Skip to main content
SpringerLink
Log in

Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth

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

Abstract

The Cdc42p GTPase regulates multiple signal transduction pathways through its interactions with downstream effectors. Specific functional domains within Cdc42p are required for guanine-nucleotide binding, interactions with downstream effectors, and membrane localization. However, little is known about how Cdc42p is clustered at polarized growth sites or is extracted from membranes by Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) at specific times in the cell cycle. To address these points, localization studies were performed in Saccharomyces cerevisiae using green fluorescent protein (GFP)-tagged Cdc42p and the RhoGDI Rdi1p. GFP-Rdi1p localized to polarized growth sites at specific times of the cell cycle but not to other sites of Cdc42p localization. Overexpression of Rdi1p led to loss of GFP-Cdc42p from internal and plasma membranes. This effect was mediated through the Cdc42p Rho-insert domain, which was also implicated in interactions with the Bni1p scaffold protein. These data suggested that Rdi1p functions in cell cycle-specific Cdc42p membrane detachment. Additional genetic and time-lapse microscopy analyses implicated nucleotide binding in the clustering of Cdc42p. Taken together, these results provide insight into the complicated nature of the relationships between Cdc42p localization, nucleotide binding, and protein–protein interactions.

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. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Benard V, Bohl BP, Bokoch GM (1999) Characterization of Rac and Cdc42 activation in chemoattraction-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 274:13198–13204

    PubMed  Google Scholar 

  • 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 

  • 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 

  • Brown JL, Jaquenoud M, Gulli M-P, Chant J, Peter M (1997) Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast. Genes Dev 11:2972–2982

    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 

  • Chen G-C, Kim Y-J, Chan CSM (1997) The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae. Genes Dev 11:2958–2971

    CAS  PubMed  Google Scholar 

  • Cvrcková F, De Virgilio C, Manser E, Pringle JR, Nasmyth K (1995) Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast. Genes Dev 9:1817–1830

    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 

  • Diebold BA, Bokoch GM (2001) Molecular basis for Rac2 regulation of phagocyte NADPH oxidase. Nat Immunol 2:211–215

    Article  CAS  PubMed  Google Scholar 

  • Eitzen G, Thorngren N, Wickner W (2001) Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking. EMBO J 20:5650–5656

    Article  CAS  PubMed  Google Scholar 

  • Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology. Nature 420:629–635

    Article  CAS  PubMed  Google Scholar 

  • Evangelista M, Blundell K, Longtine MS, Chow CJ, Adames N, Pringle JR, Peter M, Boone C (1997) Bni1p, a yeast formin linking Cdc42p and the actin cytoskeleton during polarized morphogenesis. Science 276:118–122

    PubMed  Google Scholar 

  • Finegold AA, Johnson DI, Farnsworth CC, Gelb MH, Judd SR, Glomset JA, Tamanoi F (1991) Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product, but not the DPR1 gene product. Proc Natl Acad Sci USA 88:4448–4452

    CAS  PubMed  Google Scholar 

  • Gyuris J, Golemis E, Chertkov H, Brent R (1993) Cdi, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791–803

    CAS  PubMed  Google Scholar 

  • Hart MJ, Maru Y, Leonard D, Witte ON, Evans T, Cerione RA (1992) A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like protein CDC42Hs. Science 258:812–815

    CAS  PubMed  Google Scholar 

  • Hoffman GR, Nassar N, Cerione RA (2000) Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 100:345–356

    Article  CAS  PubMed  Google Scholar 

  • Hori Y, Kikuchi A, Isomura M, Katayama M, Miura Y, Fujioka H, Kaibuchi K, Takai Y (1991) Post-translational modifications of the C-terminal region of rho are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins. Oncogene 6:515–522

    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 

  • Johnson DI, Pringle JR (1990) Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol 111:143–152

    CAS  PubMed  Google Scholar 

  • Joneson T, Bar-Sagi D (1998) A Rac1 effector site controlling mitogenesis through superoxide production. J Biol Chem 273:17991–17994

    Article  CAS  PubMed  Google Scholar 

  • Koch G, Tanaka K, Masuda T, Yamochi W, Nonaka H, Takai Y (1997) Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae. Oncogene 15:417–422

    Article  CAS  PubMed  Google Scholar 

  • Leonard D, Hart MJ, Platko JV, Eva A, Henzel W, Evans T, Cerione RA (1992) The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein. J Biol Chem 267:22860–22868

    CAS  PubMed  Google Scholar 

  • Masuda T, Tanaka K, Nonaka H, Yamochi W, Maeda A, Takai Y (1994) Molecular cloning and characterization of yeast rho GDP dissociation inhibitor. J Biol Chem 269:19713–19718

    CAS  PubMed  Google Scholar 

  • McCallum SJ, Wu WJ, Cerione RA (1996) Identification of a putative effector for Cdc42Hs with high sequence similarity to the RasGAP-related protein IQGAP1 and a Cdc42Hs binding partner with similarity to IQGAP2. J Biol Chem 271:21732–21737

    Article  CAS  PubMed  Google Scholar 

  • Merla A, Johnson DI (2000) The Cdc42p GTPase is targeted to the site of cell division in the fission yeast Schizosaccharomyces pombe. Eur J Cell Biol 79:469–477

    CAS  PubMed  Google Scholar 

  • Merla A, Johnson DI (2001) The Schizosaccharomyces pombe Cdc42p GTPase signals through Pak2p and the Mkh1p–Pek1p–Spm1p MAP kinase pathway. Curr Genet 39:205–209

    Article  CAS  PubMed  Google Scholar 

  • Miller PJ, Johnson DI (1997) Characterization of the S. cerevisiae cdc42-1 ts allele and new temperature-conditional-lethal cdc42 alleles. Yeast 13:561–572

    Article  CAS  PubMed  Google Scholar 

  • Murray JM, Johnson DI (2001) The Cdc42p GTPase and its regulators Nrf1p and Scd1p are involved in endocytic trafficking in the fission yeast Schizosaccharomyces pombe. J Biol Chem 276:3004–3009

    Article  CAS  PubMed  Google Scholar 

  • Müller O, Johnson DI, Mayer A (2001) Cdc42p functions at the docking stage of yeast vacuole membrane fusion. EMBO J 20:5657–5665

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Nomanbhoy TK, Cerione RA (1996) Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J Biol Chem 271:10004–10009

    Article  CAS  PubMed  Google Scholar 

  • Nomanbhoy TK, Erikson JW, Cerione RA (1999) Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer. Biochemistry 38:1744–1750

    Article  CAS  PubMed  Google Scholar 

  • Pruyne DW, Schott DH, Bretscher A (1998) Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast. J Cell Biol 143:1931–1945

    PubMed  Google Scholar 

  • Richman TJ, Johnson DI (2000) Saccharomyces cerevisiae Cdc42p GTPase is involved in preventing the recurrence of bud emergence during the cell cycle. Mol Cell Biol 20:8548–8559

    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 

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

    Google Scholar 

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

    Google Scholar 

  • Stevenson BJ, Ferguson B, De Virgilio C, Bi E, Pringle JR, Ammerer G, Sprague GF (1995) Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae. Genes Dev 9:2949–2963

    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 

  • Vallen EA, Caviston J, Bi E (2000) Roles of Hof1p, Bni1p, Bnr1p, and Myo1p in cytokinesis in Saccharomyces cerevisiae. Mol Biol Cell 11:593–611

    CAS  PubMed  Google Scholar 

  • Vetter IR, Wittinghofer A (2001) Signal transduction—the guanine nucleotide-binding switch in three dimensions. Science 294:1299–1304

    Article  CAS  PubMed  Google Scholar 

  • Walker SJ, Brown HA (2002) Specificity of Rho insert-mediated activation of phospholipase D1. J Biol Chem 277:26260–26267

    Article  CAS  PubMed  Google Scholar 

  • Walker SJ, Wu W-J, Cerione RA, Brown HA (2000) Activation of phospholipase D1 by Cdc42 requires the Rho insert region. J Biol Chem 275:15665–15668

    Article  CAS  PubMed  Google Scholar 

  • White WH, Johnson DI (1997) Characterization of synthetic-lethal mutants reveals a role for the Saccharomyces cerevisiae guanine-nucleotide exchange factor Cdc24p in vacuole function and Na+ tolerance. Genetics 147:43–55

    CAS  PubMed  Google Scholar 

  • Wu WJ, Leonard DA, Cerione RA, Manor D (1997) Interaction between Cdc42Hs and RhoGDI is mediated through the Rho insert region. J Biol Chem 272:26153–26158

    Article  CAS  PubMed  Google Scholar 

  • Wu WJ, Lin R, Cerione RA, Manor D (1998) Transformation activity of Cdc42 requires a region unique to Rho-related proteins. J Biol Chem 273:16655–16658

    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, O’Brien JM, Ouellette LA, Church WR, Johnson DI (1991) Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity. Mol Cell Biol 11:3537–3544

    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 Anthony Bretscher for valuable reagents, Herman Chen and Patti McClard for expert technical assistance, and members of the Johnson Laboratory for valuable discussions and critical comments on this manuscript. K.C.C. was supported in part by an undergraduate fellowship from the UVM-Howard Hughes HELiX Program. This research was supported by National Science Foundation grant MCB-0110138.

Author information

Author notes

  1. Tamara J. Richman

    Present address: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., FL 8, Worcester, MA 01605-2324, USA

Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, 95 Carrigan Dr., 202 Stafford Hall, Burlington, VT 05405, USA

    Tamara J. Richman, Kurt A. Toenjes, Sergio E. Morales, Karen C. Cole, Ben T. Wasserman, Chad M. Taylor, Jacob A. Koster, Matthew F. Whelihan & Douglas I. Johnson

Authors
  1. Tamara J. Richman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kurt A. Toenjes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sergio E. Morales
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karen C. Cole
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ben T. Wasserman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chad M. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jacob A. Koster
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Matthew F. Whelihan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. 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

Richman, T.J., Toenjes, K.A., Morales, S.E. et al. Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth. Curr Genet 45, 339–349 (2004). https://doi.org/10.1007/s00294-004-0505-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-004-0505-9

Keywords

Search

Navigation