Gamma–E–crystallin Elo mutation disrupts the specific interaction between lens MIP/Aquaporin–0 and gamma–E–crystallin.

Abstract

Abstract: : Purpose: Major Intrinsic Protein (MIP)/Aquaporin 0 is a water channel protein required for lens transparency, We have previously demonstrated that MIP C–terminal domain interacts with gamma E–crystallin in yeast cells. Our goal is to characterize the interaction between MIP and gamma crystallins in mammalian cells and to demonstrate the physiological significance of the MIP–gamma E crystallin interaction. Methods: Protein–protein interaction between MIP and gamma crystallins was examined by co–expressing unmodified MIP, or EGFP or myc–tagged MIP and HcRed or EGFP–tagged gamma crystallin in rabbit kidney epithelial cell line RK13, followed by imaging with confocal fluorescence microscopy, with or without prior immuno–staining of cells with anti–MIP or anti–myc antibody and Cy3–labeled secondary antibody. Results: We demonstrated that both unmodified MIP and myc–tagged MIP interacted with EGFP–tagged gamma–E–crystallin in individual mammalian cells and that this interaction resulted in the recruitment of gamma–E–crystallin from the cytoplasm to the plasma membrane. We also found that MIP did not interact with gamma–D–crystallin, another closely related member of the gamma crystallin family. In addition, MIP–gamma E crystallin interaction was disrupted when MIP C–terminal domain was linked to the bulky EGFP. On the contrary, MIP–gamma E crystallin interaction was not affected when MIP C–terminal was linked to the short myc tag. Furthermore, we found that MIP did not interact with a naturally–occurring gamma–E–crystallin mutant (Elo) that is associated with a mouse dominant genetic cataract. Conclusions: Our results demonstrate that the C–terminal domain of MIP plays an essential role in its interaction with gamma E crystallin. They provide evidence for the physiological significance of the MIP–gamma E crystallin interaction. Our results also suggest that perturbation of MIP and gamma–E crystallin interaction may be involved in genetic cataract.

Keywords: protein structure/function • crystallins • microscopy: confocal/tunneling