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-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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY531265 and AY531266
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
To monitor the enrichment of an additional Biogenesis of lysosome-related organelles complex-1 (BLOC-1) subunit besides Pallidin, we raised a new antibody to the Dysbindin subunit
Summary
DNA Constructs—Yeast two-hybrid plasmids encoding human Pallidin or Muted proteins fused in frame to Gal DNA-binding or activation domains have been described previously [20]. Following extensive washing with Buffer A, bound proteins were eluted in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM dithiothreitol containing the protease inhibitor mixture. The precipitated proteins were collected by centrifugation at 13,000 ϫ g for 30 min, washed twice with (NH4)2SO4 at 40% saturation, and resuspended in Buffer B (300 mM Tris-HCl, pH 7.5, 1 mM EGTA, 0.5 mM MgCl2, 1 mM dithiothreitol) containing the protease inhibitor mixture. Liver cytosol (0.2 ml, ϳ6 mg of total protein) was applied to a Superose 6 column (1 ϫ 60 cm) equilibrated and eluted at 4 °C with Buffer B with protease inhibitors at a flow rate of 0.4 ml/min. The signal obtained for BLOS3 was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (as a housekeeping control)
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