All 20 single cysteine substitution mutants in the sequence Y136-M155 of bovine rhodopsin have been prepared and modified with a sulfhydryl-specific nitroxide reagent. This sequence contains the C-D interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra were analyzed, both in the dark and after photoexcitation. Accessibility data show that the rhodopsin polypeptide crosses an aqueous/hydrophobic boundary near V138 and H152. The nitroxide mobilities inferred from the spectra are consistent with a model where the C helix extends to at least residue C140, with much of the helix surface in contact with protein rather than lipid near the cytoplasmic surface of the membrane. Upon photoexcitation, electron paramagnetic resonance spectral changes are observed at sites on the putative C helix surface that are in contact with the protein and at specific sites in the C-D interhelical loop. A simple interpretation of these results is that photoexcitation involves a rigid body movement of the C helix relative to the others in the helix bundle.

Rhodopsin, the dim light photoreceptor ofthe rod cell, is an integral membrane protein that is glycosylated at Asn-2 and Asn-15. Here we report experiments on the role of the glycosylation in rhodopsin folding and function. Nonglycosylated opsin was prepared by expression of a wild-type bovine opsin gene in COS-1 cells in the presence of tunicamycin, an inhibitor of asparagine-linked glycosylation. The nonglycosylated opsin folded correctly as shown by its normal palmitoylation, transport to the cell surface, and the formation of the characteristic rhodopsin chromophore (Aa, 500 nm) with 11-cis-retinal. However, the nonglycosylated rhodopsin showed strikingly low light-dependent activation of GT at concentration levels comparable with those of glycosylated rhodopsin. Amino acid replacements at positions 2 and 15 and the cognate tripeptide consensus sequence [Asn-2 -* Gln, Gly-3 -+ Cys (Pro), Thr-4 -+ Lys, Asn-15 Ala (Cys, Glu, Lys, Gln, Arg), Lys-16 -* Cys (Arg), Thr-17 Met (Val)] showed that the substitutions at Asn-2, Gly-3, and Thr-4 had no sgnificant effect on the folding, cellular transport, and/or function of rhodopsin, whereas those at Asn-15 and Lys-16 caused poor folding and were defective in transport to the cell surface.Further, mutant pigments with amino acid replacements at Asn-15 and Thr-17 activated Gr very poorly. We conclude that Asn-15 glycosylation is important in signal tanduction.Asparagine-linked (N-linked) glycosylation of membrane and secreted proteins is observed frequently, although the role that glycosylation may serve is not always evident (1-3). Bovine rhodopsin is glycosylated at Asn residues 2 and 15 (Fig. 1) by the hexasaccharide sequence Man3GlcNac3 (4, 5). We have now examined the role of N-linked glycosylation in rhodopsin folding and function. We expressed the wild-type bovine opsin gene (6) in the presence of the glycosylation inhibitor tunicamycin (TM). The resulting nonglycosylated opsin was normally palmitoylated, was transported to the cell surface, and formed the characteristic rhodopsin chromophore with 11-cisretinal. However, the nonglycosylated rhodopsin showed strikingly diminished light-dependent activation of transducin (GT) when compared with glycosylated rhodopsin. We next studied opsin mutants that contained amino acid replacements in the regions ofthe two glycosylation sites (Fig. 1). Mutations at or near Asn-2 had little effect on cell-surface expression, chromophore formation, and/or GT activation. In contrast, mutations at Asn-15 and Lys-16 resulted in opsins that were defective in cellular transport and formed little or no chromophore with il-cis-retinal. The mutations at Asn-15 and Thr-17 resulted in pigments that were defective in signal transduction. These results show that while glycosylation of rhodopsin is not required for its folding to an apparently correct ground-state structure, it is necessary for full activity in signal transduction.

Previously, bovine rhodopsin has been shown to be palmitoylated at cysteine residues 322 and 323.

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Previous mutagenesis studies have indicated the requirement of a tertiary structure in the intradiscal region with a disulfide bond between Cys-110 and Cys-187 for the correct assembly and/or function of rhodopsin. We have now studied a rhodopsin mutant in which only the natural intradiscal cysteines at positions 110, 185, and 187 are present while all the remaining seven cysteines in the wild-type bovine rhodopsin have been replaced by serines. The proteins formed on expression of this mutant in COS-1 cells bind 11-cis-retinal only partially to form the rhodopsin chromophore. We show that this is due to the presence of both correctly folded chromophore-forming opsin and misfolded opsins. Methods have been devised for the separation of the correctly folded and misfolded forms by selective elution from immunoaffinity adsorbants. Using several criteria, which include SDS-PAGE as well as UV/visible and CD spectroscopy, we find that the correctly folded mutant protein is indistinguishable in its spectral properties from the wild-type rhodopsin. Further, reaction of sulfhydryl groups in the correctly folded mutant pigment with N-ethylmaleimide indicates that alkylation of a single sulfhydryl requires denaturation or illumination, while reaction with an additional two sulfhydryl groups occurs only after reduction. The misfolded mutant opsins are characterized by reduced alpha-helical content, sulfhydryl reactivity under native conditions in the dark, and also the presence of a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)