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
. 1990 Nov;87(22):8711–8715. doi: 10.1073/pnas.87.22.8711

Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins.

N Suzuki 1, H R Choe 1, Y Nishida 1, Y Yamawaki-Kataoka 1, S Ohnishi 1, T Tamaoki 1, T Kataoka 1
PMCID: PMC55029  PMID: 2247439

Abstract

A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the Sa. cerevisiae RAS2 protein. The NH2-terminal 657 amino acids were found to be dispensable for the activation. However, almost all 2-amino acid insertions in the middle 600 residues comprising leucine-rich repeats and deletions in the COOH-terminal 66 residues completely abolished activation by the RAS2 protein, whereas insertion mutations in the other regions generally had no effect. Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the Sa. cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein. We found that the fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the Sa. cerevisiae cyclase rendered the catalytic domain of the Sc. pombe cyclase, which otherwise did not respond to RAS proteins, activatable by the RAS2 protein. Thus the leucine-rich repeats and the COOH terminus of the Sa. cerevisiae adenylate cyclase appear to be required for interaction with RAS proteins.

Full text

PDF
8711

Images in this article

8713Image
on p.8713
8714Image
on p.8714
8714Image
on p.8714

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. Barany F. Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4202–4206. doi: 10.1073/pnas.82.12.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  4. Beckner S. K., Hattori S., Shih T. Y. The ras oncogene product p21 is not a regulatory component of adenylate cyclase. Nature. 1985 Sep 5;317(6032):71–72. doi: 10.1038/317071a0. [DOI] [PubMed] [Google Scholar]
  5. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  6. Broek D., Samiy N., Fasano O., Fujiyama A., Tamanoi F., Northup J., Wigler M. Differential activation of yeast adenylate cyclase by wild-type and mutant RAS proteins. Cell. 1985 Jul;41(3):763–769. doi: 10.1016/s0092-8674(85)80057-x. [DOI] [PubMed] [Google Scholar]
  7. Colicelli J., Field J., Ballester R., Chester N., Young D., Wigler M. Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase. Mol Cell Biol. 1990 Jun;10(6):2539–2543. doi: 10.1128/mcb.10.6.2539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Field J., Broek D., Kataoka T., Wigler M. Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jun;7(6):2128–2133. doi: 10.1128/mcb.7.6.2128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Field J., Vojtek A., Ballester R., Bolger G., Colicelli J., Ferguson K., Gerst J., Kataoka T., Michaeli T., Powers S. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell. 1990 Apr 20;61(2):319–327. doi: 10.1016/0092-8674(90)90812-s. [DOI] [PubMed] [Google Scholar]
  10. Field J., Xu H. P., Michaeli T., Ballester R., Sass P., Wigler M., Colicelli J. Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae. Science. 1990 Jan 26;247(4941):464–467. doi: 10.1126/science.2405488. [DOI] [PubMed] [Google Scholar]
  11. Fukui Y., Kozasa T., Kaziro Y., Takeda T., Yamamoto M. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell. 1986 Jan 31;44(2):329–336. doi: 10.1016/0092-8674(86)90767-1. [DOI] [PubMed] [Google Scholar]
  12. Gray H. B., Jr, Ostrander D. A., Hodnett J. L., Legerski R. J., Robberson D. L. Extracellular nucleases of Pseudomonas BAL 31. I. Characterization of single strand-specific deoxyriboendonuclease and double-strand deoxyriboexonuclease activities. Nucleic Acids Res. 1975 Sep;2(9):1459–1492. doi: 10.1093/nar/2.9.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hashimoto C., Hudson K. L., Anderson K. V. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell. 1988 Jan 29;52(2):269–279. doi: 10.1016/0092-8674(88)90516-8. [DOI] [PubMed] [Google Scholar]
  14. Heideman W., Casperson G. F., Bourne H. R. Adenylyl cyclase in yeast. Hydrodynamic properties and activation by trypsin. J Biol Chem. 1987 May 25;262(15):7087–7091. [PubMed] [Google Scholar]
  15. Hofsteenge J., Kieffer B., Matthies R., Hemmings B. A., Stone S. R. Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats. Biochemistry. 1988 Nov 15;27(23):8537–8544. doi: 10.1021/bi00423a006. [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. Kataoka T., Broek D., Wigler M. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell. 1985 Dec;43(2 Pt 1):493–505. doi: 10.1016/0092-8674(85)90179-5. [DOI] [PubMed] [Google Scholar]
  18. Kataoka T., Powers S., Cameron S., Fasano O., Goldfarb M., Broach J., Wigler M. Functional homology of mammalian and yeast RAS genes. Cell. 1985 Jan;40(1):19–26. doi: 10.1016/0092-8674(85)90304-6. [DOI] [PubMed] [Google Scholar]
  19. Kataoka T., Powers S., McGill C., Fasano O., Strathern J., Broach J., Wigler M. Genetic analysis of yeast RAS1 and RAS2 genes. Cell. 1984 Jun;37(2):437–445. doi: 10.1016/0092-8674(84)90374-x. [DOI] [PubMed] [Google Scholar]
  20. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  21. Lopez J. A., Chung D. W., Fujikawa K., Hagen F. S., Davie E. W., Roth G. J. The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2135–2139. doi: 10.1073/pnas.85.7.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lopez J. A., Chung D. W., Fujikawa K., Hagen F. S., Papayannopoulou T., Roth G. J. Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5615–5619. doi: 10.1073/pnas.84.16.5615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McFarland K. C., Sprengel R., Phillips H. S., Köhler M., Rosemblit N., Nikolics K., Segaloff D. L., Seeburg P. H. Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science. 1989 Aug 4;245(4917):494–499. doi: 10.1126/science.2502842. [DOI] [PubMed] [Google Scholar]
  24. Ratzkin B., Carbon J. Functional expression of cloned yeast DNA in Escherichia coli. Proc Natl Acad Sci U S A. 1977 Feb;74(2):487–491. doi: 10.1073/pnas.74.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reinke R., Krantz D. E., Yen D., Zipursky S. L. Chaoptin, a cell surface glycoprotein required for Drosophila photoreceptor cell morphogenesis, contains a repeat motif found in yeast and human. Cell. 1988 Jan 29;52(2):291–301. doi: 10.1016/0092-8674(88)90518-1. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Takahashi N., Takahashi Y., Putnam F. W. Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1906–1910. doi: 10.1073/pnas.82.7.1906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Titani K., Takio K., Handa M., Ruggeri Z. M. Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5610–5614. doi: 10.1073/pnas.84.16.5610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Toda T., Uno I., Ishikawa T., Powers S., Kataoka T., Broek D., Cameron S., Broach J., Matsumoto K., Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. doi: 10.1016/0092-8674(85)90305-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  32. Yamawaki-Kataoka Y., Tamaoki T., Choe H. R., Tanaka H., Kataoka T. Adenylate cyclases in yeast: a comparison of the genes from Schizosaccharomyces pombe and Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5693–5697. doi: 10.1073/pnas.86.15.5693. [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