Palmitoylation of bovine opsin and its cysteine mutants in COS cells.
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.
Karnik
S S
SS
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Ridge
K D
KD
Bhattacharya
S
S
Khorana
H G
HG
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
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