Abstract
Abstract: :
Purpose: Other studies have indicated that lens intrinsic membrane protein MP19 is a transmembrane protein and spans the membrane with four transmembrane segments. Also, because of this four transmembrane nature of the molecule, MP19 has been placed into the tetraspanin superfamily. We have carried out investigations into the nature of MP19 transmembrane–spanning regions and propose a different topological model for the arrangement of MP19 in the lens. Methods: Using PCR, MP19 cDNA was truncated to yield separate fragments coding for the first 25, 36, 64, 79, 92, 110, 120, and 136 amino acids of the MP19 polypeptide. These PCR fragments were cloned into a tetracycline–regulated mammalian expression 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 either the 5’– or 3’–end of an EGFP coding cDNA, or with EGFP at both ends of the MP19 truncations. These vectors expressed each of the MP19 truncated fragments fused to EGFP. Each of the prepared plasmids was transfected into TRx–293 cells. Cloned cell lines from each of these transfections were obtained and used in the studies. The fluorescent expressed protein was viewed using confocal microscopy and western blot analysis. Results: Cell lines expressing intact MP19/EGFP or EGFP/MP19 fusion protein were observed to traffic MP19 to the cell membrane, where it appeared to sequester in areas very similar to lipid rafts. All of the MP19/EGFP truncations also appeared to traffic EGFP to the cell membrane. However, depending on the size of the truncation fragment, cell lines expressing protein with EGFP at the NH2–terminal end, or at both ends did not traffic to the cell membrane, rather it remained soluble in the cytoplasm, as did EGFP expressed alone. Again, depending on the size of the MP19/EGFP fragment, certain truncations were cleaved by Proteinase K (showing that the EGFP was on the outside of the cell membrane), while other truncations required triton–X (to permeablize the membrane) before Proteinase K would cleave the EGFP (indicating that the EGFP was on the inside of the cell membrane). Conclusions: Our studies have resulted in a modified topology for the association of MP19 with the fiber cell membrane. We have concluded that MP19 makes 2.5 passes through the cell membrane, with the first 18–20 amino acids integrated into the membrane as a signal anchor. This leaves large segments of MP19 on the cytoplasmic and extracellular sides of the membrane for possible interactions with other molecules.
Keywords: protein structure/function • cell membrane/membrane specializations • gene/expression