Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Jan 11;21(1):69–77. doi: 10.1093/nar/21.1.69

Glucose repression of lactose/galactose metabolism in Kluyveromyces lactis is determined by the concentration of the transcriptional activator LAC9 (K1GAL4) [corrected]

W Zachariae 1, P Kuger 1, K D Breunig 1
PMCID: PMC309066  PMID: 8441621

Abstract

In the budding yeast Kluyveromyces lactis glucose repression of genes involved in lactose and galactose metabolism is primarily mediated by LAC9 (or K1GAL4) the homologue of the well-known Saccharomyces cerevisiae transcriptional activator GAL4. Phenotypic difference in glucose repression existing between natural strains are due to differences in the LAC9 gene (Breunig, 1989, Mol.Gen.Genet. 261, 422-427). Comparison between the LAC9 alleles of repressible and non-repressible strains revealed that the phenotype is a result of differences in LAC9 gene expression. A two-basepair alteration in the LAC9 promoter region produces a promoter-down effect resulting in slightly reduced LAC9 protein levels under all growth conditions tested. In glucose/galactose medium any change in LAC9 expression drastically affects expression of LAC9 controlled genes e.g. those encoding beta-galactosidase or galactokinase revealing a strong dependence of the kinetics of induction on the LAC9 concentration. We propose that in tightly repressible strains the activator concentration drops below a critical threshold that is required for induction to occur. A model is presented to explain how small differences in activator levels are amplified to produce big changes in expression levels of metabolic genes.

Full text

PDF
69

Images in this article

71Image
on p.71
75Image
on p.75

Selected References

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

  1. Bajwa W., Torchia T. E., Hopper J. E. Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases. Mol Cell Biol. 1988 Aug;8(8):3439–3447. doi: 10.1128/mcb.8.8.3439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhat P. J., Oh D., Hopper J. E. Analysis of the GAL3 signal transduction pathway activating GAL4 protein-dependent transcription in Saccharomyces cerevisiae. Genetics. 1990 Jun;125(2):281–291. doi: 10.1093/genetics/125.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Breunig K. D. Glucose repression of LAC gene expression in yeast is mediated by the transcriptional activator LAC9. Mol Gen Genet. 1989 Apr;216(2-3):422–427. doi: 10.1007/BF00334386. [DOI] [PubMed] [Google Scholar]
  6. Breunig K. D., Kuger P. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site. Mol Cell Biol. 1987 Dec;7(12):4400–4406. doi: 10.1128/mcb.7.12.4400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brunner A., de Cobos A. T., Griffiths D. E. The isolation and genetic characterization of extrachromosomal chloramphenicol and oligomycin-resistant mutants from the petite-negative yeast Kluyveromyces lactis. Mol Gen Genet. 1977 Apr 29;152(3):183–191. doi: 10.1007/BF00268816. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Chasman D. I., Kornberg R. D. GAL4 protein: purification, association with GAL80 protein, and conserved domain structure. Mol Cell Biol. 1990 Jun;10(6):2916–2923. doi: 10.1128/mcb.10.6.2916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dickson R. C., Gerardot C. J., Martin A. K. Genetic evidence for similar negative regulatory domains in the yeast transcription activators GAL4 and LAC9. Nucleic Acids Res. 1990 Sep 11;18(17):5213–5217. doi: 10.1093/nar/18.17.5213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dohmen R. J., Strasser A. W., Höner C. B., Hollenberg C. P. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast. 1991 Oct;7(7):691–692. doi: 10.1002/yea.320070704. [DOI] [PubMed] [Google Scholar]
  12. Entian K. D. Glucose repression: a complex regulatory system in yeast. Microbiol Sci. 1986 Dec;3(12):366–371. [PubMed] [Google Scholar]
  13. Finley R. L., Jr, Chen S., Ma J., Byrne P., West R. W., Jr Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes. Mol Cell Biol. 1990 Nov;10(11):5663–5670. doi: 10.1128/mcb.10.11.5663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Flick J. S., Johnston M. Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Sep;10(9):4757–4769. doi: 10.1128/mcb.10.9.4757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giniger E., Varnum S. M., Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. doi: 10.1016/0092-8674(85)90336-8. [DOI] [PubMed] [Google Scholar]
  16. Green J. B., Smith J. C. Growth factors as morphogens: do gradients and thresholds establish body plan? Trends Genet. 1991 Aug;7(8):245–250. doi: 10.1016/0168-9525(91)90323-I. [DOI] [PubMed] [Google Scholar]
  17. Griggs D. W., Johnston M. Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8597–8601. doi: 10.1073/pnas.88.19.8597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jacobs E., Clad A. Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap. Anal Biochem. 1986 May 1;154(2):583–589. doi: 10.1016/0003-2697(86)90033-3. [DOI] [PubMed] [Google Scholar]
  19. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Johnston S. A., Hopper J. E. Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6971–6975. doi: 10.1073/pnas.79.22.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene. 1983 Nov;25(2-3):333–341. doi: 10.1016/0378-1119(83)90238-x. [DOI] [PubMed] [Google Scholar]
  22. Kuger P., Gödecke A., Breunig K. D. A mutation in the Zn-finger of the GAL4 homolog LAC9 results in glucose repression of its target genes. Nucleic Acids Res. 1990 Feb 25;18(4):745–751. doi: 10.1093/nar/18.4.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kuzhandaivelu N., Jones W. K., Martin A. K., Dickson R. C. The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene. Mol Cell Biol. 1992 May;12(5):1924–1931. doi: 10.1128/mcb.12.5.1924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lamphier M. S., Ptashne M. Multiple mechanisms mediate glucose repression of the yeast GAL1 gene. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5922–5926. doi: 10.1073/pnas.89.13.5922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lohr D., Hopper J. E. The relationship of regulatory proteins and DNase I hypersensitive sites in the yeast GAL1-10 genes. Nucleic Acids Res. 1985 Dec 9;13(23):8409–8423. doi: 10.1093/nar/13.23.8409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matern H., Holzer H. Catabolite inactivation of the galactose uptake system in yeast. J Biol Chem. 1977 Sep 25;252(18):6399–6402. [PubMed] [Google Scholar]
  27. Meyer J., Walker-Jonah A., Hollenberg C. P. Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Nov;11(11):5454–5461. doi: 10.1128/mcb.11.11.5454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mylin L. M., Bhat J. P., Hopper J. E. Regulated phosphorylation and dephosphorylation of GAL4, a transcriptional activator. Genes Dev. 1989 Aug;3(8):1157–1165. doi: 10.1101/gad.3.8.1157. [DOI] [PubMed] [Google Scholar]
  29. Nehlin J. O., Carlberg M., Ronne H. Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J. 1991 Nov;10(11):3373–3377. doi: 10.1002/j.1460-2075.1991.tb04901.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nehlin J. O., Ronne H. Yeast MIG1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. EMBO J. 1990 Sep;9(9):2891–2898. doi: 10.1002/j.1460-2075.1990.tb07479.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Riley M. I., Hopper J. E., Johnston S. A., Dickson R. C. GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon. Mol Cell Biol. 1987 Feb;7(2):780–786. doi: 10.1128/mcb.7.2.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Ruzzi M., Breunig K. D., Ficca A. G., Hollenberg C. P. Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):991–997. doi: 10.1128/mcb.7.3.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Salmeron J. M., Jr, Johnston S. A. Analysis of the Kluyveromyces lactis positive regulatory gene LAC9 reveals functional homology to, but sequence divergence from, the Saccharomyces cerevisiae GAL4 gene. Nucleic Acids Res. 1986 Oct 10;14(19):7767–7781. doi: 10.1093/nar/14.19.7767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Salmeron J. M., Jr, Langdon S. D., Johnston S. A. Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80. Mol Cell Biol. 1989 Jul;9(7):2950–2956. doi: 10.1128/mcb.9.7.2950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Salmeron J. M., Jr, Leuther K. K., Johnston S. A. GAL4 mutations that separate the transcriptional activation and GAL80-interactive functions of the yeast GAL4 protein. Genetics. 1990 May;125(1):21–27. doi: 10.1093/genetics/125.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Selleck S. B., Majors J. E. In vivo DNA-binding properties of a yeast transcription activator protein. Mol Cell Biol. 1987 Sep;7(9):3260–3267. doi: 10.1128/mcb.7.9.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  39. St Johnston D., Nüsslein-Volhard C. The origin of pattern and polarity in the Drosophila embryo. Cell. 1992 Jan 24;68(2):201–219. doi: 10.1016/0092-8674(92)90466-p. [DOI] [PubMed] [Google Scholar]
  40. Torchia T. E., Hamilton R. W., Cano C. L., Hopper J. E. Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes. Mol Cell Biol. 1984 Aug;4(8):1521–1527. doi: 10.1128/mcb.4.8.1521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Torchia T. E., Hopper J. E. Genetic and molecular analysis of the GAL3 gene in the expression of the galactose/melibiose regulon of Saccharomyces cerevisiae. Genetics. 1986 Jun;113(2):229–246. doi: 10.1093/genetics/113.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. WICKERHAM L. J., BURTON K. A. Occurrence of yeast mating types in nature1,3. J Bacteriol. 1952 Apr;63(4):449–451. doi: 10.1128/jb.63.4.449-451.1952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Webster T. D., Dickson R. C. The organization and transcription of the galactose gene cluster of Kluyveromyces lactis. Nucleic Acids Res. 1988 Aug 25;16(16):8011–8028. doi: 10.1093/nar/16.16.8011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wray L. V., Jr, Witte M. M., Dickson R. C., Riley M. I. Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):1111–1121. doi: 10.1128/mcb.7.3.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yocum R. R., Johnston M. Molecular cloning of the GAL80 gene from Saccharomyces cerevisiae and characterization of a gal80 deletion. Gene. 1984 Dec;32(1-2):75–82. doi: 10.1016/0378-1119(84)90034-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES