title: HOPS proofreads the trans-SNARE complex for yeast vacuole fusion.
abstract: The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the K(m) value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Q(c)). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Q(a)) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Starai
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Hickey
authors.forename: William
authors.initials: W
authors.lastname: Wickner
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D020816:Amino Acid Motifs; D046912:Multiprotein Complexes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D050600:SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate