Determination of Rab5 Activity in the Cell by Effector Pull-Down Assay
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 wordsRab5 Rabaptin-5 EEA1 Rabenosyn-5 Endocytosis Endosome
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.)
- 17.Ji X, Johnson WW, Sesay MA et al (1994) Structure and function of the xenobiotic substrate binding site of a glutathione S-transferase as revealed by X-ray crystallographic analysis of product complexes with the diastereomers of 9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene. Biochemistry 33:1043–1052CrossRefPubMedGoogle Scholar