STRUCTURAL & FUNCTIONAL ANALYSES OF YEAST RAS PROTEINS

对酵母RAS基因的研究有助于我们的理解

Studies of the RAS genes of yeast contribute to our understanding of the function of rasoncogenes in two significant ways. First, elucidation of the involvement of these yeast genes in the regulation of the production of second messengers (cAMP and inositol phosphates) might shed light on the function of ras proteins. Second, biosynthesis of ras proteins including their post-translational modification by fatty acid and localization at the plasma membrane can be elucidated using yeast as a model system because combined genetic and biochemical means are available. These two aspects will be investigated by concentrating on the following experiments. I. Biosynthesis of yeast RAS proteins; (1) Exact chemical nature of the fatty acid acylation as well as a modification which occurs prior to the fatty acid acylation will be analyzed by isolating peptides containing these modifications. (2) DPR1 gene involved in the biosynthesis will be characterized by determining the structure of the gene and characterizing the product of the gene. Other genes Involved In the biosynthesis will be sought. (3) Enzymes responsible for fatty acid acylation of RAS proteins will be identified. (4) Sequences on the RAS proteins responsible for their modification and localization will be characterized by a variety of experiments Including in vitro mutagenesis. II. Function of yeast RAS proteins: (1) Fatty acid acylated RAS proteins will be purified from the plasma membrane of yeast cells and their GTP binding and GTPase activities will be characterized. (2) Involvement of the RAS proteins on yeast adenylate cyclase will be characterized by reconstituting the system with the purified fatty acid acylated RAS proteins and membranes lacking RAS1 and RAS2 proteins. Presence of the components of the adenylate cyclase other than the RAS proteins and the catalytic subunit of adenylate cyclase will be investigated by fractionating the system. (3) An in vitro system to study the involvement of RAS proteins on yeast PI turnover will be developed. (4) Cross-linking experiments using membranes will be carried out to gain insights Into the physical interactions between the RAS proteins and components of the adenylate cyclase and the PI turnover systems.