ISOPRENYLATION OF RAS AND OTHER PROTEINS IN YEAST

ras蛋白通过一系列的反应被翻译后修饰

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.