CD2AP, Rabip4, and Rabip4′: Analysis of Interaction with Rab4a and Regulation of Endosomes Morphology

In this chapter, we describe various approaches that allow us to study interactions between the small GTPase Rab4a and its two effectors, Rabip4 and CD2AP. Two complementary approaches, one using the yeast two‐hybrid system and the other using a GST pull‐down assay, are described. We document the studies of the localization of these proteins by cellular fractionation. Finally, we develop cellular imaging techniques to study the morphology of vesicular structures containing Rab4a. We show that the coexpression of Rab4a with its effectors affects Rab4a‐containing structures, giving a clear indication of their interaction in the mammalian cellular context.