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.
