Abstract
Abstract: :
Purpose: To determine the stability and fate of WT and rescued P23H rhodopsins within cells. Methods: Mutant and WT and P23H opsins were expressed separately in tetracycline–inducible stable HEK293 cell lines in the presence of 11–cis retinal. After induction, opsin biogenesis was stopped by the removal of tetracycline. At fixed times thereafter, the amounts of folded protein were immunoaffinity purified and quantitated by UV–visible spectroscopy. The proteins were also examined by immunoblotting. In other experiments, WT and P23H opsin cells were induced in the absence or in presence of lysosomal inhibitors. The proteins were immunoaffinity purified and analyzed by UV–visible spectroscopy and immunoblotting. Results: Upon removal of tetracycline, the amounts of folded P23H pigment decreased from cells more quickly than the amounts of folded WT– suggesting a cellular mechanism for the recognition and degradation of membrane proteins. By immunoblots, there was no evidence for selective degradation of a particular glycosylated form of P23H. A distinct 28 kDa form of opsin was noted in all samples. This form of opsin was not recognized by a proximal N–terminal antibody. When lysosomal activity was inhibited concomitantly with the induction of opsin expression, there was a small but reproducible increase in the amount of folded P23H rhodopsin. Conclusions: We demonstrate that rescued P23H rhodopsin is more unstable than WT within HEK293 cells. This provides further evidence that although the P23H protein can be induced to fold and stabilized with 11–cis retinal, the mutant pigment is structurally different from WT rhodopsin. Our data also suggest that lysosomal degradation may participate in the degradation of rhodopsin. These findings are consistent with a post–ER protein quality control system that participates in the surveillance and eventual removal of defective membrane proteins. We will discuss the implications of these results for the pathogenesis of RP.
Keywords: retinal degenerations: cell biology • opsins • protein purification and characterization