REGULATION AND FUNCTION OF THE YEAST HEAT SHOCK RESPONSE

描述：这项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 以芽殖酵母为模型系统。酵母有两类丰富的但 功能不同的热休克70K蛋白(Hsp70)--四个SSA和两个 SSB蛋白-以及与E.ColiDNAJ相关的几种蛋白。这些 蛋白质在体内的功能是细胞中的分子伴侣 蛋白质成熟过程与热休克的调控 回应。使用遗传、分子和生物化学方法 研究人员将分析这两组胞浆的功能 HSP70蛋白质在翻译、蛋白质折叠、蛋白质降解和 热休克反应的调节。 有五个具体目标。首先要考察的是， SSB蛋白在体内的蛋白质合成。SSB蛋白是 与翻译核糖体相关，高效是必需的 翻译。遗传、分子和生化方法将是 用于试图了解SSB蛋白在体内的作用机制 翻译过程。其中包括：分析SSA/SSB嵌合体 确定对SSB的特定职能至关重要的区域 蛋白质.冷的高复制和基因外抑制物的分析 Ssb1ssb2突变体的敏感表型； 与SSB相互作用.测定SSB的化学计量比 关于核糖体.核糖体相互作用性质的定义 SSB与翻译核糖体；以及SSB的作用机制 翻译。 第二个特定的目的是研究SSA蛋白在 体内蛋白质折叠和蛋白质降解。这些蛋白质在细胞周期中的作用 蛋白质成熟度将通过检测ssa的影响来研究。 几个测试蛋白质折叠上的突变。SSA的作用 蛋白质分解中的蛋白质将通过测量 SSA基因突变对特定底物降解的影响 基于泛素的蛋白质分解系统。人与人之间的遗传交互作用 将检查SSA突变体和UBI突变体以寻找更多 伴侣蛋白和蛋白分解之间的联系。 第三个具体目标是定义SSA和 SSB蛋白。SSA和SSB蛋白将被提纯并 以生物化学为特征的，如肽刺激的 ATPase活性和多肽结合。 第四个具体目标是确定DNAJ样蛋白是否 酵母的一部分(Sis1和Ydj1)与特定的Hsp70成对发挥作用。 将在突变体之间进行遗传和表型比较 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。这些措施包括 刺激SSA蛋白的ATPase活性及复性 两种Hsp70的测试多肽底物。 最终的具体目标是检查基因表达的调节。 在热震的时候。SSA蛋白在热休克蛋白基因调控中的作用 表达将以SSA中适当的突变为特征 基因，以及参与这一反应的HSF磷酸化将 要下定决心。SSB基因减少的机制 热冲击时的表达也将被确定。

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.