MEMBRANE DYNAMICS AND STEROL BIOSYNTHESIS IN YEAST

胆固醇是细胞膜的重要结构成分， 合成重要代谢物的前体，包括胆汁 酸、维生素D和性激素。不幸的是，胆固醇也是一种 疾病中动脉壁上形成的沉积物的主要成分 动脉硬化。这些沉淀物阻塞血管，使血管减少。 弹性，增加高血压、中风和 心脏病，从而导致世界上最大的 美国。胆固醇的合成受复杂的调控 HMG-CoA还原酶的数量、活性和周转率。这种酶 是内质网的一种完整的膜蛋白，因此 担任监测和应对类固醇水平的理想职位 膜流动性。这项研究的长期目标是检查 HMG-CoA还原酶与细胞膜的相互作用 确定这些相互作用在调节两种细胞 膜的合成和HMG-CoA还原酶本身。 HMG-CoA还原酶与细胞膜的结合是 至少有两个过程：调节蛋白质的半衰期 对类固醇和诱导特化膜合成的响应 HMG-CoA还原酶水平。该提案中提出的实验包括 旨在分析遗传易驯化的生物体中的这些过程， 酿酒酵母。在过去的资助期间，我们有 鉴定了HMG-CoA还原酶蛋白的一个区域，该区域是 膜生物发生的诱导。此外，我们还鉴定了变种人 无法组装膜以响应增加的HMG-CoA 还原酶水平。为扩展这些研究，我们会：(1)完成 HMG-CoA还原酶和HMG-CoA还原酶膜诱导信号的详细分析 识别与这个蛋白质区域相互作用的蛋白质；(2)继续我们的 膜诱导信号介导的机制分析 通过以下方式进行特异膜组装：a)克隆我们 以前隔离的程序集、组织或 HMG-CoA还原酶诱导的膜的降解，b)识别基因 其转录在高水平的细胞中被诱导或上调 HMG-CoA还原酶，以及c)分析HMG-CoA中存在的蛋白质 还原酶诱导的膜及其与HMG-CoA还原酶的关系。 这些目标将通过结合细胞生物学的实验实现， 生物化学、古典遗传学和分子遗传学。 这些实验的结果将揭示出 膜生物发生的调节，并提供对 膜缔合作用与HMG-CoA活性的关系 还原酶，以及对膜合成的诱导。此信息具有 了解胆固醇调节的医学意义 合成，还可能发现临床治疗的新方法 旨在控制体内胆固醇的生物合成。

Cholesterol is an important structural component of cellular membranes and a precursor for the synthesis of important metabolites, including bile acids, vitamin D, and sex hormones. Unfortunately, cholesterol is also a major component of the deposits that form on arterial walls in the disease atherosclerosis. These deposits block blood vessels and decrease their elasticity, increasing susceptibility to high blood pressure, stroke, and heart disease, thus contributing to the leading cause of death in the United States. Cholesterol synthesis is controlled by complex regulation of the amount, activity, and turnover of HMG-CoA reductase. This enzyme is an integral membrane protein of the endoplasmic reticulum and, thus occupies an ideal position to monitor and respond to sterol levels or membrane fluidity. The long-term objective of this research is to examine the interactions of HMG-CoA reductase with cellular membranes and to determine the role of these interactions in regulation of both cellular membrane synthesis and of HMG-CoA reductase itself. Association of HMG-CoA reductase with cellular membranes is required for at least two processes: regulation of the protein's half-life in response to sterols and induction of specialized membrane synthesis in response to HMG-CoA reductase levels. The experiments presented in this proposal are designed to analyze these processes in the genetically tractable organism, Saccharomyces cerevisiae. During the past funding period, we have identified a region of the HMG-CoA reductase protein that is necessary for induction of membrane biogenesis. In addition, we have identified mutants that are unable to assemble membranes in response to increased HMG-CoA reductase levels. To extend these studies, we will: (1) Complete a detailed analysis of the membrane-inducing signal in HMG-CoA reductase and identify proteins that interact with this protein region; (2) Continue our analysis of the mechanisms by which this membrane-inducing signal mediates specific membrane assembly by: a) cloning the essential genes which we previously isolated that are needed for the assembly, organization, or degradation of HMG-CoA reductase-induced membranes, b) identifying genes whose transcription is induced or elevated in cells with high levels of HMG-CoA reductase, and c) analyzing the proteins present in HMG-CoA reductase-induced membranes and their association with HMG-CoA reductase. These goals will be achieved by experiments that integrate cell biology, biochemistry, and classical and molecular genetics. Results of these experiments will uncover general features of the regulation of membrane biogenesis and provide specific insights into the relationships between membrane association, the activity of HMG-CoA reductase, and the induction of membrane synthesis. This information has medical significance for understanding the regulation of cholesterol synthesis and may also uncover novel approaches for clinical therapies aimed at controlling cholesterol biosynthesis in vivo.