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)

Abstract

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-coilforming 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.

Snapin promotes HIV ‐1 transmission from dendritic cells by dampening TLR 8 signaling

“…The BLOC‐1 complex belongs to the endosomal protein sorting machinery and was first identified through its association with Hermansky–Pudlak syndrome (HPS), a group of heterogeneous autosomal‐recessive disorders caused by mutations in genes affecting the biogenesis of lysosomal‐related organelles (LROs). BLOC‐1 is an octameric complex containing two stable sub‐complexes, pallidin‐Cappuccino‐BLOS1 and dysbindin‐Snapin‐BLOS2 (Starcevic & Dell'Angelica, 2004). We examined the effect of BLOC‐1 subunits on HIV‐1 trans ‐infection and found that knockdown of four members, specifically muted, dysbindin, BLOS3, and the dynein motor protein Snapin had a significant effect on HIV‐1 trans ‐infection (Fig 3A).…”

BLOC-1 Is Required for Cargo-specific Sorting from Vacuolar Early Endosomes toward Lysosome-related Organelles

“…Tyrp1 was enriched at the surface of BLOC-1-deficient melanocytes relative to controls, as shown by IFM analysis of primary melanocytes labeled for Tyrp1 before fixation (Figure 6a) and quantitated by flow cytometry of melan-mu and melan-rp cells relative to BLOC-1-reconstituted cells ( Figure 6b; the more modest Tyrp1 surface expression in melan-rp relative to melan-mu likely reflects residual BLOC-1 activity in rp cells; Starcevic and Dell'Angelica, 2004; our unpublished data). Nevertheless, BLOC-1-deficient cells had lower total cellular Tyrp1 levels (ϳ60% that of BLOC-1-reconstituted cells; Figure 6c), indicating that the increased surface levels reflect Tyrp1 missorting and not increased expression; the decreased Tyrp1 cellular levels in these immortalized BLOC-1-deficient melanocytes may reflect dysregulated expression, because Tyrp1 half-life, measured by metabolic pulse/chase analysis, was unchanged compared with controls (our unpublished data).…”

“…The genes disrupted in human HPS-7 (Li et al, 2003) and HPS-8 (Morgan et al, 2006) and in the mouse HPS models pallid, muted, reduced pigmentation (rp), cappuccino, and sandy encode five of the eight known subunits of a stable protein complex known as biogenesis of lysosome-related organelles complex (BLOC)-1 (Falcon-Perez et al, 2002;Moriyama and Bonifacino, 2002;Ciciotte et al, 2003;Li et al, 2003;Gwynn et al, 2004;Starcevic and Dell'Angelica, 2004). To date, no specific subcellular function has been assigned to BLOC-1.…”

“…Those LROs that are most severely affected in all forms of HPS-pigment cell melanosomes, platelet dense granules, and lung lamellar bodies-are unique in that they coexist with bona fide lysosomes in their respective cell types (Dell'Angelica et al, 2000;Marks and Seabra, 2001). The 15 known HPS-associated genes have been identified, and although the products of most are thought to participate in trafficking events that are uniquely required to form this class of LRO, the function of only a few is understood in detail.The genes disrupted in human HPS-7 (Li et al, 2003) and HPS-8 (Morgan et al, 2006) and in the mouse HPS models pallid, muted, reduced pigmentation (rp), cappuccino, and sandy encode five of the eight known subunits of a stable protein complex known as biogenesis of lysosome-related organelles complex (BLOC)-1 (Falcon-Perez et al, 2002;Moriyama and Bonifacino, 2002;Ciciotte et al, 2003;Li et al, 2003;Gwynn et al, 2004;Starcevic and Dell'Angelica, 2004). To date, no specific subcellular function has been assigned to BLOC-1.…”

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

“…The GST-OCA2 fusion constructs were expressed in Escherichia coli and purified as described previously (Starcevic and Dell'Angelica, 2004). Detergent extracts of MNT-1 or HeLa cells were prepared in MNT-1 buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 1 MgCl 2 , 1 mM NaF, and 0.5% NP-40) or HeLa buffer (25 mM HEPES, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1 mM NaF, 0.5 mM MgCl 2 , and 0.5% Triton X-100), respectively, containing protease inhibitor mixture [1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 10 g/ml leupeptin, 5 g/ml aprotinin, and 1 g/l pepstatin A].…”