Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation.
Mutants of Saccharomyces cerevisiae deficient in mitochondrial aldehyde dehydrogenase (ALDH) activity were isolated by chemical mutagenesis with ethyl methanesulfonate. The mutants were selected by their inability to grow on ethanol as the sole carbon source. The ALDH mutants were distinguished from alcohol dehydrogenase mutants by an aldehyde indicator plate test and by immunoscreening. The ALDH gene was isolated from a yeast genomic DNA library on a 5.7-kb insert of a recombinant DNA plasmid by functional complementation of the aldh mutation in S. cerevisiae. An open reading frame which specifies 533 codons was found within the 2.0-kb BamHI-BstEII fragment in the 5.7-kb genomic insert which can encode a protein with a molecular weight of 58,630. The N-terminal portion of the protein contains many positively charged residues which may serve as a signal sequence that targets the protein to the mitochondria. The amino acid sequence of the proposed mature yeast enzyme shows 30% identity to each of the known ALDH sequences from eukaryotes or prokaryotes. The amino acid residues corresponding to mammalian cysteine 302 and glutamates 268 and 487, implicated to be involved at the active site, were conserved. S. cerevisiae ALDH was found to be localized in the mitochondria as a tetrameric enzyme. Thus, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.
Saigal
D
D
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Cunningham
S J
SJ
Farrés
J
J
Weiner
H
H
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006801:Humans; D007526:Isoelectric Point; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D000444:Aldehyde Dehydrogenase