REGULATION AND FUNCTION OF THE YEAST HEAT SHOCK RESPONSE

我们的长期目标是深入了解 位于细胞质中的应激蛋白在细胞过程中并更好地 了解编码这些基因的基因表达的调节 蛋白质，尤其是在指数生长过程中。更具体地说，我们建议 利用基因和生物化学技术的组合来研究 酵母菌必需SSA亚家族基因的功能 由SSAI、SSA2、SSA3和SSA4 4个基因组成的酿酒酵母 编码hsp70蛋白。成员之间的职能关系 这个在结构和功能上相关的基因家族 下定决心。无论它们在功能上是相同的还是不同的 通过分析含有不同SSA基因突变的菌株进行了研究 以及表达不同数量的亚家族的特定成员。 将分离SSAL的点突变和菌株的表型 携带这些突变，包括蛋白质易位 内质网(ER)和线粒体、蛋白分解、内吞和 将分析热冲击响应的调节，以获得更好的 了解SSA蛋白在这些过程中的作用。第二站点 将分离SSA突变的返回体以获得关于 SSA基因参与的相互作用的蛋白质和过程。我们 将克隆和分析能够抑制人类免疫缺陷的基因 SSA基因突变的影响。将研究SSAL的功能， 以前体蛋白转位为模型系统 分离的线粒体和微生物体。在这些研究中，SSAI突变体 已被分离的基因以及第二位点抑制基因突变 将被用来更好地了解社援协会在 蛋白质易位。 SSA1的表达，特别是在正常指数生长过程中 研究以更好地理解基础砷的调节机制 以及应激诱导的表达。我们已经确定了一个上游 抑制SSAL表达的抑制序列(URS)。变种人 在基于URS的镇压中存在缺陷的人将被隔离并分析 希望分离出含有编码基因突变的突变体 URS结合蛋白。基于城市轨道交通的城市轨道交通运行机制 抑制将使用体外方法和相互作用进行分析。 具有天然启动子的热休克转录因子 之后将对热冲击进行研究。

The long term objective is to gain insight into the role of the cytoplasmically located stress proteins in cellular processes and to better understand the regulation of expression of the genes that encode these proteins especially during exponential growth. More specifically we propose to study, using a combination of genetic and biochemical techniques, the function of the essential SSA subfamily of genes of Saccharomyces cerevisiae which is composed of 4 genes, SSAI, SSA2, SSA3 and SSA4, each encoding an hsp7O protein. The functional relationship between members of this structurally and functionally related family of genes will be determined. Whether they are functionally identical or distinct will be studied, by analyzing strains containing mutations in different SSA genes and expressing differing amounts of particular members of the subfamily. Point mutations in SSAL will be isolated and the phenotypes of strains carrying these mutations, including protein translocation in the endoplasmic reticulum (ER) and mitochondria, proteolysis, endocytosis and regulation of the heat shock response will be analyzed to gain a better understanding of the roles of SSA proteins in these processes. Second-site revertants of SSA mutations will be isolated to gain information about interacting proteins and processes in which the SSA genes are involved. We will clone and analyze the genes which are capable of suppressing the effects of mutations in SSA genes. The function of SSAL will be studied, using as a model system the translocation of precursor proteins into isolated mitochondria and microsomes. In these studies the SSAI mutants that have been isolated as well as the second-site suppressor mutations will be used to gain a better understanding of the role played by SSAl in protein translocation. The expression of SSA1, particularly during normal exponential growth will be studied to better understand the mechanism of regulation of basal as well as stressed-induced expression. We have identified an upstream repressing sequence (URS) that represses expression of SSAL. Mutants that are defective in URS-based repression will be isolated and analyzed in hopes of isolating mutants containing mutations in the gene that encodes the URS-binding protein. The mechanism of action of the URS-based repression will be analyzed using in vitro methods and the interaction of the heat shock transcription factor with a natural promoter both before and after a heat shock will be studied.