Rab GTPases pp 47-60 | Cite as

High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins

  • Ashwini K. Mishra
  • David G. LambrightEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)


Measurement of intrinsic as well as GTPase-Activating Protein (GAP)-catalyzed GTP hydrolysis is central to understanding the molecular mechanism and function of GTPases in diverse cellular processes. For the Rab GTPase family, which comprises at least 60 distinct proteins in humans, putative GAPs have been identified from both eukaryotic organisms and pathogenic bacteria. A major obstacle has involved identification of target substrates and determination of the specificity for the Rab family. Here, we describe a sensitive, high-throughput method to quantitatively profile GAP activity for Rab GTPases in microplate format based on detection of inorganic phosphate released after GTP hydrolysis. The method takes advantage of a well-characterized fluorescent phosphate sensor, requires relatively low protein concentrations, and can in principle be applied to any GAP-GTPase system.

Key words

GTPase Rab GTPase Phosphate-binding protein Phosphate PBP-MDCC GTP hydrolysis GAP reaction High throughput GAP assay 



This work was supported by an NIH grant GM056324 to DGL.


  1. 1.
    Bourne HR, Sanders DA, McCormick F (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348(6297):125–132. doi: 10.1038/348125a0 CrossRefPubMedGoogle Scholar
  2. 2.
    Bos JL, Rehmann H, Wittinghofer A (2007) GEFs and GAPs: critical elements in the control of small G proteins. Cell 129(5):865–877CrossRefPubMedGoogle Scholar
  3. 3.
    Bernards A, Settleman J (2004) GAP control: regulating the regulators of small GTPases. Trends Cell Biol 14(7):377–385. doi: 10.1016/j.tcb.2004.05.003 CrossRefPubMedGoogle Scholar
  4. 4.
    Ligeti E, Welti S, Scheffzek K (2012) Inhibition and termination of physiological responses by GTPase activating proteins. Physiol Rev 92(1):237–272. doi: 10.1152/physrev.00045.2010 CrossRefPubMedGoogle Scholar
  5. 5.
    Barr F, Lambright DG (2010) Rab GEFs and GAPs. Curr Opin Cell Biol 22:461–470CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Eberth A, Dvorsky R, Becker CF, Beste A, Goody RS, Ahmadian MR (2005) Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins. Biol Chem 386(11):1105–1114. doi: 10.1515/BC.2005.127 CrossRefPubMedGoogle Scholar
  7. 7.
    Scheffzek K, Ahmadian MR, Wittinghofer A (1998) GTPase-activating proteins: helping hands to complement an active site. Trends Biochem Sci 23(7):257–262CrossRefPubMedGoogle Scholar
  8. 8.
    Gideon P, John J, Frech M, Lautwein A, Clark R, Scheffler JE, Wittinghofer A (1992) Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity. Mol Cell Biol 12(5):2050–2056PubMedCentralPubMedGoogle Scholar
  9. 9.
    Marshall CB, Meiri D, Smith MJ, Mazhab-Jafari MT, Gasmi-Seabrook GM, Rottapel R, Stambolic V, Ikura M (2012) Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts. Methods 57(4):473–485. doi: 10.1016/j.ymeth.2012.06.014 CrossRefPubMedGoogle Scholar
  10. 10.
    Mazhab-Jafari MT, Marshall CB, Smith M, Gasmi-Seabrook GM, Stambolic V, Rottapel R, Neel BG, Ikura M (2010) Real-time NMR study of three small GTPases reveals that fluorescent 2′(3′)-O-(N-methylanthraniloyl)-tagged nucleotides alter hydrolysis and exchange kinetics. J Biol Chem 285(8):5132–5136. doi: 10.1074/jbc.C109.064766 CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Nixon AE, Brune M, Lowe PN, Webb MR (1995) Kinetics of inorganic phosphate release during the interaction of p21ras with the GTPase-activating proteins, p120-GAP and neurofibromin. Biochemistry 34(47):15592–15598CrossRefPubMedGoogle Scholar
  12. 12.
    Brune M, Hunter JL, Corrie JE, Webb MR (1994) Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33(27):8262–8271CrossRefPubMedGoogle Scholar
  13. 13.
    Webb MR (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 89(11):4884–4887CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Self AJ, Hall A (1995) Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates. Methods Enzymol 256:67–76CrossRefPubMedGoogle Scholar
  15. 15.
    Shutes A, Der CJ (2005) Real-time in vitro measurement of GTP hydrolysis. Methods 37(2):183–189. doi: 10.1016/j.ymeth.2005.05.019 CrossRefPubMedGoogle Scholar
  16. 16.
    Mishra AK, Del Campo CM, Collins RE, Roy CR, Lambright DG (2013) The Legionella pneumophila GTPase activating protein LepB accelerates Rab1 deactivation by a non-canonical hydrolytic mechanism. J Biol Chem 288(33):24000–24011. doi: 10.1074/jbc.M113.470625 CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Yu Q, Hu L, Yao Q, Zhu Y, Dong N, Wang DC, Shao F (2013) Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP. Cell Res 23(6):775–787. doi: 10.1038/cr.2013.54 CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Nottingham RM, Pusapati GV, Ganley IG, Barr FA, Lambright DG, Pfeffer SR (2012) RUTBC2 protein, a Rab9A effector and GTPase-activating protein for Rab36. J Biol Chem 287(27):22740–22748. doi: 10.1074/jbc.M112.362558 CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Dong N, Zhu Y, Lu Q, Hu L, Zheng Y, Shao F (2012) Structurally distinct bacterial TBC-like GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses. Cell 150(5):1029–1041. doi: 10.1016/j.cell.2012.06.050 CrossRefPubMedGoogle Scholar
  20. 20.
    Davey JR, Humphrey SJ, Junutula JR, Mishra AK, Lambright DG, James DE, Stockli J (2012) TBC1D13 is a RAB35 specific GAP that plays an important role in GLUT4 trafficking in adipocytes. Traffic 13(10):1429–1441. doi: 10.1111/j.1600-0854.2012.01397.x CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Nottingham RM, Ganley IG, Barr FA, Lambright DG, Pfeffer SR (2011) RUTBC1 protein, a Rab9A effector that activates GTP hydrolysis by Rab32 and Rab33B proteins. J Biol Chem 286(38):33213–33222. doi: 10.1074/jbc.M111.261115 CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Chotard L, Mishra AK, Sylvain MA, Tuck S, Lambright DG, Rocheleau CE (2010) TBC-2 regulates RAB-5/RAB-7-mediated endosomal trafficking in Caenorhabditis elegans. Mol Biol Cell 21(13):2285–2296. doi: 10.1091/mbc.E09-11-0947 CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Ingmundson A, Delprato A, Lambright DG, Roy CR (2007) Legionella pneumophila proteins that regulate Rab1 membrane cycling. Nature 450(7168):365–369. doi: 10.1038/nature06336 CrossRefPubMedGoogle Scholar
  24. 24.
    Sklan EH, Serrano RL, Einav S, Pfeffer SR, Lambright DG, Glenn JS (2007) TBC1D20 is a Rab1 GTPase-activating protein that mediates hepatitis C virus replication. J Biol Chem 282(50):36354–36361CrossRefPubMedGoogle Scholar
  25. 25.
    Mukhopadhyay A, Pan X, Lambright DG, Tissenbaum HA (2007) An endocytic pathway as a target of tubby for regulation of fat storage. EMBO Rep 8(10):931–938CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Pan X, Eathiraj S, Munson M, Lambright DG (2006) TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442(7100):303–306. doi: 10.1038/nature04847 CrossRefPubMedGoogle Scholar
  27. 27.
    Eathiraj S, Pan X, Ritacco C, Lambright DG (2005) Structural basis of family-wide Rab GTPase recognition by rabenosyn-5. Nature 436(7049):415–419. doi: 10.1038/nature03798 CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Mishra A, Eathiraj S, Corvera S, Lambright DG (2010) Structural basis for Rab GTPase recognition and endosome tethering by the C2H2 zinc finger of Early Endosomal Autoantigen 1 (EEA1). Proc Natl Acad Sci U S A 107(24):10866–10871. doi: 10.1073/pnas.1000843107 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Pharmacology, Program in Molecular MedicineUniversity of Massachusetts Medical SchoolWorcesterUSA

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