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J. Fan, A.K. Donovan, D.R. Ledee, P.S. Zelenka, R.N. Fariss, A.B. Chepelinksy; Specific Interaction Between Lens MIP/Aquaporin-0 and E-Crystallin Results in E-Crystallin Recruitment to the Plasma Membrane . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2135.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Major Intrinsic Protein (MIP)/Aquaporin 0 is required for lens transparency and is specifically expressed in the lens fiber membranes. Our goal is to identify and characterize proteins that interact with MIP and to elucidate the role of these interactions in MIP functions. Methods: A MIP cDNA fragment encoding the C-terminal 74 amino acids was used as the bait cDNA to screen a rat lens cDNA yeast two-hybrid library. Interaction between the full-length MIP and the identified binding protein γE-crystallin was confirmed in mammalian cells by immuno pulldown of FLAG-tagged γE-crystallin and EGFP tagged MIP from co-transfected rabbit kidney epithelial cells (RK13). MIP/γE-crystallin interaction was further characterized by confocal fluorescence microscopy in RK13 cells expressing MIP and γE-crystallin as an EGFP- and HcRed (a red fluorescent protein)-tagged protein, respectively. Results: The yeast two-hybrid screening identified γE-crystallin as a protein interacting with MIP. By immuno pulldown, we demonstrated that γE-crystallin interacts specifically with MIP in mammalian cells and that this interaction occurs only when MIP C-terminus is free. By using confocal fluorescence microscopy, we demonstrated that γE-crystallin is recruited to the plasma membrane when EGFP-MIP and HcRed-γE-crystallin are co-expressed in kidney epithelial cells and that this recruitment requires the MIP C-terminus to be free. Conclusions: These experiments provide the first demonstration of MIP interaction with other lens proteins at the molecular level and may suggest a structural role for MIP in the organization of γ-crystallins in lens fibers.
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