We have used Drosophila mutants which are deficient in one or both of the arrestins present in photoreceptor cells to critically test the requirements for arrestin in the stabilization of Rh1 metarhodopsin under in vitro and in vivo conditions. Heads from flies illuminated with blue light were homogenized to obtain membranes or micellar extracts, and the amount of metarhodopsin present was quantitated by spectroscopic methods. Compared to wild-type, ~64% Rh1 metarhodopsin was recovered in flies deficient in arrestin-1 (arr11 mutant), ~38% in flies deficient in arrestin-2 (arr23 mutant), and ~6% in flies deficient in both arrestin-1 and arrestin-2 (arr11, arr23 double mutant). In contrast, no decrease was observed in the amounts of Rh1 metarhodopsin recovered from illuminated flies which were deficient either in the eye-specific phosphatase (rdgC mutant) or in the eye- specific phospholipase C (norpA(H24) and norpA(H52) mutants). Further, reconstitution experiments in total head homogenates showed that metarhodopsin produced in the arr11, arr23 double mutant could be stabilized upon the addition of exogenous arrestin-2. These studies provide definitive evidence that arrestin binding stabilizes Rh1 metarhodopsin under in vitro conditions. To test whether arrestin was also required to stabilize metarhodopsin in intact photoreceptor cells, metarhodopsin was generated in arr11, arr23 double mutant flies by in vivo illumination, and after a wait period of 20 min, converted back into rhodopsin by further illumination with red light. Quantitation of the regenerated rhodopsin in extracts from Drosophila heads showed no significant change in the level of rhodopsin recovered by this illumination protocol. Together, these experiments demonstrate that in disrupted photoreceptor cells, metarhodopsin is not stabilized unless arrestin is present, but in intact photoreceptor cells, significant metarhodopsin stabilization occurs even in the absence of bound arrestin.
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