Skip to main content
NCBI 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
. 1995 Mar 28;92(7):2984–2988. doi: 10.1073/pnas.92.7.2984

Ras membrane targeting is essential for glucose signaling but not for viability in yeast.

S Bhattacharya 1, L Chen 1, J R Broach 1, S Powers 1
PMCID: PMC42343  PMID: 7708760

Abstract

Ras proteins are small GTP binding proteins that serve as critical relays in a variety of signal transduction pathways in eukaryotic cells. Like most metazoan Ras proteins, yeast Ras is post-translationally modified by addition of a farnesyl and a palmitoyl moiety, and these modifications are required for targeting the protein to the cytoplasmic face of the plasma membrane and for biological activity of the protein. We have constructed mutants of the yeast (Saccharomyces cerevisiae) Ras that are farnesylated in vivo but are not palmitoylated. These mutant proteins are not localized to the plasma membrane but function in the cell as well as the wild-type protein. Such mutants are viable but fail to induce a transient increase in intracellular cAMP concentration in response to glucose addition, although this deficiency does not yield a marked growth phenotype. These results are consistent with the hypothesis that the essential role of the farnesyl moiety on yeast Ras is to enhance productive interaction between Ras and its essential downstream target, adenylyl cyclase, rather than to localize Ras to the plasma membrane.

Full text

PDF
2984

Images in this article

2986Fig. 1
on p.2986
2986Fig. 2
on p.2986
2986Fig. 3
on p.2986
2987Fig. 4
on p.2987

Selected References

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

  1. Broek D., Toda T., Michaeli T., Levin L., Birchmeier C., Zoller M., Powers S., Wigler M. The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell. 1987 Mar 13;48(5):789–799. doi: 10.1016/0092-8674(87)90076-6. [DOI] [PubMed] [Google Scholar]
  2. Buday L., Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell. 1993 May 7;73(3):611–620. doi: 10.1016/0092-8674(93)90146-h. [DOI] [PubMed] [Google Scholar]
  3. Buss J. E., Sefton B. M. Direct identification of palmitic acid as the lipid attached to p21ras. Mol Cell Biol. 1986 Jan;6(1):116–122. doi: 10.1128/mcb.6.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buss J. E., Solski P. A., Schaeffer J. P., MacDonald M. J., Der C. J. Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal. Science. 1989 Mar 24;243(4898):1600–1603. doi: 10.1126/science.2648572. [DOI] [PubMed] [Google Scholar]
  5. Cadwallader K. A., Paterson H., Macdonald S. G., Hancock J. F. N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization. Mol Cell Biol. 1994 Jul;14(7):4722–4730. doi: 10.1128/mcb.14.7.4722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clark S. G., Stern M. J., Horvitz H. R. C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature. 1992 Mar 26;356(6367):340–344. doi: 10.1038/356340a0. [DOI] [PubMed] [Google Scholar]
  8. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. doi: 10.1146/annurev.bi.61.070192.002035. [DOI] [PubMed] [Google Scholar]
  9. Clarke S., Vogel J. P., Deschenes R. J., Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4643–4647. doi: 10.1073/pnas.85.13.4643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deschenes R. J., Broach J. R. Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function. Mol Cell Biol. 1987 Jul;7(7):2344–2351. doi: 10.1128/mcb.7.7.2344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deschenes R. J., Stimmel J. B., Clarke S., Stock J., Broach J. R. RAS2 protein of Saccharomyces cerevisiae is methyl-esterified at its carboxyl terminus. J Biol Chem. 1989 Jul 15;264(20):11865–11873. [PubMed] [Google Scholar]
  12. Egan S. E., Giddings B. W., Brooks M. W., Buday L., Sizeland A. M., Weinberg R. A. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993 May 6;363(6424):45–51. doi: 10.1038/363045a0. [DOI] [PubMed] [Google Scholar]
  13. Engelberg D., Simchen G., Levitzki A. In vitro reconstitution of cdc25 regulated S. cerevisiae adenylyl cyclase and its kinetic properties. EMBO J. 1990 Mar;9(3):641–651. doi: 10.1002/j.1460-2075.1990.tb08156.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fujiyama A., Tamanoi F. RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus. J Biol Chem. 1990 Feb 25;265(6):3362–3368. [PubMed] [Google Scholar]
  15. Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goodman L. E., Judd S. R., Farnsworth C. C., Powers S., Gelb M. H., Glomset J. A., Tamanoi F. Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9665–9669. doi: 10.1073/pnas.87.24.9665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gutierrez L., Magee A. I., Marshall C. J., Hancock J. F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. doi: 10.1002/j.1460-2075.1989.tb03478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  19. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  20. Itoh T., Kaibuchi K., Masuda T., Yamamoto T., Matsuura Y., Maeda A., Shimizu K., Takai Y. The post-translational processing of ras p21 is critical for its stimulation of mitogen-activated protein kinase. J Biol Chem. 1993 Feb 15;268(5):3025–3028. [PubMed] [Google Scholar]
  21. Jones S., Vignais M. L., Broach J. R. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras. Mol Cell Biol. 1991 May;11(5):2641–2646. doi: 10.1128/mcb.11.5.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Kuroda Y., Suzuki N., Kataoka T. The effect of posttranslational modifications on the interaction of Ras2 with adenylyl cyclase. Science. 1993 Jan 29;259(5095):683–686. doi: 10.1126/science.8430318. [DOI] [PubMed] [Google Scholar]
  24. Leevers S. J., Paterson H. F., Marshall C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994 Jun 2;369(6479):411–414. doi: 10.1038/369411a0. [DOI] [PubMed] [Google Scholar]
  25. Li N., Batzer A., Daly R., Yajnik V., Skolnik E., Chardin P., Bar-Sagi D., Margolis B., Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. doi: 10.1038/363085a0. [DOI] [PubMed] [Google Scholar]
  26. Marshall C. J. Protein prenylation: a mediator of protein-protein interactions. Science. 1993 Mar 26;259(5103):1865–1866. doi: 10.1126/science.8456312. [DOI] [PubMed] [Google Scholar]
  27. Mazón M. J., Gancedo J. M., Gancedo C. Phosphorylation and inactivation of yeast fructose-bisphosphatase in vivo by glucose and by proton ionophores. A possible role for cAMP. Eur J Biochem. 1982 Oct;127(3):605–608. doi: 10.1111/j.1432-1033.1982.tb06915.x. [DOI] [PubMed] [Google Scholar]
  28. Mbonyi K., Beullens M., Detremerie K., Geerts L., Thevelein J. M. Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1988 Aug;8(8):3051–3057. doi: 10.1128/mcb.8.8.3051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mitts M. R., Grant D. B., Heideman W. Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein. Mol Cell Biol. 1990 Aug;10(8):3873–3883. doi: 10.1128/mcb.10.8.3873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Munder T., Küntzel H. Glucose-induced cAMP signaling in Saccharomyces cerevisiae is mediated by the CDC25 protein. FEBS Lett. 1989 Jan 2;242(2):341–345. doi: 10.1016/0014-5793(89)80498-3. [DOI] [PubMed] [Google Scholar]
  31. Nakafuku M., Obara T., Kaibuchi K., Miyajima I., Miyajima A., Itoh H., Nakamura S., Arai K., Matsumoto K., Kaziro Y. Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1374–1378. doi: 10.1073/pnas.85.5.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Page B. D., Snyder M. CIK1: a developmentally regulated spindle pole body-associated protein important for microtubule functions in Saccharomyces cerevisiae. Genes Dev. 1992 Aug;6(8):1414–1429. doi: 10.1101/gad.6.8.1414. [DOI] [PubMed] [Google Scholar]
  33. Porfiri E., Evans T., Chardin P., Hancock J. F. Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchange. J Biol Chem. 1994 Sep 9;269(36):22672–22677. [PubMed] [Google Scholar]
  34. Powers S., Kataoka T., Fasano O., Goldfarb M., Strathern J., Broach J., Wigler M. Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins. Cell. 1984 Mar;36(3):607–612. doi: 10.1016/0092-8674(84)90340-4. [DOI] [PubMed] [Google Scholar]
  35. Powers S., Michaelis S., Broek D., Santa Anna S., Field J., Herskowitz I., Wigler M. RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor. Cell. 1986 Nov 7;47(3):413–422. doi: 10.1016/0092-8674(86)90598-2. [DOI] [PubMed] [Google Scholar]
  36. Purwin C., Leidig F., Holzer H. Cyclic AMP-dependent phosphorylation of fructose-1,6-bisphosphatase in yeast. Biochem Biophys Res Commun. 1982 Aug 31;107(4):1482–1489. doi: 10.1016/s0006-291x(82)80166-6. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  39. Rozakis-Adcock M., Fernley R., Wade J., Pawson T., Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. doi: 10.1038/363083a0. [DOI] [PubMed] [Google Scholar]
  40. Sass P., Field J., Nikawa J., Toda T., Wigler M. Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9303–9307. doi: 10.1073/pnas.83.24.9303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schafer W. R., Rine J. Protein prenylation: genes, enzymes, targets, and functions. Annu Rev Genet. 1992;26:209–237. doi: 10.1146/annurev.ge.26.120192.001233. [DOI] [PubMed] [Google Scholar]
  42. Stokoe D., Macdonald S. G., Cadwallader K., Symons M., Hancock J. F. Activation of Raf as a result of recruitment to the plasma membrane. Science. 1994 Jun 3;264(5164):1463–1467. doi: 10.1126/science.7811320. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Van Aelst L., Boy-Marcotte E., Camonis J. H., Thevelein J. M., Jacquet M. The C-terminal part of the CDC25 gene product plays a key role in signal transduction in the glucose-induced modulation of cAMP level in Saccharomyces cerevisiae. Eur J Biochem. 1990 Nov 13;193(3):675–680. doi: 10.1111/j.1432-1033.1990.tb19386.x. [DOI] [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