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.
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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.”
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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).
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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.
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Supplementary Methods, Supplementary Results, Supplementary Figures 1–12 and Supplementary Table 1 (PDF 5285 kb)
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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
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DOI: https://doi.org/10.1038/nchembio.386
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