Purpose: Lens intrinsic membrane protein MP19 is the second most abundant major protein of the lens fiber cell membrane and appears to be specific to the lens. Different mutations of this protein are known to cause cataract in both humans and mice. To date, the function of MP19 in the lens is not known, nor is the mechanism by which the protein migrates to the cell membrane. The goal of this study was to determine whether or not MP19 distributes to the cell membrane directed by a peptide signal within the sequence of the molecule.
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 that, upon induction with tetracycline, allows expression of cDNA inserts within the vector. These vectors expressed each of the MP19 truncated fragments fused to EGFP. Each of the prepared plasmids was transfected into T-REx-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 (with EGFP fused to the COOH-terminal end of MP19, MP19G) fusion protein were observed to traffic MP19 to the cell membrane, where it appeared to sequester in rather large pools. All of the MP19 truncations (with EGFP fused to the COOH-terminal end of each truncation; MP19-25G, MP19-36G, and MP19-64G) appeared to also traffic EGFP to the cell membrane. MP19-25G and MP19-36G did not distribute uniformly on the membrane, but appeared to localize into smaller, punctate "spots" of fluorescent material. MP19-64G distributed on the membrane similarly to MP19-25G and MP19-36G, however, the punctate areas of fluorescent material were considerably larger and similar to that demonstrated by intact MP19G. Western blot analysis of isolated total membranes, intrinsic membranes, and lipid rafts showed that MP19G and MP19-64G were associated with the intrinsic membrane fraction while MP19-25G and MP19-36G were at least 75% associated with the intrinsic membrane fraction. All of the preparations appeared to be at least 50% associated with membrane lipid rafts. However, when EGFP/MP19-25 and EGFP/MP19-36 (with EGFP fused to the NH2-terminal end of the truncated peptide, GMP19-25 or GMP19-36) were expressed, the fusion protein was observed to remain completely soluble in the cytoplasm, identical to expressed EGFP alone. Western blots of these two fusion proteins also indicated that the product did not associate with the cell membrane. In contrast, when EGFP/MP19 (with EGFP fused to the NH2-terminal end of intact MP19, GMP19) was expressed, the fusion protein did integrate into the cell membrane, identical to MP19G. Western blot analysis revealed that GMP19 also associated with lipid rafts, identical to intact MP19G.
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 integrate into the membrane. With the addition of more amino acids, the polypeptide distributes in the membrane similarly 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 integration sequence. Also, at least part of these 25 amino acids must integrate into the cell membrane, but not extend through the cell membrane.