Identification and characterization of JRAB/MICAL-L2, a junctional Rab13-binding protein.

The Rab family small G proteins are localized to distinct subsets of intracellular membranes and play a key role in membrane traffic through the interaction with their specific effector protein(s). Rab13 is identified as a plaque protein at tight junctions (TJs) and has been shown to regulate the assembly of functional TJs in epithelial cells. We have demonstrated that Rab13 mediates the endocytic recycling of integral TJ protein occludin, and identified a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) as a Rab13 effector protein using a yeast two-hybrid system. JRAB/MICAL-L2 has a calponin-homology domain in the N-terminus, a LIM domain in the middle, and a coiled-coil domain at the C-terminus, and specifically binds to the GTP-bound form of Rab13 via its C-terminus. It is localized to TJs in epithelial cells and distributed along stress fibers in fibroblasts. In epithelial cells, JRAB/MICAL-L2 as well as Rab13 mediates the endocytic recycling of occludin, but not transferrin receptor, and the formation of functional TJs. This chapter describes the procedures for the isolation of JRAB/MICAL-L2 and the analysis of its functions.