Skip to main content
SpringerLink
Log in

Separate membrane targeting and anchoring domains function in the localization of the S. cerevisiae Cdc24p guanine nucleotide exchange factor

  • Research Article
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

The Saccharomyces cerevisiae Cdc24p guanine nucleotide exchange factor (GEF) activates the Cdc42p GTPase to a GTP-bound state. Cdc42p and Cdc24p co-localize at polarized growth sites during the cell cycle; and analysis of Cdc24p carboxyl-terminal truncation and site-specific mutations identified a 56-amino-acid domain as being necessary and sufficient for localization to these sites. This domain, however, was unable to anchor Cdc24p at these sites. Anchoring was restored by fusing the targeting domain to either the Cdc24p carboxyl-terminal PC domain that interacts with the Bem1p scaffold protein or the Cdc42p KKSKKCTIL membrane-anchoring domain. Mutant analysis and protein solubilization data indicated that anchoring required Bem1p, the Rsr1p/Bud1p GTPase, and the potential transmembrane protein YGR221Cp/Tos2p. These data are consistent with Cdc24p localization being a function of both membrane-specific targeting and subsequent anchoring within a multi-protein complex. Given the highly conserved roles of GEFs in Cdc42p signaling pathways, it is likely that similar targeting and anchoring mechanisms exist for Rho GEFs in other eukaryotes.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1A, B
Fig. 2A–C
Fig. 3

Similar content being viewed by others

References

  • Bender A, Pringle JR (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

    CAS  PubMed  Google Scholar 

  • Bi E, Pringle JR (1996) ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae. Mol Cell Biol 16:5264–5275

    CAS  PubMed  Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    CAS  PubMed  Google Scholar 

  • Bishop AL, Hall A (2000) Rho GTPases and their effector proteins. Biochem J 348:241–255

    Article  PubMed  Google Scholar 

  • Blondel M, Alepuz PM, Huang LS, Shaham S, Ammerer G, Peter M (1999) Nuclear export of Far1p in response to pheromones requires the export receptor Msn5p/Ste21p. Genes Dev 13:2284–2300

    Article  CAS  PubMed  Google Scholar 

  • Bose I, Irazoqui JE, Moskow JJ, Bardes ESG, Zyla TR, Lew DJ (2001) Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p. J Biol Chem 276:7176–7186

    Article  CAS  PubMed  Google Scholar 

  • Butty AC, Perrinjaquet N, Petit A, Jaquenoud M, Segall JE, Hofmann K, Zwahlen C, Peter M (2002) A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization. EMBO J 21:1565–1576

    Article  CAS  PubMed  Google Scholar 

  • Davis CR, Richman TJ, Deliduka SB, Blaisdell JO, Collins CC, Johnson DI (1998) Analysis of the mechanisms of action of the Saccharomyces cerevisiae dominant lethal cdc42 G12V and dominant negative cdc42 D118A mutations. J Biol Chem 273:849–858

    Article  CAS  PubMed  Google Scholar 

  • Drees BL, Sundin B, Brazeau E, Caviston JP, Chen GC, Guo W, Kozminski KG, Lau MW, Moskow JJ, Tong A, Schenkman LR, McKenzie A, Brennwald P, Longtine M, Bi E, Chan C, Novick P, Boone C, Pringle JR, Davis TN, Fields S, Drubin DG (2001) A protein interaction map for cell polarity development. J Cell Biol 154:549–571

    Article  CAS  PubMed  Google Scholar 

  • Erickson JW, Cerione RA (2001) Multiple roles for Cdc42 in cell regulation. Curr Opin Cell Biol 13:153–157

    Google Scholar 

  • Gulli MP, Peter M (2001) Temporal and spatial regulation of Rho-type guanine nucleotide exchange factors: the yeast perspective. Genes Dev 15:365–379

    Article  CAS  PubMed  Google Scholar 

  • Gulli M-P, Jaquenoud M, Shimada Y, Niederhäuser G, Wiget P, Peter M (2000) Phosphorylation of the Cdc42 exchange factor Cdc24 by the PAK-like kinase Cla4 may regulate polarized growth in yeast. Mol Cell 6:1155–1167

    CAS  PubMed  Google Scholar 

  • Harlan JE, Hajduk PJ, Yoon HS, Fesik SW (1994) Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate. Nature 371:168–170

    Article  CAS  PubMed  Google Scholar 

  • Ito T, Matsui Y, Ago T, Ota K, Sumimoto H (2001) Novel modular domain PB1 recognizes PC motif to mediate functional protein–protein interactions. EMBO J 20:3938–3946

    Article  CAS  PubMed  Google Scholar 

  • Johnson DI (1999) Cdc42: an essential Rho-type GTPase controlling eukaryotic cell polarity. Microbiol Mol Biol Rev 63:54–105

    CAS  PubMed  Google Scholar 

  • Kubiseski TJ, Chook YM, Parris WE, Rozakis-Adcock M, Pawson T (1997) High affinity binding of the pleckstrin homology domain of mSos1 to phosphatidylinositol (4,5)-bisphosphate. J Biol Chem 272:1799–1804

    Article  CAS  PubMed  Google Scholar 

  • Lodder AL, Lee TK, Ballester R (1999) Characterization of the Wsc1 protein, a putative receptor in the stress response of Saccharomyces cerevisiae. Genetics 152:1487–1499

    CAS  PubMed  Google Scholar 

  • Miyamoto S, Ohya Y, Ohsumi Y, Anraku Y (1987) Nucleotide sequence of the CLS4 (CDC24) gene of Saccharomyces cerevisiae. Gene 54:125–132

    Article  CAS  PubMed  Google Scholar 

  • Miyamoto S, Ohya Y, Sano Y, Sakaguchi S, Iida H, Anraku Y (1991) A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca2+-modulated bud assembly. Biochem Biophys Res Comm 181:604–610

    CAS  PubMed  Google Scholar 

  • Murray D, Honig B (2002) Electrostatic control of the membrane targeting of C2 domains. Mol Cell 9:145–154

    CAS  PubMed  Google Scholar 

  • Neame SJ, Uff CR, Sheikh H, Wheatley SC, Isacke CM (1995) CD44 exhibits a cell type dependent interaction with Triton X-100 insoluble, lipid rich, plasma membrane domains. J Cell Sci 108:3127–3135

    CAS  PubMed  Google Scholar 

  • Nern A, Arkowitz RA (1999) A Cdc24p-Far1p-Gβγ protein complex required for yeast orientation during mating. J Cell Biol 144:1187–1202

    Article  CAS  PubMed  Google Scholar 

  • Nern A, Arkowitz RA (2000) Nucleocytoplasmic shuttling of the Cdc42p exchange factor Cdc24p. J Cell Biol 148:1115–1122

    Article  CAS  PubMed  Google Scholar 

  • Park HO, Bi E, Pringle JR, Herskowitz I (1997) Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity. Proc Natl Acad Sci USA 94:4463–4468

    Article  CAS  PubMed  Google Scholar 

  • Richman TJ, Sawyer MM, Johnson DI (1999) The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast. J Biol Chem 274:16861–16870

    Article  CAS  PubMed  Google Scholar 

  • Richman TJ, Sawyer MM, Johnson DI (2002) Saccharomyces cerevisiae Cdc42p localizes to cellular membranes and clusters at sites of polarized growth. Eukaryot Cell 1:458–468

    Article  CAS  PubMed  Google Scholar 

  • Rizo J, Sudhof TC (1998) C2-domains, structure and function of a universal Ca2+-binding domain. J Biol Chem 273:15879–15882

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Schmidt A, Hall A (2002) Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Gene Dev 16:1587–1609

    Article  CAS  Google Scholar 

  • Schmitz AAP, Govek EE, Bottner B, VanAelst L (2000) Rho GTPases: signaling, migration, and invasion. Exp Cell Res 261:1–12

    CAS  PubMed  Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Soisson SM, Nimnual AS, Uy M, Bar-Sagi D, Kuriyan J (1998) Crystal structure of the Dbl and pleckstrin homology domains from the human son of sevenless protein. Cell 95:259–268

    CAS  PubMed  Google Scholar 

  • Terasawa H, Noda Y, Ito T, Hatanaka H, Ichikawa S, Ogura K, Sumimoto H, Inagaki F (2001) Structure and ligand recognition of the PB1 domain: a novel protein module binding to the PC motif. EMBO J 20:3947–3956

    Article  CAS  PubMed  Google Scholar 

  • Toenjes KA, Sawyer MM, Johnson DI (1999) The guanine-nucleotide-exchange factor Cdc24p is targeted to the nucleus and polarized growth sites. Curr Biol 9:1183–1186

    Article  CAS  PubMed  Google Scholar 

  • Zheng Y (2001) Dbl family guanine nucleotide exchange factors. Trends Biochem Sci 26:724–732

    CAS  PubMed  Google Scholar 

  • Zheng Y, Cerione R, Bender A (1994) Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. J Biol Chem 269:2369–2372

    CAS  PubMed  Google Scholar 

  • Zheng Y, Bender A, Cerione RA (1995) Interactions among proteins involved in bud-site selection and bud-site assembly in Saccharomyces cerevisiae. J Biol Chem 270:626–630

    Article  CAS  PubMed  Google Scholar 

  • Ziman M, Johnson DI (1994) Genetic evidence for a functional interaction between S. cerevisiae CDC24 and CDC42. Yeast 10:463–474

    CAS  PubMed  Google Scholar 

  • Ziman M, Preuss D, Mulholland J, O’Brien JM, Botstein D, Johnson DI (1993) Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. Mol Biol Cell 4:1307–1316

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Alan Bender for sharing valuable reagents, Rachel Jeffrey for technical assistance, the Vermont Cancer Center DNA Sequencing Facility, and Gary Ward and members of the Johnson Laboratory for valuable discussions and critical comments on this manuscript. This work was supported by a HELiX Undergraduate Fellowship (D.S.) and NSF grants MCB-0076826 and MCB-0110138.

Author information

Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, VT 05405, USA

    Kurt A. Toenjes, David Simpson & Douglas I. Johnson

Authors
  1. Kurt A. Toenjes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Simpson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Douglas I. Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Douglas I. Johnson.

Additional information

Communicated by S. Hohmann

Rights and permissions

Reprints and permissions

About this article

Cite this article

Toenjes, K.A., Simpson, D. & Johnson, D.I. Separate membrane targeting and anchoring domains function in the localization of the S. cerevisiae Cdc24p guanine nucleotide exchange factor. Curr Genet 45, 257–264 (2004). https://doi.org/10.1007/s00294-004-0485-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-004-0485-9

Keywords

Search

Navigation