RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11

ABSTRACT The small GTPase Rab11 plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth, and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain of one of its effectors, e.g., the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, e.g., to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 visualized endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking: tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.