Rab GTPases pp 99-106 | Cite as

Kinetic Activation of Rab8 Guanine Nucleotide Exchange Factor Rabin8 by Rab11

  • Shanshan Feng
  • Bin Wu
  • Johan Peränen
  • Wei GuoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)


The Rab family of small GTPases acts as molecular switches that control various stages of vesicular transport. Rab8 functions in exocytic trafficking from the trans-Golgi network (TGN) and recycling endosomes to the plasma membrane. Rabin8 is a major guanine nucleotide exchange factor (GEF) for Rab8. It activates Rab8 by catalyzing its GDP release for subsequent GTP loading. However, how Rabin8 itself is activated in cells is unclear. Recently, it was found that Rabin8 is a downstream effector of Rab11, which controls vesicle exit from the recycling endosomes. Rab11, in its GTP-bound form, stimulates the GEF activity of Rabin8. The Rab11–Rabin8–Rab8 interactions thus couple vesicle generation from the donor compartment to its delivery to plasma membrane. Here we describe the methods we used to express and purify several Rab proteins, and to assay for the effect of Rab11 in the kinetic activation of Rabin8 GEF activity.

Key words

GTPase Guanine nucleotide exchange Rab11 Rabin8 Rab8 



W.G. is supported by National Institutes of General Medical Sciences R01 grants GM111128. S.F. was supported in part by an American Heart Association postdoctoral fellowship. The figures are presented in this paper with the permission from Proceedings of the National Academy of Sciences.


  1. 1.
    Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117CrossRefPubMedGoogle Scholar
  2. 2.
    Mizuno-Yamasaki E, Rivera-Molina F, Novick P (2012) GTPase networks in membrane traffic. Annu Rev Biochem 81:637–659CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Ortiz D, Medkova M, Walch-Solimena C, Novick P (2002) Ypt32 recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory vesicles; evidence for a Rab cascade in yeast. J Cell Biol 157:1005–1015CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Westlake CJ, Baye LM, Nachury MV, Wright KJ, Ervin KE, Phu L, Chalouni C, Beck JS, Kirkpatrick DS, Slusarski DC, Sheffield VC, Scheller RH, Jackson PK (2011) Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. Proc Natl Acad Sci U S A 108:2759–2764CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Knodler A, Feng S, Zhang J, Zhang X, Das A, Peranen J, Guo W (2010) Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc Natl Acad Sci U S A 107:6346–6351CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Feng S, Knödler A, Ren J, Zhang J, Zhang X, Hong Y, Huang S, Peränen J, Guo W (2012) A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis. J Biol Chem 287:15602–15609CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Bryant DM, Datta A, Rodríguez-Fraticelli AE, Peränen J, Martín-Belmonte F, Mostov KE (2010) A molecular network for de novo generation of the apical surface and lumen. Nat Cell Biol 12:1035–1045CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Das A, Guo W (2011) Rabs and the exocyst in ciliogenesis, tubulogenesis and beyond. Trends Cell Biol 21:383–386CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Hattula K, Furuhjelm J, Arffman A, Peränen J (2002) A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport. Mol Biol Cell 13:3268–3280CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Shanshan Feng
    • 1
  • Bin Wu
    • 1
  • Johan Peränen
    • 2
  • Wei Guo
    • 1
    Email author
  1. 1.Department of BiologyUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Institute of BiotechnologyUniversity of HelsinkiHelsinkiFinland

Personalised recommendations