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. 1995 Jan;15(1):52–57. doi: 10.1128/mcb.15.1.52

The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

Y Hu 1, T G Cooper 1, G B Kohlhaw 1
PMCID: PMC231907  PMID: 7799961

Abstract

The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

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Selected References

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  1. Andreadis A., Hsu Y. P., Kohlhaw G. B., Schimmel P. Nucleotide sequence of yeast LEU2 shows 5'-noncoding region has sequences cognate to leucine. Cell. 1982 Dec;31(2 Pt 1):319–325. doi: 10.1016/0092-8674(82)90125-8. [DOI] [PubMed] [Google Scholar]
  2. Armaleo D., Fischer M., Gross S. R. Effect of alpha-isopropylmalate on the synthesis of RNA and protein in Neurospora. Mol Gen Genet. 1985;200(2):346–349. doi: 10.1007/BF00425447. [DOI] [PubMed] [Google Scholar]
  3. Beltzer J. P., Chang L. F., Hinkkanen A. E., Kohlhaw G. B. Structure of yeast LEU4. The 5' flanking region contains features that predict two modes of control and two productive translation starts. J Biol Chem. 1986 Apr 15;261(11):5160–5167. [PubMed] [Google Scholar]
  4. Bogonez E., Satrústegui J., Machado A. Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae. J Gen Microbiol. 1985 Jun;131(6):1425–1432. doi: 10.1099/00221287-131-6-1425. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Brisco P. R., Cunningham T. S., Kohlhaw G. B. Cloning, disruption and chromosomal mapping of yeast LEU3, a putative regulatory gene. Genetics. 1987 Jan;115(1):91–99. doi: 10.1093/genetics/115.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brisco P. R., Kohlhaw G. B. Regulation of yeast LEU2. Total deletion of regulatory gene LEU3 unmasks GCN4-dependent basal level expression of LEU2. J Biol Chem. 1990 Jul 15;265(20):11667–11675. [PubMed] [Google Scholar]
  8. 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]
  9. Emr S. D., Vassarotti A., Garrett J., Geller B. L., Takeda M., Douglas M. G. The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal. J Cell Biol. 1986 Feb;102(2):523–533. doi: 10.1083/jcb.102.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Falco S. C., Dumas K. S., Livak K. J. Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase. Nucleic Acids Res. 1985 Jun 11;13(11):4011–4027. doi: 10.1093/nar/13.11.4011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. HOLZER H., SCHNEIDER S. Anreicherung und Trennung einer DPN-spezifischen und einer TPN-spezifischen Glutaminsäure-dehydrogenase aus Hefe. Biochem Z. 1957;329(5):361–369. [PubMed] [Google Scholar]
  14. Hinnebusch A. G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev. 1988 Jun;52(2):248–273. doi: 10.1128/mr.52.2.248-273.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kidd G. L., Gross S. R. Specific regulatory interconnection between the leucine and histidine pathways of Neurospora crassa. J Bacteriol. 1984 Apr;158(1):121–127. doi: 10.1128/jb.158.1.121-127.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  17. Köhrer K., Domdey H. Preparation of high molecular weight RNA. Methods Enzymol. 1991;194:398–405. doi: 10.1016/0076-6879(91)94030-g. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Olshan A. R., Gross S. R. Role of the leu-3 cistron in the regulation of the synthesis of isoleucine and valine biosynthetic enzymes of Neurospora. J Bacteriol. 1974 May;118(2):374–384. doi: 10.1128/jb.118.2.374-384.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peters M. H., Beltzer J. P., Kohlhaw G. B. Expression of the yeast LEU4 gene is subject to four different modes of control. Arch Biochem Biophys. 1990 Jan;276(1):294–298. doi: 10.1016/0003-9861(90)90041-v. [DOI] [PubMed] [Google Scholar]
  22. Petersen J. G., Holmberg S. The ILV5 gene of Saccharomyces cerevisiae is highly expressed. Nucleic Acids Res. 1986 Dec 22;14(24):9631–9651. doi: 10.1093/nar/14.24.9631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Polacco J. C., Gross S. R. The product of the leu-3 cistron as a regulatory element for the production of the leucine biosynthetic enzymes of Neurospora. Genetics. 1973 Jul;74(3):443–459. doi: 10.1093/genetics/74.3.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roon R. J., Even H. L. Regulation of the nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenases of Saccharomyces cerevisiae. J Bacteriol. 1973 Oct;116(1):367–372. doi: 10.1128/jb.116.1.367-372.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sze J. Y., Kohlhaw G. B. Purification and structural characterization of transcriptional regulator Leu3 of yeast. J Biol Chem. 1993 Feb 5;268(4):2505–2512. [PubMed] [Google Scholar]
  26. Sze J. Y., Remboutsika E., Kohlhaw G. B. Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions. Mol Cell Biol. 1993 Sep;13(9):5702–5709. doi: 10.1128/mcb.13.9.5702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Wolfner M., Yep D., Messenguy F., Fink G. R. Integration of amino acid biosynthesis into the cell cycle of Saccharomyces cerevisiae. J Mol Biol. 1975 Aug 5;96(2):273–290. doi: 10.1016/0022-2836(75)90348-4. [DOI] [PubMed] [Google Scholar]
  29. Zhou K. M., Bai Y. L., Kohlhaw G. B. Yeast regulatory protein LEU3: a structure-function analysis. Nucleic Acids Res. 1990 Jan 25;18(2):291–298. doi: 10.1093/nar/18.2.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zhou K., Brisco P. R., Hinkkanen A. E., Kohlhaw G. B. Structure of yeast regulatory gene LEU3 and evidence that LEU3 itself is under general amino acid control. Nucleic Acids Res. 1987 Jul 10;15(13):5261–5273. doi: 10.1093/nar/15.13.5261. [DOI] [PMC free article] [PubMed] [Google Scholar]

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