METABOLIC EFFECTS OF ALTERING REDUNDANT TARGETING SIGNALS FOR YEAST MITOCHONDRIAL MALATE-DEHYDROGENASE

Eukaryotic cells contain highly homologous isozymes of malate dehydrog enase which catalyze the same reaction in different cellular compartme nts. To examine whether the metabolic functions of these isozymes are interchangeable, we have altered the cellular localization of mitochon drial malate dehydrogenase (MDH1) in yeast. Since a previous study sho wed that removal of the targeting presequence from MDH1 does not preve nt mitochondrial import in vivo, we tested the role of a putative cryp tic targeting sequence near the amino terminus of the mature polypepti de. Three residues in this region were changed to residues present in analogous positions in the other two yeast MDH isozymes. Alone, these replacements did not affect activity or localization of MDH1 but, in c ombination with deletion of the presequence, prevented mitochondrial i mport in vivo. Measurable levels of the resulting cytosolic form of MD H1 were low with expression from a centromere-based plasmid but were c omparable to normal cellular levels with expression from a multicopy p lasmid. The cytosolic form of MDH1 restored the ability of a Delta MDH 1 disruption strain to grow on ethanol or acetate, suggesting that mit ochondrial localization of MDH1 is not essential for its function in t he TCA cycle. This TCA cycle function observed for the cytosolic form of MDH1 is unique to that isozyme since overexpression of MDH2 and of a cytosolic form of MDH3 in a Delta MDH1 strain failed to restore grow th. Finally, only partial restoration of growth of a Delta MDH2 disrup tion mutant was attained with the cytosolic form of MDH1, suggesting t hat MDH2 may also have unique metabolic functions. (C) 1997 Academic P ress.