The effects of replacing Sst2 with the heterologous RGS4 on polarization and mating in yeast.
RGS proteins stimulate the deactivation of heterotrimeric G-proteins. The yeast RGS protein Sst2 is regulated at both the transcriptional and posttranscriptional levels. We replaced the SST2 gene with the distantly related human RGS4 gene, which consists of the catalytic domain and an N-terminal membrane attachment peptide, and replaced the native promoter (P(SST2)) with the heterologous tetracycline-repressible promoter (P(TET)). We then measured the effect of the substitutions on pheromone sensitivity, mating, and polarization. Although the pheromone sensitivity was essentially normal, there were differences in mating and polarization. In particular, the RGS4-substituted strains did not form multiple mating projections at high levels of alpha-factor, but instead formed a single malformed projection, which frequently gave rise to a bud. We provide evidence that this phenotype arose because unlike Sst2, RGS4 did not localize to the projection. We use mathematical modeling to argue that localization of Sst2 to the projection prevents excess G-protein activation during the pheromone response. In addition, modeling and experiments demonstrate that the dose of Sst2 influences the frequency of mating projection formation.
Tanaka
Hiromasa
H
Department of Developmental and Cell Biology, University of California, Irvine, California, USA.
Yi
Tau-Mu
TM
D016764:Cell Polarity / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D010641:Phenotype; D010675:Pheromones / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects; D020710:RGS Proteins / Q000378:metabolism*; D012098:Reproduction / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics
D020690:GTPase-Activating Proteins; D050505:Mutant Proteins; D010675:Pheromones; D020710:RGS Proteins; C094301:SST2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C098799:RGS4 protein; D019204:GTP-Binding Proteins; D013752:Tetracycline