ASSEMBLY AND FUNCTION OF A CYTOSOLIC FORM OF NADH-SPECIFIC ISOCITRATEDEHYDROGENASE IN YEAST

Mitochondrial NAD-dependent isocitrate dehydrogenase catalyzes a rate- limiting step in the tricarboxylic acid cycle. Yeast isocitrate dehydr ogenase is an octomer composed of two subunits (IDH1 and IDH2) encoded by different genes and possessing independent mitochondrial targeting presequences. Oligonucleotide-directed mutagenesis was used to remove the presequences from each gene and from both genes carried on centro mere-based expression plasmids. Effects on cellular localization were examined in a yeast strain containing chromosomal disruptions of IDH1 and IDH2 loci. Each subunit was found to be dependent upon its prese q uence for mitochondrial localization, and the subunits are independent ly imported into mitochondria under most growth conditions. Furthermor e, an active holoenzyme can be assembled in the cytosol and this ''cyt osolic'' form of isocitrate dehydrogenase can reverse the acetate(-) g rowth phenotype characteristic of the Delta IDH1/Delta IDH2 disruption strain, indicating functional replacement of the mitochondrial enzyme . However, transformants containing plasmids lacking either the IDH1 o r IDH2 presequence coding regions were unexpectedly found to be capabl e of growth on acetate medium. Further investigation demonstrated that cellular localization of the IDH1 subunit can be biased by this strin gent growth pressure.