GTPase Protocols pp 157-165 | Cite as
Analysis and Preparation of Stable Complexes between Rab GTPases, Rab Escort Protein, and Rab Geranylgeranyl Transferase
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Abstract
Rab proteins are small Ras-like GTPases that regulate vesicular trafficking events in the cell. More than 50 Rabs have been described in mammalian cells (1), each with a specific subcellular localization reflecting the functional specificity of Rabs to specific trafficking steps (2, 3, 4, 5). Rabs contain two cysteine residues at or near the carboxyl terminus, arranged in a variety of motifs. Both cysteine residues are modified by the attachment of geranylgeranyl groups via thioether bonds, in a reaction catalyzed by Rab geranylgeranyl transferase (also known as GGTase type II, RGGT) (6). This enzyme is a tightly bound heterodimer, composed of a 60-kDa α-subunit and a 38-kDa β-subunit, both related to the α- and β-subunits of the other known protein prenyltransferases, farnesyl transferase and caax geranylgeranyl transferase (also known as GGTase type I).
Keywords
Ternary Complex Smart System Geranylgeranyl Pyrophosphate Sodium HEPES Fast Performance Liquid ChromatographyReferences
- 1.Pereira-Leal, J. and Seabra, M. C. (2000) The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. J. Mol. Biol. 301, 1077–1087.PubMedCrossRefGoogle Scholar
- 2.Lazar, T., Gotte, M., and Gallwitz, TD. (1997) Vesicular transport: how many Ypt/Rab-GTPases make a eukaryotic cell? Trends Biochem. Sci. 22, 468–472.CrossRefGoogle Scholar
- 3.Novick, P. and Zerial, M. (1997) The diversity of Rab proteins in vesicle transport. Curr. Opin. Cell Biol. 9, 496–504.PubMedCrossRefGoogle Scholar
- 4.Schimmoller, F., Simon, I., and Pfeffer, S. R. (1998) Rab GTPases, directors of vesicle docking. J. Biol. Chem. 273, 22,161–22,164.PubMedCrossRefGoogle Scholar
- 5.Chavrier, P. and Goud, B. (1999) The role of ARF and Rab GTPases in membrane transport. Curr. Opin. Cell Biol. 11, 466–475.PubMedCrossRefGoogle Scholar
- 6.Seabra, M. C. (2000) Biochemistry of Rab geranylgeranyl transferase, in The Enzymes (Tamanoi, E and Sigman, D., eds.), vol. XXI, Academic Press, New York, NY, pp. 131–154.Google Scholar
- 7.Andres, D. A., Seabra, M. C., Brown, M. S., Armstrong, S. A., Smeland, T. E., Cremers, E P., et al. (1993) cDNA cloning of component A of Rab geranylgeranyl transferase and demonstration of its role as a Rab escort protein. Cell 73, 1091–1099.PubMedCrossRefGoogle Scholar
- 8.Alexandrov, K., Horiuchi, H., Steele-Mortimer, O., Seabra, M. C., and Zerial, M. (1994) Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated Rab proteins to their target membranes. EMBO J. 13, 5262–5273.PubMedGoogle Scholar
- 9.Armstrong, S. A., Brown, M. S., Goldstein, J. L., and Seabra, M. C. (1995) Preparation of recombinant Rab geranylgeranyltransferase and Rab escort proteins. Methods Enzymol. 257, 30–41.PubMedCrossRefGoogle Scholar
- 10.Seabra, M. C. and James, G. L. (1998) Prenylation assays for small GTPases. Methods Mol. Biol. 84, 251–260.PubMedGoogle Scholar
- 11.Anant, J. S., Desnoyers, L., Machius, M., Demeler, B., Hansen, J. C., Westover, K. D., et al. (1998) Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex. Biochemistry 37, 12,559–12,568.PubMedCrossRefGoogle Scholar
- 12.Alexandrov, K., Simon, I., Iakovenko, A., Holz, B., Goody, R. S., and Scheidig, A. J. (1998) Moderate discrimination of REP-1 between Rab7 x GDP and Rab7×GTP arises from a difference of an order of magnitude in dissociation rates. FEBS Lett. 425, 460–464.PubMedCrossRefGoogle Scholar
- 13.Shen, F. and Seabra, M. C. (1996) Mechanism of digeranylation of Rab proteins.J. Biol. Chem. 271, 3692–3698.CrossRefGoogle Scholar