Biogenesis of lysosome-related organelles complex 3 (BLOC-3): a complex containing the Hermansky-Pudlak syndrome (HPS) proteins HPS1 and HPS4

Abstract

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.

Fig. 1.

(A and B) Identification of endogenous HPS4 (A) and HPS3 (B) proteins from HeLa cells by immunoprecipitation recapture. Cells metabolically labeled with [³⁵S]Met and [³⁵S]Cys were lysed in the presence of 1% (wt/vol) Triton X-100 and immunoprecipitated with control rabbit IgG, rabbit antibody HP3c to HPS3, or rabbit antibodies HP4b and HP4c to HPS4. After extensive washing, each first immunoprecipitate (1^st IP) was denatured by heating in the presence of SDS and DTT, diluted, and immunoprecipitated again (2^nd IP) by using the indicated antibodies. Washed immunoprecipitates were analyzed by 4–20% SDS/PAGE followed by fluorography. (C) Cytosolic and microsomal membrane fractions from metabolically labeled HeLa cells were prepared by differential centrifugation and subjected to immunoprecipitation recapture by using HP3c antibody to HPS3 and HP4c antibody to HPS4.

Fig. 2.

Sedimentation-velocity analysis of HPS proteins. Cytosol from metabolically labeled HeLa cells was fractionated by centrifugation on a linear 5–20% (wt/vol) sucrose gradient. Each fraction was subjected to immunoprecipitation recapture by using antibody HP3c to detect HPS3, antibody HP4c to detect HPS4, or a combination of HP4c antibody in the first immunoprecipitation (1^st IP) and anti-HPS1 in the second immunoprecipitation (2^nd IP) to detect HPS1 protein associated to HPS4. Not shown are fractions 1, 16, 18, and 20, which contained none of these proteins. The positions in the gradient of standard proteins of known sedimentation coefficient (in Svedberg units) are indicated at the top.

Fig. 3.

Coimmunoprecipitation of HPS1 and HPS4. HeLa cells metabolically labeled with [³⁵S]Met and [³⁵S]Cys were lysed under nondenaturing conditions. The cleared lysate was subjected to a first immunoprecipitation (1^st IP) by using control rabbit IgG or purified rabbit antibodies to HPS4 (either HP4b or HP4c antibodies), HPS1, or HPS3. After extensive washing, the immunoprecipitates were treated by heating in the presence of SDS and DTT, diluted, and subjected to a second immunoprecipitation (2^nd IP) by using the indicated antibodies. Final immunoprecipitates were analyzed by SDS/PAGE and fluorography.

Fig. 4.

Intracellular distribution of markers of late endosomes and lysosomes in fibroblasts from wild-type and mutant mice. Skin fibroblasts from wild-type C57BL-6J (A, C, and E) and HPS1-deficient pale-ear (B, D, and F–H) mice were grown on glass coverslips. Cells were stained by using mAb 1D4B to Lamp-1 (A and B) or mAb 6C4 to LBPA (E and F), loaded with Texas red-conjugated dextran (C and D) or transiently transfected with plasmid encoding human HPS1 and subsequently stained with rabbit antibodies to HPS1 (G) together with mAb 1D4B to Lamp-1 (H). The perimeters of selected cells are highlighted by white lines that were drawn based on an overexposed version of the same image. (Bars, 20 μm.)

Fig. 5.

(Upper) Coat-color phenotype of 2-week-old mice carrying the pale-ear and/or pallid mutations as compared with control wild-type mice of the same age. Homozygous pale-ear mice (ep/ep) display a characteristic reduction in pigmentation in the tail and ears, whereas homozygous pallid mice (pa/pa) display a generalized pigment dilution. (Lower) Result of RT-PCR analysis to verify the presence of wild-type (normal) and mutant (ep) Hps1 alleles in two mice known to be homozygous for the pallid mutation (as inferred by DNA sequencing).