ISOPRENYLATION OF RAS AND OTHER PROTEINS IN YEAST

ras蛋白通过一系列反应后修饰 包括法尼基化，三个C-末端氨基酸的去除， 羧甲基化法呢基化是合成法呢基化的关键步骤。 改性法尼基化和香叶基香叶基化是两种类型的 涉及胆固醇中间体的修饰。这些修改 发生在各种蛋白质上，其中大多数是小GTP结合 proteins.在本申请中，我们建议进一步了解 利用两种酵母系统，S. cerevisiae和S.粟球。我们的重点将是表征酶， 催化法尼基化（FT酶）和香叶基香叶基化（GGT酶）。 I.法尼基化。S.将表征酿酒酵母FTase。活性 每个亚基将被研究，并且酶的重建 将尝试从子单元。DPR 1和RAM 2的体外诱变 编码FTase亚基的基因将被进行，突变蛋白将 被描述。此外，最近发现的FTase抑制剂 将被定性。 二.香叶基香叶基化。S.酿酒酵母GGT酶I和II将是 并与FTase进行了比较。使用GGT酶I，实验 将进行涉及嵌合基因和诱变的研究，以确定 CDC 43上的区域，编码特定GGT酶的GGT酶亚基的基因 我正常工作。对于GGT酶II，酶的结构以及底物 将进行调查。 三. S.粟酒S. pombe GGTase I will be 使用cwg 2+基因表征; CDC 43对应物。S.粟酒谷氨酰转肽酶将 与S相比。酿酒酵母GGT酶哺乳动物对应的cwg 2 + 将获得。其他异戊烯基转移酶基因将被确定。色葡萄 pombe系统将用于体内鉴定 异戊二烯基转移酶的底物。 四.蛋白质异戊二烯化的生物学意义。S.酿酒酵母 腺苷酸环化酶系统将被用来解决的意义， 法尼基化对ras蛋白功能的影响。S.粟酒β-葡聚糖 合成酶系统将建立，以解决的意义， 香叶基香叶基化

The ras proteins are posttranslationally modified by a series of reaction involving farnesylation, three C-terminal amino acid removal and carboxymethylation. The farnesylation is a critical step in the modification. Farnesylation and geranylgeranylation are two types of modification involving cholesterol intermediates. These modifications occur on a variety of proteins, a majority of them small GTP binding proteins. In this application, we propose to further gain understanding about this modification by taking advantage of two yeast systems, S. cerevisiae and S. pombe. Our emphasis will be to characterize enzymes that catalyze farnesylation (FTase) and geranylgeranylation (GGTase). I. Farnesylation. S. cerevisiae FTase will be characterized. Activity of each subunit will be investigated and the reconstitution of the enzyme from the subunit will be attempted. In vitro mutagenesis of DPR1 and RAM2 genes encoding FTase subunits will be carried out and mutant proteins will be characterized. In addition, recently identified inhibitors of FTase will be characterized. II. Geranylgeranylation. S. cerevisiae GGTase I and II will be characterized and compared with FTase. With GGTase I, experiments involving chimeric genes and mutagenesis will be carried out to define a region on CDC43, a gene encoding a subunit of GGTase that specifies GGTase I function. With GGTase II, structure of the enzyme as well as substrate recognition will be investigated. III. Protein prenylation in S. pombe. S. pombe GGTase I will be characterized using cwg2+ gene; a CDC43 counterpart. S. pombe GGTase will be compared with S. cerevisiae GGTase l. Mammalian counterpart of cwg2+ will be obtained. Other prenyltransferase genes will be identified. The S. pombe system will be exploited for the identification of in vivo substrates of prenyltransferases. IV. Biological significance of protein prenylation. S. cerevisiae adenylate cyclase system will be employed to address the significance of farnesylation on the function of ras proteins. S. pombe beta-glucan synthase system will be established to address the significance of geranylgeranylation.