Isolation, Nucleotide Sequence Analysis, and Disruption of the MDH2 Gene from Saccharomyces cerevisiae: Evidence for Three Isozymes of Yeast Malate Dehydrogenase

Karyl I. Minard, Lee McAlister-Henn

Research output: Contribution to journalArticle

80 Scopus citations

Abstract

The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000. Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2. Transformation and complementation of the acetate growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library. The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins. Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source. Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDH1 gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source. The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.

Original languageEnglish (US)
Pages (from-to)370-380
Number of pages11
JournalMolecular and cellular biology
Volume11
Issue number1
DOIs
StatePublished - Jan 1991

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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