The Hermansky-Pudlak syndromes (HPS) are heritable diseases characterized by oculocutaneous albinism (with associated visual impairment and susceptibility to skin and ocular cancers), excessive bleeding and other variable symptoms. The symptoms reflect the malformation of cell type-specific lysosome-related organelles (LROs) such as melanosomes, the organelles of skin and ocular pigment cells within which melanin pigments are synthesized. The genes that are defective in HPS and its mouse models encode subunits of five protein complexes ? Biogenesis of Lysosome-related Organelles Complex (BLOC)-1, -2 and -3, AP-3 and HOPS ? that regulate membrane trafficking during LRO maturation. This proposal seeks to define how these complexes cooperate to regulate transport to and from melanosomes of fusion proteins, or SNAREs, and thereby define the essential roles of these proteins in fusion events during the biogenesis of melanosomes and other LROs. Most newly synthesized melanogenic membrane proteins are delivered to maturing melanosomes via tubular transport carriers that form from early endosomes in a BLOC-1-dependent manner and that are then targeted to maturing melanosomes by BLOC-2. These transport carriers harbor the SNARE subunit syntaxin 13 (STX13), but STX13 is not incorporated into the melanosome membrane and thus does not likely drive tubule fusion with melanosomes. Rather, the vesicular SNARE VAMP7 drives fusion, but how either SNARE is sorted into the transport carriers and how STX13 and VAMP7 are segregated during fusion with melanosomes is not known. We hypothesize that: (1) cis-SNARE complexes containing VAMP7 and STX13 are sorted from early endosomes into the tubules via partially redundant interactions with BLOC-1 and AP-3; (2) this complex is dissembled at the maturing melanosome; and (3) HOPS stabilizes a complex containing VAMP7 and a distinct syntaxin, permitting transient fusion of the tubules with melanosomes and allowing inactive STX13 to return to endosomes upon tubule retraction. After the carriers fuse, VAMP7 ? now on melanosomes ? must be returned to its endosomal site of origin to participate in new rounds of forward transport. Based on preliminary data, we hypothesize that (4) VAMP7 is recycled from melanosomes in distinct tubular structures via BLOC-3- dependent recruitment of cytoskeletal motors, including Myosin VI. Our hypotheses create a unifying model for the involvement of all HPS protein complexes in SNARE cycling between endosomes and melanosomes that should apply to all LRO-containing cell types, and might explain how promiscuous endosomal SNARE interactions are regulated to promote directional membrane transport among distinct endosomal compartments in all vertebrate cells.
Our Specific Aims are: 1. to test whether AP-3 and BLOC-1 cooperate in SNARE complex sorting into melanosome-bound tubules; 2. to test whether HOPS facilitates SNARE formation during BLOC-1-dependent melanosome maturation; and 3. to test whether BLOC-3 activation of RAB proteins activates Myosin VI-dependent recycling of VAMP7 from melanosomes.
Hermansky-Pudlak syndrome is a group of genetic disorders that are particularly common in Puerto Rico and are characterized by oculocutaneous albinism, excessive bleeding, and in many cases a fatal lung fibrosis and/or immune deficiency. The symptoms result from the failure to form certain specialized membrane structures, or organelles, within affected cell types. This proposal uses the melanosome, a target organelle within pigment cells, as a model system to understand how the products of the genes that are disrupted in Hermansky-Pudlak syndrome patients normally function together to generate specialized organelles.
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