Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Jun;11(6):3307–3316. doi: 10.1128/mcb.11.6.3307

The CYC8 and TUP1 proteins involved in glucose repression in Saccharomyces cerevisiae are associated in a protein complex.

F E Williams 1, U Varanasi 1, R J Trumbly 1
PMCID: PMC360183  PMID: 2038333

Abstract

Mutations of yeast CYC8 or TUP1 genes greatly reduce the degree of glucose repression of many genes and affect other regulatory pathways, including mating type. The predicted CYC8 protein contains 10 copies of the 34-amino-acid tetratricopeptide repeat unit, and the predicted TUP1 protein has six repeated regions found in the beta subunit of heterotrimeric G proteins. The absence of DNA-binding motifs and the presence of these repeated domains suggest that the CYC8 and TUP1 proteins function via protein-protein interaction with transcriptional regulatory proteins. We raised polyclonal antibodies against TrpE-CYC8 and TrpE-TUP1 fusion proteins expressed in Escherichia coli. The CYC8 and TUP1 proteins from yeast cells were detected as closely spaced doublets on Western immunoblots of sodium dodecyl sulfate-polyacrylamide gels. Western blots of nondenaturing gels revealed that both proteins are associated in a high-molecular-weight complex with an apparent size of 1,200 kDa. In extracts from delta cyc8 strains, the size of the complex is reduced to 830 kDa. The CYC8 and TUP1 proteins were coprecipitated by either antiserum, further supporting the conclusion that they are associated with each other. The complex could be reconstituted in vitro by mixing extracts from strains with complementary mutations in the CYC8 and TUP1 genes.

Full text

PDF
3307

Images in this article

3308Image
on p.3308
3309Image
on p.3309
3310Image
on p.3310
3311Image
on p.3311
3312Image
on p.3312
3313Image
on p.3313
3314Image
on p.3314

Selected References

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

  1. Amati B. B., Gasser S. M. Chromosomal ARS and CEN elements bind specifically to the yeast nuclear scaffold. Cell. 1988 Sep 23;54(7):967–978. doi: 10.1016/0092-8674(88)90111-0. [DOI] [PubMed] [Google Scholar]
  2. Andersson L. -O., Borg H., Mikaelsson M. Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity. FEBS Lett. 1972 Feb 1;20(2):199–202. doi: 10.1016/0014-5793(72)80793-2. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Carlson M., Osmond B. C., Neigeborn L., Botstein D. A suppressor of SNF1 mutations causes constitutive high-level invertase synthesis in yeast. Genetics. 1984 May;107(1):19–32. doi: 10.1093/genetics/107.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Celenza J. L., Carlson M. A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science. 1986 Sep 12;233(4769):1175–1180. doi: 10.1126/science.3526554. [DOI] [PubMed] [Google Scholar]
  6. Clos J., Westwood J. T., Becker P. B., Wilson S., Lambert K., Wu C. Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation. Cell. 1990 Nov 30;63(5):1085–1097. doi: 10.1016/0092-8674(90)90511-c. [DOI] [PubMed] [Google Scholar]
  7. Dalrymple M. A., Petersen-Bjorn S., Friesen J. D., Beggs J. D. The product of the PRP4 gene of S. cerevisiae shows homology to beta subunits of G proteins. Cell. 1989 Sep 8;58(5):811–812. doi: 10.1016/0092-8674(89)90930-6. [DOI] [PubMed] [Google Scholar]
  8. Ey P. L., Ashman L. K. The use of alkaline phosphatase-conjugated anti-immunoglobulin with immunoblots for determining the specificity of monoclonal antibodies to protein mixtures. Methods Enzymol. 1986;121:497–509. doi: 10.1016/0076-6879(86)21050-2. [DOI] [PubMed] [Google Scholar]
  9. Ferguson B., Krippl B., Andrisani O., Jones N., Westphal H., Rosenberg M. E1A 13S and 12S mRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA. Mol Cell Biol. 1985 Oct;5(10):2653–2661. doi: 10.1128/mcb.5.10.2653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Firestone G. L., Winguth S. D. Immunoprecipitation of proteins. Methods Enzymol. 1990;182:688–700. doi: 10.1016/0076-6879(90)82054-6. [DOI] [PubMed] [Google Scholar]
  11. Fong H. K., Hurley J. B., Hopkins R. S., Miake-Lye R., Johnson M. S., Doolittle R. F., Simon M. I. Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2162–2166. doi: 10.1073/pnas.83.7.2162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gasser S. M., Laemmli U. K. Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell. 1986 Aug 15;46(4):521–530. doi: 10.1016/0092-8674(86)90877-9. [DOI] [PubMed] [Google Scholar]
  13. Hardy W. R., Strauss J. H. Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies. J Virol. 1988 Mar;62(3):998–1007. doi: 10.1128/jvi.62.3.998-1007.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hartley D. A., Preiss A., Artavanis-Tsakonas S. A deduced gene product from the Drosophila neurogenic locus, enhancer of split, shows homology to mammalian G-protein beta subunit. Cell. 1988 Dec 2;55(5):785–795. doi: 10.1016/0092-8674(88)90134-1. [DOI] [PubMed] [Google Scholar]
  15. Hirano T., Kinoshita N., Morikawa K., Yanagida M. Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+. Cell. 1990 Jan 26;60(2):319–328. doi: 10.1016/0092-8674(90)90746-2. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Lemontt J. F., Fugit D. R., Mackay V. L. Pleiotropic Mutations at the TUP1 Locus That Affect the Expression of Mating-Type-Dependent Functions in SACCHAROMYCES CEREVISIAE. Genetics. 1980 Apr;94(4):899–920. doi: 10.1093/genetics/94.4.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lemontt J. F. Pathways of ultraviolet mutability in Saccharomyces cerevisiae. III. Genetic analysis and properties of mutants resitant to ultraviolet-induced forward mutation. Mutat Res. 1977 May;43(2):179–204. doi: 10.1016/0027-5107(77)90003-3. [DOI] [PubMed] [Google Scholar]
  20. MacConnell W. P., Verma I. M. Expression of FBJ-MSV oncogene (fos) product in bacteria. Virology. 1983 Dec;131(2):367–374. doi: 10.1016/0042-6822(83)90504-4. [DOI] [PubMed] [Google Scholar]
  21. Neigeborn L., Carlson M. Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations. Genetics. 1987 Feb;115(2):247–253. doi: 10.1093/genetics/115.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Olesen J., Hahn S., Guarente L. Yeast HAP2 and HAP3 activators both bind to the CYC1 upstream activation site, UAS2, in an interdependent manner. Cell. 1987 Dec 24;51(6):953–961. doi: 10.1016/0092-8674(87)90582-4. [DOI] [PubMed] [Google Scholar]
  23. Rothstein R. J., Sherman F. Genes affecting the expression of cytochrome c in yeast: genetic mapping and genetic interactions. Genetics. 1980 Apr;94(4):871–889. doi: 10.1093/genetics/94.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ruggieri R., Tanaka K., Nakafuku M., Kaziro Y., Toh-e A., Matsumoto K. MSI1, a negative regulator of the RAS-cAMP pathway in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8778–8782. doi: 10.1073/pnas.86.22.8778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rüther U., Müller-Hill B. Easy identification of cDNA clones. EMBO J. 1983;2(10):1791–1794. doi: 10.1002/j.1460-2075.1983.tb01659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schamhart D. H., Ten Berge A. M., Van De Poll K. W. Isolation of a catabolite repression mutant of yeast as a revertant of a strain that is maltose negative in the respiratory-deficient state. J Bacteriol. 1975 Mar;121(3):747–752. doi: 10.1128/jb.121.3.747-752.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schultz J., Carlson M. Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Oct;7(10):3637–3645. doi: 10.1128/mcb.7.10.3637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schultz J., Marshall-Carlson L., Carlson M. The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae. Mol Cell Biol. 1990 Sep;10(9):4744–4756. doi: 10.1128/mcb.10.9.4744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sikorski R. S., Boguski M. S., Goebl M., Hieter P. A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell. 1990 Jan 26;60(2):307–317. doi: 10.1016/0092-8674(90)90745-z. [DOI] [PubMed] [Google Scholar]
  30. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Spindler K. R., Rosser D. S., Berk A. J. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol. 1984 Jan;49(1):132–141. doi: 10.1128/jvi.49.1.132-141.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stark H. C., Fugit D., Mowshowitz D. B. Pleiotropic properties of a yeast mutant insensitive to catabolite repression. Genetics. 1980 Apr;94(4):921–928. doi: 10.1093/genetics/94.4.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thrash-Bingham C., Fangman W. L. A yeast mutation that stabilizes a plasmid bearing a mutated ARS1 element. Mol Cell Biol. 1989 Feb;9(2):809–816. doi: 10.1128/mcb.9.2.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Trumbly R. J. Cloning and characterization of the CYC8 gene mediating glucose repression in yeast. Gene. 1988 Dec 15;73(1):97–111. doi: 10.1016/0378-1119(88)90316-2. [DOI] [PubMed] [Google Scholar]
  35. Trumbly R. J. Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis. J Bacteriol. 1986 Jun;166(3):1123–1127. doi: 10.1128/jb.166.3.1123-1127.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Whiteway M., Hougan L., Dignard D., Thomas D. Y., Bell L., Saari G. C., Grant F. J., O'Hara P., MacKay V. L. The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein. Cell. 1989 Feb 10;56(3):467–477. doi: 10.1016/0092-8674(89)90249-3. [DOI] [PubMed] [Google Scholar]
  38. Wickner R. B. Mutants of Saccharomyces cerevisiae that incorporate deoxythymidine-5'-monophosphate into deoxyribonucleic acid in vivo. J Bacteriol. 1974 Jan;117(1):252–260. doi: 10.1128/jb.117.1.252-260.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Williams F. E., Trumbly R. J. Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6500–6511. doi: 10.1128/mcb.10.12.6500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yochem J., Byers B. Structural comparison of the yeast cell division cycle gene CDC4 and a related pseudogene. J Mol Biol. 1987 May 20;195(2):233–245. doi: 10.1016/0022-2836(87)90646-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES