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.
