Kinetic Activation of Rab8 Guanine Nucleotide Exchange Factor Rabin8 by Rab11
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 wordsGTPase 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.
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