Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic protein probes

Nature Chemical Biology volume 6, pages 534–540 (2010)Cite this article

Subjects

An Erratum to this article was published on 01 August 2010

This article has been updated

Abstract

Post-translationally isoprenylated proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these protein probes, we have demonstrated that RabGDI and the related Rab escort protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab–GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Quantitative analysis of interaction of Rab7-NF and Rab7NBD-G with REP-1 and GDI-1.
Figure 2: Kinetics of dissociation of Rab–REP and Rab–GDI complexes.
Figure 3: DrrA-mediated displacement of GDI-1.
Figure 4: Models of modulation of Rab recycling and targeting of Rabs to membranes by the state of bound nucleotide.

Similar content being viewed by others

Change history

  • 19 July 2010

    In the version of this article initially published, one of the corresponding authors (Y.-W.W.) was not included in the Additional information section. The error has been corrected in the HTML and PDF versions of the article to read “Correspondence and requests for materials should be addressed to R.S.G. or Y.-W.W.”

References

  1. Pfeffer, S.R. Rab GTPases: specifying and deciphering organelle identity and function. Trends Cell Biol. 11, 487–491 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Anant, J.S. et al. Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex. Biochemistry 37, 12559–12568 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Goldberg, J. Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 95, 237–248 (1998).

    Article  CAS  PubMed  Google Scholar 

  5. Goody, R.S., Rak, A. & Alexandrov, K. The structural and mechanistic basis for recycling of Rab proteins between membrane compartments. Cell. Mol. Life Sci. 62, 1657–1670 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Rak, A. et al. Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase. Science 302, 646–650 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Wu, Y.W., Tan, K.T., Waldmann, H., Goody, R.S. & Alexandrov, K. Interaction analysis of prenylated Rab GTPase with Rab escort protein and GDP dissociation inhibitor explains the need for both regulators. Proc. Natl. Acad. Sci. USA 104, 12294–12299 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sivars, U., Aivazian, D. & Pfeffer, S.R. Yip3 catalyses the dissociation of endosomal Rab-GDI complexes. Nature 425, 856–859 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Sivars, U., Aivazian, D. & Pfeffer, S. Purification and properties of Yip3-PRA1 as a Rab GDI displacement factor. Methods Enzymol. 403, 348–356 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Figueroa, C., Taylor, J. & Vojtek, A.B. Prenylated Rab acceptor protein is a receptor for prenylated small GTPases. J. Biol. Chem. 276, 28219–28225 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Seabra, M.C. Nucleotide dependence of Rab geranylgeranylation. Rab escort protein interacts preferentially with GDP-bound Rab. J. Biol. Chem. 271, 14398–14404 (1996).

    Article  CAS  PubMed  Google Scholar 

  12. Kalinin, A. et al. Expression of mammalian geranylgeranyltransferase type-II in Escherichia coli and its application for in vitro prenylation of Rab proteins. Protein Expr. Purif. 22, 84–91 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Shapiro, A.D. & Pfeffer, S.R. Quantitative analysis of the interactions between prenyl Rab9, GDP dissociation inhibitor-alpha, and guanine nucleotides. J. Biol. Chem. 270, 11085–11090 (1995).

    Article  CAS  PubMed  Google Scholar 

  14. Muir, T.W. Semisynthesis of proteins by expressed protein ligation. Annu. Rev. Biochem. 72, 249–289 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Gitler, C., Zarmi, B. & Kalef, E. Use of cationic detergents to enhance reactivity of protein sulfhydryls. Methods Enzymol. 251, 366–375 (1995).

    Article  CAS  PubMed  Google Scholar 

  16. Sanford, J.C., Pan, Y. & Wessling-Resnick, M. Prenylation of Rab5 is dependent on guanine nucleotide binding. J. Biol. Chem. 268, 23773–23776 (1993).

    CAS  PubMed  Google Scholar 

  17. Seabra, M.C. Nucleotide dependence of Rab geranylgeranylation. Rab escort protein interacts preferentially with GDP-bound Rab. J. Biol. Chem. 271, 14398–14404 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Machner, M.P. & Isberg, R.R. A bifunctional bacterial protein links GDI displacement to Rab1 activation. Science 318, 974–977 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Ingmundson, A., Delprato, A., Lambright, D.G. & Roy, C.R. Legionella pneumophila proteins that regulate Rab1 membrane cycling. Nature 450, 365–369 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Schoebel, S., Oesterlin, L.K., Blankenfeldt, W., Goody, R.S. & Itzen, A. RabGDI displacement by DrrA from Legionella is a consequence of its guanine nucleotide exchange activity. Mol. Cell 36, 1060–1072 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Dursina, B.E. et al. A genetically encodable microtag for chemoenzymatic derivatization and purification of recombinant proteins. Protein Expr. Purif. 39, 71–81 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Alexandrov, K. et al. Moderate discrimination of REP-1 between Rab7 x GDP and Rab7 x GTP arises from a difference of an order of magnitude in dissociation rates. FEBS Lett. 425, 460–464 (1998).

    Article  CAS  PubMed  Google Scholar 

  23. Chavrier, P. et al. Hypervariable C-terminal domain of rab proteins acts as a targeting signal. Nature 353, 769–772 (1991).

    Article  CAS  PubMed  Google Scholar 

  24. Ali, B.R., Wasmeier, C., Lamoreux, L., Strom, M. & Seabra, M.C. Multiple regions contribute to membrane targeting of Rab GTPases. J. Cell Sci. 117, 6401–6412 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Ali, B.R. & Seabra, M.C. Targeting of Rab GTPases to cellular membranes. Biochem. Soc. Trans. 33, 652–656 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Beranger, F., Paterson, H., Powers, S., de Gunzburg, J. & Hancock, J.F. The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization. Mol. Cell. Biol. 14, 744–758 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hutt, D.M., Da-Silva, L.F., Chang, L.H., Prosser, D.C. & Ngsee, J.K. PRA1 inhibits the extraction of membrane-bound Rab GTPase by GDI1. J. Biol. Chem. 275, 18511–18519 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Soldati, T., Shapiro, A.D., Svejstrup, A.B. & Pfeffer, S.R. Membrane targeting of the small GTPase Rab9 is accompanied by nucleotide exchange. Nature 369, 76–78 (1994).

    Article  CAS  PubMed  Google Scholar 

  29. Ullrich, O., Horiuchi, H., Bucci, C. & Zerial, M. Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange. Nature 368, 157–160 (1994).

    Article  CAS  PubMed  Google Scholar 

  30. Pylypenko, O. et al. Structure of doubly prenylated Ypt1-GDI complex and the mechanism of GDI-mediated Rab recycling. EMBO J. 25, 13–23 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sato, Y., Fukai, S., Ishitani, R. & Nureki, O. Crystal structure of the Sec4p.Sec2p complex in the nucleotide exchanging intermediate state. Proc. Natl. Acad. Sci. USA 104, 8305–8310 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lippé, R., Miaczynska, M., Rybin, V., Runge, A. & Zerial, M. Functional synergy between Rab5 effector Rabaptin-5 and exchange factor Rabex-5 when physically associated in a complex. Mol. Biol. Cell 12, 2219–2228 (2001).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Mattera, R. & Bonifacino, J.S. Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes. EMBO J. 27, 2484–2494 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhu, H. et al. Rabaptin-5-independent membrane targeting and Rab5 activation by Rabex-5 in the cell. Mol. Biol. Cell 18, 4119–4128 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Durek, T. et al. Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase. J. Am. Chem. Soc. 126, 16368–16378 (2004).

    Article  CAS  PubMed  Google Scholar 

  36. Wu, Y.W. et al. A protein fluorescence amplifier: continuous fluorometric assay for rab geranylgeranyltransferase. Chembiochem. 7, 1859–1861 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Itzen (Max Planck Institute of Molecular Physiology, Dortmund, Germany) for providing DrrA proteins and helpful discussions on DrrA nucleotide exchange. T. Durek (Institute for Molecular Bioscience, The University of Queensland, Australia) is acknowledged for help in protein ligation. This work was supported in part by the Deutsche Forschungsgemeinschaft (grants to K.A., R.S.G. and H.W. in the program SFB 642).

Author information

Authors and Affiliations

  1. Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Yao-Wen Wu, Lena K Oesterlin & Roger S Goody

  2. Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Kui-Thong Tan & Herbert Waldmann

  3. Institute for Molecular Bioscience and Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia

    Kirill Alexandrov

Authors
  1. Yao-Wen Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lena K Oesterlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kui-Thong Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Herbert Waldmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kirill Alexandrov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Roger S Goody
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.-W.W. and R.S.G. designed the research and wrote the manuscript. Y.-W.W. and L.K.O. performed the research. Y.-W.W., L.K.O., K.A., K.-T.T. and H.W. contributed new reagents and analytical tools.

Corresponding authors

Correspondence to Yao-Wen Wu or Roger S Goody.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Results, Supplementary Figures 1–12 and Supplementary Table 1 (PDF 5285 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, YW., Oesterlin, L., Tan, KT. et al. Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic protein probes. Nat Chem Biol 6, 534–540 (2010). https://doi.org/10.1038/nchembio.386

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.386

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing