Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function.Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1-deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS. INTRODUCTIONHermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by hypopigmentation, prolonged bleeding, and sometimes ceroid accumulation, lung fibrosis, and/or immune defects leading to premature death Wei, 2006). HPS or a similar disorder in mice results from mutations in any of at least 15 genes (Wei, 2006). All of these genes are ubiquitously expressed, but their mutation in HPS affects mainly the generation and function of selected tissuespecific lysosome-related organelles (LROs;Bonifacino, 2004;Di Pietro and Dell'Angelica, 2005). 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...

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

SUMMARYCopper is a cofactor for many cellular enzymes and transporters 1 . To load onto secreted and endomembrane cuproproteins, copper is translocated from the cytosol into membrane-bound organelles by ATP7A or ATP7B transporters, the genes for which are mutated in the copper imbalance syndromes, Menkes and Wilson disease, respectively 2 . Endomembrane cuproproteins are thought to stably incorporate copper upon transit through the trans Golgi network (TGN), within which ATP7A3 accumulates by dynamic cycling through early endocytic compartments4. Here we show that the pigment cell-specific cuproenzyme tyrosinase acquires copper only transiently and inefficiently within the TGN of melanocytes. To catalyze melanin synthesis, tyrosinase is subsequently reloaded with copper within specialized organelles called melanosomes. Copper is supplied to melanosomes by ATP7A, a cohort of which localizes to melanosomes in a Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1)-dependent manner. These results indicate that cell type-specific localization of a metal transporter is required to sustain metallation of an endomembrane cuproenzyme, providing a mechanism for exquisite spatial control of metalloenzyme activity. Moreover, as BLOC-1 subunits are mutated in subtypes of the genetic disease, HermanskyPudlak syndrome (HPS), these results also show that defects in copper transporter localization contribute to hypopigmentation, and hence perhaps other systemic defects, in HPS.Copper is essential for all cells, but is particularly important for vertebrate pigmentation as a cofactor for tyrosinase 5 . Tyrosinase is expressed in epidermal melanocytes and ocular pigment cells, and catalyzes the initial steps of melanin biosynthesis within melanosomes 6 . Mutations in either of two copper binding sites in tyrosinase ablate enzymatic activity and result in