Control of adaptation to mating pheromone by G protein beta subunits of Saccharomyces cerevisiae.

Abstract

The STE4 gene of the yeast Saccharomyces cerevisiae encodes the beta subunit of a heterotrimeric G protein that mediates response to mating pheromones and influences recovery from pheromone-induced growth arrest. To explore how G beta subunits regulate response and recovery (adaptation), we isolated and characterized signaling-defective STE4 alleles (STE4sd). STE4sd mutations resulted in amino acid substitutions in the N-terminal region of Ste4p, proximal to the first of seven repeat units conserved in G protein beta subunits. Genetic tests indicated that STE4sd mutations disrupted functions of Ste4p required for inducing pheromone responses. Wild-type cells that overexpressed STE4sd alleles displayed apparently normal initial responses to pheromone as judged by quantitative mating, G1 arrest and transcriptional assays. However, after undergoing initial G1 arrest, wild-type cells overexpressing STE4sd alleles recovered more quickly from division arrest, suggestive of a hyperadaptive phenotype. Because hyperadaptation occurred when STE4sd alleles were overexpressed in cells lacking Sst1p (Bar1p), Sst2p or the C-terminal domain of the alpha-factor receptor, this phenotype did not involve three principal modes of adaptation in yeast. However, hyperadaptation was abolished when STE4sd mutations were combined in cis with a deletion that removes a segment of Ste4p (residues 310-346) previously implicated in adaptation to pheromone. These results indicate that G beta subunits possess two independent activities, one required for triggering pheromone response and another that promotes adaptation. Potential models for G beta subunit-mediated adaptation are discussed.

Unnatural Amino Acid Replacement in a Yeast G Protein-Coupled Receptor in Its Native Environment

“…The mutants F55 TAG (Figure E) and F204 TAG (Figure Q) formed dose-dependent halos in the absence of Bpa, although these halos were “fuzzy”, referring to the poor definition of the borders and reduced clarity of the zone of inhibition compared to the WT control. The fuzzy nature of these halos is most likely due to a decreased receptor number or to more rapid desensitization of the Bpa mutant receptor, resulting in a recovery from the growth arrest response , . Immunoblot analysis indicated that, in the absence of Bpa, the protein expression level of the F55 TAG mutant was below the limit of detection (Figure A) and the expression of the F204 TAG mutant was very low (see Figure S1 in the Supporting Information).…”