title: Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes.
abstract: The mammalian homotypic fusion and vacuole protein sorting (HOPS) complex is comprised of six subunits: VPS11, VPS16, VPS18, VPS39, VPS41 and the Sec1/Munc18 (SM) family member VPS33A. Human HOPS has been predicted to be a tethering complex required for fusion of intracellular compartments with lysosomes, but it remains unclear whether all HOPS subunits are required. We showed that the whole HOPS complex is required for fusion of endosomes with lysosomes by monitoring the delivery of endocytosed fluorescent dextran to lysosomes in cells depleted of individual HOPS proteins. We used the crystal structure of the VPS16/VPS33A complex to design VPS16 and VPS33A mutants that no longer bind each other and showed that, unlike the wild-type proteins, these mutants no longer rescue lysosome fusion with endosomes or autophagosomes in cells depleted of the endogenous proteins. There was no effect of depleting either VIPAR or VPS33B, paralogs of VPS16 and VPS33A, on fusion of lysosomes with either endosomes or autophagosomes and immunoprecipitation showed that they form a complex distinct from HOPS. Our data demonstrate the necessity of recruiting the SM protein VPS33A to HOPS via its interaction with VPS16 and that HOPS proteins, but not VIPAR or VPS33B, are essential for fusion of endosomes or autophagosomes with lysosomes.
authors.affiliation: Cambridge Institute for Medical Research and Department of Clinical Biochemistry, Wellcome Trust/MRC Building, University of Cambridge, Cambridge, CB2 0XY, UK.
authors.forename: Lena
authors.initials: L
authors.lastname: Wartosch
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, CB2 1QN, UK.
authors.forename: Ufuk
authors.initials: U
authors.lastname: Günesdogan
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Graham
authors.affiliation: Cambridge Institute for Medical Research and Department of Clinical Biochemistry, Wellcome Trust/MRC Building, University of Cambridge, Cambridge, CB2 0XY, UK.
authors.forename: J Paul
authors.initials: JP
authors.lastname: Luzio
mesh_terms: D000595:Amino Acid Sequence; D001343:Autophagy; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010588:Phagosomes / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C424439:VPS16 protein, human; C424440:VPS33A protein, human; D033921:Vesicular Transport Proteins
keywords: CORVET; HOPS; SM protein; VPS16; VPS33A; autophagy; endocytosis; lysosomes; tethering factor