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T Chen, X Li, Z Li, Y Yang, RL Church; Localization of Lens Intrinsic Membrane Proteins MP19, Cx50, and Mutant MP19To3 Using Fluorescent Expression Vectors . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4637.
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Purpose: MP19 and connexin50 (Cx50) are known to be intrinsic proteins of the lens fiber cell membrane. A specific mutation of MP19, termed MP19To3 leads to heritable cataract. The goals of this study were to determine the specific localization of MP19 and Cx50 in the cell membrane, and to determine whether the MP19To3 protein migrates to the cell membrane in a similar fashion to normal MP19. Methods: MP19, Cx50, and MP19To3 cDNAs were cloned into two different types of expression vectors. The first vector 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 three 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. 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 were transfected into mouse TRx-293 and human lens epithelial cells using FuGene 6. The fluorescent cells were viewed using confocal microscopy. Results: Each of the transfected plasmids expressed fluorescent protein in both cell lines. MP19 and Cx50 were observed to transport to the cell membrane. When compared to the distribution of a separate fusion protein consisting of a signal peptide that targets to cellular membranes fused to EGFP, both MP19 and Cx50 did not distribute uniformly on the membrane, but appeared to localize into "spots" or pools of fluorescent material around the cell membrane. Little co-localization of MP19 and Cx50 was observed. In contrast, MP19To3 protein appeared to not distribute to the cell membrane, it instead appeared to collect in vesicles within the cell. Conclusion: The distribution of MP19 and Cx50 around the cell membrane appeared to be distinct. Little co-localization of the two proteins was observed, indicating the possibly that the two lens membrane proteins function completely different from one another. The fact that the mutant MP19To3 did not traffic to the membrane, instead appearing to be trapped in vesicles within the cell may shed light on the cause of the cataract and microphthalmia observed in the To3 mutant animal.
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