γE-crystallin Recruitment to the Plasma Membrane by Specific Interaction between Lens MIP/Aquaporin-0 and γE-crystallin

Jianguo Fan, Anna K. Donovan, Dolena R. Ledee, Peggy S. Zelenka, Robert N. Fariss, Ana B. Chepelinsky
2004 Investigative Ophthalmology and Visual Science  
PURPOSE. Major intrinsic protein (MIP), also called aquaporin-0, is essential for lens transparency and is specifically expressed in the lens fiber cell membranes. The goal of the current study was to identify and characterize proteins that interact with MIP and to elucidate the role of these interactions in MIP functions. METHODS. The C-terminal 74-amino-acid fragment of MIP was used as bait to screen a rat lens cDNA yeast two-hybrid library. The full-length MIP was expressed as enhanced green
more » ... fluorescent protein (EGFP)-tagged or myc-tagged proteins, and ␥Ecrystallin was expressed as FLAG-tagged or red fluorescent protein (HcRed)-tagged proteins, respectively, in the RK13 rabbit kidney epithelial cell line. Protein-protein interactions were analyzed by coimmunoprecipitation assays and visualized by confocal fluorescence microscopy. RESULTS. ␥E-Crystallin, a water-soluble protein that is specifically expressed in lens fibers, was identified as a binding protein to the MIP C-terminal peptide. Coimmunoprecipitation assays demonstrated that ␥E-crystallin interacts specifically with full-length MIP in mammalian cells. MIP did not interact with ␥D-crystallin, another member of the highly conserved ␥-crystallin gene family. Confocal fluorescence microscopy demonstrated that MIP interacted with ␥E-crystallin in individual mammalian cells and that this interaction resulted in the recruitment of ␥E-crystallin from the cytoplasm to the plasma membrane. CONCLUSIONS. These experiments provide the first demonstration of MIP interaction with other lens proteins at the molecular level and raise the possibility of a structural role of MIP in the organization of ␥-crystallins in lens fibers. (Invest Ophthalmol Vis Sci.
doi:10.1167/iovs.03-0708 pmid:14985303 fatcat:arxhlkshbredxdtxyozmfzbeva