Considerable evidence suggests key metabolic functions for compartmentalized isozymes of isocitrate dehydrogenase in eurcaryotic cells: the mitochondrial NAD+-specific enzyme as the site for control of rates of aerobic metabolism and the cytoplasmic NADP+-specific enzyme as a critical source of biosynthetic reducting equivalents. Also, through function in the isocitrate/alpha-ketoglutarate shuttle cycle, these isozymes coordinate and regulate intercompartmental communication. This research will provide direct experimental proof and measurements of these functions in addition to clarifying the number and expression of genes encoding these enzymes. The mitochondrial and cytoplasmic isozymes of isocitrate dehydrogernase will be isolated from the yeast Saccharomyces cervisiae for direct biochemical study of enzyme functions and structures and for the production of isozyme specific antisera. Immunochemical methods will be used to examine isozyme expression and to isolate genes encoding the isozymes from a yeast genomic DNA expression library. The identities of isolated genes and the physiological consequences of interruption of NAD+- or NADP+-specific isociatrate dehydrogenase function will be assessed in null mutants of yeast constructed by gene replacement. The isolated genes will also be used as hybridization probes in Northern blot analyses to examine gene expression, for nucleic acid sequence analyses, and for in vitro mutagenesis designed to produce temperature-sensitive mutations which affect various aspects of isozyme function in vivo.
Specific aims of this research are to determine (1) the number and functions of individual subunits of the mitochondrial NAD+-specific isocitrate dehydrogenase, a complex allosteric enzyme, (2) the number, cellular localization, and functions of NADP+-specific isozyme(s) in eucaryotic cells, (3) the effect of various metabolic conditions on expression of isocitrate dehydrogenase genes and isozymes, and (4) in vivo functions of these compartmentalized isozymes in corrdinating central metabolic pathways separated by the mitochondrial membrane.
