Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae.

Abstract

The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADHI and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADHI and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly liasic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions ofADHI and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III.The alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae represent a functionally diverse enzyme family. ADH I, the classical fermentative isozyme, is responsible for the last step in the yeast glycolytic pathway, the reduction of acetaldehyde to ethanol (23,28). ADH II, the oxidative isozyme, is highly repressed by fermentative growth and is derepressed in the absence of a fermentable sugar such as glucose. ADH2, the structural gene for ADH II, is regulated at the transcriptional level by catabolite repression through a specific positive effector encoded by the ADRI gene (8, 10). The function of ADH II in the cell is to oxidize ethanol, formed during fermentation, to acetaldehyde, which can then be metabolized via the tricarboxylic cycle in the mitochondria and also serves as an intermediate in gluconeogenesis.ADH III was discovered by Lutsdorf and Megnet (23) and was shown to purify with a particulate fraction from S. cerevisiae. Sugar et al. (35) further characterized this activity and confirmed its location in the mitochondrion. Wills and Phelps (45) obtained evidence that the size of the native enzyme was the same as that of ADH I. Despite the same apparent molecular weight, the native ADH III enzyme migrates in an unusual pattern on nondenaturing polyacrylamide gels, suggesting charge heterogeneity on the subunits or formation of heterotetramers synthesized by two different genes or both.The metabolic function of ADH III is unknown, but indications are that it does not function fermentatively: strains containing ADH III as their only ADH isozyme cannot survive as petites, which must obtain their energy through fermentation (45). A role in respiratory metabolism is thus suggested, which would be consistent with a mitochondrial location. Molecular cloning of the structural gene for ADH III would aid in elucidating its structure and function. The structural genes encoding ADH I and ADH II, ADHI and ADH2, respectively, have been cloned and sequenced (3,30,41,42), allowing comparisons to be made * Corresponding author. ...

Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1

“…The next ORF, transcribed divergently from meaA, was shown to encode a polypeptide highly similar to catalase subunits from various sources (Murthy e t al., 1981;Schroeder e t al., 1982;Bell e t al., 1986;Furuta etal., 1986;Orr etal., 1990;Bol etal., 1991) with identities ranging from 40 to 57%, and was designated katA. The gene downstream of katA encodes a polypeptide with similarity to NAD-dependent ADHs belonging to different classes having specificity for shortchain alcohols ( 2 4 3 4 % identity; Russell e t al., 1983;Russell & Hall, 1983;Young & Pilgrim, 1985;Gwynne e t al., 1987 ;Saliola e t al., 1990) and was designated adbA. The polypeptide translated from the next ORF, transcribed divergently from adbA did not reveal strong identity to known proteins and was designated orf2.…”

“…The specificity of the product of a d b A is unknown, but the enzyme reveals highest similarity with ADHs having specificity for short-chain alcohols Russell & Hall, 1983;Young & Pilgrim, 1985;Gwynne et al, 1987;Saliola e t al., 1990), which are most active on ethanol. ADHs are usually readily stained in gels (Megnet, 1967).…”

The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

“…The mitochondiral components, Aat1 and Mdh1, convert mitochondrial malate into aspartate, with the reverse reactions occurring on the cytoplasmic side of the mitochondrial membrane. The ethanol-acetaldehyde shuttle, consisting of mitochondrial alcohol dehydrogenase (Adh3) (Young and Pilgrim 1985;Bakker et al 2000), and cytosolic alcohol dehydrogenase (Adh1/Adh2) (Thielen and Ciriacy 1991), creates a symmetrical shuttle system for the interconversion of ethanol and acetaldehyde between the mitochondrial and cytoplasmic pools. Because both ethanol and acetaldehyde diffuse freely across biological membranes, it cannot exchange NADH and NAD against a concentration gradient.…”

Synthesis and Assembly of Mitochondrial Proteins

“…Proteins: (1) 70 kDa protein (yeast), Hase et al (1983); (2) cytochrome c peroxidase (yeast), Kaput et al (1982), Reid et al (1982); (3) cytochrome b 2 (yeast), Guiard (1985), Gasser et al (1982b); (4) Rieske Fe/S of bc, complex (N. crassa), Harnisch et al (1985), Hartl et al (1986); (5) cytochrome d (yeast), Sadler et al (1984), Gasser et al (1982b); (6) cytochrome oxidase V (yeast), Koerner et aJ. (1985); (7) cytochrome oxidase V (N. crassa), Sachs et al (1986); (8) cytochrome oxidase IV (bovine), Lomax et al (1984); (9) cytochrome P-450 (SCC) (bovine), Morohashi et al (1984); (10) cytochrome oxidase VI (yeast), Wright et al (1984) ; (11) ATPase IX (N. crassa), Viebrock et al (1982), Schmidt et al (1984); (12) ATPase IX-Pl (bovine), Gay and Walker (1985); (13) ATPase IX-P2 (bovine), Gay and Walker (1985); (14) cytochrome oxidase IV (yeast), Maarse et al (1984); (15) citrate synthase (yeast), Suissa et al (1984); (16) Ornithine aminotransferase (rat), Mueckler and Pitot (1985), Simmaco et al (1986); (17) aspartate aminotransferase (chicken), Jaussi et al (1985) ; (18) aspartate aminotransferase (porcine), Joh et al (1985); (19) Mn-superoxide dismutase (yeast), Marres et al (1985); (20) ATPase F, 5 subunit (N. crassa), Kruse and Sebald (1984); (21) 5-aminolevulinate synthase (yeast), Keng et al (1986), Urban-Grimal et al (1986); (22) alcohol dehydrogenase iso-III (yeast), Young and Pilgrim (1985); (23) Ornithine carbamoyltransferase (human), Horwich et al (1984); (24) Ornithine carbamoyltransferase (rat), Mclntyre et al (1...…”