FLUORESCENCE LABELING OF THE PALMITOYLATION SITES OF RHODOPSIN

Two tandem cysteine residues in the carboxyl-terminal region of-rhodop sin have been shown to be covalently linked to palmitate via thioester bonds (Ovchinnikov, Y. A., et al. (1988) FEBS Lett. 230, 1-5). We hav e synthesized a fluorescent analogue of palmitoyl coenzyme A (16-(9-an throyloxy)hexadecanoyl coenzyme A ester) and incorporated the fluoresc ent derivative of palmitate into the protein in high yield (>40%) thro ugh pretreatment of bovine rod outer segments with 1 M hydroxylamine a nd subsequent incubation with the fluorescent label. Covalent incorpor ation of label into protein was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic digestion of labeled rhodopsin in the di sc membrane with papain and thermolysin verified the C-terminal locati on of the label. Treatment of SDS-solubilized, labeled rod outer segme nts with 10% beta-mercaptoethanol provided evidence that partial depal mitoylation may induce the formation of rhodopsin aggregates. Labeled, unbleached rhodopsin was purified by chromatography over hydroxyapati te and concanavalin A-agarose and reconstituted into dimyristoylphosph atidylcholine vesicles. SDS gels of the rhodopsin vesicle preparation verified that all unbound fluorescent label had been removed and that the thioester bond linking probe to protein was not labile.