Haploid cells of a and alpha mating type in the yeast Saccharomyces cerevisiae secrete peptide pheromones called a and alpha-factor, respective binding of the pheromones to receptors or cells of the opposite mating type activates an intracellular response pathway, which leads to cell cycle arrest in the GI phase of the cell cycle, morphological changes, and induction of genes involved in many aspects of pheromone response and mating. Many components of the pheromone response pathway have been identified, including the receptors, three subunits of a G protein, several kinases, and a transcription factor. In addition, desensitization of the response pathway has been shown to act at multiple levels of the pathway. The yeast system shows many similarities to mammalian signal transduction systems. Mutations in components of these systems are implicated in some cancer and disease states. Yeast provides a system in which such a pathway can be analyzed using a combination of genetic and biochemical approaches.
The aims of the proposed research are to elucidate several aspects of this process. Biochemical assays of the alpha subunit (Scg1) of the G protein will be developed, including assays of the guanine nucleotide dissociation rate, the GTPase activity, and the conformational shift to the active state. A comparison of the biochemical and the phenotypic effects of mutations that alter these activities will elucidate the mechanism of G protein action. A critical role of the Galpha subunit is to interact with the pheromone receptors. Because the two receptors are not homologous to one another, the specificity for the two interactions may differ. Mutations in the receptor-binding domain are being constructed and analyzed for phenotypic effects to localize the receptor binding domain and to identify the residues involved in the specificity for the interactions with the two receptors. Allele-specific suppressors of these mutations will be isolated to localize the receptor domains involved in the G protein interaction. In the yeast pathway, the Beta/gamma subunit of the G protein is proposed to interact with the downstream effector, which has not been identified. Two genetic approaches have identified several candidate genes for the effector. These genes will be characterized to determine their role in the pathway and to determine whether they identify the effector. The Sst2 product plays an important role in desensitization of the response but its mechanism of action is not know. Sst2 mutants with altered function are being isolated, and suppressors of these mutations will be isolated to identify the target of Sst2 action. Other experiments are aimed at determining where in the pathway Sst2 acts with respect to known components. Finally, new mutants have been isolated that may identify new components of the pathway that play negative roles. These mutants will be characterized and the genes will be cloned to investigate their mechanism of action and where they act in the pathway. These experiments will elucidate several aspects of the mechanism by which cells respond to pheromone and by which desensitization to pheromone occurs.
