Localization of lens intrinsic membrane protein MP19 and mutant protein MP19(To3) using fluorescent expression vectors.
OBJECTIVE
[corrected] MP19 is the second most abundant major intrinsic protein of the lens fiber cell membrane. A specific heritable mutation at amino acid 15 in the MP19 protein, termed MP19To3, results in total cataract and microphthalmia in the mouse. The goals of this study were to determine the specific localization of MP19 in the cell membrane and to determine whether the mutant MP19To3 protein migrates to the cell membrane in a similar fashion to normal MP19.


METHODS
MP19 and MP19To3 cDNAs were cloned into two different sets of expression vectors. The first set was composed of two vectors, pEGFP-N1 and pDsRed2-N1. The first vector expressed green fluorescent protein and the second expressed a red fluorescent protein when transfected into mammalian cells. The two lens membrane protein cDNAs were separately cloned into the vectors so that the cDNA was at the 5'-end of the fluorescent protein coding DNA. These vectors expressed each of the lens proteins fused to the fluorescent protein upon transfection into mammalian cell cultures. The second vector set was a single vector, pcDNA4/TO which must be induced in the transfected cells by tetracycline in order to express the cloned cDNAs. Each of the membrane cDNAs coupled to the fluorescent protein coding region was cut out of the first vector set and cloned into pcDNA4/TO and stable clones were isolated. Each of the prepared plasmids was transfected into human and chick embryo lens epithelial cells and human T-RexTM-293 cells. The fluorescent cells were viewed using confocal and episcopic-fluorescence microscopy.


RESULTS
Each of the transfected plasmids expressed fluorescent protein in all three cell lines. MP19 was observed to transport to the cell membrane. When compared to the distribution of another, separate fusion protein consisting of a signal peptide that targets to cell membranes fused to EGFP, MP19 did not distribute uniformly on the membrane, but appeared to localize into "spots" or pools of fluorescent material around the cell membrane. In contrast, MP19To3 protein appeared to not distribute to the cell membrane; it instead appeared to collect in a particular subcellular compartment within the cell.


CONCLUSIONS
The distribution of MP19 and MP19To3 in the cell appeared to be quite distinct. MP19 was observed to distribute to the cell membrane while MP19To3 did not. The fact that the MP19To3 did not traffic to the membrane, instead appearing to be trapped within a subcellular compartment within the cell sheds further light on the cause of the cataract and microphthalmia observed in the MP19To3 mutation, and further sheds information on the pathway of MP19 transport to the cell membrane.
Chen
Tong
T
Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA, USA.
Li
XiaLian
X
Yang
Yu
Y
Church
Robert L
RL
D000818:Animals; D001692:Biological Transport; D002386:Cataract / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002462:Cell Membrane; D002642:Chick Embryo; D004847:Epithelial Cells / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008850:Microphthalmos / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D011993:Recombinant Fusion Proteins; D014162:Transfection; D000097573:Red Fluorescent Protein
D005136:Eye Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C044539:LIM2 protein, human; C489025:Lim2 protein, mouse