title: The Habc domain of the SNARE Vam3 interacts with the HOPS tethering complex to facilitate vacuole fusion.
abstract: Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the Habc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 Habc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the Habc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the Habc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the Habc domain. In agreement, membranes carrying Vam3ΔHabc fuse poorly unless an excess of HOPS is provided. These data suggest that the Habc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion.
authors.affiliation: From the Department of Biology/Chemistry, Biochemistry Section and.
authors.forename: Anna
authors.initials: A
authors.lastname: Lürick
authors.affiliation: the Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Kuhlee
authors.affiliation: From the Department of Biology/Chemistry, Biochemistry Section and.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Bröcker
authors.affiliation: the Department of Biology/Chemistry, Structural Biology, University of Osnabrück, 49076 Osnabrück, Germany and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kümmel
authors.affiliation: the Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Raunser
authors.affiliation: From the Department of Biology/Chemistry, Biochemistry Section and cu@uos.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Ungermann
mesh_terms: D001665:Binding Sites; D015151:Immunoblotting; D008561:Membrane Fusion; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D050765:Qa-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C107706:VAM3 protein, S cerevisiae; C080045:VPS16 protein, S cerevisiae; C065454:VPS33 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins
keywords: HOPS; Lysosome; Membrane Fusion; Membrane Reconstitution; Membrane Trafficking; SNARE Proteins; Soluble NSF Attachment Protein Receptor (SNARE); Tethering; Vacuole