STRUCTURAL AND FUNCTIONAL ANALYSES OF YEAST RAS PROTEINS

酵母 RAS 蛋白的结构和功能分析

• 批准号: 3182674
• 负责人: Fuyuhiko Tamanoi
• 金额: $ 21.71万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-06-01 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3182674
• 关键词: acylation; acyltransferase; adenylate cyclase; affinity chromatography; aminoacid analyzer; antibody; conformation; crosslink; fatty acid metabolism; fungal genetics; gene expression; guanine nucleotide binding protein; guanosine triphosphate; guanosinetriphosphatases; laboratory rabbit; monoclonal antibody; oncogenes; phosphatidylinositols; protein biosynthesis; protein sequence; protein structure function; yeasts

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.

对酵母RAS基因的研究有助于我们理解 ras癌基因的功能有两个重要的方面。 第一、 阐明这些酵母基因参与了 调节第二信使（cAMP和 磷酸肌醇）可能揭示ras的功能 proteins. 第二，ras蛋白的生物合成，包括其 脂肪酸的翻译后修饰和定位 可以使用酵母作为模型系统来阐明质膜 因为基因和生物化学的结合是可行的。 这两个方面将通过集中研究 在实验之后。 I.酵母RAS蛋白的生物合成 (1)脂肪酸酰化的确切化学性质以及 在脂肪酸酰化之前发生的改性将 通过分离含有这些修饰的肽进行分析。 (2)参与生物合成的DPR 1基因将被表征 通过确定基因的结构和表征 基因的产物。 参与生物合成的其他基因将 被寻找。 (3)负责RAS脂肪酸酰化的酶 蛋白质将被识别。 (4)RAS蛋白的序列 负责其修改和本地化的将是 通过各种实验表征，包括体外实验 诱变 二. 酵母RAS蛋白的功能：（1）脂肪酸 酰化的RAS蛋白将从细胞质膜纯化， 酵母细胞及其GTP结合和GTP酶活性将被 表征了(2)RAS蛋白在酵母中的作用 腺苷酸环化酶的特征在于重组腺苷酸环化酶。 系统与纯化的脂肪酸酰化RAS蛋白和 细胞膜缺乏RAS1和RAS2蛋白。 存在 RAS蛋白以外的腺苷酸环化酶组分， 腺苷酸环化酶催化亚基将通过以下方法研究 分离系统。 (3)一个体外系统来研究 RAS蛋白对酵母PI周转的参与将被开发。 (4)将进行使用膜的交联实验 以深入了解RAS之间的物理相互作用 腺苷酸环化酶和PI的蛋白质和组分 周转系统。