Expression and gene disruption analysis of the isocitrate dehydrogenase family in yeast.

Abstract

Mammalian and yeast cells contain three isozymes of isocitrate dehydrogenase: mitochondrial NAD- and NADP-specific enzymes and a cytosolic NADP-specific enzyme. Independent metabolic functions of these enzymes in Saccharomyces cerevisiae were examined by analyses of expression and of phenotypes displayed by mutants containing all possible combinations of isozyme gene disruptions. All three isocitrate dehydrogenases are expressed at high levels with growth on nonfermentable carbon sources, whereas the mitochondrial NADP-specific enzyme constitutes the major cellular activity with growth on glucose. Distinct growth phenotypes 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 compartments 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 does not compensate for function of the yeast NAD-specific tricarboxylic acid cycle enzyme.