gammaE-crystallin recruitment to the plasma membrane by specific interaction between lens MIP/aquaporin-0 and gammaE-crystallin.
OBJECTIVE
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 fluorescent protein (EGFP)-tagged or myc-tagged proteins, and gammaE-crystallin 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
gammaE-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 gammaE-crystallin interacts specifically with full-length MIP in mammalian cells. MIP did not interact with gammaD-crystallin, another member of the highly conserved gamma-crystallin gene family. Confocal fluorescence microscopy demonstrated that MIP interacted with gammaE-crystallin in individual mammalian cells and that this interaction resulted in the recruitment of gammaE-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 gamma-crystallins in lens fibers.
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Jianguo
J
Fan
Anna K
AK
Donovan
Dolena R
DR
Ledee
Peggy S
PS
Zelenka
Robert N
RN
Fariss
Ana B
AB
Chepelinsky
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D020346:Aquaporins; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003459:Crystallins / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D015723:Gene Library; D049452:Green Fluorescent Proteins; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D010957:Plasmids; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011817:Rabbits; D051381:Rats; D020798:Two-Hybrid System Techniques; D038222:gamma-Crystallins
D000954:Antigens, Surface; D020346:Aquaporins; D003459:Crystallins; D005136:Eye Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; C030328:aquaporin 0; C107256:crystallin gammaE; D038222:gamma-Crystallins; D049452:Green Fluorescent Proteins