Abstract
Trans-phosphorylation of rhodopsin refers to a reaction in which a rhodopsin kinase molecule that has been activated by a light-activated rhodopsin molecule collides with and phosphorylates a second molecule of rhodopsin that has not been activated by light. It has been invoked as a mechanism for high-gain phosphorylation, a phenomenon that is observed at low bleaching levels where up to several hundred moles of phosphate are added to the rhodopsin pool per mole of photolyzed rhodopsin. Trans-phosphorylation is an appealing mechanism to propose for high-gain phosphorylation, but it has not been tested directly because of the difficulty inherent in unambiguous identification of light-activated and dark forms of rhodopsin present in the same reaction mixture. We report here a direct assay for trans-phosphorylation of rhodopsin. The assay is based on the use of a split receptor mutant of rhodopsin, SR(1−4/5−7), in which the fully functional protein is assembled from two separately expressed fragments. Because of different electrophoretic mobilities, SR(1−4/5−7) and wild-type rhodopsin can be monitored independently for phosphorylation while in the same reaction mixture. Thus, if wild-type rhodopsin is exposed to light and then incubated in the dark with SR(1−4/5−7), ATP, and rhodopsin kinase, phosphorylation of SR(1−4/5−7) would be a clear demonstration that trans-phosphorylation has occurred. Despite numerous attempts using several different experimental configurations, we have been unable to detect trans-phosphorylation of dark rhodopsin with this system.