Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Two subclasses of guanine exchange factor (GEF) domains revealed by comparison of activities of chimeric genes constructed from CDC25, SDC25 and BUD5 in Saccharomyces cerevisiae

Abstract

Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p. Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization. We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p. We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation. Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins. In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways. This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.

This is a preview of subscription content, log in to check access.

References

  1. Bender A, Pringle JC (1989) Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci USA 86:9976–9980

  2. Bowtell D, Fu P, Simon M, Senior P (1992) Identification of murine homologs of the Drosophila Son of sevenless gene: potential activators of ras. Proc Natl Sci USA 89:6511–6515

  3. Boy-Marcotte E, Jacquet M (1982) A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant. Gene 20:433–440

  4. Boy-Marcotte E, Damak F, Camonis J, Garreau H, Jacquet M (1989) The C-terminal part of a gene partially homologous to CDC25 gene suppresses the cdc25-5 mutation in Saccharomyces cerevisiae. Gene 77:21–30

  5. Boy-Marcotte E, Buu A, Soustelle C, Poullet P, Parmeggiani A, Jacquet M (1993) The C-terminal part of the CDC25 gene product has Ras-nucleotide exchange activity when present in a chimeric SDC25-CDC25 protein. Curr Genet 23:397–401

  6. Broach JR, Strathern JN, Hicks JB (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–133

  7. Broek D, Toda T, Michaeli T, Levin L. Birchmeir C, Zeller M, Powers S, Wigler M (1987) The Saccharomyces cerevisiae CDC25 gene product regulates the Ras/adenylate cyclase pathway. Cell 48:789–800

  8. Camonis JH, Kalékine M, Gondré B, Garreau H, Boy-Marcotte E, Jacquet M (1986) Characterization, cloning and sequence analysis of the CDC25 gene which controls the cAMP level of Saccharomyces cerevisiae. EMBO J 5:375–380

  9. Camonis JH, Jacquet M (1988). A new RAS mutation that suppresses the CDC25 gene requirement for growth of Saccharomyces cerevisiae. Mot Cell Biol 8:2980–2983

  10. Camonis JH, Cassan M, Rousset JP (1990) Of mice and yeast: versatile vectors which permit gene expression in both budding yeast and higher eukaryotic cells. Gene 86:263–268

  11. Cen H, Papageorge A, Zippel R, Lowy D, Zhang K (1992) Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24. EMBO J 11:4007–4015

  12. Chant J, Herskowitz I (1991) Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell 65:1203–1212

  13. Chant J, Corrado K, Pringle JR, Herskowitz I (1991) Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1 Cell 65:1213–1224

  14. Chardin P, Camonis JH, Gale NW, Van Aelst L, Schlessinger J, Wigler MH, Bar-Sagi D (1993) Human Sos1: a guanine nucleotide exchange factor that binds to Grb2. Science 260:1338–1343

  15. Créchet JB, Poullet P, Mistou M, Parmeggiani A, Camonis J, Boy-Marcotte E, Damak F, Jacquet M (1990) Enchancement of the GDP to GTP exchange of ras proteins by the carboxyterminal domain of SDC25 gene product. Science 248:866–868

  16. Damak F. Boy-Marcotte M, Le-Roscouet D, Guilbaud R, Jacquet M (1991) SDC25, a CDC25 like gene, which contains a RASactivating domain is a dispensable gene of Saccharomyces cerevisiae. Mol Cell Biol 11:202–212

  17. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequences and analysis programs for the VAX. Nucleic Acids Res 12:387–395

  18. Garreau H, Camonis JH, Guitton C, Jacquet M (1990) The Saccharomyces cerevisiae CDC25 gene product is a 180kD polypeptide and is associated to a membrane fraction. FEBS Letters 269:53–59

  19. Goldberg D, Marbach I, Gross E, Levitzki A, Simchen G (1993) A Candida albicans homolog of CDC25 is functional in Saccharomyces cerevisiae. Eur J Biochem 213:195–205

  20. Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163–167

  21. Hughes DA, Fukui Y, Yamamoto M (1990) Homologous activators of ras in fission and budding yeasts. Nature 344:355–357

  22. Jacquet M, Buhler JM, Iborra F, Francingues-Gaillard MC, Soustelle C (1991) The MAT locus revisited within a 9.8 kb fragment of chromosome III containing BUD5 and two new open reading frames. Yeast 7:881–888

  23. Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M (1989) A ras-related gene with transformation suppressor activity. Cell 56:77–84

  24. Lai CC, Boguski M, Broek D, Powers S (1993) Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins. Mol Cell Biol 13:1345–1352

  25. Martegani E, Vanoni M, Zippel R, Cocetti P, Brambilla R, Ferrari C, Sturani E, Alberghina L (1992) Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae ras activator. EMBO J 11:2151–2157

  26. Maruta H, Holden J, Sizeland A, D'Abaco G (1991) The residues of Ras and Rap proteins that determine their GAP specificities. J Biol Chem 266:11661–11668

  27. Matsumoto K, Uno I, Oshima Y, Ishikawa T (1982) Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMP-dependent protein kinase. Proc Natl Acad Sci USA 79:2355–2359

  28. Petitjean A, Hilger F, Tatchell K (1990) Comparison of thermosensitive alleles of the CDC25 gene involved in the CAMP metabolism of Saccharomyces cerevisiae. Genetics 124:797–806

  29. Pompon D, Nicolas A (1989) Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions. Gene 83:15–24

  30. Powers S, O'Neill K, Wigler M (1989) Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild type RAS in S. cerevisiae. Mol Cell Biol 9:390–395

  31. Powers S, Gonzales E, Christensen T, Cubert J, Broek D (1991) Functional cloning of BUD5, a CDC25-related gene from S. cerevisiae that suppress a dominant-negative RAS2 mutant. Cell 65:1225–1231

  32. Pringle JR, Preston RA, Adams AEM, Stearns T, Drubin DG, Haarer BK, Jones EW (1989) Fluorescence microscopy methods for yeast. Meth Cell Biol 31:357–435

  33. Ruggieri R, Bender A, Matsui Y, Powers S, Takai Y, Pringle JR, Matsumoto K (1992) RSR1, a ras-like gene homologous to Krev-1 (smg21 A/rap1 A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae. Mol Cell Biol 12:758–766

  34. Schweighoffer F, Faure M, Fath I, Chevallier-Multon MC, Apiou F, Dutrillaux B, Sturani E, Jacquet M, Tocqué B (1993) Identification of a human guanine nucleotide-releasing factor (H-GRF55) specific for Ras proteins. Oncogene 8:1477–1485

  35. Sherman F., Fink GR, Lawrence CW (1974) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Gold spring Harbor, New York

  36. Shou C, Farnsworth C, Neel BG, Feig LA (1992) Molecular cloning of cDNAs encoding a guanine nucleotide releasing factor for ras p21. Nature 358:351–354

  37. Simon MA, Bowtell DL, Dodson GS, Laverty TR, Rubin GM (1991) Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67:701–716

  38. Van Aelst L, Boy-Marcotte E, Camonis JH, Thevelein JM, Jacquet M (1990) The C-terminal part of the CDC25 gene product plays a key role for signal transduction in the glucose-induced modulation of the cAMP level in Saccharomyces cerevisiae. Eur J Biochem 193:675–680

  39. Vieira J, Messing J (1987) Production of single stranded plasmid DNA. Methods in Enzymol 153:3–11

  40. Wei W, Mosteller RD, Sanyal P, Gonzales E, McKinney D, Dasgupta C, Li P, Liu BX, Broek D (1992) Identification of a mammalian gene structurally and functionally related to the CDC25 gene of Saccharomyces cerevisiae. Proc Natl Sci USA 89:7100–7104

  41. Wickner RB, Koh TJ, Crowley JC, O'Neil J, Kaback DB (1987) Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae. Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperature. Yeast 3:51–57

  42. Yon J, Fried M (1989) Precise gene fusion by PCR. Nucleic Acids Res 17:4895

Download references

Author information

Correspondence to Michel Jacquet.

Additional information

Communicated by C.P. Hollenberg

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Camus, C., Boy-Marcotte, E. & Jacquet, M. Two subclasses of guanine exchange factor (GEF) domains revealed by comparison of activities of chimeric genes constructed from CDC25, SDC25 and BUD5 in Saccharomyces cerevisiae . Molec. Gen. Genet. 245, 167–176 (1994). https://doi.org/10.1007/BF00283264

Download citation

Key words

  • Saccharomyces cerevisiae
  • Cell cycle
  • Bud site selection
  • Guanine exchange factor
  • Ras