Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Aug;11(8):3931–3939. doi: 10.1128/mcb.11.8.3931

In vivo assembly of yeast mitochondrial alpha-ketoglutarate dehydrogenase complex.

B Repetto 1, A Tzagoloff 1
PMCID: PMC361187  PMID: 2072900

Abstract

The assembly of alpha-ketoglutarate dehydrogenase complex (KGDC) has been studied in wild-type Saccharomyces cerevisiae and in respiratory-deficient strains (pet) with mutations in KGD1 and KGD2, the structural genes for alpha-ketoglutarate dehydrogenase (KE1) and dihydrolipoyl transsuccinylase (KE2) components, respectively. Mutants unable to express KE1 or KE2 form partial complexes similar to those reported in earlier studies on the resolution and reconstitution of bacterial and mammalian KGDC. Thus mutants lacking KE1 assemble a high-molecular-weight subcomplex consisting of a KE2 core particle with bound dihydrolipoyl dehydrogenase (E3). Similarly, mitochondrial extracts of mutants lacking KE2 contain dimeric KE1 and E3. These components, however, are not associated with each other. The partial complexes detected in the mutants are capable of reconstituting normal KGDC when supplied with the missing subunit. Complete restoration of overall alpha-ketoglutarate dehydrogenase activity is achieved by mixing appropriate ratios of mitochondrial extracts from mutants deficient in KE1 and KE2. The reconstitution of enzymatic activity correlates with binding of KE1 to the KE2-E3 particle to form a complex with the same sedimentation properties as wild-type KGDC. Overexpression of KE2 relative to KE1 results in a preponderance of incompletely assembled complexes with substoichiometric contents of KE1. Formation of a complex with a full complement of KE1 therefore depends on a balanced output of KE1 and KE2 from their respective genes. Biochemical screens of a pet mutant collection have led to the identification of a new gene required for the expression of enzymatically active KGDC. Mitochondria of the mutant have all of the catalytic subunits of KGDC. Sedimentation analysis of these components indicates that while the mutant has a stable KE2-E3 subcomplex, the interaction of KE1 with KE2 core is much weaker in the mutant than in the wild type. The gene product responsible for this phenotype, therefore, appears to function at a late stage of assembly of KGDC, most likely by posttranslational modification of one of the subunits.

Full text

PDF
3931

Images in this article

3932Image
on p.3932
3933Image
on p.3933
3933Image
on p.3933

Selected References

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

  1. Bates D. L., Danson M. J., Hale G., Hooper E. A., Perham R. N. Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Nature. 1977 Jul 28;268(5618):313–316. doi: 10.1038/268313a0. [DOI] [PubMed] [Google Scholar]
  2. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. Hirabayashi T., Harada T. Isolation and properties of -ketoglutarate dehydrogenase complex from baker's yeast (Saccharomyces cerevisiae). Biochem Biophys Res Commun. 1971 Dec 17;45(6):1369–1375. doi: 10.1016/0006-291x(71)90172-0. [DOI] [PubMed] [Google Scholar]
  7. Hirashima M., Hayakawa T., Koike M. Mammalian alpha-keto acid dehydrogenase complexes. II. An improved procedure for the preparation of 2-oxoglutarate dehydrogenase complex from pig heart muscle. J Biol Chem. 1967 Mar 10;242(5):902–907. [PubMed] [Google Scholar]
  8. KOIKE M., REED L. J., CARROLL W. R. alpha-Keto acid dehydrogenation complexes. I. Purification and properties of pyruvate and alpha-ketoglutarate dehydrogenation complexes of Escherichia coli. J Biol Chem. 1960 Jul;235:1924–1930. [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. 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]
  11. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  12. MUKHERJEE B. B., MATTHEWS J., HORNEY D. L., REED L. J. RESOLUTION AND RECONSTITUTION OF THE ESCHERICHIA COLI ALPHA-KETOGLUTARATE DEHYDROGENASE COMPLEX. J Biol Chem. 1965 May;240:2268–2269. [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Pettit F. H., Hamilton L., Munk P., Namihira G., Eley M. H., Willms C. R., Reed L. J. Alpha-keto acid dehydrogenase complexes. XIX. Subunit structure of the Escherichia coli alpha-ketoglutarate dehydrogenase complex. J Biol Chem. 1973 Aug 10;248(15):5282–5290. [PubMed] [Google Scholar]
  17. Pettit F. H., Reed L. J. Pyruvate dehydrogenase complex from bovine kidney and heart. Methods Enzymol. 1982;89(Pt 500):376–386. doi: 10.1016/s0076-6879(82)89067-8. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Repetto B., Tzagoloff A. Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase. Mol Cell Biol. 1990 Aug;10(8):4221–4232. doi: 10.1128/mcb.10.8.4221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Tanaka N., Koike K., Hamada M., Otsuka K. I., Suematsu T., Koike M. Mammalian -keto acid dehydrogenase complexes. VII. Resolution and reconstitution of the pig heart 2-oxoglutarate dehydrogenase complex. J Biol Chem. 1972 Jun 25;247(12):4043–4049. [PubMed] [Google Scholar]
  26. 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]
  27. Tzagoloff A., Dieckmann C. L. PET genes of Saccharomyces cerevisiae. Microbiol Rev. 1990 Sep;54(3):211–225. doi: 10.1128/mr.54.3.211-225.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Willms C. R., Oliver R. M., Henney H. R., Jr, Mukherjee B. B., Reed L. J. Alpha-keto acid dehydrogenase complexes. VI. Dissociation and reconstitution of the dihydrolipoyl transacetylase of Escherichia coli. J Biol Chem. 1967 Mar 10;242(5):889–897. [PubMed] [Google Scholar]
  29. Yeaman S. J. The 2-oxo acid dehydrogenase complexes: recent advances. Biochem J. 1989 Feb 1;257(3):625–632. doi: 10.1042/bj2570625. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES