Fluorescence studies of the location and membrane accessibility of the palmitoylation sites of rhodopsin

Fluorescent fatty acid labels have been incorporated into the palmitoylation sites of rhodopsin and used to probe the membrane accessibility and location of these sites. The fluorescence properties of anthroyloxy and pyrenyl fatty acids bound to rhodopsin were investigated in a reconstituted vesicle system. Collisional quenching of fluorescence by stearic acid (DSA) labeled with doxyls in the 16, 12, and 5 positions was used to determine the membrane accessibility and disposition of the modifying fatty acids. To properly determine the membrane concentration of these quenchers, the dependence of the Stern-Volmer parameters on both quencher and vesicle concentration was determined. An analysis of these dependences provided a correction for partitioning of the quencher between the aqueous phase and the membrane. After this correction, the relative effectiveness of doxyl quenchers was 16-DSA > 12-DSA > 5-DSA. Parallel studies on free anthroyloxy and pyrenyl fatty acids incorporated into the reconstituted system showed the same dependence on quencher position. These results indicate that the labels at the palmitoylation sites of rhodopsin are situated in the membrane much as a free fatty acid. This anchoring of the palmitates in the membrane results in the formation of a fourth cytoplasmic loop.