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（包括胆汁）的前体 酸、维生素D和性激素。 胆固醇也是一种 在疾病中动脉壁上形成的沉积物的主要成分 动脉粥样硬化 这些沉积物阻塞血管， 弹性，增加对高血压，中风的易感性， 心脏病，从而导致死亡的主要原因， 美国的 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， 占据了监测和响应固醇水平的理想位置， 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细胞内 膜合成和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， 和诱导专门的膜合成， HMG-CoA还原酶水平。 该提案中提出的实验是 旨在分析这些过程中的遗传听话的有机体， 酿酒酵母 在过去的资助期内，我们 鉴定了HMG-CoA还原酶蛋白的一个区域， 诱导膜生物发生。 另外，我们还发现了 它们不能响应增加的HMG-CoA组装膜 还原酶水平。 为了进一步研究，我们会：（1）完成一项 详细分析HMG-CoA还原酶中的膜诱导信号， 鉴定与该蛋白质区域相互作用的蛋白质;（2）继续我们的 分析这种膜诱导信号介导 特异性膜组装：a）克隆我们 以前隔离的组件、组织或 HMG-CoA还原酶诱导的膜的降解，B）鉴定基因 其转录在具有高水平转录因子的细胞中被诱导或升高。 HMG-CoA还原酶，和c）分析HMG-CoA中存在的蛋白质 还原酶诱导的膜及其与HMG-CoA还原酶的结合。 这些目标将通过整合细胞生物学， 生物化学，经典和分子遗传学。 这些实验的结果将揭示 调节膜生物发生，并提供具体的见解， 膜结合、HMG-CoA活性 还原酶和膜合成的诱导。 这一信息 了解胆固醇调节的医学意义 合成，也可能发现新的临床治疗方法 旨在控制体内胆固醇生物合成。

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.