Abstract
The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids.
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- 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]
- Bhat P. J., Hopper J. E. Overproduction of the GAL1 or GAL3 protein causes galactose-independent activation of the GAL4 protein: evidence for a new model of induction for the yeast GAL/MEL regulon. Mol Cell Biol. 1992 Jun;12(6):2701–2707. doi: 10.1128/mcb.12.6.2701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bhat P. J., Hopper J. E. The mechanism of inducer formation in gal3 mutants of the yeast galactose system is independent of normal galactose metabolism and mitochondrial respiratory function. Genetics. 1991 Jun;128(2):233–239. doi: 10.1093/genetics/128.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Broach J. R. Galactose regulation in Saccharomyces cerevisiae. The enzymes encoded by the GAL7, 10, 1 cluster are co-ordinately controlled and separately translated. J Mol Biol. 1979 Jun 15;131(1):41–53. doi: 10.1016/0022-2836(79)90300-0. [DOI] [PubMed] [Google Scholar]
- Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Douglas H. C., Hawthorne D. C. Regulation of genes controlling synthesis of the galactose pathway enzymes in yeast. Genetics. 1966 Sep;54(3):911–916. doi: 10.1093/genetics/54.3.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fukasawa T., Segawa T., Nogi Y. Uridine diphosphate glucose-4-epimerase and galactose-1-phosphate uridylyltransferase from Saccharomyces cerevisiae. Methods Enzymol. 1982;89(Pt 500):584–592. doi: 10.1016/s0076-6879(82)89101-5. [DOI] [PubMed] [Google Scholar]
- Hashimoto H., Kikuchi Y., Nogi Y., Fukasawa T. Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4. Mol Gen Genet. 1983;191(1):31–38. doi: 10.1007/BF00330886. [DOI] [PubMed] [Google Scholar]
- 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]
- Leuther K. K., Johnston S. A. Nondissociation of GAL4 and GAL80 in vivo after galactose induction. Science. 1992 May 29;256(5061):1333–1335. doi: 10.1126/science.1598579. [DOI] [PubMed] [Google Scholar]
- 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]
- Nogi Y., Fukasawa T. Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae. Mol Cell Biol. 1989 Jul;9(7):3009–3017. doi: 10.1128/mcb.9.7.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nogi Y., Fukasawa T. Nucleotide sequence of the yeast regulatory gene GAL80. Nucleic Acids Res. 1984 Dec 21;12(24):9287–9298. doi: 10.1093/nar/12.24.9287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nogi Y. GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae. J Bacteriol. 1986 Jan;165(1):101–106. doi: 10.1128/jb.165.1.101-106.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parthun M. R., Jaehning J. A. Purification and characterization of the yeast transcriptional activator GAL4. J Biol Chem. 1990 Jan 5;265(1):209–213. [PubMed] [Google Scholar]
- Rose M. D., Broach J. R. Cloning genes by complementation in yeast. Methods Enzymol. 1991;194:195–230. doi: 10.1016/0076-6879(91)94017-7. [DOI] [PubMed] [Google Scholar]
- 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]
- Shimada H., Fukasawa T. Controlled transcription of the yeast regulatory gene GAL80. Gene. 1985;39(1):1–9. doi: 10.1016/0378-1119(85)90100-3. [DOI] [PubMed] [Google Scholar]
- St John T. P., Davis R. W. Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell. 1979 Feb;16(2):443–452. doi: 10.1016/0092-8674(79)90020-5. [DOI] [PubMed] [Google Scholar]
- St John T. P., Davis R. W. The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol. 1981 Oct 25;152(2):285–315. doi: 10.1016/0022-2836(81)90244-8. [DOI] [PubMed] [Google Scholar]
- Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
- Tajima M., Nogi Y., Fukasawa T. Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast. 1985 Sep;1(1):67–77. doi: 10.1002/yea.320010108. [DOI] [PubMed] [Google Scholar]
- 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]
- Tsuyumu S., Adams B. G. Dilution kinetic studies of yeast populations: in vivo aggregation of galactose utilizing enzymes and positive regulator molecules. Genetics. 1974 Jul;77(3):491–505. doi: 10.1093/genetics/77.3.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vernet T., Dignard D., Thomas D. Y. A family of yeast expression vectors containing the phage f1 intergenic region. Gene. 1987;52(2-3):225–233. doi: 10.1016/0378-1119(87)90049-7. [DOI] [PubMed] [Google Scholar]
- Yun S. J., Hiraoka Y., Nishizawa M., Takio K., Titani K., Nogi Y., Fukasawa T. Purification and characterization of the yeast negative regulatory protein GAL80. J Biol Chem. 1991 Jan 15;266(2):693–697. [PubMed] [Google Scholar]