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Localization of AtROP4 and AtROP6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis

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Abstract

The small GTPases of the Rho family play a key role in actin cytoskeletal organization. In plants, a novel Rho subfamily, called ROP (Rho of plants), has been found. In Arabidopsis, 12 ROP GTPases have been identified which differ mainly at their C-termini. To test the localization of two members of this subfamily (AtROP4 and AtROP6), we have generated translational fusions with the green fluorescent protein (GFP). Microscopic analysis of transiently transfected BY2 cells revealed a predominant localization of AtROP4 in the perinuclear region, while AtROP6 was localized almost exclusively to the plasma membrane. Swapping of the AtROP4 and AtROP6 C-termini produced a change in localization. As RhoGDIs are known to bind to the C-terminus of GTPases of the Rho family, we searched for ArabidopsisRhoGDI genes. We identified the AtRhoGDI1gene and mapped it to chromosome 3. AtRhoGDI1 encodes a 22.5 kDa protein which contains highly conserved amino acids in the isoprene binding pocket and exhibits 29% to 37% similarity to known mammalian RhoGDI homologues. The AtRhoGDI1 gene was expressed in all tissues studied. Using the yeast two-hybrid system, we showed specific interaction of AtRhoGDI1 with both AtROP4 and AtROP6 as well as with their GTP-locked mutants, but not with a GTPase of the RAB family. Recombinant GST-AtRhoGDI1 could bind GFP-AtROP4 from transgenic tobacco BY2 cell extracts, confirming the interaction observed with the two-hybrid system.

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References

  • Abo, A., Webb, M.R., Grogan, A. and Segal, A.W. 1994. Activation of NADPH oxidase involves the dissociation of p21-rac from its inhibitory GDP-GTP exchange protein (RhoGDI) followed by its translocation to the plasma membrane. Biochem J. 298: 585–591.

    Google Scholar 

  • Adamson, P., Marshall, C.J., Hall, A. and Tilbrook, P.A. 1992. Posttranslational modifications of p21-RHO proteins. J. Biol. Chem. 267: 20033–20038.

    Google Scholar 

  • Adra, C.N., Manor, D., Ko, J.L., Zhu, S., Horiuchi, T., Van Aelst, L., Cerione, R.A. and Lim, B. 1997. RhoGDI-gamma: a GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas. Proc. Natl. Acad. Sci. USA 94: 4279–4284.

    Google Scholar 

  • Bischoff, F., Molendijk, A., Rajendrakumar, C.S.V. and Palme, K. 1999. GTP-binding proteins in plants. Cell. Mol. Life Sci. 55: 233–256.

    Google Scholar 

  • Brennwald, P. and Novick, P. 1993. Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4. Nature 362: 560–563.

    Google Scholar 

  • Chavrier, P., Gorvel, J.P., Stelzer, E., Simons, K., Gruenberg, J. and Zerial, M. 1991. Hypervariable C-terminal domain of Rab proteins acts as a targeting signal. Nature 353: 769–772.

    Google Scholar 

  • Chiu, W.L., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H. and Sheen, J. 1996. Engineered GFP as a vital reporter in plants. Curr. Biol. 6: 325–330.

    Google Scholar 

  • Connerton, I.F. 1994. Expression of Membrane Proteins in Yeast, Portland Press, pp. 177–218.

  • Creusot, F., Fouilloux, E., Dron, M., Lafleuriel, J., Picard, G., Billault, A., Le Paslier, D., Cohen, D., Chaboute, M.E., Durr, A., Fleck, J., Gigot, C., Camilleri, C., Bellini, C., Caboche, M. and Bouchez, D. 1995. The CIC library: a large insect YAC library for genome mapping in Arabidopsis thaliana. Plant J. 8: 763–770.

    Google Scholar 

  • Delmer, D.P., Pear, J.R., Andrawis, A. and Stalker, D.M. 1995. Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers. Mol. Gen. Genet. 248: 43–51.

    Google Scholar 

  • Ecker, J.R. 1990. PFGE und YAC analysis of the Arabidopsis genome. Methods 1: 186–194.

    Google Scholar 

  • Estojak, J., Brent, R. and Golemis, E.A. 1995. Correlation of twohybrid affinity data with in vitro measurements. Mol. Cell. Biol. 15: 5820–5829.

    Google Scholar 

  • Feldbrügge, M., Sprenger, M., Dinkelbach, M., Yazaki, K., Harter, K. and Weisshaar, B. 1994. Functional analysis of a lightresponsive plant bZIP transcriptional regulator. Plant Cell 6: 1607–1621.

    Google Scholar 

  • Finley, R.L. and Brent, R. 1995. Interaction trap cloning with yeast. In: B.D. Hames and D.M. Glover (Eds.), DNA Cloning, Expression Systems: A Practical Approach, Oxford University Press, Oxford, pp. 169–203.

    Google Scholar 

  • Gamborg, O.L., Miller, R.A. and Ojima, K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151–158.

    Google Scholar 

  • Glomset, J.A. and Farnsworth, C.C. 1994. Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes. Annu. Rev. Cell Biol. 10: 181–205.

    Google Scholar 

  • Gosser, Y.Q., Nomanbhoy, T.K., Aghazadeh, B., Manor, D., Combs, C., Cerione, R.A. and Rosen, M.K. 1997. C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases. Nature 387: 814–819.

    Google Scholar 

  • Guillemont, J.C., Kruskal, B.A., Adra, C.N., Zhu, S., Ko, J.L., Burch, P., Nocka, K., Seetoo, K., Simons, E. and Lim, B. 1996. Targeted disruption of guanosine diphosphate-dissociation inhibitor for Rho-related proteins, GDID4: normal hematopoietic differentiation but subtle defect in superoxide production by macrophages derived from in vitro embryonal stem cell differentiation. Blood 88: 2722–2731.

    Google Scholar 

  • Gyuris, J., Golemis, E., Chertkov, H. and Brent, R. 1993. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75: 791–803.

    Google Scholar 

  • Haizel, T., Merkle, T., Pay, A., Fejes, E. and Nagy, F. 1997. Characterization of proteins that interact with the GTP-bound form of the regulatory GTPase Ran in Arabidopsis. Plant J. 11: 93–103.

    Google Scholar 

  • Hall, A. 1998. Rho GTPases and the actin cytoskeleton. Science 279: 509–514.

    Google Scholar 

  • Hancock, J.F. and Hall, A. 1993. A novel role for RhoGDI as an inhibitor of GAP proteins. EMBO J. 12: 1915–1921.

    Google Scholar 

  • Haseloff, J., Siemering, K.R., Prasher, D.C. and Hodge, S. 1997. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA 94: 2122–2127.

    Google Scholar 

  • Janoueix-Lerosey, I., Jollivet, F., Camonis, J., Marche, P.N. and Goud, B. 1995. Two-hybrid system screen with the small GTPbinding protein Rab6: identification of a novel mouse GDP dissociation inhibitor isoform and two other potential partners of Rab6. J. Biol. Chem. 270: 14801–14808.

    Google Scholar 

  • John, J., Rensland, H., Schlichting, I., Vetter, I., Borasio, G.D., Goody, R.S. and Wittinghofer, A. 1993. Kinetic and structural analysis of the Mg2C-binding site of the guanine nucleotide binding protein p21 H-ras. J. Biol. Chem. 268: 923–929.

    Google Scholar 

  • Joshi, C.P. 1987. An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl. Acids Res. 15: 6643–6653.

    Google Scholar 

  • Kost, B., Lemichez, E., Spielhofer, P., Hong, Y., Tolias, K., Carpenter, C. and Chua, N.-H. 1999. RAC homologues and compartimentalized phosphatidylinositol 4,5-bisphosphate act in a common pathway to regulate polar pollen tube growth. J. Cell Biol. 145: 317–330.

    Google Scholar 

  • Krengel, U., Schlichting, I., Scherer, A., Schumann, R., Frech, M., John, J., Kabsch, W., Pai, E.F. and Wittinghofer, A. 1990. Threedimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules. Cell 62: 539–548.

    Google Scholar 

  • Li, H., Wu, G., Ware, D., Davis, K.R. and Yang, Z. 1998. Arabidopsis Rho-related GTPases: differential gene expression in pollen and polar localization in fission yeast. Plant Physiol. 118: 407–417.

    Google Scholar 

  • Lin, Y. and Yang, Z. 1997. Inhibition of pollen tube elongation by microinjected anti-Rop1Ps antibodies suggests a crucial role for Rho-type GTPases in the control of tip growth. Plant Cell 9: 1647–1659.

    Google Scholar 

  • Lin, Y., Wang, Y., Zhu, J. and Yang, Z. 1996. Localization of a Rho GTPase implies a role in tip growth and movement of the generative cell in pollen tubes. Plant Cell 8: 293–303.

    Google Scholar 

  • Loraine, A.E., Yalovsky, S., Fabry, S. and Gruissem, W. 1996. Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells. Plant Physiol. 110: 1337–1347.

    Google Scholar 

  • Ma, H. 1994. GTP-binding proteins in plants: new members of an old family. Plant Mol. Biol. 26: 1611–1636.

    Google Scholar 

  • Maas, C., Reichel, C., Schell, J. and Steinbiss, H.H. 1995. Preparation and transformation of monocot protoplasts. Meth. Cell Biol. 50: 383–399.

    Google Scholar 

  • Mariot, P., O'Sullivan, A.J., Brown, A.M. and Tatham, P.E. 1996. Rho guanine nucleotide dissociation inhibitor protein (RhoGDI) inhibits exocytosis in mast cells. EMBO J. 15: 6476–6482.

    Google Scholar 

  • Moores, S.L., Schaber, M.D., Mosser, S.D., Rands, E., Ohara, M.B., Garsky, V.M., Mashall, M.S., Pompliano, D.L. and Gibbs, J.B. 1991. Sequence dependence of protein isoprenylation. J. Biol. Chem. 266: 14603–14610.

    Google Scholar 

  • Nagata, T., Okada, K., Tabeke, I. and Mastui, C. 1981. Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Mol. Gen. Genet. 184: 161–165.

    Google Scholar 

  • Nomanbhoy, T.K. and Cerione, R.A. 1996. Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J. Biol. Chem. 271: 10004–10009.

    Google Scholar 

  • Olofsson, B. 1999. Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell Signal 11: 545–554.

    Google Scholar 

  • Picard, V., Ersdal-Badju, E., Lu, A. and Bock, S.C. 1994. A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucl. Acids Res. 22: 2587–2591.

    Google Scholar 

  • Platko, J.V., Leonard, D.A., Adra, C.N., Shaw, R.J., Cerione, R.A. and Lim, B. 1995. A single residue can modify targetbinding affinity and activity of the functional domain of the Rho-subfamily GDP dissociation inhibitors. Proc. Natl. Acad. Sci. USA 92: 2974–2978.

    Google Scholar 

  • Reichel, C., Mathur, J., Eckes, P., Langenkemper, K., Koncz, C., Schell, J., Reiss, B. and Maas, C. 1996. Enhanced green fluorescence by expression of an Aequorea victoria green fluorescent protein mutant in mono-and dicotyledonous plant cells. Proc. Natl. Acad. Sci. USA 93: 5888–5893.

    Google Scholar 

  • Ridley, A.J. 1995. Rho-related proteins: actin cytoskeleton and cell cycle. Curr. Opin. Genet. Dev. 5: 24–30.

    Google Scholar 

  • Rogers, S.O., Bendich, A.J. 1997. Extraction of total cellular DNA from plants, algae and fungi. In: S.B. Gelvin and R.A. Schilperoort (Eds.), Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht, Netherlands, pp. D1: 1–8.

    Google Scholar 

  • Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  • Sasaki, T. and Takai, Y. 1998. The Rho small g protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. Biochem. Biophys. Res. Comm. 245: 641–645.

    Google Scholar 

  • Schlichting, I., Almo, S.C., Rapp, G., Wilson, K., Petratos, K., Lentfer, A., Wittinghofer, A., Kabsch, W., Pai, E.F., Petsko, G.A. and Goody, R.S. 1990. Time-resolved X-ray crystallographic study of the conformational change in Ha-ras p21 protein on GTP hydrolysis. Nature 345: 309–315.

    Google Scholar 

  • Schmidt, R. and Dean, C. 1995. Hybridization analysis of YAC clones. Meth. Mol. Cell. Biol. 5: 309–318.

    Google Scholar 

  • Seabra, M.C. 1998. Membrane association and targeting of prenylated Ras-like GTPases. Cell Signal 10: 167–172.

    Google Scholar 

  • Stenmark, H., Valencia, A., Martinez, O., Ullrich, O., Goud, B. and Zerial, M. 1994. Distinct structural elements of rab5 define its functional specificity. EMBO J. 13: 575–583.

    Google Scholar 

  • Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H. and Takai, Y. 1997. Regulation of cell-cell adhesion by Rac and Rho small g proteins in MDCK cells. J. Cell Biol. 139: 1047–1059.

    Google Scholar 

  • Trainin, T., Shmuel, M. and Delmer, D.P. 1996. In vitro prenylation of the small GTPase Rac13 on cotton. Plant Physiol. 112: 1491–1497.

    Google Scholar 

  • Valencia, A. and Sander, C. 1995. The Ras superfamily. In: M. Zerial and L.A. Huber (Eds.), Guidebook to the Small GTPases, Oxford University Press, Oxford, pp. 12–20.

    Google Scholar 

  • Winge, P., Brembu, T. and Bones, A.M. 1997. Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana. Plant Mol. Biol. 35: 483–495.

    Google Scholar 

  • Wu, W.J., Leonard, D.A. and Manor, D. 1997. Interaction between Cdc42Hs and RhoGDI is mediated through the Rho insert region. J. Biol. Chem. 272: 26153–26158.

    Google Scholar 

  • Xia, G., Srinivasan, R., Yan, H., Chan, Y., Simanis, V. and Chua, N.-H. 1996. Identification of plant cytoskeletal, cell cycle-related and polarity-related proteins using Schizosaccharomyces pombe. Plant J. 10: 761–769.

    Google Scholar 

  • Yang, Z. and Watson, J.C. 1993. Molecular cloning and characterization of Rho a Ras-related small GTP-binding protein from the garden pea. Proc. Natl. Acad. Sci. USA 90: 8732–8736.

    Google Scholar 

  • Zalcman, G., Closson, V., Camonis, J., Honore, N., Rousseau, M., Tavitian, A. and Olofsson, B. 1996. RhoGDI-3 is a new GDP dissociation inhibitor (GDI). Identification of a non-cytosolic GDI protein interacting with the small GTP-binding proteins RhoB and RhoG. J. Biol. Chem. 271: 30366–30374.

    Google Scholar 

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Authors and Affiliations

  1. Max-Delbrück-Laboratorium in der Max-Planck Gesellschaft, Carl-von-Linné Weg 10, 50829, Köln-Vogelsang, Germany

    Arthur Molendijk & Klaus Palme

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  1. Friedrich Bischoff
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  2. Lars Vahlkamp
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  3. Arthur Molendijk
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  4. Klaus Palme
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these authors contributed equally to the work

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Bischoff, F., Vahlkamp, L., Molendijk, A. et al. Localization of AtROP4 and AtROP6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis. Plant Mol Biol 42, 515–530 (2000). https://doi.org/10.1023/A:1006341210147

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