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TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism

Abstract

Rab GTPases regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations1. The duration of the active state is limited by GTPase-activating proteins (GAPs), which accelerate the slow intrinsic rate of GTP hydrolysis. Proteins containing TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotic organisms and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis2. An exposed arginine residue is a critical determinant of GAP activity in vitro and in vivo3,4,5. It has been expected that the catalytic mechanism of TBC domains would parallel that of Ras and Rho family GAPs. Here we report crystallographic, mutational and functional analyses of complexes between Rab GTPases and the TBC domain of Gyp1p. In the crystal structure of a TBC-domain–Rab-GTPase–aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers indicates that most TBC-domain GAPs may accelerate GTP hydrolysis by a similar dual-finger mechanism.

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Figure 1: Identification of mammalian Rab GTPase substrates for the Gyp1p TBC domain.
Figure 2: Structure of the Gyp1p TBC domain in complex with Rab33–GDP–AlF3.
Figure 3: Network of polar interactions in the AlF 3 -binding site and comparison with other GAP–GTPase complexes.
Figure 4: Mutational analysis of the Gyp1p Rab interaction epitope.

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References

  1. Segev, N. Ypt/Rab GTPases: regulators of protein trafficking. Sci. STKE 2001, RE11 (2001)

    CAS  PubMed  Google Scholar 

  2. Bernards, A. GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila. Biochim. Biophys. Acta 1603, 47–82 (2003)

    CAS  PubMed  Google Scholar 

  3. Albert, S., Will, E. & Gallwitz, D. Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases. EMBO J. 18, 5216–5225 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Rak, A. et al. Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins. EMBO J. 19, 5105–5113 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Du, L. L. & Novick, P. Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p. Mol. Biol. Cell 12, 1215–1226 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Strom, M., Vollmer, P., Tan, T. J. & Gallwitz, D. A yeast GTPase-activating protein that interacts specifically with a member of the Ypt/Rab family. Nature 361, 736–739 (1993)

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Du, L. L., Collins, R. N. & Novick, P. J. Identification of a Sec4p GTPase-activating protein (GAP) as a novel member of a Rab GAP family. J. Biol. Chem. 273, 3253–3256 (1998)

    Article  CAS  PubMed  Google Scholar 

  8. Albert, S. & Gallwitz, D. Two new members of a family of Ypt/Rab GTPase activating proteins. Promiscuity of substrate recognition. J. Biol. Chem. 274, 33186–33189 (1999)

    Article  CAS  PubMed  Google Scholar 

  9. Cuif, M. H. et al. Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome. EMBO J. 18, 1772–1782 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Vollmer, P., Will, E., Scheglmann, D., Strom, M. & Gallwitz, D. Primary structure and biochemical characterization of yeast GTPase-activating proteins with substrate preference for the transport GTPase Ypt7p. Eur. J. Biochem. 260, 284–290 (1999)

    Article  CAS  PubMed  Google Scholar 

  11. Lanzetti, L. et al. The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408, 374–377 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  12. Will, E. & Gallwitz, D. Biochemical characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating protein from yeast. J. Biol. Chem. 276, 12135–12139 (2001)

    Article  CAS  PubMed  Google Scholar 

  13. Gao, X. D. et al. The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization. J. Cell Biol. 162, 635–646 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Haas, A. K., Fuchs, E., Kopajtich, R. & Barr, F. A. A. GTPase-activating protein controls Rab5 function in endocytic trafficking. Nature Cell Biol. 7, 887–893 (2005)

    Article  CAS  PubMed  Google Scholar 

  15. Miinea, C. P. et al. AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain. Biochem. J. 391, 87–93 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lafourcade, C., Galan, J. M., Gloor, Y., Haguenauer-Tsapis, R. & Peter, M. The GTPase-activating enzyme Gyp1p is required for recycling of internalized membrane material by inactivation of the Rab/Ypt GTPase Ypt1p. Mol. Cell. Biol. 24, 3815–3826 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Segev, N., Mulholland, J. & Botstein, D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell 52, 915–924 (1988)

    Article  CAS  PubMed  Google Scholar 

  18. Saraste, J., Lahtinen, U. & Goud, B. Localization of the small GTP-binding protein rab1p to early compartments of the secretory pathway. J. Cell Sci. 108, 1541–1552 (1995)

    CAS  PubMed  Google Scholar 

  19. Zheng, J. Y. et al. A novel Rab GTPase, Rab33B, is ubiquitously expressed and localized to the medial Golgi cisternae. J. Cell Sci. 111, 1061–1069 (1998)

    CAS  PubMed  Google Scholar 

  20. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E. & Sigler, P. B. GTPase mechanism of G proteins from the 1.7-Å crystal structure of transducin α-GDP-AlF4-. Nature 372, 276–279 (1994)

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Coleman, D. E. et al. Structures of active conformations of Giα1 and the mechanism of GTP hydrolysis. Science 265, 1405–1412 (1994)

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Mittal, R., Ahmadian, M. R., Goody, R. S. & Wittinghofer, A. Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPase-activating proteins. Science 273, 115–117 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Scheffzek, K. et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338 (1997)

    Article  CAS  PubMed  Google Scholar 

  24. Rittinger, K., Walker, P. A., Eccleston, J. F., Smerdon, S. J. & Gamblin, S. J. Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389, 758–762 (1997)

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Nassar, N., Hoffman, G. R., Manor, D., Clardy, J. C. & Cerione, R. A. Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP. Nature Struct. Biol. 5, 1047–1052 (1998)

    Article  CAS  PubMed  Google Scholar 

  26. Tesmer, J. J., Berman, D. M., Gilman, A. G. & Sprang, S. R. Structure of RGS4 bound to AlF4-activated Giα1: stabilization of the transition state for GTP hydrolysis. Cell 89, 251–261 (1997)

    Article  CAS  PubMed  Google Scholar 

  27. Slep, K. C. et al. Structural determinants for regulation of phosphodiesterase by a G protein at 2.0 Å. Nature 409, 1071–1077 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  28. Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R. & Wittinghofer, A. The GTPase-activating protein Rap1GAP uses a catalytic asparagine. Nature 429, 197–201 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  29. Eathiraj, S., Pan, X., Ritacco, C. & Lambright, D. G. Structural basis of family-wide Rab GTPase recognition by rabenosyn-5. Nature 436, 415–419 (2005)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. De Antoni, A., Schmitzova, J., Trepte, H. H., Gallwitz, D. & Albert, S. Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum to Golgi transport revealed by the analysis of two novel Ypt1-GAPs. J. Biol. Chem. 277, 41023–41031 (2002)

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank P. Novick for the GYP1-null strains and plasmids, and J. Saporita for assistance with yeast experiments. This work was supported by a National Institutes of Health grant.

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Correspondence to David G. Lambright.

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Competing interests

Coordinates and structure factors for the Gyp1p–Rab33–AlF3 complex have been deposited with the Protein Data Bank under the ID code 2G77. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure S1

Identification of Rab GTPase substrates for the Gyp1p TBC domain. (PDF 113 kb)

Supplementary Figure S2

Gyp1p and Rab33 form a complex in the presence of aluminum fluoride. (PDF 190 kb)

Supplementary Figure S3

Additional information relevant to structure determination. (PDF 347 kb)

Supplementary Figure S4

Conservation and variability within the interface between Gyp1p and Rab33. (PDF 307 kb)

Supplementary Figure S5

Comparison of the structures of Gyp1p and Rab33 alone and in the complex. (PDF 216 kb)

Supplementary Figure S6

Kinetics of GTP hydrolysis catalyzed by the Gyp1p and Gyp7p TBC domains. (PDF 286 kb)

Supplementary Figure S7

Western blot analysis of wild type and mutant Gyp1p expression. (PDF 145 kb)

Supplementary Table S1

Rab constructs. (PDF 37 kb)

Supplementary Table S2

Data collection, phasing, and refinement statistics. (PDF 77 kb)

Supplementary Methods

This file contains supplementary methods, including one table and eight references. (PDF 106 kb)

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Pan, X., Eathiraj, S., Munson, M. et al. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442, 303–306 (2006). https://doi.org/10.1038/nature04847

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