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V.Z. Slepak, K.S. Nair, V.I. Shestopalov, S.M. Hanson, J.B. Hurley, V.V. Gurevich; Mechanism of Light–Dependent Translocation of Arrestin and Transducin in Vertebrate Rod Photoreceptors: Passive Diffusion Restricted by Protein Interactions . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4781.
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Purpose: In the dark–adapted rod photoreceptors, transducin is present in the outer segment (OS), and arrestin is localized in the inner segment (IS). In light, arrestin re–localizes to the OS and transducin to the IS. Here we investigated the molecular mechanism underlying this re–arrangement. Methods: Mouse eyecups were incubated in a culture medium for 1–4 hours under various conditions affecting ATP or GTP content and stability of active rhodopsin. Following light or dark adaptation of the eyecups, localization of arrestin and transducin in the cells was examined by immunofluorescence microscopy. Phosphorylation status of rhodopsin was determined by mass–spectrometry. The rate of protein diffusion in live rods was measured using fluorescence recovery after photobleaching (FRAP). Retinas were fractionated by ultracentrifugation, and the association of proteins with various cellular fractions was determined by western blot. Results:Re–localization of arrestin and transducin occurs normally in ATP–depleted rod cells, whereas rhodopsin phosphorylation is completely abolished. GTP is required for transducin movement to the IS. The rate of protein diffusion between IS and OS has a time constant of approximately 2 min. Arrestin localization in the OS requires the continuous presence of active rhodopsin but does not require its phosphorylation. In dark, arrestin associated with the microtubule–rich cytoskeleton. Conclusions: Light–dependent re–localization of arrestin and transducin is an energy–independent process sustained by passive diffusion. The re–distribution of arrestin in cellular compartments is determined by direct binding of arrestin to rhodopsin in the OS in light and microtubules in the IS in dark. The nature of the OS and IS binding sites for transducin is yet to be understood.
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