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Originally published In Press as doi:10.1074/jbc.M209270200 on October 21, 2002

J. Biol. Chem., Vol. 277, Issue 52, 50415-50421, December 27, 2002
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Rab11 Family Interacting Protein 2 Associates with Myosin Vb and Regulates Plasma Membrane Recycling*

Chadwick M. HalesDagger , Jean-Pierre Vaerman§, and James R. Goldenring||

From the Dagger  Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, § Universite Catholique de Louvain, C. de Duve Institute of Cell Pathology, Brussels, Belgium, and  Department of Surgery, Vanderbilt University School of Medicine and Nashville Veterans Affairs Medical Center, Nashville, Tennessee 37232

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.

* This work was supported in part by NIDDK, National Institutes of Health Grants DK48370 and DK43405 (to J. R. G.) and by a Veterans Affairs merit award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Surgery, Vanderbilt University School of Medicine, 4160A MRB III, 465 21st St. S., Nashville, TN 37232-2733. Tel.: 615-936-3726; Fax: 615-343-1355; E-mail: jim.goldenring@vanderbilt.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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