A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity

Abstract

Electrophoretically distinguishable mutants of yeast alcohol dehydrogenase with specifically altered kinetics have been obtained using a modification of the allyl alcohol selection technique of Megnet. The modification depends on the fact that mutant cells producing no detectable ADH or only the ADH sequestered inside the mitochondria cannot become petite. Allyl alcohol is oxidized by yeast ADH to the poisonous compound acrolein, so petite cells possessing a cytoplasmic ADH will die unless the structural gene is altered to permit the enzyme to distinguish between ethanol and allyl alcohol. The independent nuclear control of yeast mitochondrial ADH, suspected by other workers, is demonstrated. The fact that strains carrying this ADH cannot become petite provides direct genetic evidence that mitochondrial and cytoplasmic NAD pools are separate in Saccharomyces cerevisiae.