Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Sep;83(17):6553–6557. doi: 10.1073/pnas.83.17.6553

Functional domains of the yeast regulatory protein GAL4.

S A Johnston, M J Zavortink, C Debouck, J E Hopper
PMCID: PMC386542  PMID: 2944111

Abstract

In the yeast Saccharomyces cerevisiae regulation of the galactose/melibiose regulon rests on a dosage-dependent functional interplay between the positive regulator of transcription, the GAL4 protein, and the negative regulator, GAL80 protein. We have used this interplay to select in vitro generated fusions between the yeast ADH1 promoter and GAL4 coding sequence that overproduce GAL4 protein, allowing the identification of GAL4 protein in crude extracts from yeast. One type of these constructions produces a GAL4 protein that lacks its normal NH2 terminus. This protein is unable to complement a gal4 lesion but still retains a domain that functionally antagonizes the negative regulatory protein. One defect in this truncated protein is its inability to be concentrated in the nucleus. However, the nuclear localization defect is complemented by full-length GAL4 protein. The truncated protein also appears to effect changes in the relative transcriptional levels of the structural genes. These observations imply that GAL4 protein consists of several domains, including ones for nuclear localization, interaction with the negative regulatory protein, and, possibly, separable transcriptional activation domains for the structural genes.

Full text

PDF
6554

Images in this article

6554Image
on p.6554
6555Image
on p.6555
6556Image
on p.6556
6556Image
on p.6556

Selected References

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

  1. Ammerer G. Expression of genes in yeast using the ADCI promoter. Methods Enzymol. 1983;101:192–201. doi: 10.1016/0076-6879(83)01014-9. [DOI] [PubMed] [Google Scholar]
  2. Batteiger B., Newhall W. J., 5th, Jones R. B. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982 Dec 30;55(3):297–307. doi: 10.1016/0022-1759(82)90089-8. [DOI] [PubMed] [Google Scholar]
  3. Bennetzen J. L., Hall B. D. The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase. J Biol Chem. 1982 Mar 25;257(6):3018–3025. [PubMed] [Google Scholar]
  4. Bloom K. S., Fitzgerald-Hayes M., Carbon J. Structural analysis and sequence organization of yeast centromeres. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):1175–1185. doi: 10.1101/sqb.1983.047.01.133. [DOI] [PubMed] [Google Scholar]
  5. Bram R. J., Kornberg R. D. Specific protein binding to far upstream activating sequences in polymerase II promoters. Proc Natl Acad Sci U S A. 1985 Jan;82(1):43–47. doi: 10.1073/pnas.82.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clarke L., Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980 Oct 9;287(5782):504–509. doi: 10.1038/287504a0. [DOI] [PubMed] [Google Scholar]
  7. Denis C. L., Ferguson J., Young E. T. mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a nonfermentable carbon source. J Biol Chem. 1983 Jan 25;258(2):1165–1171. [PubMed] [Google Scholar]
  8. Douglas H. C., Hawthorne C. D. Uninducible mutants in the gal i locus of Saccharomyces cerevisiae. J Bacteriol. 1972 Mar;109(3):1139–1143. doi: 10.1128/jb.109.3.1139-1143.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Guarente L., Yocum R. R., Gifford P. A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410–7414. doi: 10.1073/pnas.79.23.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hinnebusch A. G., Fink G. R. Positive regulation in the general amino acid control of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5374–5378. doi: 10.1073/pnas.80.17.5374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laughon A., Gesteland R. F. Isolation and preliminary characterization of the GAL4 gene, a positive regulator of transcription in yeast. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6827–6831. doi: 10.1073/pnas.79.22.6827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laughon A., Gesteland R. F. Primary structure of the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol. 1984 Feb;4(2):260–267. doi: 10.1128/mcb.4.2.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matsumoto K., Adachi Y., Toh-e A., Oshima Y. Function of positive regulatory gene gal4 in the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae: evidence that the GAL81 region codes for part of the gal4 protein. J Bacteriol. 1980 Feb;141(2):508–527. doi: 10.1128/jb.141.2.508-527.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matsumoto K., Toh-e A., Oshima Y. Genetic control of galactokinase synthesis in Saccharomyces cerevisiae: evidence for constitutive expression of the positive regulatory gene gal4. J Bacteriol. 1978 May;134(2):446–457. doi: 10.1128/jb.134.2.446-457.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Metzenberg R. L., Chia W. Genetic control of phosphorus assimilation in Neurospora crassa: dose-dependent dominance and recessiveness in constitutive mutants. Genetics. 1979 Nov;93(3):625–643. doi: 10.1093/genetics/93.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nogi Y., Shimada H., Matsuzaki Y., Hashimoto H., Fukasawa T. Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. II. The isolation and dosage effect of the regulatory gene GAL80. Mol Gen Genet. 1984;195(1-2):29–34. doi: 10.1007/BF00332719. [DOI] [PubMed] [Google Scholar]
  21. Perlman D., Hopper J. E. Constitutive synthesis of the GAL4 protein, a galactose pathway regulator in Saccharomyces cerevisiae. Cell. 1979 Jan;16(1):89–95. doi: 10.1016/0092-8674(79)90190-9. [DOI] [PubMed] [Google Scholar]
  22. Post-Beittenmiller M. A., Hamilton R. W., Hopper J. E. Regulation of basal and induced levels of the MEL1 transcript in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jul;4(7):1238–1245. doi: 10.1128/mcb.4.7.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Silver P. A., Keegan L. P., Ptashne M. Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5951–5955. doi: 10.1073/pnas.81.19.5951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. West R. W., Jr, Yocum R. R., Ptashne M. Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. Mol Cell Biol. 1984 Nov;4(11):2467–2478. doi: 10.1128/mcb.4.11.2467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yocum R. R., Hanley S., West R., Jr, Ptashne M. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES