title: Functional analysis of RhoGDI inhibitory activity on vacuole membrane fusion.
abstract: RhoGDIs (Rho GDP-dissociation inhibitors) are the natural inhibitors of Rho GTPases. They interfere with Rho protein function by either blocking upstream activation or association with downstream signalling molecules. RhoGDIs can also extract membrane-bound Rho GTPases to form soluble cytosolic complexes. We have shown previously that purified yeast RhoGDI Rdi1p, can inhibit vacuole membrane fusion in vitro. In the present paper we functionally dissect Rdi1p to discover its mode of regulating membrane fusion. Overexpression of Rdi1p in vivo profoundly affected cell morphology including increased actin patches in mother cells indicative of polarity defects, delayed ALP (alkaline phosphatase) sorting and the presence of highly fragmented vacuoles indicative of membrane fusion defects. These defects were not caused by the loss of typical transport and fusion proteins, but rather were linked to the reduction of membrane localization and activation of Cdc42p and Rho1p. Subcellular fractionation showed that Rdi1p is predominantly a cytosolic monomer, free of bound Rho GTPases. Overexpression of endogenous Rdi1p, or the addition of exogenous Rdi1p, generated stable cytosolic complexes. Rdi1p structure-function analysis showed that membrane association via the C-terminal β-sheet domain was required for the functional inhibition of membrane fusion. Furthermore, Rdi1p inhibited membrane fusion through the binding of Rho GTPases independent from its extraction activity.
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Logan
authors.forename: Lynden
authors.initials: L
authors.lastname: Jones
authors.forename: Daniel
authors.initials: D
authors.lastname: Forsberg
authors.forename: Alex
authors.initials: A
authors.lastname: Bodman
authors.forename: Alicia
authors.initials: A
authors.lastname: Baier
authors.forename: Gary
authors.initials: G
authors.lastname: Eitzen
mesh_terms: D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000502:physiology*; D008561:Membrane Fusion; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D014617:Vacuoles / Q000502:physiology*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D020730:Guanine Nucleotide Dissociation Inhibitors; C534963:Rdi1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C051302:RHO1 protein, S cerevisiae; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins