REGULATION OF ADENYLATE CYCLASE ACTIVITY IN YEAST

酵母中腺苷酸环化酶活性的调节

• 批准号: 3467670
• 负责人: WARREN no middle name HEIDEMAN
• 金额: $ 9.14万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467670
• 关键词: Saccharomyces cerevisiae; adenylate cyclase; affinity chromatography; crosslink; enzyme complex; enzyme mechanism; fungal genetics; gel electrophoresis; genetic manipulation; guanine nucleotide binding protein; immunochemistry; laboratory rabbit; molecular cloning; mutant; oncogenes; phosphorylation; Saccharomyces cerevisiae; adenylate cyclase; affinity chromatography; crosslink; enzyme complex; enzyme mechanism; fungal genetics; gel electrophoresis; genetic manipulation; guanine nucleotide binding protein; immunochemistry; laboratory rabbit; molecular cloning; mutant; oncogenes; phosphorylation

The long term goal of this project is to understand, at a molecular level, how adenylate cyclase is regulated in the yeast, S. cerevisiae. Intracellular levels of cAMP have been shown to play a major role in the regulation of growth in yeast. Production of cAMP is catalyses by adenylate cyclase system is thus relevant not only to mammalian adenylate cyclase, which plays a crucial role in many homeostatic mechanisms, but to the proteins encoded by the animal ras oncogenes as well. We propose to identify the proteins that make up the enzyme complex, assign functions to these proteins, and determine the locations of functional domains on the catalytic protein. In order to identify proteins associated with adenylate cyclase, we will rely on biochemical techniques, including coprecipitation using antibodies to adenylate cyclase, crosslinking with bifunctional agents, and affinity purification using segments of the catalytic subunit overexpressed in E. coli. We will examine the functional roles of these proteins by using molecular genetic techniques to produce yeast strains which lack each protein, but which retain the other components of the system. We will determine how these changes in the subunit composition of adenylate cyclase affect the biochemical properties of the enzyme, as well as the phenotypes of the altered yeast. In order to produce the altered strains, we will clone the genes for proteins associated with adenylate cyclase, and use the cloned DNA to disrupt the chromosomal copy of each gene in wild type yeast. To examine the system in more detailed, we will identify the regions of the catalytic protein that are important in interactions with the RAS proteins, the cell membrane, and other proteins. Specific, in frame deletions will be made in the coding region of the CYR1 gene, encoding the catalytic polypeptide, and the resulting truncated enzyme will be examined for losses in function. An alternate strategy will be to express isolated segments of the catalytic protein at high levels in wild type yeast. If these segments fold properly to produce functional domains, they will compete with the homologous domains on the normal enzyme, and prevent interactions with proteins that normally associate with adenylate cyclase. This will provide positive evidence for the locations of functional domains within the catalytic polypeptide.

该项目的长期目标是了解 分子水平，如何在酵母中调节腺苷酸环化酶， S. cerevisiae。 细胞内cAMP的水平已显示起主要作用 在调节酵母的增长中。 营地的生产是 因此 哺乳动物腺苷酸环化酶，在许多人中起着至关重要的作用 稳态机制，但对动物编码的蛋白质 RAS癌基因。 我们建议鉴定构成酶的蛋白质 复杂，将功能分配给这些蛋白质，并确定 催化蛋白上功能结构域的位置。 为了 为了鉴定与腺苷酸环化酶相关的蛋白质，我们将 依靠生化技术，包括使用 腺苷酸环化酶的抗体，与双功能交联 代理和使用催化片段的亲和力纯化 大肠杆菌中过表达的亚基。 我们将检查功能 这些蛋白质的作用通过使用分子遗传技术 产生缺乏每种蛋白质的酵母菌菌株，但保留 系统的其他组件。 我们将确定这些 腺苷酸环化酶亚基组成的变化会影响 酶的生化特性以及表型 改变的酵母。 为了产生改变的菌株，我们 将克隆与腺苷酸盐相关的蛋白质的基因 并使用克隆的DNA破坏染色体副本 野生型酵母中的每个基因。 在更多中检查系统 详细说明，我们将确定催化蛋白的区域 在与RAS蛋白相互作用的相互作用中很重要 膜和其他蛋白质。 具体，在框架中删除将 在CYR1基因的编码区域中制作，编码 催化多肽，所得的截短酶将是 检查功能损失。 替代策略将是 高水平的催化蛋白的孤立段 在野生型酵母中。 如果这些段正确折叠以产生 功能领域，他们将与同源域竞争 在正常酶上，并防止与蛋白质相互作用 通常与腺苷酸环化酶相关。 这将提供 功能领域位置的积极证据 催化多肽。