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:

Analysis of the eukaryotic prenylome by isoprenoid affinity tagging

Nature Chemical Biology volume 5, pages 227–235 (2009)Cite this article

Abstract

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl–tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.

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: Overview of the prenyltransferase-catalyzed protein prenylation reactions and lipid donors.
Figure 2: Evaluation of the incorporation of synthetic isoprenoids by wild-type prenyltransferases into GTPases.
Figure 3: Enrichment of endogenous RabGTPases prenylated with biotin-geranyl using streptavidin affinity chromatography.
Figure 4: Structural analysis of the BGPP-FTase complex.
Figure 5: Engineering of FTase and GGTase-I mutants capable of using BGPP as a lipid donor.
Figure 6: Labeling of the complete set of mammalian prenylation substrates in the lysates of COS-7 cells treated with inhibitors of protein prenylation.
Figure 7: Identification and quantification of RabGTPases in COS-7 cells.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Walsh, C.T., Garneau-Tsodikova, S. & Gatto, G.J. Jr. Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Edn Engl. 44, 7342–7372 (2005).

    Article  CAS  Google Scholar 

  2. Meri, S. & Baumann, M. Proteomics: posttranslational modifications, immune responses and current analytical tools. Biomol. Eng. 18, 213–220 (2001).

    Article  CAS  Google Scholar 

  3. Prescher, J.A. & Bertozzi, C.R. Chemistry in living systems. Nat. Chem. Biol. 1, 13–21 (2005).

    Article  CAS  Google Scholar 

  4. Gelb, M.H. Protein prenylation, et cetera: signal transduction in two dimensions. Science 275, 1750–1751 (1997).

    Article  CAS  Google Scholar 

  5. McTaggart, S.J. Isoprenylated proteins. Cell. Mol. Life Sci. 63, 255–267 (2006).

    Article  CAS  Google Scholar 

  6. Casey, P.J. & Seabra, M.C. Protein prenyltransferases. J. Biol. Chem. 271, 5289–5292 (1996).

    Article  CAS  Google Scholar 

  7. Maurer-Stroh, S. et al. Towards complete sets of farnesylated and geranylgeranylated proteins. PLoS Comput. Biol. 3, e66 (2007).

    Article  Google Scholar 

  8. Armstrong, S.A., Brown, M.S., Goldstein, J.L. & Seabra, M.C. Preparation of recombinant Rab geranylgeranyltransferase and Rab escort proteins. Methods Enzymol. 257, 30–41 (1995).

    Article  CAS  Google Scholar 

  9. Wherlock, M., Gampel, A., Futter, C. & Mellor, H. Farnesyltransferase inhibitors disrupt EGF receptor traffic through modulation of the RhoB GTPase. J. Cell Sci. 117, 3221–3231 (2004).

    Article  CAS  Google Scholar 

  10. Greenwood, J., Steinman, L. & Zamvil, S.S. Statin therapy and autoimmune disease: from protein prenylation to immunomodulation. Nat. Rev. Immunol. 6, 358–370 (2006).

    Article  CAS  Google Scholar 

  11. Konstantinopoulos, P.A., Karamouzis, M.V. & Papavassiliou, A.G. Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets. Nat. Rev. Drug Discov. 6, 541–555 (2007).

    Article  CAS  Google Scholar 

  12. Denoyelle, C. et al. Molecular mechanism of the anti-cancer activity of cerivastatin, an inhibitor of HMG-CoA reductase, on aggressive human breast cancer cells. Cell. Signal. 15, 327–338 (2003).

    Article  CAS  Google Scholar 

  13. Lackner, M.R. et al. Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors. Cancer Cell 7, 325–336 (2005).

    Article  CAS  Google Scholar 

  14. Watanabe, M. et al. Inhibitors of protein geranylgeranyltransferase I and Rab geranylgeranyltransferase identified from a library of allenoate-derived compounds. J. Biol. Chem. 283, 9571–9579 (2008).

    Article  CAS  Google Scholar 

  15. Hancock, J.F. Reticulocyte lysate assay for in vitro translation and posttranslational modification of Ras proteins. Methods Enzymol. 255, 60–65 (1995).

    Article  CAS  Google Scholar 

  16. Peter, M., Chavrier, P., Nigg, E.A. & Zerial, M. Isoprenylation of rab proteins on structurally distinct cysteine motifs. J. Cell Sci. 102, 857–865 (1992).

    CAS  PubMed  Google Scholar 

  17. Benetka, W., Koranda, M., Maurer-Stroh, S., Pittner, F. & Eisenhaber, F. Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo. BMC Biochem. 7, 6 (2006).

    Article  Google Scholar 

  18. Nguyen, U.T. et al. Exploiting the substrate tolerance of farnesyltransferase for site-selective protein derivatization. ChemBioChem 8, 408–423 (2007).

    Article  CAS  Google Scholar 

  19. Kho, Y. et al. A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl. Acad. Sci. USA 101, 12479–12484 (2004).

    Article  CAS  Google Scholar 

  20. Dursina, B. et al. Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids. J. Am. Chem. Soc. 128, 2822–2835 (2006).

    Article  CAS  Google Scholar 

  21. Baron, R. et al. RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody. Proc. Natl. Acad. Sci. USA 97, 11626–11631 (2000).

    Article  CAS  Google Scholar 

  22. Troutman, J.M., Roberts, M.J., Andres, D.A. & Spielmann, H.P. Tools to analyze protein farnesylation in cells. Bioconjug. Chem. 16, 1209–1217 (2005).

    Article  CAS  Google Scholar 

  23. Lane, K.T. & Beese, L.S. Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I. J. Lipid Res. 47, 681–699 (2006).

    Article  CAS  Google Scholar 

  24. Turek-Etienne, T.C., Strickland, C.L. & Distefano, M.D. Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase. Biochemistry 42, 3716–3724 (2003).

    Article  CAS  Google Scholar 

  25. Guo, Z. et al. Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation. EMBO J. 27, 2444–2456 (2008).

    Article  CAS  Google Scholar 

  26. Reigard, S.A. et al. Interplay of isoprenoid and peptide substrate specificity in protein farnesyltransferase. Biochemistry 44, 11214–11223 (2005).

    Article  CAS  Google Scholar 

  27. Krzysiak, A.J. et al. Combinatorial modulation of protein prenylation. ACS Chem. Biol. 2, 385–389 (2007).

    Article  CAS  Google Scholar 

  28. Cassidy, P.B., Dolence, J.M. & Poulter, C.D. Continuous fluorescence assay for protein prenyltransferases. Methods Enzymol. 250, 30–43 (1995).

    Article  CAS  Google Scholar 

  29. Cox, A.D. et al. The CAAX peptidomimetic compound B581 specifically blocks farnesylated, but not geranylgeranylated or myristylated, oncogenic ras signaling and transformation. J. Biol. Chem. 269, 19203–19206 (1994).

    CAS  PubMed  Google Scholar 

  30. McGuire, T.F., Qian, Y., Vogt, A., Hamilton, A.D. & Sebti, S.M. Platelet-derived growth factor receptor tyrosine phosphorylation requires protein geranylgeranylation but not farnesylation. J. Biol. Chem. 271, 27402–27407 (1996).

    Article  CAS  Google Scholar 

  31. Delahunty, C.M. & Yates, J.R. III . MudPIT: multidimensional protein identification technology. Biotechniques 43 563, 565, 567 (2007).

  32. Lutcke, A. et al. Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization. J. Cell Biol. 121, 553–564 (1993).

    Article  CAS  Google Scholar 

  33. Florens, L. et al. A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520–526 (2002).

    Article  CAS  Google Scholar 

  34. Zybailov, B., Coleman, M.K., Florens, L. & Washburn, M.P. Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling. Anal. Chem. 77, 6218–6224 (2005).

    Article  CAS  Google Scholar 

  35. Chen, B.C., Sundeen, J.E., Guo, P., Bednarz, M.S. & Zhao, R. Novel triethylsilane mediated reductive N-alkylation of amines: improved synthesis of 1-(4-imidazolyl)methyl-4-sulfonylbenzodiazepines new farnesyltransferase inhibitors. Tetrahedron Lett. 42, 1245–1246 (2001).

    Article  Google Scholar 

  36. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  37. McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. & Read, R.J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458–464 (2005).

    Article  Google Scholar 

  38. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  39. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  40. Schuttelkopf, A.W. & van Aalten, D.M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported in part by grant Deutsche Forschungsgemeinschaft AL 484/7-2 to K.A. and grant Sonderforschungsbereich 642 to K.A., D.W., R.S.G. and H.W. U.T.T.N. was supported by the predoctoral fellowship of Fonds der chemischen Industrie. R.S.B. and C. Deraeve thank the Alexander von Humboldt Stiftung for a scholarship. We thank R. Heuann (Ruhr-Universität Bochum) for supplying mouse brains. The authors gratefully acknowledge M. Terbeck, A. Sander, T. Rogowsky, S. Thuns and N. Lupilova for excellent technical assistance. The authors are very grateful to T. Bergbrede and the Dortmund Protein Facility at the Max Planck Institute. The use of beamlines at the Swiss Light Source (Paul Scherrrer Institute) and the help of the X-ray communities at the Max Planck Institute of Molecular Physiology and the Max Planck Insitute of Medical Research with data collection is gratefully acknowledged. We thank A. Barnekow (University of Münster) for the generous gift of Rab6A antibody.

Author information

Authors and Affiliations

  1. Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Uyen T T Nguyen, Zhong Guo, Christine Delon, Yaowen Wu, Celine Deraeve, Robin S Bon, Wulf Blankenfeldt, Roger S Goody, Herbert Waldmann & Kirill Alexandrov

  2. Biomolekulare Massenspektrometrie Proteincenter, Ruhr-Universität Bochum, Bochum, Germany

    Benjamin Fränzel & Dirk Wolters

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

    Kirill Alexandrov

Authors
  1. Uyen T T Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhong Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christine Delon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yaowen Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Celine Deraeve
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Benjamin Fränzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robin S Bon
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wulf Blankenfeldt
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Roger S Goody
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Herbert Waldmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Dirk Wolters
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Kirill Alexandrov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kirill Alexandrov.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 2405 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nguyen, U., Guo, Z., Delon, C. et al. Analysis of the eukaryotic prenylome by isoprenoid affinity tagging. Nat Chem Biol 5, 227–235 (2009). https://doi.org/10.1038/nchembio.149

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.149

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