The set of experiments in this research proposal explore the physiological signals and specific products involved in the upstream control of the RAS/cAMP pathway in S. cerevisiae. The long-term objective is to understand the biochemical details of this sensory/signal transduction pathway in terms of (a) the molecules that are sensed, and (b) the gene products and mechanisms involved in communicating this sensory information to RAS. Insights into the control of yeast RAS activity are of potential medical significance because of the structural and functional homology of yeast and mammalian ras proteins, and the role of ras genes in human tumors. There is considerable indirect evidence suggesting that the activity of yeast RAS gene products reflects the nutritional state of the cell. I will develop biochemical assays for RAS activity that reflect in vivo conditions, and with these assays directly determine which nutrients control the activity of RAS. Mutants defective in the metabolism of these nutrients will be employed to help identify the metabolites or other molecules that actually are being monitored by the RAS pathway. Very little is known about the identity or function of proteins that control RAS activity. The CDC25 gene product is the only protein that has been identified in yeast which controls RAS activity. Antibodies to CDC25 protein will be produced and used to examine any possible covalent modifications that might be involved in the regulation of CDC25 function. To analyze the functional domains of CDC25, I will overexpress various segments of CDC25 and analyze their phenotypic and biochemical effects. Phenotypic effects will be exploited in genetic screens to identify gene products that regulate CDC25 function. To identify new gene products involved in the regulation of RAS, I will use novel genetic screens that employ particular mutant alleles of RAS2 that elicit a strong phenotype, but are unlike the RAS alleles currently employed in genetic screens. The activity of the corresponding altered protein or overproduced protein encoded by these particular alleles is known to be significantly modulated by CDC25 activity, and in principle by the activity of other upstream regulators. Genes of interest will be cloned by complementation and characterized by the construction and phenotypic testing of mutant alleles. To ascertain the biochemical function of the gene products, I will pursue different approaches including sequencing, testing the effect of mutant alleles on biochemical assays for nutrient-controlled RAS activity, and examining the effect of mutant alleles on the state of RAS and CDC25 proteins.
