Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking.
Rho GTPases, which control polarized cell growth through cytoskeletal reorganization, have recently been implicated in the control of endo- and exocytosis. We now report that both Rho1p and Cdc42p have a direct role in mediating the docking stage of homotypic vacuole fusion. Vacuoles prepared from strains with temperature-sensitive alleles of either Rho1p or Cdc42p are thermolabile for fusion. RhoGDI (Rdi1p), which extracts Rho1p and Cdc42p from the vacuole membrane, blocks vacuole fusion. The Rho GTPases can not fulfill their function as long as priming and Ypt7p-dependent tethering are inhibited. However, reactions that are reversibly blocked after docking by the calcium chelator BAPTA have passed the point of sensitivity to Rdi1p. Extraction and removal of Ypt7p, Rho1p and Cdc42p from docked vacuoles (by Gdi1p, Gyp7p and Rdi1p) does not impede subsequent membrane fusion, which is still sensitive to GTPgammaS. Thus, multiple GTPases act in a defined sequence to regulate the docking steps of vacuole fusion.
Eitzen
G
G
Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844, USA.
Thorngren
N
N
Wickner
W
W
D000483:Alleles; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008561:Membrane Fusion; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000502:physiology*; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D020703:ras GTPase-Activating Proteins / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D002614:Chelating Agents; D005656:Fungal Proteins; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; C439608:Gyp7 protein, S cerevisiae; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; C079005:YPT7 protein, S cerevisiae; C051302:RHO1 protein, S cerevisiae; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; D020691:rab GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid