Cdc42p functions at the docking stage of yeast vacuole membrane fusion.
Membrane fusion reactions have been considered to be primarily regulated by Rab GTPases. In the model system of homotypic vacuole fusion in the yeast Saccharomyces cerevisiae, we show that Cdc42p, a member of the Rho family of GTPases, has a direct role in membrane fusion. Genetic evidence suggested a relationship between Cdc42p and Vtc1p/Nrf1p, a central part of the vacuolar membrane fusion machinery. Vacuoles from cdc42 temperature-sensitive mutants are deficient for fusion at the restrictive temperature. Specific amino acid changes on the Cdc42p protein surface in these mutants define the putative interaction domain that is crucial for its function in membrane fusion. Affinity-purified antibodies to this domain inhibited the in vitro fusion reaction. Using these antibodies in kinetic analyses and assays for subreactions of the priming, docking and post-docking phase of the reaction, we show that Cdc42p action follows Ypt7p-dependent tethering, but precedes the formation of trans-SNARE complexes. Thus, our data define an effector binding domain of Cdc42p by which it regulates the docking reaction of vacuole fusion.
Müller
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O
Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39, 72076 Tübingen, Germany.
Johnson
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DI
Mayer
A
A
D000251:Adenosine Triphosphatases; D000483:Alleles; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D046911:Macromolecular Substances; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000502:physiology; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D014617:Vacuoles / Q000502:physiology*; D033921:Vesicular Transport Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000502:physiology
D002352:Carrier Proteins; D002614:Chelating Agents; D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C075137:SEC17 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; C499587:VTC1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; C079005:YPT7 protein, S cerevisiae; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; D020691:rab GTP-Binding Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium