Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Apr;178(7):1842–1849. doi: 10.1128/jb.178.7.1842-1849.1996

The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

V D Dang 1, C Bohn 1, M Bolotin-Fukuhara 1, B Daignan-Fornier 1
PMCID: PMC177877  PMID: 8606156

Abstract

In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.

Full Text

The Full Text of this article is available as a PDF (428.7 KB).

Selected References

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

  1. Benjamin P. M., Wu J. I., Mitchell A. P., Magasanik B. Three regulatory systems control expression of glutamine synthetase in Saccharomyces cerevisiae at the level of transcription. Mol Gen Genet. 1989 Jun;217(2-3):370–377. doi: 10.1007/BF02464906. [DOI] [PubMed] [Google Scholar]
  2. Boles E., Lehnert W., Zimmermann F. K. The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant. Eur J Biochem. 1993 Oct 1;217(1):469–477. doi: 10.1111/j.1432-1033.1993.tb18266.x. [DOI] [PubMed] [Google Scholar]
  3. Bowman S. B., Zaman Z., Collinson L. P., Brown A. J., Dawes I. W. Positive regulation of the LPD1 gene of Saccharomyces cerevisiae by the HAP2/HAP3/HAP4 activation system. Mol Gen Genet. 1992 Jan;231(2):296–303. doi: 10.1007/BF00279803. [DOI] [PubMed] [Google Scholar]
  4. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  5. Bussey H., Kaback D. B., Zhong W., Vo D. T., Clark M. W., Fortin N., Hall J., Ouellette B. F., Keng T., Barton A. B. The nucleotide sequence of chromosome I from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3809–3813. doi: 10.1073/pnas.92.9.3809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang L. F., Gatzek P. R., Kohlhaw G. B. Total deletion of yeast LEU4: further evidence for a second alpha-isopropylmalate synthase and evidence for tight LEU4-MET4 linkage. Gene. 1985;33(3):333–339. doi: 10.1016/0378-1119(85)90241-0. [DOI] [PubMed] [Google Scholar]
  7. Coschigano P. W., Miller S. M., Magasanik B. Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae. Mol Cell Biol. 1991 Sep;11(9):4455–4465. doi: 10.1128/mcb.11.9.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daignan-Fornier B., Nguyen C. C., Reisdorf P., Lemeignan B., Bolotin-Fukuhara M. MBR1 and MBR3, two related yeast genes that can suppress the growth defect of hap2, hap3 and hap4 mutants. Mol Gen Genet. 1994 Jun 3;243(5):575–583. doi: 10.1007/BF00284206. [DOI] [PubMed] [Google Scholar]
  9. Daignan-Fornier B., Valens M., Lemire B. D., Bolotin-Fukuhara M. Structure and regulation of SDH3, the yeast gene encoding the cytochrome b560 subunit of respiratory complex II. J Biol Chem. 1994 Jun 3;269(22):15469–15472. [PubMed] [Google Scholar]
  10. Dang V. D., Valens M., Bolotin-Fukuhara M., Daignan-Fornier B. A genetic screen to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p. Yeast. 1994 Oct;10(10):1273–1283. doi: 10.1002/yea.320101004. [DOI] [PubMed] [Google Scholar]
  11. Daugherty J. R., Rai R., el Berry H. M., Cooper T. G. Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae. J Bacteriol. 1993 Jan;175(1):64–73. doi: 10.1128/jb.175.1.64-73.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dickinson J. R., Dawes I. W. The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae. J Gen Microbiol. 1992 Oct;138(10):2029–2033. doi: 10.1099/00221287-138-10-2029. [DOI] [PubMed] [Google Scholar]
  13. Dubois E. L., Grenson M. Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1974 Sep 9;60(1):150–157. doi: 10.1016/0006-291x(74)90185-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Fondrat C., Kalogeropoulos A. Approaching the function of new genes by detection of their potential upstream activation sequences in Saccharomyces cerevisiae: application to chromosome III. Curr Genet. 1994 May;25(5):396–406. doi: 10.1007/BF00351777. [DOI] [PubMed] [Google Scholar]
  16. Forsburg S. L., Guarente L. Communication between mitochondria and the nucleus in regulation of cytochrome genes in the yeast Saccharomyces cerevisiae. Annu Rev Cell Biol. 1989;5:153–180. doi: 10.1146/annurev.cb.05.110189.001101. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Friden P., Schimmel P. LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence. Mol Cell Biol. 1988 Jul;8(7):2690–2697. doi: 10.1128/mcb.8.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gallwitz D., Sures I. Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 May;77(5):2546–2550. doi: 10.1073/pnas.77.5.2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Grenson M., Dubois E., Piotrowska M., Drillien R., Aigle M. Ammonia assimilation in Saccharomyces cerevisiae as mediated by the two glutamate dehydrogenases. Evidence for the gdhA locus being a structural gene for the NADP-dependent glutamate dehydrogenase. Mol Gen Genet. 1974;128(1):73–85. doi: 10.1007/BF00267295. [DOI] [PubMed] [Google Scholar]
  23. Guarente L., Lalonde B., Gifford P., Alani E. Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae. Cell. 1984 Feb;36(2):503–511. doi: 10.1016/0092-8674(84)90243-5. [DOI] [PubMed] [Google Scholar]
  24. Hahn S., Pinkham J., Wei R., Miller R., Guarente L. The HAP3 regulatory locus of Saccharomyces cerevisiae encodes divergent overlapping transcripts. Mol Cell Biol. 1988 Feb;8(2):655–663. doi: 10.1128/mcb.8.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Haselbeck R. J., McAlister-Henn L. Function and expression of yeast mitochondrial NAD- and NADP-specific isocitrate dehydrogenases. J Biol Chem. 1993 Jun 5;268(16):12116–12122. [PubMed] [Google Scholar]
  26. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  27. Hu Y., Cooper T. G., Kohlhaw G. B. The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation. Mol Cell Biol. 1995 Jan;15(1):52–57. doi: 10.1128/mcb.15.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Kammann M., Laufs J., Schell J., Gronenborn B. Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Res. 1989 Jul 11;17(13):5404–5404. doi: 10.1093/nar/17.13.5404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Keng T., Richard C., Larocque R. Structure and regulation of yeast HEM3, the gene for porphobilinogen deaminase. Mol Gen Genet. 1992 Aug;234(2):233–243. doi: 10.1007/BF00283844. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. McNabb D. S., Xing Y., Guarente L. Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding. Genes Dev. 1995 Jan 1;9(1):47–58. doi: 10.1101/gad.9.1.47. [DOI] [PubMed] [Google Scholar]
  35. Miller S. M., Magasanik B. Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae. J Bacteriol. 1990 Sep;172(9):4927–4935. doi: 10.1128/jb.172.9.4927-4935.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Minehart P. L., Magasanik B. Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain. Mol Cell Biol. 1991 Dec;11(12):6216–6228. doi: 10.1128/mcb.11.12.6216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Minehart P. L., Magasanik B. Sequence of the GLN1 gene of Saccharomyces cerevisiae: role of the upstream region in regulation of glutamine synthetase expression. J Bacteriol. 1992 Mar;174(6):1828–1836. doi: 10.1128/jb.174.6.1828-1836.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moye W. S., Amuro N., Rao J. K., Zalkin H. Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase. J Biol Chem. 1985 Jul 15;260(14):8502–8508. [PubMed] [Google Scholar]
  39. 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]
  40. Nagasu T., Hall B. D. Nucleotide sequence of the GDH gene coding for the NADP-specific glutamate dehydrogenase of Saccharomyces cerevisiae. Gene. 1985;37(1-3):247–253. doi: 10.1016/0378-1119(85)90279-3. [DOI] [PubMed] [Google Scholar]
  41. Olesen J. T., Guarente L. The HAP2 subunit of yeast CCAAT transcriptional activator contains adjacent domains for subunit association and DNA recognition: model for the HAP2/3/4 complex. Genes Dev. 1990 Oct;4(10):1714–1729. doi: 10.1101/gad.4.10.1714. [DOI] [PubMed] [Google Scholar]
  42. Perlman P. S., Mahler H. R. Intracellular localization of enzymes in yeast. Arch Biochem Biophys. 1970 Jan;136(1):245–259. doi: 10.1016/0003-9861(70)90348-6. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. 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]
  47. Rohde M., Lim F., Wallace J. C. Electron microscopic localization of pyruvate carboxylase in rat liver and Saccharomyces cerevisiae by immunogold procedures. Arch Biochem Biophys. 1991 Oct;290(1):197–201. doi: 10.1016/0003-9861(91)90608-l. [DOI] [PubMed] [Google Scholar]
  48. Roon R. J., Even H. L., Larimore F. Glutamate synthase: properties of the reduced nicotinamide adenine dinucleotide-dependent enzyme from Saccharomyces cerevisiae. J Bacteriol. 1974 Apr;118(1):89–95. doi: 10.1128/jb.118.1.89-95.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rosenkrantz M., Kell C. S., Pennell E. A., Devenish L. J. The HAP2,3,4 transcriptional activator is required for derepression of the yeast citrate synthase gene, CIT1. Mol Microbiol. 1994 Jul;13(1):119–131. doi: 10.1111/j.1365-2958.1994.tb00407.x. [DOI] [PubMed] [Google Scholar]
  50. Ruby S. W., Szostak J. W., Murray A. W. Cloning regulated yeast genes from a pool of lacZ fusions. Methods Enzymol. 1983;101:253–269. doi: 10.1016/0076-6879(83)01019-8. [DOI] [PubMed] [Google Scholar]
  51. Schmitt M. E., Brown T. A., Trumpower B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990 May 25;18(10):3091–3092. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  53. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sze J. Y., Woontner M., Jaehning J. A., Kohlhaw G. B. In vitro transcriptional activation by a metabolic intermediate: activation by Leu3 depends on alpha-isopropylmalate. Science. 1992 Nov 13;258(5085):1143–1145. doi: 10.1126/science.1439822. [DOI] [PubMed] [Google Scholar]
  55. Walker M. E., Val D. L., Rohde M., Devenish R. J., Wallace J. C. Yeast pyruvate carboxylase: identification of two genes encoding isoenzymes. Biochem Biophys Res Commun. 1991 May 15;176(3):1210–1217. doi: 10.1016/0006-291x(91)90414-3. [DOI] [PubMed] [Google Scholar]
  56. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES