title: Evidence for adenylate cyclase as a scaffold protein for Ras2-Ira interaction in Saccharomyces cerevisie.
abstract: Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.
authors: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza Della Scienza 2, 20126 Milan, Italy; SysBio Centre of Systems Biology, Piazza della Scienza 2, 20126 Milan, Italy.
authors: Sonia
authors: S
authors: Colombo
authors: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza Della Scienza 2, 20126 Milan, Italy.
authors: Chiara
authors: C
authors: Paiardi
authors: Functional Biology, KU Leuven, Kasteelpark Arenberg 31, Box 2433, 3001 Heverlee, Belgium.
authors: Katrien
authors: K
authors: Pardons
authors: Functional Biology, KU Leuven, Kasteelpark Arenberg 31, Box 2433, 3001 Heverlee, Belgium.
authors: Joris
authors: J
authors: Winderickx
authors: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza Della Scienza 2, 20126 Milan, Italy; SysBio Centre of Systems Biology, Piazza della Scienza 2, 20126 Milan, Italy. Electronic address: enzo.martegani@unimib.it.
authors: Enzo
authors: E
authors: Martegani
mesh_terms: D000262:Adenylyl Cyclases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; C065725:IRA1 protein, S cerevisiae; C064960:IRA2 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases
keywords: Cyr1; Ras2-GTP; Yeast; cAMP; cAMP/PKA pathway