Structure and regulation of KGD1, the structural gene for yeast alpha-ketoglutarate dehydrogenase.
Nuclear respiratory-defective mutants of Saccharomyces cerevisiae have been screened for lesions in the mitochondrial alpha-ketoglutarate dehydrogenase complex. Strains assigned to complementation group G70 were ascertained to be deficient in enzyme activity due to mutations in the KGD1 gene coding for the alpha-ketoglutarate dehydrogenase component of the complex. The KGD1 gene has been cloned by transformation of a representative kgd1 mutant, C225/U1, with a recombinant plasmid library of wild-type yeast nuclear DNA. Transformants containing the gene on a multicopy plasmid had three- to four-times-higher alpha-ketoglutarate dehydrogenase activity than did wild-type S. cerevisiae. Substitution of the chromosomal copy of KGD1 with a disrupted allele (kgd1::URA3) induced a deficiency in alpha-ketoglutarate dehydrogenase. The sequence of the cloned region of DNA which complements kgd1 mutants was found to have an open reading frame of 3,042 nucleotides capable of coding for a protein of Mw 114,470. The encoded protein had 38% identical residues with the reported sequence of alpha-ketoglutarate dehydrogenase from Escherichia coli. Two lines of evidence indicated that transcription of KGD1 is catabolite repressed. Higher steady-state levels of KGD1 mRNA were detected in wild-type yeast grown on the nonrepressible sugar galactose than in yeast grown on high glucose. Regulation of KGD1 was also studied by fusing different 5'-flanking regions of KGD1 to the lacZ gene of E. coli and measuring the expression of beta-galactosidase in yeast. Transformants harboring a fusion of 693 nucleotides of the 5'-flanking sequence expressed 10 times more beta-galactosidase activity when grown under derepressed conditions. The response to the carbon source was reduced dramatically when the same lacZ fusion was present in a hap2 or hap3 mutant. The promoter element(s) responsible for the regulated expression of KGD1 has been mapped to the -354 to -143 region. This region contained several putative activation sites with sequences matching the core element proposed to be essential for binding of the HAP2 and HAP3 regulatory proteins.
Repetto
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B
Department of Biological Sciences, Columbia University, New York, New York 10027.
Tzagoloff
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A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005947:Glucose / Q000502:physiology; D007655:Ketoglutarate Dehydrogenase Complex / Q000235:genetics*; D007658:Ketone Oxidoreductases / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D004271:DNA, Fungal; D012333:RNA, Messenger; D014157:Transcription Factors; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D001616:beta-Galactosidase; D005947:Glucose