In Hermansky-Pudlak syndrome (HPS), defects in the lysosome-related organelles (melanosomes, platelet dense bodies, and lysosomes) result in albinism, prolonged bleeding, and lysosomal ceroid pigment deposition. HPS type 1 is caused by defects in the HPS1 gene, which encodes a protein of unknown function. HPS type 2 is caused by defects in the beta3A subunit of the AP-3 complex, which functions in endosomal-lysosomal protein trafficking. Significantly, 30-50 percent of HPS patients have no HPS1 or AP-3 defects. Fifteen mouse models of HPS mapping to distinct chromosomal loci have been described, underscoring its marked genetic heterogeneity. All show striking clinical correlations with human HPS. Pale ear (ep) encodes mouse HPS1. Defects in the AP-3 beta3A and delta subunits cause the pearl (pe) and mocha (mh) mutations, respectively. Ashen (ash) and pallid (pa) result from defects in vesicle fusion and docking proteins. Clearly, cloning the remaining mouse mutations will provide additional candidate genes for human HPS, elucidate mechanisms of organelle biogenesis and protein and vesicle trafficking, and identify novel pathways common to the biogenesis of melanosomes, dense bodies, and lysosomes. Here, we will: 1. Identify the gene defects in two mouse models of HPS, reduced pigmentation (rp) and cappuccino (cno); characterize the encoded proteins; identify binding ligands for each; and test for defects in human HPS cell panels. 2. Knockout the TRAPP protein-related R26W gene. A viral insertion at the R26W locus in mice results in clonal inactivation of the gene in a subset melanocytes, producing patchy pigmentation defects. Notably, the R26W phenotype is exacerbated when combined with the HPS mutation rp (R26W /+, rp/+). We will generate a true R26W null mutation, test homozygotes for the HPS phenotype, test for direct interactions between the R26W and rp gene products, identify additional binding partners, and test human HPS cell panels for R26W defects. 3. Generate a conditional knockout of ankyrin (ANK3), a major membrane skeleton protein present in the lysosome-related organelles. Specifically, we test the hypotheses that (a) defects in rp and cno cause HPS in humans; (b) R26W is an HPS candidate gene that interacts with rp during organelle biogenesis; and (c) Ank3 is an HPS candidate gene required for organelle structural integrity and function.
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