Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase.
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.
Minard
K I
KI
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
McAlister-Henn
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003062:Codon; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007527:Isoenzymes / Q000235:genetics*; D008291:Malate Dehydrogenase / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D003062:Codon; D005656:Fungal Proteins; D007527:Isoenzymes; D008291:Malate Dehydrogenase