Analysis of the Gal3 Signal Transduction Pathway Activating Gal4 Protein-Dependent Transcription in Saccharomyces Cerevisiae (original) (raw)

Abstract

The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho--are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.

Full Text

The Full Text of this article is available as a PDF (4.0 MB).

Selected References

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

  1. Aguilera A. Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae. Mol Gen Genet. 1986 Aug;204(2):310–316. doi: 10.1007/BF00425515. [DOI] [PubMed] [Google Scholar]
  2. Aguilera A., Zimmermann F. K. Isolation and molecular analysis of the phosphoglucose isomerase structural gene of Saccharomyces cerevisiae. Mol Gen Genet. 1986 Jan;202(1):83–89. doi: 10.1007/BF00330521. [DOI] [PubMed] [Google Scholar]
  3. 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]
  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. Blume K. G., Beutler E. Galactokinase from human erythrocytes. Methods Enzymol. 1975;42:47–53. doi: 10.1016/0076-6879(75)42091-2. [DOI] [PubMed] [Google Scholar]
  6. Bostian K. A., Lemire J. M., Cannon L. E., Halvorson H. O. In vitro synthesis of repressible yeast acid phosphatase: identification of multiple mRNAs and products. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4504–4508. doi: 10.1073/pnas.77.8.4504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Ciriacy M., Breitenbach I. Physiological effects of seven different blocks in glycolysis in Saccharomyces cerevisiae. J Bacteriol. 1979 Jul;139(1):152–160. doi: 10.1128/jb.139.1.152-160.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cirillo V. P. Galactose transport in Saccharomyces cerevisiae. I. Nonmetabolized sugars as substrates and inducers of the galactose transport system. J Bacteriol. 1968 May;95(5):1727–1731. doi: 10.1128/jb.95.5.1727-1731.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clifton D., Weinstock S. B., Fraenkel D. G. Glycolysis mutants in Saccharomyces cerevisiae. Genetics. 1978 Jan;88(1):1–11. doi: 10.1093/genetics/88.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DOUGLAS H. C., CONDIE F. The genetic control of galactose utilization in Saccharomyces. J Bacteriol. 1954 Dec;68(6):662–670. doi: 10.1128/jb.68.6.662-670.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  14. Dieckmann C. L., Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
  15. 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]
  16. Hopper J. E., Broach J. R., Rowe L. B. Regulation of the galactose pathway in Saccharomyces cerevisiae: induction of uridyl transferase mRNA and dependency on GAL4 gene function. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2878–2882. doi: 10.1073/pnas.75.6.2878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Kawasaki G., Fraenkel D. G. Cloning of yeast glycolysis genes by complementation. Biochem Biophys Res Commun. 1982 Oct 15;108(3):1107–1122. doi: 10.1016/0006-291x(82)92114-3. [DOI] [PubMed] [Google Scholar]
  20. Kew O. M., Douglas H. C. Genetic co-regulation of galactose and melibiose utilization in Saccharomyces. J Bacteriol. 1976 Jan;125(1):33–41. doi: 10.1128/jb.125.1.33-41.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kleid D. G., Yansura D., Small B., Dowbenko D., Moore D. M., Grubman M. J., McKercher P. D., Morgan D. O., Robertson B. H., Bachrach H. L. Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine. Science. 1981 Dec 4;214(4525):1125–1129. doi: 10.1126/science.6272395. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lee C. Y. Phosphoglucose isomerase from mouse and Drosophila melanogaster. Methods Enzymol. 1982;89(Pt 500):559–562. doi: 10.1016/s0076-6879(82)89096-4. [DOI] [PubMed] [Google Scholar]
  24. Maitra P. K. Glucose and fructose metabolism in a phosphoglucoisomeraseless mutant of Saccharomyces cerevisiae. J Bacteriol. 1971 Sep;107(3):759–769. doi: 10.1128/jb.107.3.759-769.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Oh D., Hopper J. E. Transcription of a yeast phosphoglucomutase isozyme gene is galactose inducible and glucose repressible. Mol Cell Biol. 1990 Apr;10(4):1415–1422. doi: 10.1128/mcb.10.4.1415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Russell D. W., Jensen R., Zoller M. J., Burke J., Errede B., Smith M., Herskowitz I. Structure of the Saccharomyces cerevisiae HO gene and analysis of its upstream regulatory region. Mol Cell Biol. 1986 Dec;6(12):4281–4294. doi: 10.1128/mcb.6.12.4281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rymond B. C., Zitomer R. S., Schümperli D., Rosenberg M. The expression in yeast of the Escherichia coli galK gene on CYC1::galK fusion plasmids. Gene. 1983 Nov;25(2-3):249–262. doi: 10.1016/0378-1119(83)90229-9. [DOI] [PubMed] [Google Scholar]
  31. SPIEGELMAN S., SUSSMAN R. R., PINSKA E. On the cytoplasmic nature of "long-term adaptation" in yeast. Proc Natl Acad Sci U S A. 1950 Nov;36(11):591–606. doi: 10.1073/pnas.36.11.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. Szkutnicka K., Tschopp J. F., Andrews L., Cirillo V. P. Sequence and structure of the yeast galactose transporter. J Bacteriol. 1989 Aug;171(8):4486–4493. doi: 10.1128/jb.171.8.4486-4493.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Tschopp J. F., Emr S. D., Field C., Schekman R. GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae. J Bacteriol. 1986 Apr;166(1):313–318. doi: 10.1128/jb.166.1.313-318.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Tsuyumu S., Adams B. G. Population analysis of the deinduction kinetics of galactose long-term adaptation mutants of yeast. Proc Natl Acad Sci U S A. 1973 Mar;70(3):919–923. doi: 10.1073/pnas.70.3.919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zakian V. A., Scott J. F. Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA. Mol Cell Biol. 1982 Mar;2(3):221–232. doi: 10.1128/mcb.2.3.221. [DOI] [PMC free article] [PubMed] [Google Scholar]