EXPRESSION AND GENE DISRUPTION ANALYSIS OF THE ISOCITRATE DEHYDROGENASE FAMILY IN YEAST

Mammalian and yeast cells contain three isozymes of isocitrate dehydro genase: mitochondrial NAD- and NADP-specific enzymes and a cytosolic N ADP-specific enzyme. Independent metabolic functions of these enzymes in Saccharomyces cerevisiae were examined by analyses of expression an d of phenotypes displayed by mutants containing all possible combinati ons of isozyme gene disruptions. All three isocitrate dehydrogenases a re expressed at high levels with growth on nonfermentable carbon sourc es, whereas the mitochondrial NADP-specific enzyme constitutes the maj or cellular activity with growth on glucose. Distinct growth phenotype s are observed for mutants expressing a single isozyme, and expression of at least one isozyme is necessary for glutamate-independent growth . The NADP-specific tricarboxylic acid cycle isocitrate dehydrogenase from Escherichia coli was expressed in mitochondrial and cytosolic com partments of the yeast disruption mutants using plasmids carrying gene fusions of yeast promoters and a mitochondrial targeting presequence with the bacterial coding sequence. The bacterial enzyme is competent for restoration of NADP-specific functions in either compartment but d oes not compensate for function of the yeast NAD-specific tricarboxyli c acid cycle enzyme.