We have shown that the transport of anions across the mitochondrial membrane is involved in the regulation of the inducible alcohol dehydrogenase (ADH-II) of yeast. This regulation is in turn tied in with the regulation of glycolysis through another set of regulatory genes. We have been able to rule out the transport mechanisms involved in the malate-aspartate shuttle, and have accumulated some evidence that the ATP-ADP shuttle is not involved. The shuttle with the greatest effect may be the dicarboxylate shuttle, but other shuttle systems may be more directly involved and will be investigated during this project. Mutants enhancing the efficiency of the malate-asparatate shuttle have been isolated from cells grown under conditions in which the transport of reducing equivalents into the mitochondrion are rate-limiting. We are now able to produce mutants that both enhance and interfere with this and other shuttles. The effect of these mutants on ADH regulation and the genetics and physiology of the mutants themselves will be investigated. They are, to our knowledge, the first found in any organism that affect these fundamental cellular processes. At the same time, we will clone CCR1, one of the two most important regulatory genes of ADH. This gene is also involved in the regulation of a number of glycolytic enzymes. A search for small molecules with anti-inducer or apo-repressor properties will continue, and the interaction of these small molecules with regulatory proteins will be investigated. The work will employ the discipline of biochemistry, physiology, genetics and molecular biology. An increase in our understanding of the regulation of glycolysis in yeast will have application to many other types of cells, because the process is of such fundamental importance to cellular physiology.
