REGULATION AND FUNCTION OF THE YEAST HEAT SHOCK RESPONSE

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DESCRIPTION: The long term goal of this research is to understand the function and regulation of the heat shock response in eukaryotes, using budding yeast as a model system. Yeast has two classes of abundant but functionally distinct heat shock 70K proteins (hsp70s)-four SSA and two SSB proteins-and several proteins related to E. coli DnaJ. These proteins function in vivo as molecular chaperones in the cellular processes of protein maturation and regulation of the heat shock response. Using genetic, molecular, and biochemical approaches the investigator will analyze the function of the two groups of cytosolic hsp70 proteins in translation, protein folding, proteolysis, and regulation of the heat shock response. There are five specific aims. The first is to examine the function of the SSB proteins in protein synthesis in vivo. SSB proteins are associated with translating ribosomes and are required for efficient translation. Genetic, molecular, and biochemical approaches will be used in an attempt to get at the mechanism of action of SSB proteins in the translation process. These include: analysis of SSA/SSB chimeras to define regions critical for the specific functions of the SSB proteins; analysis of high copy and extragenic suppressors of the cold- sensitive phenotype of ssb1ssb2 mutants; identification of proteins that interact with Ssbs; determination of the stoichiometry of Ssbs with respect to ribosomes; definition of the nature of the interaction of Ssbs with translating ribosomes; and the mechanism of Ssb action in translation. The second specific aim is to examine the function of SSA proteins in protein folding and proteolysis in vivo. The role of these proteins in protein maturation will be studied by examining the effects of ssa mutations on the folding of several test proteins. The role of SSA proteins in proteolysis will be studied by measuring the effect of mutations in the SSA genes on the degradation of specific substrates of the ubiquitin based proteolysis system. The genetic interactions between ssa mutants and ubi mutants will be examined to look for additional links between the chaperones and proteolysis. The third specific aim is to define the differences between the SSA and SSB proteins. The SSA and SSB proteins will be purified and characterized biochemically for such parameters as peptide-stimulated ATPase activity and peptide binding. The fourth specific aim is to determine whether the DnaJ-like proteins of yeast (Sis1 and Ydj1) function in pairs with specific Hsp70s. Genetic and phenotypic comparisons will be made between mutants in the SSA, SSB, and dnaJ-like genes. Biochemical assays will be performed with purified chaperones to assess the roles of the DnaJ proteins in several functions of the SSA and SSB proteins. These include stimulation of the ATPase activity of the SSA protein and the refolding of test polypeptide substrates by both Hsp70s. The final specific aim is to examine the regulation of gene expression upon heat shock. The role of SSA proteins in the regulation of HSP gene expression will be characterized with appropriate mutations in SSA genes, and the involvement of HSF-phosphorylation in this response will be determined. The mechanism responsible for the decrease in SSB gene expression upon heat shock will also be determined.