title: Palmitoylation of bovine opsin and its cysteine mutants in COS cells.
abstract: Previously, bovine rhodopsin has been shown to be palmitoylated at cysteine residues 322 and 323. Here we report on palmitoylation of bovine opsin in COS-1 cells following expression of the synthetic wild-type opsin gene and several of its cysteine mutants in the presence of [3H]palmitic acid. Two moles of palmitic acid are introduced per wild-type opsin molecule in thioester linkages. Palmitoylation is abolished when both Cys-322 and Cys-323 are replaced by serine residues. Replacement of Cys-322 by serine prevents palmitoylation at Cys-323, whereas replacement of the latter with serine allows palmitoylation at Cys-322. Opsin mutants that evidently do not contain a Cys-110/Cys-187 disulfide bond and presumably remain in the endoplasmic reticulum are not palmitoylated. Replacement of Cys-140 or Cys-185 reduces the extent of palmitoylation of the opsin. Lack of palmitoylation at Cys-322 and/or Cys-323 does not affect 11-cis-retinal binding, absorption maximum or extinction coefficient of the chromophore, the bleaching behavior of the chromophore, or the light-dependent binding and activation of transducin. Mutants containing serine substitutions at Cys-140 or Cys-323 showed reduced light-dependent phosphorylation by rhodopsin kinase.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: S S
authors.initials: SS
authors.lastname: Karnik
authors.forename: K D
authors.initials: KD
authors.lastname: Ridge
authors.forename: S
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D013053:Spectrophotometry; D014162:Transfection
chemical_list: D005136:Eye Proteins; D010169:Palmitic Acids; D011994:Recombinant Proteins; D017299:Rod Opsins; D019308:Palmitic Acid; D012694:Serine; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D003545:Cysteine