Lysosome-related organelles (LROs) are cell type-specific subcellular compartments that derive from the endosomal system but that serve unique and vital physiologic functions. They include melanosomes in which melanins are synthesized in skin melanocytes and in developing retinal pigment epithelial cells. The specific functions of LROs are conferred by cell type-specific resident transmembrane proteins, such as melanogenic enzymes and transporters, that are specifically targeted to newly forming LROs. Mutations in genes encoding the cellular machinery responsible for this intracellular targeting underlie the Hermansky-Pudlak syndromes (HPS), a group of genetic disorders in which defective LRO formation leads to several symptoms including oculocutaneous albinism with its associated defective vision and skin and eye hypopigmentation. The known isoforms of HPS result from mutations in genes that encode subunits of four obligate multisubunit complexes – AP-3 and BLOC-1, -2, and -3 – that facilitate protein sorting and membrane remodeling to promote cargo delivery from early endosomal intermediates to maturing melanosomes. We have shown that BLOC-1, together with AP-3, the microtubule motor KIF13A, and additional partners, functions to generate recycling endosome-like tubular transport carriers from early endosomes that extend along microtubules and transiently fuse with maturing melanosomes to deliver their cargoes. BLOC-2 and the lipid phosphatidylinositol-4- phosphate (PtdIns4P) function downstream of BLOC-1 to target the tubules to maturing melanosomes, but the molecular function of BLOC-2 is not known. Moreover, the spatiotemporal relationship between each of these complexes and PtdIns4P during tubule formation, extension, and targeting is not understood. Finally, it is not known how the ubiquitous AP-3 and BLOCs are adapted in specific cell types to target the tubules to LROs instead of for recycling. This competing renewal proposal seeks to fill these knowledge gaps using a combination of live cell imaging, biochemical and electron microscopy approaches, together with genetic manipulation in cultured melanocytes. If successful, our aims will establish new paradigms of tubular transport from endosomes to melanosomes that will extend to additional LROs and to ubiquitous endolysosomal compartments, identify potential targets of mutations in HPS patients who currently lack a molecular diagnosis, and provide potential therapeutic insights for the more debilitating HPS symptoms. The aims are: 1. Test whether BLOC-1 and its interacting partners associate with membrane tubules during their formation, extension, and/or tethering using live cell imaging coupled with electron microscopy analyses; 2. Test whether BLOC-2 and PtdIns4P regulate the dynamic association of tubular membrane transport carriers with microtubules using live cell imaging, biochemical, and unbiased proteomic approaches; 3. Test whether RAB30 confers cell-type specificity in BLOC-1-de...