The long-term aim of this proposal is to learn how the pathway that leads to the synthesis of sterols such as cholesterol and ergosterol is regulated. In particular, we want to determine which products of this pathway act as feedback regulators of the pathway. In addition, we will study the mechanism that serves to coordinate the control of several steps in this pathway. Our investigations will begin with the structural gene for 3-hydroxy-3-methylglutaryl Coenzyme A reductase in Saccharomyces cerevisiae. This enzyme is rate-limiting for sterol biosynthesis in all organisms studied. The regulation of this gene will be studied by constructing recombinant clones that fuse the reductase structural gene to the E. coli B-galactosidase gene. Expression of the reductase gene can then be assayed by convenient in vivo and in vitro assays. We will determine if yeast have both sterol and nonsterol regulators of reductase, analogous to the regulation of reductase in mammalian cells. If so, we will identify the regulators, and in particular the nonsterol regulator, by assaying mutants of the sterol biosynthetic pathway for those that no longer make the regulator and thus lose feedback control of reductase activity. In addition, the use of gene fusions should allow us to isolate mutants defective in regulation of reductase synthesis by their effects on synthesis of the fusion protein. These mutants will allow us to determine to what extent the genes for other proteins in this pathway are coordinately regulated with reductase. Experiments with animal cell reductase indicate that the enzyme is subject to a variety of post-translational influences on activity levels. We will raise antisera against yeast reductase and use this antisera to monitor covalent modifications of reductase or changes in its half-life under conditions that influence the activity level of reductase. If we find in vitro evidence for post-translational regulation, we will determine the magnitude of the in vivo role of these effects by isolating mutants deficient in these forms on regulation. Heart disease is the leading cause of death in the United States. The control of cholesterol metabolism is one of several important elements in treating patients with dangerously high cholesterol levels. Effective pharmacological treatment of these patients will be most practical when all of the regulators of reductase activity have been identified.
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