog DTNBP1 causes a novel form of HPS called HPS-7. Dysbindin is a ubiquitously expressed protein that binds to alpha- and beta-dystrobrevins, components of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells. We also show that dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1; refs. 9-11), which regulates trafficking to lysosome-related organelles and includes the proteins pallidin, muted and cappuccino, which are associated with HPS in mice. These findings show that BLOC-1 is important in producing the HPS phenotype in humans, indicate that dysbindin has a role in the biogenesis of lysosome-related organelles and identify unexpected interactions between components of DPC and BLOC-1.

Hermansky-Pudlak syndrome (HPS) defines a group of autosomal recessive disorders characterized by deficiencies in lysosome-related organelles such as melanosomes and platelet-dense granules. Several HPS genes encode proteins of unknown function including HPS1, HPS3, and HPS4. Here we have identified and characterized endogenous HPS3 and HPS4 proteins from HeLa cells. Both proteins were found in soluble and membrane-associated forms. Sedimentation-velocity and coimmunoprecipitation experiments revealed that HPS4 but not HPS3 associates with HPS1 in a complex, which we term biogenesis of lysosome-related organelles complex 3 (BLOC-3). Mutant fibroblasts deficient in either HPS1 or HPS4 displayed abnormal localization of lysosomes and late endosomes, which were less concentrated at the juxtanuclear region in mutant cells than in control fibroblasts. The coat-color phenotype of young homozygous double-mutant mice deficient in subunits of BLOC-3 (HPS1) and BLOC-1 (pallidin) was indistinguishable from that of BLOC-1 single mutants. Taken together, these observations suggest that HPS1 and HPS4 are components of a protein complex that regulates the intracellular localization of lysosomes and late endosomes and may function in a BLOC-1-dependent pathway for melanosome biogenesis.

Recent studies have led to the identification of a group of genes required for normal biogenesis of lysosome-related organelles such as melanosomes and platelet-dense granules. Two of these genes, which are defective in the pallid and muted mutant mouse strains, encode small, coiled-coil-forming proteins that display no homology to each other or to any known protein. We report that these two proteins, pallidin and muted, are components of a novel protein complex. We raised antibodies that allow for detection of pallidin from a wide variety of mammalian cells. Endogenous pallidin was distributed in both soluble and peripheral membrane protein fractions. Size-exclusion chromatography and sedimentation velocity analyses indicated that the bulk of cytosolic pallidin is a component of an asymmetric protein complex with a molecular mass of approximately 200 kDa. We named this complex BLOC-1 (for biogenesis of lysosome-related organelles complex 1). Steady-state pallidin protein levels were reduced in fibroblasts derived from muted and reduced pigmentation mice, suggesting that the genes defective in these two mutant strains could encode components of BLOC-1 that are required for pallidin stability. Co-immunoprecipitation and immunodepletion experiments using an antibody to muted confirmed that this protein is a subunit of BLOC-1. Yeast two-hybrid analyses revealed that pallidin is capable of self-association through a region that contains its two coiled-coil forming domains. Unlike AP-3-deficient pearl fibroblasts, which display defects in intracellular zinc storage, zinc distribution was not noticeably affected in pallid or muted fibroblasts. Interestingly, immunofluorescence and in vitro binding experiments demonstrated that pallidin/BLOC-1 is able to associate with actin filaments. We propose that BLOC-1 mediates the biogenesis of lysosome-related organelles by a mechanism that may involve self-assembly and interaction with the actin cytoskeleton.

Hermansky-Pudlak syndrome (HPS) defines a group of autosomal recessive disorders characterized by defects in lysosome-related organelles such as melanosomes and platelet dense granules. The genes that are defective in each of the different forms of HPS in humans, or in HPS-like disorders in mice, are thought to encode components of a putative molecular machinery required for the formation of specialized organelles of the lysosomal system. This review discusses the biochemical and functional properties of the products of identified HPS genes, which include subunits of the AP-3 complex and the novel proteins HPS1p, HPS3p, HPS4p, pallidin and muted.

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, approximately 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.