REGULATION AND FUNCTION OF THE YEAST HEAT SHOCK RESPONSE

描述：本研究的长期目标是了解 真核生物中热休克反应的功能和调节， 芽殖酵母作为模型系统。 酵母有两类丰富的，但 功能不同的热休克70 K蛋白（hsp 70）-四个SSA和两个 SSB蛋白以及与E. coli DnaJ. 这些 蛋白质在体内作为细胞中的分子伴侣起作用 蛋白质成熟过程和热休克调节 反应 利用遗传、分子和生物化学方法， 研究者将分析两组细胞质的功能， hsp 70蛋白在翻译、蛋白质折叠、蛋白质水解和 调节热休克反应。 有五个具体目标。 第一是考察 SSB蛋白在体内蛋白质合成中的作用。 SSB蛋白是 与翻译核糖体相关，并且是高效 翻译. 遗传、分子和生物化学方法将是 用于试图了解SSB蛋白的作用机制 翻译过程。 这些包括：SSA/SSB嵌合体的分析 确定对SSB特定功能至关重要的区域 蛋白质;分析高拷贝和基因外抑制剂的冷- ssb 1 ssb 2突变体的敏感表型;鉴定 与Ssbs相互作用;测定Ssbs与 关于核糖体;定义的相互作用的性质 Ssb与翻译核糖体;和机制的Ssb行动， 翻译. 第二个具体目标是检查SSA蛋白在细胞中的功能。 蛋白质折叠和体内蛋白质水解。 这些蛋白质在 蛋白质成熟将通过检测SSA的作用来研究。 几种测试蛋白质的折叠突变。 SSA的作用 蛋白质在蛋白质水解将通过测量的影响， SSA基因突变对特定底物降解的影响 基于泛素的蛋白水解系统。 基因之间的相互作用 ssa突变体和ubi突变体将被检查以寻找额外的 分子伴侣和蛋白水解之间的联系。 第三个具体目标是确定SSA和 SSB蛋白。 SSA和SSB蛋白将被纯化， 生物化学特征的参数，如肽刺激 ATP酶活性和肽结合。 第四个具体目标是确定DnaJ样蛋白是否 酵母（Sis 1和Ydj 1）的功能与特定的热休克蛋白70对。 遗传和表型比较将在突变体之间进行， SSA、SSB和dnaJ样基因。 将进行生化分析 用纯化的伴侣蛋白来评估DnaJ蛋白在 SSA和SSB蛋白的几种功能。 这些包括 SSA蛋白的ATP酶活性的刺激和复性 的测试多肽底物。 最后的具体目标是检查基因表达的调节 在热休克时。 SSA蛋白在HSP基因调控中的作用 表达将用SSA中的适当突变来表征 基因，并且在这种反应中涉及HSF-磷酸化， 被确定。 SSB基因减少的机制 还将确定热冲击时的表达。

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.