title: Identification and characterisation of the RalA-ERp57 interaction: evidence for GDI activity of ERp57.
abstract: RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57's active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H(2)O(2) inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI).
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, Australia.
authors.forename: Adam
authors.initials: A
authors.lastname: Brymora
authors.forename: Iain G
authors.initials: IG
authors.lastname: Duggin
authors.forename: Leise A
authors.initials: LA
authors.lastname: Berven
authors.forename: Ellen M
authors.initials: EM
authors.lastname: van Dam
authors.forename: Basil D
authors.initials: BD
authors.lastname: Roufogalis
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D020725:ral GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020730:Guanine Nucleotide Dissociation Inhibitors; D010455:Peptides; D006153:Guanosine Diphosphate; D020725:ral GTP-Binding Proteins; C513196:PDIA3 protein, rat; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human