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Rab GTPases pp 259-270 | Cite as

Determination of Rab5 Activity in the Cell by Effector Pull-Down Assay

  • Yaoyao Qi
  • Zhimin Liang
  • Zonghua Wang
  • Guodong Lu
  • Guangpu LiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)

Abstract

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

Key words

Rab5 Rabaptin-5 EEA1 Rabenosyn-5 Endocytosis Endosome 

Notes

Acknowledgments

We thank M. Caleb Marlin for helpful comments and Fig. 1 illustration, and John Colicelli for the generous gift of pGEX-2 T/Rabenosyn-5:R5BD. This work was supported in part by the NIH grant R01 GM074692 (to G.L.) and a scholarship from the China Scholarship Council (to Y.Q.)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Yaoyao Qi
    • 1
    • 2
  • Zhimin Liang
    • 2
  • Zonghua Wang
    • 1
  • Guodong Lu
    • 1
  • Guangpu Li
    • 3
    Email author
  1. 1.Key Laboratory of Biopesticides and Chemical Biology, Ministry of EducationFujian Agriculture & Forestry UniversityFuzhouChina
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  3. 3.Department of Biochemistry and Molecular Biology, Peggy and Charles Stephenson Cancer CenterUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA

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