ISOPRENYLATION OF RAS AND OTHER PROTEINS IN YEAST

Ras蛋白通过一系列反应进行翻译后修饰。 包括法尼化，三个C末端氨基酸的去除和 羧甲基化。法尼化是生物合成中的关键步骤。 修改。法尼化和香叶基香叶化是两种类型的 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 蛋白质。在本申请中，我们建议进一步了解 关于这一改进，利用了两个酵母系统，S. Cerevisiae和S.pombe。我们的重点将是确定酶的特性 催化法尼化(FTase)和香叶基香叶化(GGTase)。 I.法尼斯化。对酿酒酵母FTase进行了性质研究。的活动 将研究每个亚基和酶的重组 将尝试从子单元。DPR1和RAM2的体外诱变 编码FTase亚基的基因将被执行，突变蛋白将被 被刻画出来。此外，最近发现的FTase抑制剂 将被描述为。 二、Geranylgeranyation。酿酒酵母GGTase I和II将是 并与FTase进行了表征和比较。使用GGTase I，实验 将进行涉及嵌合基因和突变的研究，以确定 CDC43上的区域，编码GGTase亚基的基因，指定GGTase 我起作用了。在GGTase II中，酶的结构以及底物 将对认可度进行调查。 庞氏链霉菌中的蛋白质戊烯基化。S.Pombe GGTase I将成为 以cwg2+基因为特征的；CDC43的对应物。S.Pombe GGTase将 与酿酒酵母GGTase L进行比较。Cwg2+在哺乳动物中的同源 将会被获得。其他异戊烯基转移酶基因将被鉴定出来。S。 POMBE系统将被用于体内鉴定 异戊烯基转移酶的底物。 四、蛋白质预烯基化的生物学意义。酿酒酵母 腺苷环化酶系统将被用来解决 法尼化对ras蛋白功能的影响。蓬贝β-葡聚糖 将建立合成酶系统，以解决 香叶香叶。

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.