Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Jul;10(7):3551–3561. doi: 10.1128/mcb.10.7.3551

Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate.

S P Gangloff 1, D Marguet 1, G J Lauquin 1
PMCID: PMC360790  PMID: 1972545

Abstract

We have isolated genomic clones complementing the aconitase-deficient strain (glu1-1) of Saccharomyces cerevisiae. Identification of the aconitase gene was established by enzymatic assays and molecular analyses. The corresponding mRNA has been characterized, and its direction of transcription has been determined. The complete nucleotide sequence revealed strong amino acid homologies with the sequences of some peptides isolated from the mammalian protein. Disruption of the gene by deletion-insertion led to glutamate auxotrophy. Expression of the aconitase gene was sensitive to glucose repression and was synergistically down regulated by glucose and glutamate.

Full text

PDF

Images in this article

3553Image
on p.3553
3554Image
on p.3554
3555Image
on p.3555
3559Image
on p.3559

Selected References

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

  1. 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]
  2. Beinert H. Iron-sulphur clusters: agents of electron transfer and storage, and direct participants in enzymic reactions. Tenth Keilin memorial lecture. Biochem Soc Trans. 1986 Jun;14(3):527–533. doi: 10.1042/bst0140527. [DOI] [PubMed] [Google Scholar]
  3. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  4. Chatton B., Walter P., Ebel J. P., Lacroute F., Fasiolo F. The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. J Biol Chem. 1988 Jan 5;263(1):52–57. [PubMed] [Google Scholar]
  5. Duntze W., Neumann D., Gancedo J. M., Atzpodien W., Holzer H. Studies on the regulation and localization of the glyoxylate cycle enzymes in Saccharomyces cerevisiae. Eur J Biochem. 1969 Aug;10(1):83–89. doi: 10.1111/j.1432-1033.1969.tb00658.x. [DOI] [PubMed] [Google Scholar]
  6. Emptage M. H., Kent T. A., Kennedy M. C., Beinert H., Münck E. Mössbauer and EPR studies of activated aconitase: development of a localized valence state at a subsite of the [4Fe-4S] cluster on binding of citrate. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4674–4678. doi: 10.1073/pnas.80.15.4674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. GORNALL A. G., BARDAWILL C. J., DAVID M. M. Determination of serum proteins by means of the biuret reaction. J Biol Chem. 1949 Feb;177(2):751–766. [PubMed] [Google Scholar]
  10. Hahm K. S., Gawron O., Piszkiewicz D. Amino acid sequence of a peptide containing an essential cysteine residue of pig heart aconitase. Biochim Biophys Acta. 1981 Feb 27;667(2):457–461. doi: 10.1016/0005-2795(81)90211-7. [DOI] [PubMed] [Google Scholar]
  11. Hanson R. S., Cox D. P. Effect of different nutritional conditions on the synthesis of tricarboxylic acid cycle enzymes. J Bacteriol. 1967 Jun;93(6):1777–1787. doi: 10.1128/jb.93.6.1777-1787.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hu N., Messing J. The making of strand-specific M13 probes. Gene. 1982 Mar;17(3):271–277. doi: 10.1016/0378-1119(82)90143-3. [DOI] [PubMed] [Google Scholar]
  13. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kennedy M. C., Spoto G., Emptage M. H., Beinert H. The active site sulfhydryl of aconitase is not required for catalytic activity. J Biol Chem. 1988 Jun 15;263(17):8190–8193. [PubMed] [Google Scholar]
  15. Kennedy S. C., Rauner R., Gawron O. On pig heart aconitase. Biochem Biophys Res Commun. 1972 May 26;47(4):740–745. doi: 10.1016/0006-291x(72)90554-2. [DOI] [PubMed] [Google Scholar]
  16. Kim K. S., Rosenkrantz M. S., Guarente L. Saccharomyces cerevisiae contains two functional citrate synthase genes. Mol Cell Biol. 1986 Jun;6(6):1936–1942. doi: 10.1128/mcb.6.6.1936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Li J. M., Hopper A. K., Martin N. C. N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae. J Cell Biol. 1989 Oct;109(4 Pt 1):1411–1419. doi: 10.1083/jcb.109.4.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McAlister-Henn L., Thompson L. M. Isolation and expression of the gene encoding yeast mitochondrial malate dehydrogenase. J Bacteriol. 1987 Nov;169(11):5157–5166. doi: 10.1128/jb.169.11.5157-5166.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Najarian D., Dihanich M. E., Martin N. C., Hopper A. K. DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translational starts. Mol Cell Biol. 1987 Jan;7(1):185–191. doi: 10.1128/mcb.7.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Natsoulis G., Hilger F., Fink G. R. The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S. cerevisiae. Cell. 1986 Jul 18;46(2):235–243. doi: 10.1016/0092-8674(86)90740-3. [DOI] [PubMed] [Google Scholar]
  23. Ogur M., Coker L., Ogur S. Glutamate auxotrophs in Saccharomyces 1. I. The biochemical lesion in the glt-1 mutants-2. Biochem Biophys Res Commun. 1964;14:193–197. doi: 10.1016/0006-291x(64)90254-2. [DOI] [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. Plank D. W., Howard J. B. Identification of the reactive sulfhydryl and sequences of cysteinyl-tryptic peptides from beef heart aconitase. J Biol Chem. 1988 Jun 15;263(17):8184–8189. [PubMed] [Google Scholar]
  26. Plank D. W., Kennedy M. C., Beinert H., Howard J. B. Cysteine labeling studies of beef heart aconitase containing a 4Fe, a cubane 3Fe, or a linear 3Fe cluster. J Biol Chem. 1989 Dec 5;264(34):20385–20393. [PubMed] [Google Scholar]
  27. Polakis E. S., Bartley W. Changes in the enzyme activities of Saccharomyces cerevisiae during aerobic growth on different carbon sources. Biochem J. 1965 Oct;97(1):284–297. doi: 10.1042/bj0970284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rickey T. M., Lewin A. S. Extramitochondrial citrate synthase activity in bakers' yeast. Mol Cell Biol. 1986 Feb;6(2):488–493. doi: 10.1128/mcb.6.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  30. Robbins A. H., Stout C. D. Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal. Proc Natl Acad Sci U S A. 1989 May;86(10):3639–3643. doi: 10.1073/pnas.86.10.3639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rose I. A., O'Connell E. L. Mechanism of aconitase action. I. The hydrogen transfer reaction. J Biol Chem. 1967 Apr 25;242(8):1870–1879. [PubMed] [Google Scholar]
  32. Rosenkrantz M. S., Dingman D. W., Sonenshein A. L. Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine. J Bacteriol. 1985 Oct;164(1):155–164. doi: 10.1128/jb.164.1.155-164.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rosenkrantz M., Alam T., Kim K. S., Clark B. J., Srere P. A., Guarente L. P. Mitochondrial and nonmitochondrial citrate synthases in Saccharomyces cerevisiae are encoded by distinct homologous genes. Mol Cell Biol. 1986 Dec;6(12):4509–4515. doi: 10.1128/mcb.6.12.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Ruzicka F. J., Beinert H. The soluble "high potential" type iron-sulfur protein from mitochondria is aconitase. J Biol Chem. 1978 Apr 25;253(8):2514–2517. [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Satrustegui J., Machado A. The synthesis of yeast matrix mitochondrial enzymes is regulated by different levels of mitochondrial function. Arch Biochem Biophys. 1977 Dec;184(2):355–363. doi: 10.1016/0003-9861(77)90362-9. [DOI] [PubMed] [Google Scholar]
  38. Scholze H. Studies on aconitase species from Saccharomyces cerevisiae, porcine and bovine heart, obtained by a modified isolation method. Biochim Biophys Acta. 1983 Aug 16;746(3):133–137. doi: 10.1016/0167-4838(83)90066-3. [DOI] [PubMed] [Google Scholar]
  39. Sharp P. M., Li W. H. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987 Feb 11;15(3):1281–1295. doi: 10.1093/nar/15.3.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Slonimski P. P., Perrodin G., Croft J. H. Ethidium bromide induced mutation of yeast mitochondria: complete transformation of cells into respiratory deficient non-chromosomal "petites". Biochem Biophys Res Commun. 1968 Feb 15;30(3):232–239. doi: 10.1016/0006-291x(68)90440-3. [DOI] [PubMed] [Google Scholar]
  41. Smith T. F., Waterman M. S., Fitch W. M. Comparative biosequence metrics. J Mol Evol. 1981;18(1):38–46. doi: 10.1007/BF01733210. [DOI] [PubMed] [Google Scholar]
  42. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  43. Villafranca J. J., Mildvan A. S. The mechanism of aconitase action. I. Preparation, physical properties of the enzyme, and activation by iron (II). J Biol Chem. 1971 Feb 10;246(3):772–779. [PubMed] [Google Scholar]
  44. Wu M., Tzagoloff A. Mitochondrial and cytoplasmic fumarases in Saccharomyces cerevisiae are encoded by a single nuclear gene FUM1. J Biol Chem. 1987 Sep 5;262(25):12275–12282. [PubMed] [Google Scholar]
  45. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  46. Zheng L., Andrews P. C., Hermodson M. A., Dixon J. E., Zalkin H. Cloning and structural characterization of porcine heart aconitase. J Biol Chem. 1990 Feb 15;265(5):2814–2821. [PubMed] [Google Scholar]
  47. Zitomer R. S., Montgomery D. L., Nichols D. L., Hall B. D. Transcriptional regulation of the yeast cytochrome c gene. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3627–3631. doi: 10.1073/pnas.76.8.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 1986 Jun;5(6):1335–1342. doi: 10.1002/j.1460-2075.1986.tb04364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES