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Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo

Abstract

Lipidated Rho and Rab GTP-binding proteins are transported between membranes in complex with solubilizing factors called 'guanine nucleotide dissociation inhibitors' (GDIs). Unloading from GDIs using GDI displacement factors (GDFs) has been proposed but remains mechanistically elusive. PDEδ is a putative solubilizing factor for several prenylated Ras-subfamily proteins. Here we report the structure of fully modified farnesylated Rheb-GDP in complex with PDEδ. The structure explains the nucleotide-independent binding of Rheb to PDEδ and the relaxed specificity of PDEδ. We demonstrate that the G proteins Arl2 and Arl3 act in a GTP-dependent manner as allosteric release factors for farnesylated cargo. We thus describe a new transport system for farnesylated G proteins involving a GDI-like molecule and an unequivocal GDF. Considering the importance of PDEδ for proper Ras and Rheb signaling, this study is instrumental in developing a new target for anticancer therapy.

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Figure 1: Overall structural analysis.
Figure 2: Ternary complex model and conformational change.
Figure 3: GppNHp-bound Arl2 or Arl3 disrupts the F-Rheb–PDEδ complex.
Figure 4: Arl2-GppNHp actively displaces farnesylated cargo via a ternary complex.
Figure 5: The effect of expressing PDEδ and Arl2Q70L on Rheb and N-Ras cellular steady-state localization.
Figure 6: Fluorescence lifetime measurements of PDEδ interaction.

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Acknowledgements

A.W., H.W. and P.I.H.B. thank the German Research Foundation (DFG) for financial support (SFB 642). A.W. and H.W. thank the European Research Council for financial support (ERC Grants 268782 and 268309). We thank A. Itzen and R.S. Goody for help with the stopped flow and discussion, D. Kuhlmann and C. Koerner for expert technical assistance. We thank I. Vetter. and R. Gaspar for scientific discussion and I. Schlichting for X-ray data collection.

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S.A.I. crystallized and solved the crystal structures, analyzed the data, designed the biochemical experiments and wrote the paper with A.W.; Y.-X.C. synthesized and produced the modified full-length proteins and peptides and contributed in the biochemical experiments and analysis; A.R. performed the biochemical experiments and purified proteins; A.C. performed and analyzed the experiments in cells; M.B. performed and designed the FCS experiments; L.G. purified proteins; G.T. and H.W. supervised Y.-X.C.; P.I.H.B. supervised A.C. and M.B.; and A.W. supervised the project and wrote the paper with S.I.

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Correspondence to Alfred Wittinghofer.

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PDEδ Conformational change (WittinghoferMovie.mov) (MOV 1188 kb)

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Ismail, S., Chen, YX., Rusinova, A. et al. Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo. Nat Chem Biol 7, 942–949 (2011). https://doi.org/10.1038/nchembio.686

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