Skip to main content
Genetics logoLink to Genetics
. 1996 Sep;144(1):57–69. doi: 10.1093/genetics/144.1.57

Mutants of Saccharomyces Cerevisiae with Defects in Acetate Metabolism: Isolation and Characterization of Acn(-) Mutants

M T McCammon 1
PMCID: PMC1207517  PMID: 8878673

Abstract

The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn(-) (``ACetate Nonutilizing'') mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Bieber L. L., Markwell M. A. Peroxisomal and microsomal carnitine acetyltransferases. Methods Enzymol. 1981;71(Pt 100):351–358. doi: 10.1016/0076-6879(81)71044-9. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Carlson M. Regulation of sugar utilization in Saccharomyces species. J Bacteriol. 1987 Nov;169(11):4873–4877. doi: 10.1128/jb.169.11.4873-4877.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chelstowska A., Butow R. A. RTG genes in yeast that function in communication between mitochondria and the nucleus are also required for expression of genes encoding peroxisomal proteins. J Biol Chem. 1995 Jul 28;270(30):18141–18146. doi: 10.1074/jbc.270.30.18141. [DOI] [PubMed] [Google Scholar]
  5. Cooper T. G., Beevers H. Mitochondria and glyoxysomes from castor bean endosperm. Enzyme constitutents and catalytic capacity. J Biol Chem. 1969 Jul 10;244(13):3507–3513. [PubMed] [Google Scholar]
  6. Cupp J. R., McAlister-Henn L. Kinetic analysis of NAD(+)-isocitrate dehydrogenase with altered isocitrate binding sites: contribution of IDH1 and IDH2 subunits to regulation and catalysis. Biochemistry. 1993 Sep 14;32(36):9323–9328. doi: 10.1021/bi00087a010. [DOI] [PubMed] [Google Scholar]
  7. Cupp J. R., McAlister-Henn L. NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae. J Biol Chem. 1991 Nov 25;266(33):22199–22205. [PubMed] [Google Scholar]
  8. Daum G., Gasser S. M., Schatz G. Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13075–13080. [PubMed] [Google Scholar]
  9. De Virgilio C., Bürckert N., Barth G., Neuhaus J. M., Boller T., Wiemken A. Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae. Yeast. 1992 Dec;8(12):1043–1051. doi: 10.1002/yea.320081207. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dmochowska A., Dignard D., Maleszka R., Thomas D. Y. Structure and transcriptional control of the Saccharomyces cerevisiae POX1 gene encoding acyl-coenzyme A oxidase. Gene. 1990 Apr 16;88(2):247–252. doi: 10.1016/0378-1119(90)90038-s. [DOI] [PubMed] [Google Scholar]
  12. Dommes V., Baumgart C., Kunau W. H. Degradation of unsaturated fatty acids in peroxisomes. Existence of a 2,4-dienoyl-CoA reductase pathway. J Biol Chem. 1981 Aug 25;256(16):8259–8262. [PubMed] [Google Scholar]
  13. Erdmann R., Veenhuis M., Mertens D., Kunau W. H. Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5419–5423. doi: 10.1073/pnas.86.14.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gancedo C., Delgado M. A. Isolation and characterization of a mutant from Saccharomyces cerevisiae lacking fructose 1,6-bisphosphatase. Eur J Biochem. 1984 Mar 15;139(3):651–655. doi: 10.1111/j.1432-1033.1984.tb08053.x. [DOI] [PubMed] [Google Scholar]
  15. Gancedo J. M. Carbon catabolite repression in yeast. Eur J Biochem. 1992 Jun 1;206(2):297–313. doi: 10.1111/j.1432-1033.1992.tb16928.x. [DOI] [PubMed] [Google Scholar]
  16. Gangloff S. P., Marguet D., Lauquin G. J. Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate. Mol Cell Biol. 1990 Jul;10(7):3551–3561. doi: 10.1128/mcb.10.7.3551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goldstein A., Lampen J. O. Beta-D-fructofuranoside fructohydrolase from yeast. Methods Enzymol. 1975;42:504–511. doi: 10.1016/0076-6879(75)42159-0. [DOI] [PubMed] [Google Scholar]
  18. Hansen R. J., Hinze H., Holzer H. Assay of phosphoenolpyruvate carboxykinase in crude yeast extracts. Anal Biochem. 1976 Aug;74(2):576–584. doi: 10.1016/0003-2697(76)90240-2. [DOI] [PubMed] [Google Scholar]
  19. Hartig A., Simon M. M., Schuster T., Daugherty J. R., Yoo H. S., Cooper T. G. Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae. Nucleic Acids Res. 1992 Nov 11;20(21):5677–5686. doi: 10.1093/nar/20.21.5677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Haselbeck R. J., McAlister-Henn L. Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase. J Biol Chem. 1991 Feb 5;266(4):2339–2345. [PubMed] [Google Scholar]
  21. Keys D. A., McAlister-Henn L. Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae. J Bacteriol. 1990 Aug;172(8):4280–4287. doi: 10.1128/jb.172.8.4280-4287.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Kispal G., Evans C. T., Malloy C., Srere P. A. Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme. J Biol Chem. 1989 Jul 5;264(19):11204–11210. [PubMed] [Google Scholar]
  24. Kispal G., Rosenkrantz M., Guarente L., Srere P. A. Metabolic changes in Saccharomyces cerevisiae strains lacking citrate synthases. J Biol Chem. 1988 Aug 15;263(23):11145–11149. [PubMed] [Google Scholar]
  25. Kispal G., Sumegi B., Dietmeier K., Bock I., Gajdos G., Tomcsanyi T., Sandor A. Cloning and sequencing of a cDNA encoding Saccharomyces cerevisiae carnitine acetyltransferase. Use of the cDNA in gene disruption studies. J Biol Chem. 1993 Jan 25;268(3):1824–1829. [PubMed] [Google Scholar]
  26. Koonin E. V. Yeast protein controlling inter-organelle communication is related to bacterial phosphatases containing the Hsp 70-type ATP-binding domain. Trends Biochem Sci. 1994 Apr;19(4):156–157. doi: 10.1016/0968-0004(94)90275-5. [DOI] [PubMed] [Google Scholar]
  27. Kulbe K. D., Bojanovski M. 3-Phosphoglycerate kinase from bovine liver and yeast. Methods Enzymol. 1982;90(Pt E):115–120. doi: 10.1016/s0076-6879(82)90116-1. [DOI] [PubMed] [Google Scholar]
  28. Lee S. Y., Rasheed S. A simple procedure for maximum yield of high-quality plasmid DNA. Biotechniques. 1990 Dec;9(6):676–679. [PubMed] [Google Scholar]
  29. Liao X. S., Small W. C., Srere P. A., Butow R. A. Intramitochondrial functions regulate nonmitochondrial citrate synthase (CIT2) expression in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):38–46. doi: 10.1128/mcb.11.1.38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liao X., Butow R. A. RTG1 and RTG2: two yeast genes required for a novel path of communication from mitochondria to the nucleus. Cell. 1993 Jan 15;72(1):61–71. doi: 10.1016/0092-8674(93)90050-z. [DOI] [PubMed] [Google Scholar]
  31. Littlewood B. S. Methods for selecting auxotrophic and temperature-sensitive mutants in yeasts. Methods Cell Biol. 1975;11:273–285. doi: 10.1016/s0091-679x(08)60328-3. [DOI] [PubMed] [Google Scholar]
  32. Loftus T. M., Hall L. V., Anderson S. L., McAlister-Henn L. Isolation, characterization, and disruption of the yeast gene encoding cytosolic NADP-specific isocitrate dehydrogenase. Biochemistry. 1994 Aug 16;33(32):9661–9667. doi: 10.1021/bi00198a035. [DOI] [PubMed] [Google Scholar]
  33. Ma H., Bloom L. M., Walsh C. T., Botstein D. The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Dec;9(12):5643–5649. doi: 10.1128/mcb.9.12.5643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. McCammon M. T., Dowds C. A., Orth K., Moomaw C. R., Slaughter C. A., Goodman J. M. Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae. J Biol Chem. 1990 Nov 25;265(33):20098–20105. [PubMed] [Google Scholar]
  36. Middleton B. 3-Ketoacyl-CoA thiolases of mammalian tissues. Methods Enzymol. 1975;35:128–136. doi: 10.1016/0076-6879(75)35148-3. [DOI] [PubMed] [Google Scholar]
  37. Minard K. I., McAlister-Henn L. Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase. Mol Cell Biol. 1991 Jan;11(1):370–380. doi: 10.1128/mcb.11.1.370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Oliver S. G. From DNA sequence to biological function. Nature. 1996 Feb 15;379(6566):597–600. doi: 10.1038/379597a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Ronne H. Glucose repression in fungi. Trends Genet. 1995 Jan;11(1):12–17. doi: 10.1016/s0168-9525(00)88980-5. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  44. Small W. C., Brodeur R. D., Sandor A., Fedorova N., Li G., Butow R. A., Srere P. A. Enzymatic and metabolic studies on retrograde regulation mutants of yeast. Biochemistry. 1995 Apr 25;34(16):5569–5576. doi: 10.1021/bi00016a031. [DOI] [PubMed] [Google Scholar]
  45. Steffan J. S., McAlister-Henn L. Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase. J Biol Chem. 1992 Dec 5;267(34):24708–24715. [PubMed] [Google Scholar]
  46. Sumegi B., McCammon M. T., Sherry A. D., Keys D. A., McAlister-Henn L., Srere P. A. Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast. Biochemistry. 1992 Sep 22;31(37):8720–8725. doi: 10.1021/bi00152a006. [DOI] [PubMed] [Google Scholar]
  47. Sumegi B., Porpaczy Z., McCammon M. T., Sherry A. D., Malloy C. R., Srere P. A. Regulatory consequences of organization of citric acid cycle enzymes. Curr Top Cell Regul. 1992;33:249–260. doi: 10.1016/b978-0-12-152833-1.50019-8. [DOI] [PubMed] [Google Scholar]
  48. Thevelein J. M. Fermentable sugars and intracellular acidification as specific activators of the RAS-adenylate cyclase signalling pathway in yeast: the relationship to nutrient-induced cell cycle control. Mol Microbiol. 1991 Jun;5(6):1301–1307. doi: 10.1111/j.1365-2958.1991.tb00776.x. [DOI] [PubMed] [Google Scholar]
  49. Thevelein J. M., Hohmann S. Trehalose synthase: guard to the gate of glycolysis in yeast? Trends Biochem Sci. 1995 Jan;20(1):3–10. doi: 10.1016/s0968-0004(00)88938-0. [DOI] [PubMed] [Google Scholar]
  50. Thomas B. J., Rothstein R. Elevated recombination rates in transcriptionally active DNA. Cell. 1989 Feb 24;56(4):619–630. doi: 10.1016/0092-8674(89)90584-9. [DOI] [PubMed] [Google Scholar]
  51. Tolbert N. E. Isolation of subcellular organelles of metabolism on isopycnic sucrose gradients. Methods Enzymol. 1974;31:734–746. doi: 10.1016/0076-6879(74)31077-4. [DOI] [PubMed] [Google Scholar]
  52. Trumbly R. J. Glucose repression in the yeast Saccharomyces cerevisiae. Mol Microbiol. 1992 Jan;6(1):15–21. doi: 10.1111/j.1365-2958.1992.tb00832.x. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Veenhuis M., Mateblowski M., Kunau W. H., Harder W. Proliferation of microbodies in Saccharomyces cerevisiae. Yeast. 1987 Jun;3(2):77–84. doi: 10.1002/yea.320030204. [DOI] [PubMed] [Google Scholar]
  55. Walker M. E., Wallace J. C. Isolation of a yeast mutant deficient in pyruvate carboxylase activity. Biochem Int. 1991 Mar;23(4):697–705. [PubMed] [Google Scholar]
  56. Walsh K., Koshland D. E., Jr Determination of flux through the branch point of two metabolic cycles. The tricarboxylic acid cycle and the glyoxylate shunt. J Biol Chem. 1984 Aug 10;259(15):9646–9654. [PubMed] [Google Scholar]
  57. Williams F. E., Trumbly R. J. Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6500–6511. doi: 10.1128/mcb.10.12.6500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wills C., Melham T. Pyruvate carboxylase deficiency in yeast: a mutant affecting the interaction between the glyoxylate and Krebs cycles. Arch Biochem Biophys. 1985 Feb 1;236(2):782–791. doi: 10.1016/0003-9861(85)90684-8. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. de Winde J. H., Grivell L. A. Global regulation of mitochondrial biogenesis in Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol. 1993;46:51–91. doi: 10.1016/s0079-6603(08)61018-1. [DOI] [PubMed] [Google Scholar]
  61. van Roermund C. W., Elgersma Y., Singh N., Wanders R. J., Tabak H. F. The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions. EMBO J. 1995 Jul 17;14(14):3480–3486. doi: 10.1002/j.1460-2075.1995.tb07354.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES