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The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9- or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased approximately 3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9- or 11-cis-retinal had blue-shifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.

The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum bacteriorhodopsin. Strains were constructed that express the bacteriorhodopsin mutants I4C:H(6) and T5C:H(6), which carry a unique Cys in the N-terminal extracellular domain and a polyhistidine tag at the C terminus. Translocation of the N-terminal domain was detected using a membrane-impermeant gel shift reagent to derivatize the Cys residue of nascent radiolabeled molecules. Derivatization was assessed by gel electrophoresis of the fully elongated radiolabeled population. The time required to translocate and fully derivatize the Cys residues of I4C:H(6) and T5C:H(6) is 46 +/- 9 and 61 +/- 6 s, respectively. This is significantly shorter than the elongation times of the proteins, which are 114 +/- 26 and 169 +/- 16 s, respectively. These results establish that translocation of the bacterioopsin N terminus and insertion of the first transmembrane segment occur co-translationally and confirm the use of the assay to monitor the kinetics of polytopic membrane protein insertion in vivo.