The overall goal of this research is to examine the importance of compartmentation in control of metabolism in eucaryotic cells. A basic hypothesis to be tested is that features other than catalytic activity may be essential for optimal function of an enzyme in vivo. The focus of this work is the family of three differentially compartmentalized isozymes of malate dehydrogenases (MDH's) in Saccharomyces cerevisiae. These enzymes catalyze a reaction with a very unfavorable equilibrium for production of oxaloacetate and it has been proposed that direct interaction with other enzymes in specific pathways would facilitate correct delivery of this limiting metabolite. Three specific aims of this proposal are designed to define structural features of the mitochondrial MDH1 enzyme and of the cytosolic MDH2 enzyme that contribute to respective functions in the tricarboxylic acid cycle and in gluconeogenesis: (1) A physical association between two yeast cytosolic gluconeogenic enzymes, MDH2 and phosphoenolpyruvate carboxykinase, has been observed using the yeast two-hybrid system as an assay. The specificity, structural basis, and metabolic significance of this interaction will be examined using molecular genetic techniques and appropriate yeast mutants. (2) The two-hybrid system and other techniques will be used to test interactions between the yeast tricarboxylic acid cycle enzymes, MDH1 and citrate synthase. (3) To determine if MDH1 has structural attributes other than catalytic activity that impact function in the tricarboxylic acid cycle, a mitochondrial form of cytosolic MDH2 will be engineered and expressed in a yeast mutant lacking mitochondrial MDH1. The final specific aim (4) is designed to determine the requirements for each of the central metabolic pathways involving malate dehydrogenases in the normal developmental processes of mating and sporulation in yeast. This is technically a simple aim but it has broad implications; it will be used as a teaching tool for students desiring experience in a research laboratory. SIGNIFICANCE: Results of this research will contribute to the depth of our understanding of the mechanisms for regulation of central metabolic pathways in vivo and, consequently, to an appreciation of the molecular basis for dysfunctions at a new level of regulation. For this project, a model organism that is uniquely amenable to molecular genetic manipulation will be used. Nevertheless, the results are expected to be broadly applicable and could have a significant impact on current views about metabolic regulation as well as about the selective pressures that drive the evolution of enzyme structure and function. In addition, this research will promote the education and training of student personnel directly involved in the project.
