In Vitro and In Vivo Characterization of the Rab11-GAP Activity of Drosophila Evi5
Small GTPases of the Rab family are master regulators of vesicular trafficking. As such, they control the spatial distribution of various proteins, including proteins involved in cell signaling and the regulation of cell polarity. Misregulation of Rab proteins is associated with a large array of diseases. Surprisingly, the target of some key regulators of Rab proteins, including many GTPase-activating protein (GAP) is still unknown. Identifying the target of a specific GAP requires the combination of both in vitro and in vivo experiments to avoid any misinterpretation. Here is described the methodology we used to characterize the Rab11-GAP activity of Drosophila Evi5. We first focus on the in vitro Rab11 effector pull-down assay we developed and then we detail the in vivo characterization of Rab11 activity during Drosophila border cell migration.
Key wordsGTPase-activating protein Rab Evi5 Rab11
This work was supported by the Canadian Institute for Health Research (MOP 114899). G. Emery holds a Canada Research Chair (Tier II) in Vesicular Trafficking and Cell Signaling. CL is supported by a FRSQ scholarship.
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