Mistargeting of peroxisomal EHHADH and inherited renal Fanconi's syndrome.

BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).

Dysregulation of galectin-3. Implications for Hermansky-Pudlak syndrome pulmonary fibrosis.

The etiology of Hermansky-Pudlak syndrome (HPS) pulmonary fibrosis (HPSPF), a progressive interstitial lung disease with high mortality, is unknown. Galectin-3 is a ß-galactoside-binding lectin with profibrotic effects. The objective of this study was to investigate the involvement of galectin-3 in HPSPF. Galectin-3 was measured by ELISA, immunohistochemistry, and immunoblotting in human specimens from subjects with HPS and control subjects. Mechanisms of galectin-3 accumulation were studied by quantitative RT-PCR, Northern blot analysis, membrane biotinylation assays, and rescue of HPS1-deficient cells by transfection. Bronchoalveolar lavage galectin-3 concentrations were significantly higher in HPSPF compared with idiopathic pulmonary fibrosis or that from normal volunteers, and correlated with disease severity. Galectin-3 immunostaining was increased in HPSPF compared with idiopathic pulmonary fibrosis or normal lung tissue. Fibroblasts from subjects with HPS subtypes associated with pulmonary fibrosis had increased galectin-3 protein expression compared with cells from nonfibrotic HPS subtypes. Galectin-3 protein accumulation was associated with reduced Galectin-3 mRNA, normal Mucin 1 levels, and up-regulated microRNA-322 in HPSPF cells. Membrane biotinylation assays showed reduced galectin-3 and normal Mucin 1 expression at the plasma membrane in HPSPF cells compared with control cells, which suggests that galectin-3 is mistrafficked in these cells. Reconstitution of HPS1 cDNA into HPS1-deficient cells normalized galectin-3 protein and mRNA levels, as well as corrected galectin-3 trafficking to the membrane. Intracellular galectin-3 levels are regulated by HPS1 protein. Abnormal accumulation of galectin-3 may contribute to the pathogenesis of HPSPF.

Mutations in SLC6A19, encoding B0AT1, cause Hartnup disorder.

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B(0)AT1 (ref. 4). We isolated the human homolog of B(0)AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.

Alveolar macrophage dysregulation in Hermansky-Pudlak syndrome type 1.

RATIONALE: Individuals with Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles, develop an accelerated form of progressive fibrotic lung disease. The etiology of pulmonary fibrosis associated with HPS-1 is unknown. OBJECTIVES: To investigate the potential pathogenesis of pulmonary fibrosis in HPS-1, lung cells and proteins from individuals with HPS-1 were studied. METHODS: Forty-one subjects with HPS-1 with and without pulmonary fibrosis were evaluated with pulmonary function tests, high-resolution computed tomography scan, and bronchoscopy. Bronchoalveolar lavage cells and analytes were analyzed. MEASUREMENTS AND MAIN RESULTS: Concentrations of total bronchoalveolar lavage cells and alveolar macrophages were significantly higher in epithelial lining fluid from subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers. Concentrations of cytokines and chemokines (i.e., monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and granulocyte-macrophage colony-stimulating factor) in alveolar epithelial lining fluid were significantly higher in subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers (P < 0.001). In vitro, HPS-1 pulmonary fibrosis alveolar macrophages, which did not express HPS1 mRNA, secreted significantly higher concentrations of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and regulated upon activation, normal T cell expressed and secreted (RANTES) protein compared with normal cells (P = 0.001, P = 0.014, and P = 0.011, respectively). Pirfenidone suppressed HPS-1 alveolar macrophage cytokine and chemokine secretion in vitro in a dose-dependent manner. CONCLUSIONS: In HPS-1, alveolar inflammation predominantly involves macrophages and is associated with high lung concentrations of cytokines and chemokines. HPS-1 alveolar macrophages provide a model system in which to study the pathogenesis and treatment of HPS pulmonary fibrosis.

Identifying putative promoter regions of Hermansky-Pudlak syndrome genes by means of phylogenetic footprinting.

HPS is an autosomal recessive disorder characterized by oculocutaneous albinism and prolonged bleeding. Eight human genes are described resulting in the HPS subtypes 1-8. Certain HPS proteins combine to form Biogenesis of Lysosome-related Organelles Complexes (BLOCs), thought to function in the formation of intracellular vesicles such as melanosomes, platelet dense bodies, and lytic granules. Specifically, BLOC-2 contains the HPS3, HPS5 and HPS6 proteins. We used phylogenetic footprinting to identify conserved regions in the upstream sequences of HPS3, HPS5 and HPS6. These conserved regions were verified to have in vitro transcription activation activity using luciferase reporter assays. Transcription factor binding site analyses of the regions identified 52 putative sites shared by all three genes. When analysis was limited to the conserved footprints, seven binding sites were found shared among all three genes: Pax-5, AIRE, CACD, ZF5, Zic1, E2F and Churchill. The HPS3 conserved upstream region was sequenced in four patients with decreased fibroblast HPS3 RNA levels and only one HPS3 mutation in the coding exons and surrounding exon/intron boundaries; no mutation was found. These findings illustrate the power of phylogenetic footprinting for identifying potential regulatory regions in non-coding sequences and define the first putative promoter elements for any HPS genes.

An immunoblotting assay to facilitate the molecular diagnosis of Hermansky-Pudlak syndrome.

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.

A novel missense mutation (G43S) in the switch I region of Rab27A causing Griscelli syndrome.

The autosomal recessive Griscelli syndrome type II (GSII) is caused by mutations in the RAB27A gene. Typical clinical features include immunological impairment, silver-gray scalp hair, eyelashes and eyebrows and hypomelanosis of the skin. Rabs help determine the specificity of membrane trafficking steps within cells. In melanocytes, the GTP-bound form of Rab27A associates with the membranes of mature fully-pigmented melanosomes through its geranylgeranyl group. Once attached, Rab27A recruits the downstream effector Melanophilin (Mlph) and the actin-dependent motor protein Myosin Va (MyoVa). The molecular Rab27A/Mlph/MyoVA tripartite complex, which links melanosomes to the peripheral actin network, is required to achieve melanosome transfer to surrounding keratinocytes in the epidermis. Here we report a novel homozygous missense mutation c.127G>A, p.G43S in exon 2 of the RAB27A gene of an Afghani GSII patient. Laser scanning confocal microscopy showed that the G43S mutation, which is located in the highly conserved switch I region of Rab27A, induces perinuclear localization of melanosomes in normal melanocytes, and fails to restore melanosomes to the actin-rich periphery in GSII melanocytes. Co-immunoprecipitation studies showed that Rab27A(G43S) fails to interact with its effector Melanophilin, indicating that the switch I region functions in the recruitment of Rab effector proteins.

A new genetic isolate with a unique phenotype of syndromic oculocutaneous albinism: clinical, molecular, and cellular characteristics.

An extended, highly consanguineous Israeli Bedouin family with at least 20 individuals exhibiting a unique phenotype of oculocutaneous albinism (OCA) was identified. All known OCA genes were excluded in this family. Electron microscopic analysis of platelets revealed absence of dense bodies, suggesting a diagnosis of Hermansky-Pudlak syndrome (HPS). HPS is a rare autosomal recessive disorder of lysosome-related organelle biogenesis, clinically characterized by OCA and platelet dysfunction, sometimes accompanied by other systemic pathologies. All human HPS genes (HPS1-8) and five genes corresponding to murine HPS models were evaluated. Haplotype analysis and homozygosity mapping of the HPS loci revealed linkage to chromosome 10 in the studied family. Subsequently, a novel insertion mutation, c.1066-1067insG was identified in HPS6. Most frameshift mutations generating premature termination codon cause mRNA nonsense mediated decay (NMD), while intronless genes like HPS6 are usually not monitored by NMD. Expression analysis revealed no mRNA decay in patient's fibroblasts, hence truncated protein is most probably produced. Confocal microscopy revealed abnormal distribution of LAMP-3 (lysosomal associated membrane protein-3) in fibroblasts from the patients, indicating abnormal trafficking of lysosomal lineage organelles. So far, a single HPS-6 patient phenotypically similar to HPS-3 and HPS-5 has been identified. The HPS-6 phenotype in the studied family is unique since it resembles OCA and not HPS. Therefore, our finding broadens the phenotypic definition of HPS. Two major genetic isolates of HPS-1 and HPS-3 patients were previously diagnosed in Puerto Rico. The extended Bedouin family is the largest isolate of non-Puerto Rican HPS patients.

Association of the Hermansky-Pudlak syndrome type-3 protein with clathrin.

BACKGROUND: Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172-176. RESULTS: Clathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20 degrees C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution. CONCLUSION: HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking.

Disorders of lysosome-related organelle biogenesis: clinical and molecular genetics.

Lysosome-related organelles (LROs) are a heterogeneous group of vesicles that share various features with lysosomes, but are distinct in function, morphology, and composition. The biogenesis of LROs employs a common machinery, and genetic defects in this machinery can affect all LROs or only an individual LRO, resulting in a variety of clinical features. In this review, we discuss the main components of LRO biogenesis. We also summarize the function, composition, and resident cell types of the major LROs. Finally, we describe the clinical characteristics of the major human LRO disorders.

Platelet alpha granules in BLOC-2 and BLOC-3 subtypes of Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis that displays genetic locus heterogeneity. The eight known HPS proteins combine in functional complexes, two of which are called BLOC-2 and BLOC-3; a BLOC is a Biogenesis of Lysosome-related Organelles Complex. Organelles affected in HPS include the melanosome, resulting in hypopigmentation, and the platelet delta (dense) granule, resulting in prolonged bleeding times. Whole mount electron microscopy (EM) detects the absence of platelet delta granules and confirms the diagnosis of HPS. To date, the status of other organelles and granules in HPS platelets has not been documented. We performed ultrastructural studies on platelets of patients with different genetic forms of HPS, specifically those comprising the BLOC-2 and BLOC-3 subtypes. No differences in distribution, size or quantity of other platelet organelles and membrane structures could be detected in our patients. Since alpha and delta granules are formed from multivesicular bodies in the megakaryocyte, and since only delta granules are defective in HPS, we conclude that HPS genes function within the portion of delta granule biogenesis that has diverged from that of alpha granules. Thus, it is unlikely that the generalized bleeding diathesis of HPS is attributed to a deficiency of alpha granules.

Improper trafficking of melanocyte-specific proteins in Hermansky-Pudlak syndrome type-5.

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis resulting in melanosome dysfunction and absent platelet dense bodies. HPS patients have oculocutaneous albinism, bruising, and bleeding. HPS-5 results from deficiency of the HPS5 protein, a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2). HPS5 has an unknown function and lacks homology to known proteins. We performed ultrastructural studies of HPS-5 melanocytes revealing predominantly early-stage melanosomes with many small 3,4(OH)2-phenylalanine-positive vesicles throughout the cell body and dendrites. These findings resemble the distinct ultrastructural features of HPS-3 melanocytes; HPS3 is also a BLOC-2 component. Immunofluorescence and immunoEM studies showed decreased TYRP1 labeling in the dendrites of HPS-5 melanocytes, and the overall abundance of TYRP1 was reduced. No substantial differences were observed in the distribution or abundance of Pmel17 in HPS-5 melanocytes. In normal melanocytes, endogenous tyrosinase colocalized with Pmel17 and TYRP1 in the perinuclear area and dendritic tips; this was much reduced in HPS-5 melanocytes, particularly in the tips. We conclude that early stage melanosome formation and Pmel17 trafficking are preserved in HPS5-deficient cells. Tyrosinase and TYRP1 are mistrafficked, however, and fail to be efficiently delivered to melanosomes of HPS-5 melanocytes.

Melanocyte-specific proteins are aberrantly trafficked in melanocytes of Hermansky-Pudlak syndrome-type 3.

Hermansky-Pudlak Syndrome-type 3 (HPS-3) is a relatively mild subtype of HPS with minimal cutaneous and ocular depigmentation. The HPS-3 gene encodes a novel protein of unknown function with a predicted molecular weight of 114 kd. To assess the role of the HPS3 protein in melanization, cultured melanocytes developed from HPS-3 patients were evaluated biochemically and histologically for activity and localization of melanocyte-specific proteins. Endogenous tyrosinase activity of HPS-3 melanocytes was substantial, but tyrosinase activity and melanin synthesis was suppressed in intact melanocytes. However, the level of suppression, as well as extent to which up-regulation by isobutylmethylxanthine and cholera toxin was muted, was less that in HPS-1 melanocytes. Ultrastructurally, HPS-3 melanocytes contained morphologically normal melanosomes, predominantly of stage I and II with minimal stage III and few stage IV melanosomes. Dihydroxyphenylalanine (DOPA) histochemistry demonstrated an increase in melanization of melanosomes. Unique to HPS-3 melanocytes were numerous DOPA-positive 50-nm vesicles and tubular elements present throughout the cell body and dendrites. Tyrosinase, tyrosinase-related protein-1 (Tyrp1), dopachrome tautomerase (Dct), and LAMP1 and 3 localization in HPS-3 melanocytes, as evaluated by immunocytochemistry and confocal microscopy, demonstrated a fine, floccular distribution in contrast to the coarse, granular distribution characteristic of control melanocytes. The localization profile of other proteins expressed by melanocytes (ie, Silver/Pmel17, Melan-A/MART-1, LAMP2, Rab 27, transferrin, c-kit, adaptin-3, and the HPS1 protein) appeared normal. These results suggest that a specific subset of melanocyte proteins are aberrantly trafficked throughout the HPS-3 melanocyte and may be responsible for the reduction in melanin synthesis.

Sucrose-induced vacuolation results in increased expression of cholesterol biosynthesis and lysosomal genes.

Mammalian cells cultured in the presence of high concentrations of sucrose demonstrate large, phase-lucent, osmotically swollen vacuoles. Three normal human fibroblast cell lines exposed to 100 mM of sucrose for 24 h demonstrated increased expression of lysosomal, intracellular vesicle trafficking, cholesterol biosynthesis, and fatty acid metabolism genes. Most steps of the cholesterol biosynthesis pathway were upregulated including HMG CoA reductase, which catalyzes the rate-limiting step of cholesterol biosynthesis. The lysosomal genes neuraminidase, CLN3, and CLCN5 and the small GTP-binding proteins Rab7L1 and Arl7 were also increased. A Rab7L1-GFP fusion protein was overexpressed in human fibroblasts and was demonstrated to localize primarily to the Golgi apparatus, and in some cells to the membranes bounding vesicles in the perinuclear region. Increased levels of the transcription factor C/EBP were found in nuclear extracts from cells exposed to sucrose for 12 h, relative to matched controls suggesting regulation of gene expression following sucrose-induced vacuolation may be coordinated, at least in part, by the transcription factor C/EBP. Sucrose-induced vacuolation is a useful model in which to study the regulation of lysosomal gene expression and biogenesis.

Lysosomal cystine storage augments apoptosis in cultured human fibroblasts and renal tubular epithelial cells.

Nephropathic cystinosis is a lethal disorder of lysosomal cystine storage due to defective lysosomal cystine transport. How lysosomal cystine causes this multisystemic disorder culminating in end-stage renal disease is not known, because the cystine is isolated from cellular metabolism by the lysosomal membrane. It is here reported that in both normal and nephropathic cystinotic fibroblasts and cultured renal proximal tubule epithelial cells, increased lysosomal cystine causes an increased rate of apoptosis. In nephropathic cystinotic fibroblasts, the rate of apoptosis is 14.8% after exposure to TNF-alpha versus 7.8% in control normal fibroblasts. Anti-Fas antibodies and UV exposure induced apoptosis in 18.1% and 17.4% of nephropathic cystinotic fibroblasts, respectively, versus 5.2% and 7.1% in normal fibroblasts when analyzed by CaspACE (P < 0.05). Similar results were found when the cells were analyzed by TdT-mediated dUTP nick end labeling (TUNEL). When the cystine content of normal fibroblasts is increased by exposure to cystine dimethylester (CDME), the apoptotic rate is increased to the rate seen in nephropathic cystinotic cells. Decreasing the cystinotic cells' cystine content by use of cysteamine results in normalization of the apoptotic rate. Renal proximal tubule epithelial (RPTE) cells are much more sensitive to CDME than fibroblasts, reaching 43.8% apoptosis 6 h after exposure to CDME alone, compared with 38.2% when exposed to TNF-alpha alone. Serum withdrawal causes an apoptotic rate of 8.7% in nephropathic cystinotic fibroblasts, compared with 6.1% in normal fibroblasts. That rate increases to 37.3% in normal fibroblasts after CDME exposure. Fibroblasts from two cystinotic variants, benign ocular and intermediate cystinosis, do not display increased apoptosis with increased lysosomal cystine. It is concluded that enhanced apoptosis resulting from lysosomal cystine storage may lead to inappropriate cell death and decreased cell numbers in many tissues and hence contribute to the nephropathic cystinotic phenotype. The variant forms may represent co-segregation or linkage of rare alleles that confer resistance to apoptosis, moderating the cell loss and causing the milder disease expression.

Cellular, molecular and clinical characterization of patients with Hermansky-Pudlak syndrome type 5.

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelles such as melanosomes and platelet dense granules. Seven genes are now associated with HPS in humans. An accurate diagnosis of each HPS subtype has important prognostic and treatment implications. Here we describe the cellular, molecular, and clinical aspects of the recently identified HPS-5 subtype. We first analyzed the genomic organization and the RNA expression pattern of HPS5, located on chromosome 11p14, and demonstrated tissue-specific expression of at least three alternatively spliced HPS5 mRNA transcripts, coding for HPS5A and HPS5B proteins, that differ at their 5'-ends. Genetic screening of 15 unassigned HPS patients yielded six new HPS5 mutations in four patients. Clinically, our HPS-5 patients exhibited iris transillumination, variable hair and skin pigmentation, and absent platelet dense bodies, but not pulmonary fibrosis or granulomatous colitis. In two patients with homozygous missense mutations, hemizygosity was ruled out by gene-dosage multiplex polymerase chain reaction, and immunocytochemical analyses of their fibroblasts supported the HPS-5 diagnosis. Specifically, LAMP-3 distribution was restricted to the perinuclear region in HPS-5 fibroblasts, in contrast to the normal LAMP-3 distribution, which extended to the periphery. This specific intracellular vesicle distribution in fibroblasts, in combination with the clinical features, will improve the characterization of the HPS-5 subtype.

Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex.

Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.