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T. Chen, X. Li, Y. Yang, R.L. Church; Does Lens Intrinsic Membrane Protein MP19 Contain a Membrane Transport and Insertion Signal? . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4482.
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Purpose: Lens intrinsic membrane protein MP19 is the second most abundant major protein of the lens fiber cell membrane. MP19 transports to the cell membrane through the classic secretory protein pathway. The goal of this study was to determine whether or not MP19 distributes to the cell membrane directed by a signal. Methods: Using PCR, MP19 cDNA was truncated to yield separate fragments coding for the first 25, 36, and 64 amino acids of the MP19 polypeptide chain. These PCR fragments were further cloned into mammalian expression vector pcDNA4/TO, a tetracycline-regulated vector which, upon induction with tetracycline allows expression of cDNA inserts within the vector. These truncated cDNA fragments were separately cloned into the vector so that the fragments were at the 5'-end of an EGFP coding cDNA. These vectors expressed each of the MP19 truncated fragments fused to EGFP. Each of the prepared plasmids was transfected into TRx-293 cells using FuGene 6. Cloned cell lines from each of these transfections were obtained and used in the studies. The fluorescent expressed protein was viewed using confocal microscopy. Proteins from the different cell lines were isolated by different membrane extraction methods and western blot analysis was carried out to further determine the localization of expressed MP19 and MP19 truncated fragments. Results: Cell lines expressing intact MP19/EGFP fusion protein were observed to traffic MP19 to the cell membrane, where it appeared to sequester in rather large pools or areas. All of the MP19 truncations appeared to traffic EGFP to the cell membrane also. MP19-25 and MP19-36 did not distribute uniformly on the membrane, but appeared to localize into very small, punctate "spots" of fluorescent material. MP19-64 distributed on the membrane very similar to MP19-25 and MP19-36, however, the punctate "spots" of flunrescent material were considerably larger. Western blot analysis of isolated total membranes, intrinsic membranes, and lipid rafts showed that MP19 and MP19-64 were associated with the intrinsic membrane fraction while MP19-25 and -36 were at least 75% associated with the intrinsic membrane fraction. All of the preparations appeared to be about 50% associated with membrane lipid rafts. Conclusions: It appears that the first 25 amino acids of the MP19 molecule are sufficient to target the protein to the cell membrane, and apparently insert into the membrane. With the addition of more amino acids, the polypeptide distributes in the membrane very similar to that of the intact MP19 molecule. It appears that the first 25 amino acids of the MP19 molecule is, indeed, a membrane signal and insertion sequence.
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