Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

Abstract

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. Thius, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.Saccharomyces cerevisiae, unlike higher eukaryotes, can metabolize as well as produce ethanol. During fermentation, the consumption of sugars results in the accumulation of ethanol. When glucose or any other fermentable sugar is absent from the culture medium, S. cerevisiae can utilize ethanol as the carbon source aerobically. The metabolism of ethanol is well understood in mammals. In the liver, cytosolic alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde and then mitochondrial aldehyde dehydrogenase (ALDH) oxidizes the intermediate to acetate (14). In S. cerevisiae, it appears that a mitochondrial alcohol dehydrogenase (ADH3) is responsible for the initial oxidation (65,67). Little is known about the role of ALDHs in the next step.Various groups have purified and characterized yeast ALDH (12,49,54). It was reported to be a tetrameric enzyme with a subunit molecular mass of 60 kDa and a low Km for acetaldehyde. Unlike the liver enzymes, the yeast enzyme is activated by K+ ions (4, 32). Whereas the mammalian enzymes have been sequenced at the cDNA (16,19,29,34) as well as the protein (24, 33, 60) level, no sequence work has been reported for the yeast enzyme.One of our interests was to study the subcellular localization of acetaldehyde metabolism in S. cerevisiae. In the mammalian liver tissue, this was studied by selectively inhibiting the cytosolic or the mitochondrial isozyme of ALDH (11,52). An alternative approach would be to introduce the enzyme into a cell deficient in ALDH activity. S. cerevisiae would be a suitable model system, as it can metabolize ethanol and could serve as a host for the expression of foreign g...

Response to acetaldehyde stress in the yeast Saccharomyces cerevisiae involves a strain‐dependent regulation of several ALD genes and is mediatedby the general stress response pathway

“…Actually, it was lower in cells grown in glucose than in cells from ethanol cultures, in agreement with previous studies on enzyme activities (Wang et al, 1998;Saigal et al, 1991). This result was obtained for all the strains considered and the differences ranged between 5-and 20-fold, as shown in panel A for C strain (approximately 18-fold).…”

“…The pattern is highly dependent on the genetic background in the case of the industrial strains, as described by other authors for laboratory strains (Boubekeur et al, 2001). ALD6, encoding the major cytoplasmic enzyme, shows higher mRNA levels under basal conditions in glucose than in ethanol, while for ALD4, which encodes the major mitochondrial enzyme, the opposite situation occurs, as expected for the reported contribution of the corresponding proteins for growth on these carbon sources (Saigal et al, 1991). But some intriguing data have also been obtained.…”

“…The major mitochondrial enzyme uses NAD + and NADP + as co-enzymes, it is activated by K + and thiols and its expression is highly glucose-repressed (Jacobson and Bernofsky, 1974). Traditionally, cytosolic enzymes have been considered responsible for the formation of acetate from glucose while the main role of mitochondrial enzymes would take place during growth on ethanol (Wang et al, 1998;Saigal et al, 1991). However, growth behaviour analysis of mutants in genes encoding aldehyde dehydrogenases have indicated that mitochondrial and cytosolic enzymes may -at least partially and under several conditions -compensate each other (Boubekeur et al, 1999(Boubekeur et al, , 2001Remize et al, 2000).…”

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

“…Comparison of the amino acid sequence of AcDH-II with those of proteins collected in the NBRF data bank revealed extended homologies with aldehyde dehydrogenases (AlDH) from other sources: mitochondrial AlDHs from human liver (44.5% identical amino acids [18]), horse liver (43.5% [23]), rat liver (43.5% [11]), bovine liver (46.5% [11]), or Saccharomyces cerevisiae (23.6% [39]); cytoplasmic AlDHs from human liver (42.3% [19]), horse liver (41.2% [51]), or rat liver (41.1% [9]); AlDHs from Aspergillus nidulans (42.6% [35]), Aspergillus niger (43.5% [31]), E. coli (40.0% [17]), or Pseudomonas oleovorans (28.6% [26]). The alignment of the amino acid sequences of AcDH-II and of six representative AlDHs is shown in Fig.…”

Aldehyde dehydrogenases: Widespread structural and functional diversity within a shared framework

“…A sequence reported from yeast (Saigal et al, 1991) has been omitted because certain portions of that sequence have been questioned by these authors.…”