MEMBRANE DYNAMICS AND STEROL BIOSYNTHESIS IN YEAST

胆固醇是细胞膜的重要结构成分， 不幸的是，它也是沉积物的主要成分， 动脉壁的动脉粥样硬化。 这些存款增加 易患高血压、中风和心脏病， 导致美国人死亡的主要原因 心血管疾病 胆固醇的合成受复合物控制 调节HMG-CoA还原酶的量、活性和周转。 这种酶是内质网的一种完整的膜蛋白， 因此占据了监测和响应甾醇水平的理想位置， 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 调节蛋白质的半衰期和诱导专门的 合成. 本提案中提出的实验旨在 分析这些在遗传上易处理的有机体中的过程， 酿酒酵母 具体目标是：（1）获得 HMG-CoA还原酶结构特征的详细图谱， 诱导膜合成;（2）鉴定参与 该信号的转导和所得膜的合成;（3） 纯化和分析响应HMG诱导的膜的组成- CoA还原酶水平;（4）研究这种酶的生物起源和周转。 膜阵列 这些目标将通过以下实验来实现， 细胞生物学，生物化学，经典和分子遗传学。 的 表达改变形式的HMG-CoA还原酶的细胞的超微结构将 进行检查以确定存在于该蛋白质中的膜诱导信号， 研究HMG-CoA还原酶诱导的HMG-CoA还原酶的生物起源和降解， 膜，并确定HMG-CoA的定位和运输 还原酶在细胞内的生命周期中发挥作用。 HMG-CoA还原酶诱导 膜将被分离并进行生物化学检查，以确定 这些膜的结构成分，并将它们与内源性 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脉冲追踪中进行了检查， 实验 经典的遗传学方法将被用来识别基因 响应HMG-CoA还原酶的膜合成所必需的 水平，以及表达响应HMG-CoA而改变的基因 还原酶水平将通过分子遗传学鉴定和分析。 技术. 结果将确定一般原则和模式， 膜生物发生，并提供具体的见解的关系 膜结合，HMG-CoA还原酶的活性， 膜合成的诱导。 这些信息具有医学意义 了解胆固醇合成的调节， 发现临床治疗的新方法， 体内胆固醇生物合成。

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.