REGULATION OF YEAST INVERTASE GENE EXPRESSION

描述：这是玛丽安·卡尔森博士研究的竞争性更新 转化酶基因SUC2在面包酵母中的葡萄糖调节。这 应用重点是发挥中枢作用的Snf1蛋白激酶复合体 在感知葡萄糖和改变的信号转导通路中的作用 转录大量葡萄糖被抑制和激活的基因。 PI当前的SUC2葡萄糖调节模型涉及三个层次 是复合体的。1)。葡萄糖的存在是通过REG1-PP1感受到的 (蛋白磷酸酶I)使2)脱磷酸化的复合体。 Snf1激酶复合体。Snf1激酶复合体至少包括Snf4， 以及SIP1、2和Gal83蛋白。当被葡萄糖脱磷时 发出信号表明它是不起作用的，但当缺乏葡萄糖时，它会磷酸化 Mig1抑制因子，使其失活。激活时，Mig1抑制因子 与UP1-SSN6通用抑制子的复合体抑制转录 南加州大学。这些层之间的联系很薄弱，尽管 有证据表明，每一种都像所描述的那样相互作用。然而，有一种 缺失因素的数量包括：建议的Snf1激酶、 葡萄糖敏感机制，以及SUC2的转录激活子 可能也是通过Snf1来调节血糖的。此应用程序建议 详细说明已知的相互作用，并确定和描述缺失的 各种因素。 将结合使用生化和遗传方法。Snf1 两种体外磷酸化研究都将提纯激酶， 特别侧重于Mig1和蛋白质-蛋白质相互作用的研究。这个 后者将使用融合蛋白进行，这将允许系留 将建筑群的一个成员连接到一根柱子上。 遗传学研究严重依赖于双杂交系统来分离 与SNF1和SNF1复合体成员以及 以详细说明相互作用的区域。这一方法将得到以下补充 突变和高拷贝抑制猎杀旨在识别 作用于Snf1激酶的上游。对于下游的分析 野生型Mig1和突变型Mig1的靶点、磷酸化改变 在推测的SNF1靶点，将在体外和体内进行研究。 此外，还使用改进的双混合方法描述了一种策略 基于以下假设确定SUC2激活子(S) 不是必需的就是多余的，因为它们从未在 原始的无诱导性突变体猎杀。

DESCRIPTION: This is a competitive renewal from Dr. Marian Carlson to study the glucose regulation of the invertase gene, SUC2, in baker's yeast. This application focuses on the Snf1 protein kinase complex that plays a central role in the signal transduction pathway that senses glucose and alters transcription of a large number of glucose repressed and activated genes. The PI's current model for glucose regulation of SUC2 involves three tiers of complexes. 1). The presence of glucose is sensed through the Reg1-PP1 (protein phosphatase I) complex which dephosphorylates 2) a member of the Snf1 kinase complex. The Snf1 kinase complex includes at least the Snf4, and the Sip1, 2, and Gal83 proteins. When dephosphorylated by the glucose signal it isnon-functional, but when glucose is absent, it phosphorylates 3) the Mig1 repressor, inactivating it. When active, the Mig1 repressor complexes with the up1-Ssn6 general repressor to repress transcription of SUC2. The link between each of these tiers is tenuous, although the evidence suggests that each interacts as described. However, there are a number of missing factors including: the proposed Snf1 kinase kinase, the glucose sensing mechanism, and the transcriptional activators of SUC2 which may well be glucose regulated through Snf1, too. This application proposes to detail the known interactions and identify and characterize the missing factors. A combination of biochemical and genetic approaches will be used. The Snf1 kinase will be purified for both in vitro phosphorylation studies, especially focused on Mig1, and protein- protein interaction studies. The latter will be carried out using fusion proteins which will allow tethering one member of the complex to a column. The genetic studies place heavy reliance on the two-hybrid system to isolate factors that interact with Snf1 and members of the SNf1 complex as well as to detail the interacting regions. This approach will be complemented with mutant and high copy suppression hunts designed to identify factors that function upstream of the Snf1 kinase. For analyses of the downstream targets, phosphorylation of both wildtype Mig1 and mutant versions altered in the putative Snf1 target sites, will be studied in vitro and in vivo. Also a strategy is described using a modified two-hybrid approach to identify the SUC2 activator(s) based upon the assumption that these proteins are either essential or redundant since they were never identified in the original SUC2 uninducible mutant hunts.