MEMBRANE DYNAMICS AND STEROL BIOSYNTHESIS IN YEAST

胆固醇是细胞膜的重要结构成分，但 不幸的是，它也是形成于 动脉壁在动脉粥样硬化疾病中的作用。这些存款增加了 易患高血压、中风和心脏病，因此 导致美国人死亡的主要原因是- 心血管疾病。胆固醇的合成受复合体的控制 调节HMG-CoA还原酶的数量、活性和周转。 这种酶是内质网的一个完整的膜蛋白， 从而占据了一个理想的位置来监测和响应类固醇水平或 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 膜合成和HMG-CoA还原酶本身。 HMG-CoA还原酶与细胞膜的结合是 调节蛋白质的半衰期和诱导特化 综合。本提案中提出的实验旨在 在基因易驯化的生物体中分析这些过程， 酿酒酵母。具体目标是：(1)获得 HMG-CoA还原酶的详细结构特征图 诱导膜合成；(2)鉴定参与细胞因子 该信号的转导和所产生的膜的合成； HMG-诱导的膜的纯化和成分分析 CoA还原酶水平；(4)研究这一生物发生和周转 膜阵列。这些目标将通过实验来实现， 细胞生物学、生物化学、经典和分子遗传学。这个 表达变异形式HMG-CoA还原酶Will的细胞超微结构 被检测以确定该蛋白质中存在的膜诱导信号， HMG-CoA还原酶的生物发生和降解研究 并测定HMG-CoA的定位和转运。 还原酶在细胞内的生命周期内。HMG-CoA还原酶诱导 膜将被分离并进行生化检查以确定 这些膜的结构组成，并与内源性进行比较 细胞内的膜。HMG-CoA还原酶的半衰期为 在一系列生化和超微结构的脉冲追逐中进行检查 实验。传统的遗传学方法将被用来识别基因 HMG-CoA还原酶对膜合成的影响 水平，以及表达改变的基因对HMG-CoA的响应 还原酶水平将通过分子遗传学进行鉴定和分析 技巧。结果将确定一般原则和模式 膜生物发生，并提供对这些关系的具体见解 膜结合力、HMG-CoA还原酶活性与 膜合成的诱导。这一信息具有医学意义 以了解胆固醇合成的调节，也可能 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, but unfortunately is also a major component of the deposits that form on arterial walls in the disease atherosclerosis. These deposits increase susceptibility to high blood pressure, stroke, and heart disease, thus contributing to the leading cause of death in the United States - cardiovascular disease. Synthesis of cholesterol 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, thus occupying 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 HMG-CoA reductase itself. Association of HMG-CoA reductase with cellular membranes is required for regulation of the protein's half-life and for induction of specialized synthesis. The experiments presented in this proposal are designed to analyze these processes in the genetically tractable organism, Saccharomyces cerevisiae. The specific goals are to: (1) obtain a detailed map of the structural features of HMG-CoA reductase that serve to induce membrane synthesis; (2) identify cellular factors involved in transduction of that signal and synthesis of the resulting membranes; (3) Purify and analyze the composition of membranes induced in response to HMG- CoA reductase levels; (4) Investigate the biogenesis and turnover of this membrane array. These goals will be achieved by experiments that integrate cell biology, biochemistry, and classical and molecular genetics. The ultrastructure of cells expressing altered forms of HMG-CoA reductase will be examined to define the membrane-inducing signal present in this protein, to study the biogenesis and degradation of HMG-CoA reductase-induced membranes, and to determine the localization and transit of HMG-CoA reductase during its lifetime within the cell. HMG-CoA reductase-induced membranes will be isolated and examined biochemically to identify structural components of these membranes and to compare them to endogenous membranes within the cell. The half-life of HMG-CoA reductase will be examined in a series of biochemical and ultrastructural pulse-chase experiments. Classical genetic approaches will be used to identify genes necessary for synthesis of membranes in response to HMG-CoA reductase levels, and genes whose expression is altered in response to HMG-CoA reductase levels will be identified and analyzed by molecular genetic techniques. Results will identify general principles and patterns 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.