Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Apr;15(4):2321–2330. doi: 10.1128/mcb.15.4.2321

Roles of URE2 and GLN3 in the proline utilization pathway in Saccharomyces cerevisiae.

S Xu 1, D A Falvey 1, M C Brandriss 1
PMCID: PMC230460  PMID: 7891726

Abstract

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, gamma-aminobutyrate, or proline when preferred nitrogen sources like glutamine, asparagine, or ammonium ions are unavailable in the environment. Utilization of alternative nitrogen sources requires the relief of nitrogen repression and induction of specific permeases and enzymes. The products of the GLN3 and URE2 genes are required for the appropriate transcription of many genes in alternative nitrogen assimilatory pathways. GLN3 appears to activate their transcription when good nitrogen sources are unavailable, and URE2 appears to repress their transcription when alternative nitrogen sources are not needed. The participation of nitrogen repression and the regulators GLN3 and URE2 in the proline utilization pathway was evaluated in this study. Comparison of PUT gene expression in cells grown in repressing or derepressing nitrogen sources, in the absence of the inducer proline, indicated that both PUT1 and PUT2 are regulated by nitrogen repression, although the effect on PUT2 is comparatively small. Recessive mutations in URE2 elevated expression of the PUT1 and PUT2 genes 5- to 10-fold when cells were grown on a nitrogen-repressing medium. Although PUT3, the proline utilization pathway transcriptional activator, is absolutely required for growth on proline as the sole nitrogen source, a put3 ure2 strain had somewhat elevated PUT gene expression, suggesting an effect of the ure2 mutation in the absence of the PUT3 product. PUT1 and PUT2 gene expression did not require the GLN3 activator protein for expression under either repressing or derepressing conditions. Therefore, regulation of the PUT genes by URE2 does not require a functional GLN3 protein. The effect of the ure2 mutation on the PUT genes is not due to increased internal proline levels. URE2 repression appears to be limited to nitrogen assimilatory systems and does not affect genes involved in carbon, inositol, or phosphate metabolism or in mating-type control and sporulation.

Full Text

The Full Text of this article is available as a PDF (359.7 KB).

Selected References

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

  1. André B. The UGA3 gene regulating the GABA catabolic pathway in Saccharomyces cerevisiae codes for a putative zinc-finger protein acting on RNA amount. Mol Gen Genet. 1990 Jan;220(2):269–276. doi: 10.1007/BF00260493. [DOI] [PubMed] [Google Scholar]
  2. Axelrod J. D., Majors J., Brandriss M. C. Proline-independent binding of PUT3 transcriptional activator protein detected by footprinting in vivo. Mol Cell Biol. 1991 Jan;11(1):564–567. doi: 10.1128/mcb.11.1.564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergman L. W. A DNA fragment containing the upstream activator sequence determines nucleosome positioning of the transcriptionally repressed PHO5 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jul;6(7):2298–2304. doi: 10.1128/mcb.6.7.2298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brandriss M. C. Evidence for positive regulation of the proline utilization pathway in Saccharomyces cerevisiae. Genetics. 1987 Nov;117(3):429–435. doi: 10.1093/genetics/117.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brandriss M. C. Isolation and preliminary characterization of Saccharomyces cerevisiae proline auxotrophs. J Bacteriol. 1979 Jun;138(3):816–822. doi: 10.1128/jb.138.3.816-822.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brandriss M. C., Krzywicki K. A. Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Oct;6(10):3502–3512. doi: 10.1128/mcb.6.10.3502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brandriss M. C., Magasanik B. Genetics and physiology of proline utilization in Saccharomyces cerevisiae: mutation causing constitutive enzyme expression. J Bacteriol. 1979 Nov;140(2):504–507. doi: 10.1128/jb.140.2.504-507.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brandriss M. C., Magasanik B. Proline: an essential intermediate in arginine degradation in Saccharomyces cerevisiae. J Bacteriol. 1980 Sep;143(3):1403–1410. doi: 10.1128/jb.143.3.1403-1410.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brandriss M. C. Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT2 gene. Mol Cell Biol. 1983 Oct;3(10):1846–1856. doi: 10.1128/mcb.3.10.1846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bricmont P. A., Daugherty J. R., Cooper T. G. The DAL81 gene product is required for induced expression of two differently regulated nitrogen catabolic genes in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Feb;11(2):1161–1166. doi: 10.1128/mcb.11.2.1161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Buckingham L. E., Wang H. T., Elder R. T., McCarroll R. M., Slater M. R., Esposito R. E. Nucleotide sequence and promoter analysis of SPO13, a meiosis-specific gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9406–9410. doi: 10.1073/pnas.87.23.9406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Carlson M., Taussig R., Kustu S., Botstein D. The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence. Mol Cell Biol. 1983 Mar;3(3):439–447. doi: 10.1128/mcb.3.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chisholm G., Cooper T. G. Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae. Mol Cell Biol. 1982 Sep;2(9):1088–1095. doi: 10.1128/mcb.2.9.1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cooper T. G., Ferguson D., Rai R., Bysani N. The GLN3 gene product is required for transcriptional activation of allantoin system gene expression in Saccharomyces cerevisiae. J Bacteriol. 1990 Feb;172(2):1014–1018. doi: 10.1128/jb.172.2.1014-1018.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Coornaert D., Vissers S., André B., Grenson M. The UGA43 negative regulatory gene of Saccharomyces cerevisiae contains both a GATA-1 type zinc finger and a putative leucine zipper. Curr Genet. 1992 Apr;21(4-5):301–307. doi: 10.1007/BF00351687. [DOI] [PubMed] [Google Scholar]
  19. Coornaert D., Vissers S., André B. The pleiotropic UGA35(DURL) regulatory gene of Saccharomyces cerevisiae: cloning, sequence and identity with the DAL81 gene. Gene. 1991 Jan 15;97(2):163–171. doi: 10.1016/0378-1119(91)90048-g. [DOI] [PubMed] [Google Scholar]
  20. Coschigano P. W., Magasanik B. The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione s-transferases. Mol Cell Biol. 1991 Feb;11(2):822–832. doi: 10.1128/mcb.11.2.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Courchesne W. E., Magasanik B. Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes. J Bacteriol. 1988 Feb;170(2):708–713. doi: 10.1128/jb.170.2.708-713.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Cunningham T. S., Cooper T. G. Expression of the DAL80 gene, whose product is homologous to the GATA factors and is a negative regulator of multiple nitrogen catabolic genes in Saccharomyces cerevisiae, is sensitive to nitrogen catabolite repression. Mol Cell Biol. 1991 Dec;11(12):6205–6215. doi: 10.1128/mcb.11.12.6205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Drillien R., Aigle M., Lacroute F. Yeast mutants pleiotropically impaired in the regulation of the two glutamate dehydrogenases. Biochem Biophys Res Commun. 1973 Jul 17;53(2):367–372. doi: 10.1016/0006-291x(73)90671-2. [DOI] [PubMed] [Google Scholar]
  25. Drillien R., Lacroute F. Ureidosuccinic acid uptake in yeast and some aspects of its regulation. J Bacteriol. 1972 Jan;109(1):203–208. doi: 10.1128/jb.109.1.203-208.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Dubois E., Vissers S., Grenson M., Wiame J. M. Glutamine and ammonia in nitrogen catabolite repression of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1977 Mar 21;75(2):233–239. doi: 10.1016/0006-291x(77)91033-6. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Han M., Kim U. J., Kayne P., Grunstein M. Depletion of histone H4 and nucleosomes activates the PHO5 gene in Saccharomyces cerevisiae. EMBO J. 1988 Jul;7(7):2221–2228. doi: 10.1002/j.1460-2075.1988.tb03061.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hirsch J. P., Henry S. A. Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis. Mol Cell Biol. 1986 Oct;6(10):3320–3328. doi: 10.1128/mcb.6.10.3320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Jauniaux J. C., Vandenbol M., Vissers S., Broman K., Grenson M. Nitrogen catabolite regulation of proline permease in Saccharomyces cerevisiae. Cloning of the PUT4 gene and study of PUT4 RNA levels in wild-type and mutant strains. Eur J Biochem. 1987 May 4;164(3):601–606. doi: 10.1111/j.1432-1033.1987.tb11169.x. [DOI] [PubMed] [Google Scholar]
  33. Koerner T. J., Hill J. E., Myers A. M., Tzagoloff A. High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 1991;194:477–490. doi: 10.1016/0076-6879(91)94036-c. [DOI] [PubMed] [Google Scholar]
  34. Kovari L., Sumrada R., Kovari I., Cooper T. G. Multiple positive and negative cis-acting elements mediate induced arginase (CAR1) gene expression in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Oct;10(10):5087–5097. doi: 10.1128/mcb.10.10.5087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Lopes J. M., Hirsch J. P., Chorgo P. A., Schulze K. L., Henry S. A. Analysis of sequences in the INO1 promoter that are involved in its regulation by phospholipid precursors. Nucleic Acids Res. 1991 Apr 11;19(7):1687–1693. doi: 10.1093/nar/19.7.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marczak J. E., Brandriss M. C. Analysis of constitutive and noninducible mutations of the PUT3 transcriptional activator. Mol Cell Biol. 1991 May;11(5):2609–2619. doi: 10.1128/mcb.11.5.2609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Marczak J. E., Brandriss M. C. Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator. Mol Cell Biol. 1989 Nov;9(11):4696–4705. doi: 10.1128/mcb.9.11.4696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. Mitchell A. P., Magasanik B. Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Dec;4(12):2758–2766. doi: 10.1128/mcb.4.12.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Park H. D., Luche R. M., Cooper T. G. The yeast UME6 gene product is required for transcriptional repression mediated by the CAR1 URS1 repressor binding site. Nucleic Acids Res. 1992 Apr 25;20(8):1909–1915. doi: 10.1093/nar/20.8.1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Perlman D., Halvorson H. O. Distinct repressible mRNAs for cytoplasmic and secreted yeast invertase are encoded by a single gene. Cell. 1981 Aug;25(2):525–536. doi: 10.1016/0092-8674(81)90071-4. [DOI] [PubMed] [Google Scholar]
  45. Rai R., Genbauffe F. S., Sumrada R. A., Cooper T. G. Identification of sequences responsible for transcriptional activation of the allantoate permease gene in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):602–608. doi: 10.1128/mcb.9.2.602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rogers D. T., Lemire J. M., Bostian K. A. Acid phosphatase polypeptides in Saccharomyces cerevisiae are encoded by a differentially regulated multigene family. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2157–2161. doi: 10.1073/pnas.79.7.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Shah H. C., Carlson G. P. Alteration by phenobarbital and 3-methyl-cholanthrene of functional and structural changes in rat liver due to carbon tetrachloride inhalation. J Pharmacol Exp Ther. 1975 Apr;193(1):281–292. [PubMed] [Google Scholar]
  49. Siddiqui A. H., Brandriss M. C. A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box. Mol Cell Biol. 1988 Nov;8(11):4634–4641. doi: 10.1128/mcb.8.11.4634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Siddiqui A. H., Brandriss M. C. The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences. Mol Cell Biol. 1989 Nov;9(11):4706–4712. doi: 10.1128/mcb.9.11.4706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Trueheart J., Boeke J. D., Fink G. R. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol Cell Biol. 1987 Jul;7(7):2316–2328. doi: 10.1128/mcb.7.7.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vallier L. G., Carlson M. New SNF genes, GAL11 and GRR1 affect SUC2 expression in Saccharomyces cerevisiae. Genetics. 1991 Nov;129(3):675–684. doi: 10.1093/genetics/129.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Vissers S., Andre B., Muyldermans F., Grenson M. Induction of the 4-aminobutyrate and urea-catabolic pathways in Saccharomyces cerevisiae. Specific and common transcriptional regulators. Eur J Biochem. 1990 Feb 14;187(3):611–616. doi: 10.1111/j.1432-1033.1990.tb15344.x. [DOI] [PubMed] [Google Scholar]
  57. Vissers S., Andre B., Muyldermans F., Grenson M. Positive and negative regulatory elements control the expression of the UGA4 gene coding for the inducible 4-aminobutyric-acid-specific permease in Saccharomyces cerevisiae. Eur J Biochem. 1989 May 1;181(2):357–361. doi: 10.1111/j.1432-1033.1989.tb14732.x. [DOI] [PubMed] [Google Scholar]
  58. Wang S. S., Brandriss M. C. Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product. Mol Cell Biol. 1987 Dec;7(12):4431–4440. doi: 10.1128/mcb.7.12.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wickner R. B. [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science. 1994 Apr 22;264(5158):566–569. doi: 10.1126/science.7909170. [DOI] [PubMed] [Google Scholar]

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

RESOURCES