Abstract
The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Galpha protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of RAS2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.
Highlights
In Saccharomyces cerevisiae the addition of glucose or other rapidly fermentable sugars to derepressed cells triggers a remarkable variety of regulatory phenomena, including many rapid changes at the post-translational and transcriptional level
Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins
The results reported in the literature about the role played by the Ras proteins in activation of cAMP synthesis are in part contradictory
Summary
W303–1A W303–1A (YEpRAS2) W303–1A (YEpTPK1) W303–1A (YCpRAS2) W303–1A (YCpRAS2val19) cdc25⌬ (YEpTPK1) LC7 LC8 PM903. GAL SUC mal MATa leu112 ura trp his ade can100 GAL SUC mal gpa2::URA3 MATa leu112 ura trp his ade can100 GAL SUC mal gpr1::LEU2 MATa ura his leu 112 MAL2 SUC2 GAL MEL hxt1-7⌬ gal2⌬ hxt1-7⌬ gal2⌬ gpa2⌬ hxt1-7⌬ gal2⌬ gpr1⌬ MATa leu 112 ura trp his ade can100 GAL SUC mal glk1::LEU2. Hxk2::LEU2 glk1::LEU2 hxk1::HIS3 hxk2::LEU2 ira2::URA3 Mata his leu ura trp ade can Mat␣ his leu ura trp ade tpk1w1 tpk2::HIS3 tpk3::TRP1 Mat␣ his leu ura trp ade tpk1w1 tpk2::HIS3 tpk3::TRP1 gpa2::URA3 Mata his leu ura trp ade can ras2::URA3 Mat␣ his leu ura trp ras1::HIS3 ras2::URA3 bcy a M.
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