Structural basis of Vps33A recruitment to the human HOPS complex by Vps16

The multisubunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for late endosome-lysosome and autophagosome-lysosome fusion in mammals. We have determined the crystal structure of the human HOPS subunit Vps33A, confirming its identity as a Sec1/Munc18 family member. We show that HOPS subunit Vps16 recruits Vps33A to the human HOPS complex and that residues 642–736 are necessary and sufficient for this interaction, and we present the crystal structure of Vps33A in complex with Vps16(642–736). Mutations at the binding interface disrupt the Vps33A–Vps16 interaction both in vitro and in cells, preventing recruitment of Vps33A to the HOPS complex. The Vps33A–Vps16 complex provides a structural framework for studying the association between Sec1/Munc18 proteins and tethering complexes.Eukaryotic cells tightly regulate the movement of macromolecules between their membrane-bound compartments. Multiple proteins and protein complexes interact to identify vesicles or organelles destined to fuse, bring them into close proximity, and then fuse their membranes, thereby allowing their contents to mix (1). Multisubunit tethering complexes modulate key steps in these fusion events by recognizing specific Rab-family small GTPases on the membrane surfaces, physically docking the membranes and then recruiting the machinery that effects the membrane fusion (2, 3).In metazoans, the multisubunit tethering complex homologous to the yeast homotypic fusion and vacuole protein sorting (HOPS) complex (4–7) is required for the maturation of endosomes (8); the delivery of cargo to lysosomes (9) and lysosome-related organelles, such as pigment granules in Drosophila melanogaster (10); and the fusion of autophagosomes with late endosomes/lysosomes (11). The mammalian HOPS complex comprises six subunits (Vps11, Vps16, Vps18, Vps33A, Vps39, and Vps41) (4–6). Homologs of HOPS components can be identified in almost all eukaryotic genomes (12) and are thought to be essential; for example, removal of the Vps33A homolog carnation (car) in Drosophila is lethal during larval development (13).HOPS components have been identified in animal models of human disease. A missense point mutation in the murine Vps33a gene gives rise to the buff mouse phenotype, characterized by pigmentation, platelet activity, and motor deficiencies (14). This phenotype closely resembles the clinical presentation of Hermansky–Pudlak syndrome (HPS) (15), and a mutation in the human VPS33A gene has been observed in a patient with HPS who lacked mutations at other known HPS loci (14). In metazoans, there is a second homolog of yeast Vps33 called Vps33B, but disruption of the VPS33B gene in humans gives rise to a clinical phenotype distinct from HPS (16).Human Vps33A is predicted to be a member of the Sec1/Munc18 (SM) family of proteins (7, 17) that, together with SNAREs, comprise the core machinery essential for membrane fusion in eukaryotes (18). Three SNAREs with glutamine residues at the center of their SNARE domain (Q_a-, Q_b-, and Q_c-SNAREs) and one with a central arginine residue (R-SNARE) associate to form a four-helical bundle, the trans-SNARE complex. Formation of this trans-SNARE complex by SNAREs on adjacent membranes drives the fusion of these membranes (18). SM proteins are essential regulators of this process, promoting membrane fusion by correctly formed (cognate) SNARE complexes (18). Although a comprehensive understanding of how SM proteins achieve this still remains elusive, it is clear that SM proteins bind directly both to individual SNAREs and to SNARE complexes (18, 19). Most SM proteins bind strongly and specifically to an N-terminal segment of their cognate Q_a-SNARE, the N-peptide, and this interaction is thought to recruit the SM protein to the site of SNARE-mediated fusion (20, 21).When considered as a whole, the HOPS complex has the functional characteristics of an SM protein: It binds SNAREs and SNARE complexes (5, 22–24), and yeast HOPS has been shown to promote SNARE-mediated membrane fusion (25, 26). Recent biochemical analysis of Vps33, the yeast Vps33A homolog, shows it to be capable of binding isolated SNARE domains and SNARE complexes but not the N-terminal domain or full cytosolic portion of the Q_a-SNARE Vam3 (23, 24). Data from the yeast HOPS complex are consistent with a model whereby Vps33 provides the SM functionality of HOPS, accelerating SNARE-mediated fusion, whereas the rest of the HOPS complex recruits Vps33 (and thus SM function) to the site of SNARE-mediated fusion (24).Although a recent EM study has defined the overall topology of the yeast HOPS complex (27), atomic resolution insights into the assembly of the HOPS complex have thus far been unavailable. Here, we present the 2.4-Å resolution structure of human Vps33A, confirming its structural identity as an SM protein. We have mapped the HOPS epitope that binds Vps33A to a helical fragment comprising residues 642–736 of Vps16, solved the structure of this complex to 2.6-Å resolution, and identified mutations at the binding interface that disrupt the Vps33A–Vps16 complex both in vitro and in cultured cells.