Abstract
Plasma membrane recycling is an important process necessary for maintaining membrane composition. The motor protein myosin Vb regulates plasma membrane recycling through its association with Rab11a. Overexpression of the tail of myosin Vb disrupts trafficking out of plasma membrane recycling systems and leads to the accumulation of Rab11a in both polarized and non-polarized cells. We have investigated the association of Rab11 family interacting protein 2 (Rab11-FIP2) with myosin Vb as an adapter protein between Rab11a and myosin Vb. Immunofluorescence studies indicated a colocalization of endogenous Rab11-FIP2 with green fluorescent protein-myosin Vb tail overexpressed in Madin-Darby canine kidney (MDCK) cells. Yeast two hybrid assays showed that amino acids 129-356 of Rab11-FIP2 were important for binding to myosin Vb tail. In vitro association assays and co-transfection experiments in both MDCK and HeLa cells confirmed this result but further refined the binding site to amino acids 129-290 of Rab11-FIP2. Like myosin Vb, functional studies indicated that Rab11-FIP2 is also important for normal plasma membrane recycling. Green fluorescent protein-Rab11-FIP2 (129-512), which lacks its amino-terminal C2 domain, functioned as a dominant negative acting truncation that caused accumulation of Rab11a and disrupted IgA trafficking in MDCK cells and transferrin trafficking in HeLa cells. The ternary association of myosin Vb and Rab11-FIP2 with Rab11a suggests that a multimeric protein complex is involved in vesicle trafficking through plasma membrane recycling systems.
Highlights
Plasma membrane recycling is an important process necessary for maintaining membrane composition
Yeast two hybrid assays showed that amino acids 129 –356 of Rab11-FIP2 were important for binding to myosin Vb tail
GFP-myosin Vb transfection blocked exit of transferrin from the recycling system vesicles in HeLa cells and polymeric IgA receptor (pIgA-R) from the apical recycling system in MadinDarby canine kidney (MDCK) cells
Summary
Vol 277, No 52, Issue of December 27, pp. 50415–50421, 2002 Printed in U.S.A. Rab Family Interacting Protein 2 Associates with Myosin Vb and Regulates Plasma Membrane Recycling*. GFP-myosin Vb transfection blocked exit of transferrin from the recycling system vesicles in HeLa cells and pIgA-R from the apical recycling system in MDCK cells All of these results indicated that myosin Vb was the motor protein responsible for transit out of plasma membrane recycling systems. We hypothesized that Rab11-FIP2 may contribute to anchoring of Rab11a-positive vesicles to myosin Vb. We report that yeast two hybrid analysis, immunolocalization experiments, and in vitro binding studies all confirm the direct association of Rab11-FIP2 with myosin Vb. In trafficking studies, a truncation of Rab11-FIP2 lacking the aminoterminal C2-domain inhibited plasma membrane recycling in both HeLa and MDCK cells. A truncation of Rab11-FIP2 lacking the aminoterminal C2-domain inhibited plasma membrane recycling in both HeLa and MDCK cells These studies show that Rab11FIP2 is a receptor/adapter protein linking myosin Vb to Rab11a and regulating the movement of vesicle cargo through the recycling endosome
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