Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Aug;10(8):4221–4232. doi: 10.1128/mcb.10.8.4221

Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase.

B Repetto 1, A Tzagoloff 1
PMCID: PMC360958  PMID: 2115121

Abstract

Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex. The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library. Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex. Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate. The expression of KGD2 was transcriptionally regulated by glucose. Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides. The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells. Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E. coli in episomal plasmids, and the resultant constructs were tested for expression of beta-galactosidase in wild-type yeast cells and in hap2 and hap3 mutants. Results of the lacZ fusion assays indicated that transcription of KGD2 is activated by the HAP2 and HAP3 proteins. The regulated expression of KGD2 was found to depend on sequences that map to a region 244 to 484 nucleotides upstream of the structural gene. This region contains two short sequence elements that differ by one nucleotide from the consensus core (5'-TN[A/G]TTGGT-3') that has been proposed to be essential for binding of the HAP activation complex. These data together with earlier reports on the regulation of the KGD1 and LPD1 genes for the alpha-ketoglutarate and dihydrolipoyl dehydrogenases indicate that all three enzyme components of the complex are catabolite repressed and subject to positive regulation by the HAP2 and HAP3 proteins.

Full text

PDF
4232

Images in this article

4227Image
on p.4227
4227Image
on p.4227
4228Image
on p.4228
4228Image
on p.4228
4229Image
on p.4229

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Browning K. S., Uhlinger D. J., Reed L. J. Nucleotide sequence for yeast dihydrolipoamide dehydrogenase. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1831–1834. doi: 10.1073/pnas.85.6.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Darlison M. G., Spencer M. E., Guest J. R. Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12. Eur J Biochem. 1984 Jun 1;141(2):351–359. doi: 10.1111/j.1432-1033.1984.tb08199.x. [DOI] [PubMed] [Google Scholar]
  6. Dickinson J. R., Roy D. J., Dawes I. W. A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae. Mol Gen Genet. 1986 Jul;204(1):103–107. doi: 10.1007/BF00330195. [DOI] [PubMed] [Google Scholar]
  7. Faye G., Kujawa C., Fukuhara H. Physical and genetic organization of petite and grande yeast mitochondrial DNA. IV. In vivo transcription products of mitochondrial DNA and localization of 23 S ribosomal RNA in petite mutants of saccharomyces cerevisiae. J Mol Biol. 1974 Sep 5;88(1):185–203. doi: 10.1016/0022-2836(74)90304-0. [DOI] [PubMed] [Google Scholar]
  8. Forsburg S. L., Guarente L. Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev. 1989 Aug;3(8):1166–1178. doi: 10.1101/gad.3.8.1166. [DOI] [PubMed] [Google Scholar]
  9. Forsburg S. L., Guarente L. Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site. Mol Cell Biol. 1988 Feb;8(2):647–654. doi: 10.1128/mcb.8.2.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  11. Hahn S., Guarente L. Yeast HAP2 and HAP3: transcriptional activators in a heteromeric complex. Science. 1988 Apr 15;240(4850):317–321. doi: 10.1126/science.2832951. [DOI] [PubMed] [Google Scholar]
  12. Hartl F. U., Pfanner N., Nicholson D. W., Neupert W. Mitochondrial protein import. Biochim Biophys Acta. 1989 Jan 18;988(1):1–45. doi: 10.1016/0304-4157(89)90002-6. [DOI] [PubMed] [Google Scholar]
  13. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  14. Jue R. A., Woodbury N. W., Doolittle R. F. Sequence homologies among E. coli ribosomal proteins: evidence for evolutionarily related groupings and internal duplications. J Mol Evol. 1980 May;15(2):129–148. doi: 10.1007/BF01732666. [DOI] [PubMed] [Google Scholar]
  15. Keng T., Guarente L. Constitutive expression of the yeast HEM1 gene is actually a composite of activation and repression. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9113–9117. doi: 10.1073/pnas.84.24.9113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Laughon A., Gesteland R. F. Isolation and preliminary characterization of the GAL4 gene, a positive regulator of transcription in yeast. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6827–6831. doi: 10.1073/pnas.79.22.6827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Myers A. M., Crivellone M. D., Koerner T. J., Tzagoloff A. Characterization of the yeast HEM2 gene and transcriptional regulation of COX5 and COR1 by heme. J Biol Chem. 1987 Dec 15;262(35):16822–16829. [PubMed] [Google Scholar]
  20. Myers A. M., Pape L. K., Tzagoloff A. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J. 1985 Aug;4(8):2087–2092. doi: 10.1002/j.1460-2075.1985.tb03896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Myers A. M., Tzagoloff A., Kinney D. M., Lusty C. J. Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene. 1986;45(3):299–310. doi: 10.1016/0378-1119(86)90028-4. [DOI] [PubMed] [Google Scholar]
  22. Myers A. M., Tzagoloff A. MSW, a yeast gene coding for mitochondrial tryptophanyl-tRNA synthetase. J Biol Chem. 1985 Dec 5;260(28):15371–15377. [PubMed] [Google Scholar]
  23. Niu X. D., Browning K. S., Behal R. H., Reed L. J. Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7546–7550. doi: 10.1073/pnas.85.20.7546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Olesen J., Hahn S., Guarente L. Yeast HAP2 and HAP3 activators both bind to the CYC1 upstream activation site, UAS2, in an interdependent manner. Cell. 1987 Dec 24;51(6):953–961. doi: 10.1016/0092-8674(87)90582-4. [DOI] [PubMed] [Google Scholar]
  25. Packman L. C., Perham R. N. Chain folding in the dihydrolipoyl acyltransferase components of the 2-oxo-acid dehydrogenase complexes from Escherichia coli. Identification of a segment involved in binding the E3 subunit. FEBS Lett. 1986 Oct 6;206(2):193–198. doi: 10.1016/0014-5793(86)80979-6. [DOI] [PubMed] [Google Scholar]
  26. Packman L. C., Perham R. N. Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components. Biochem J. 1987 Mar 1;242(2):531–538. doi: 10.1042/bj2420531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Perlman P. S., Mahler H. R. Derepression of mitochondria and their enzymes in yeast: regulatory aspects. Arch Biochem Biophys. 1974 May;162(1):248–271. doi: 10.1016/0003-9861(74)90125-8. [DOI] [PubMed] [Google Scholar]
  28. Pinkham J. L., Guarente L. Cloning and molecular analysis of the HAP2 locus: a global regulator of respiratory genes in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Dec;5(12):3410–3416. doi: 10.1128/mcb.5.12.3410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Repetto B., Tzagoloff A. Structure and regulation of KGD1, the structural gene for yeast alpha-ketoglutarate dehydrogenase. Mol Cell Biol. 1989 Jun;9(6):2695–2705. doi: 10.1128/mcb.9.6.2695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ross J., Reid G. A., Dawes I. W. The nucleotide sequence of the LPD1 gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae: comparison between eukaryotic and prokaryotic sequences for related enzymes and identification of potential upstream control sites. J Gen Microbiol. 1988 May;134(5):1131–1139. doi: 10.1099/00221287-134-5-1131. [DOI] [PubMed] [Google Scholar]
  31. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  32. Roy D. J., Dawes I. W. Cloning and characterization of the gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae. J Gen Microbiol. 1987 Apr;133(4):925–933. doi: 10.1099/00221287-133-4-925. [DOI] [PubMed] [Google Scholar]
  33. Spencer M. E., Darlison M. G., Stephens P. E., Duckenfield I. K., Guest J. R. Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase. Eur J Biochem. 1984 Jun 1;141(2):361–374. doi: 10.1111/j.1432-1033.1984.tb08200.x. [DOI] [PubMed] [Google Scholar]
  34. Spencer M. E., Guest J. R. Transcription analysis of the sucAB, aceEF and lpd genes of Escherichia coli. Mol Gen Genet. 1985;200(1):145–154. doi: 10.1007/BF00383328. [DOI] [PubMed] [Google Scholar]
  35. Stephens P. E., Darlison M. G., Lewis H. M., Guest J. R. The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component. Eur J Biochem. 1983 Jul 1;133(3):481–489. doi: 10.1111/j.1432-1033.1983.tb07490.x. [DOI] [PubMed] [Google Scholar]
  36. Stephens P. E., Lewis H. M., Darlison M. G., Guest J. R. Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12. Eur J Biochem. 1983 Oct 3;135(3):519–527. doi: 10.1111/j.1432-1033.1983.tb07683.x. [DOI] [PubMed] [Google Scholar]
  37. Trawick J. D., Rogness C., Poyton R. O. Identification of an upstream activation sequence and other cis-acting elements required for transcription of COX6 from Saccharomyces cerevisiae. Mol Cell Biol. 1989 Dec;9(12):5350–5358. doi: 10.1128/mcb.9.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tzagoloff A., Akai A., Foury F. Assembly of the mitochondrial membrane system XVI. Modified form of the ATPase proteolipid in oligomycin-resistant mutants of Saccharomyces cerevisiae. FEBS Lett. 1976 Jun 15;65(3):391–395. doi: 10.1016/0014-5793(76)80154-8. [DOI] [PubMed] [Google Scholar]
  39. Tzagoloff A., Akai A., Kurkulos M., Repetto B. Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli. J Biol Chem. 1988 Jan 15;263(2):850–856. [PubMed] [Google Scholar]
  40. Tzagoloff A., Akai A., Needleman R. B. Assembly of the mitochondrial membrane system: isolation of nuclear and cytoplasmic mutants of Saccharomyces cerevisiae with specific defects in mitochondrial functions. J Bacteriol. 1975 Jun;122(3):826–831. doi: 10.1128/jb.122.3.826-831.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES