Specific interaction between lens MIP/Aquaporin-0 and two members of the gamma-crystallin family.
OBJECTIVE
Major Intrinsic Protein (MIP)/Aquaporin 0 is required for lens transparency and is specifically expressed in lens fiber cell membranes. We have demonstrated previously that in the rat lens MIP interacts specifically with gammaE-crystallin, resulting in its recruitment to the plasma membrane. Our goal was to examine the interaction or lack of interaction between MIP and all members of the gamma-crystallin family and to provide evidence for a physiological role these interactions may play in gamma-crystallin or MIP function.


METHODS
Full length MIP was expressed as untagged, enhanced green fluorescent protein (EGFP) tagged, or myc tagged proteins. Members of the gamma-crystallin family were expressed as red fluorescent protein (HcRed) tagged proteins in the rabbit kidney epithelial cell line RK13. Co-localization of tagged proteins was analyzed by confocal fluorescence microscopy.


RESULTS
Confocal fluorescence microscopy demonstrated that gammaE- and gammaF-crystallin co-localize specifically with full length MIP in mammalian cells while other gamma-crystallins, including gammaA-, gammaB-, gammaC-, gammaD-, and gammaS-crystallin do not. As a result of this interaction, either gammaE- or gammaF-crystallin was recruited to the plasma membrane from the cytoplasm. MIP does not interact with the Elo mutant of gammaE-crystallin, which has been linked to a dominant cataract phenotype in mice.


CONCLUSIONS
These experiments demonstrate that MIP interacts selectively with gammaE- and gammaF-crystallin, and not with other gamma-crystallins. This raises the possibility of MIP playing a structural role in the organization of gamma-crystallins in rodent lens fibers and/or that gammaE- and gammaF-crystallin may have a specific role in MIP function in the rodent lens.
Fan
Jianguo
J
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-0704, USA. fanj@nei.nih.gov
Fariss
Robert N
RN
Purkiss
Andrew G
AG
Slingsby
Christine
C
Sandilands
Aileen
A
Quinlan
Roy
R
Wistow
Graeme
G
Chepelinsky
Ana B
AB
D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011817:Rabbits; D014162:Transfection; D038222:gamma-Crystallins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D020346:Aquaporins; D005136:Eye Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D016271:Proto-Oncogene Proteins c-myc; D038222:gamma-Crystallins; D000097573:Red Fluorescent Protein; C030328:aquaporin 0; C485184:enhanced green fluorescent protein