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

Abstract

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 . 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. E ukaryotic 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 lysosomerelated 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 -SNARE...

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

“…We confirm that TGFBRAP1 and VPS8 are the mammalian CORVET-specific subunits (with TGFBRAP1 as the yeast vps3 ortholog) (11,35). In contrast to previous data (16), however, our data indicate that VIPAS39 and VPS33B, two CORVET/HOPS subunit homologues not present in yeast, are not part of CORVET/HOPS as also suggested by others (12,21,36 interactions of VPS33B and/or VIPAS39 with CORVET/HOPS or the existence of VPS33B-VIPAS39-HOPS interactions in other cellular systems. However, the existence of a VPS33/ VIPAS39 complex independent of other core subunits might explain why only mutations in VPS33B and VIPAS39 but not any of other HOPS subunits are associated to ARC syndrome (21)(22)(23)33) and why all HOPS complex subunits, but not VPS33B and VIPAS39, are required for Ebola-virus infection (19).…”

The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion

“…Also the putative N-peptide was not recognized by HOPS (Fig. 1A, lane 7), even if we fused it to the SNARE domain (not shown), in agreement with the lack of an N-peptide binding site in Vps33 (11,12). We thus conclude that purified HOPS binds strongly to the H abc domain of Vam3.…”

Impairment of autophagosome-lysosome fusion in the buff mutant mice with the VPS33AD251E mutation

“…In addition, the crystal structure of human VPS33A revealed that domain 3a is unlikely to interact directly with VPS16. 28 Indeed, our data showed that the interaction between mutant VPS33A D251E and VPS16 was unaffected.…”

“…Inferred from the crystal structure of human VPS33A, the Tyr438-to-Asp (Y438D) mutation is expected to disrupt its interaction with VPS16. 28 We made a homologous Y440D mutation in mouse VPS33A. Indeed, we found that the Y440D mutation greatly reduced the interactions with VPS16, VPS39 or VPS18 by coIP assays (Fig.…”