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.
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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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Qi, Y., Liang, Z., Wang, Z., Lu, G., Li, G. (2015). Determination of Rab5 Activity in the Cell by Effector Pull-Down Assay. In: Li, G. (eds) Rab GTPases. Methods in Molecular Biology, vol 1298. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2569-8_22
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DOI: https://doi.org/10.1007/978-1-4939-2569-8_22
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2568-1
Online ISBN: 978-1-4939-2569-8
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