Light induces massive translocation of major signaling proteins between the subcellular compartments of photoreceptors. Among them is visual arrestin responsible for quenching photoactivated rhodopsin, which moves into photoreceptor outer segments during illumination. Here, for the first time, we determined the light dependency of arrestin translocation, which revealed two key features of this phenomenon. First, arrestin translocation is triggered when the light intensity approaches a critical threshold corresponding to the upper limits of the normal range of rod responsiveness. Second, the amount of arrestin entering rod outer segments under these conditions is superstoichiometric to the amount of photoactivated rhodopsin, exceeding it by at least 30-fold. We further showed that it is not the absolute amount of excited rhodopsin but rather the extent of downstream cascade activity that triggers translocation. Finally, we demonstrated that the total amount of arrestin in the rod cell is nearly 10-fold higher than previously thought and therefore sufficient to inactivate the entire pool of rhodopsin at any level of illumination. Thus, arrestin movement to the outer segment leads to an increase in the free arrestin concentration and thereby may serve as a powerful mechanism of light adaptation.

Transducin is a prototypic heterotrimeric G-protein mediating visual signaling in vertebrate photoreceptor cells. Despite its central role in phototransduction, little is known about the mechanisms that regulate its expression and maintain approximately stoichiometric levels of the ␣-and ␤␥-subunits. Here we demonstrate that the knock-out of transducin ␥-subunit leads to a major downregulation of both ␣-and ␤-subunit proteins, despite nearly normal levels of the corresponding transcripts, and fairly rapid photoreceptor degeneration. Significant fractions of the remaining ␣-and ␤-subunits were mislocalized from the light-sensitive outer segment compartment of the rod. Yet, the tiny amount of the ␣-subunit present in the outer segments of knock-out rods was sufficient to support light signaling, although with a markedly reduced sensitivity. These data indicate that the ␥-subunit controls the expression level of the entire transducin heterotrimer and that heterotrimer formation is essential for normal transducin localization. They further suggest that the production of transducin ␤-subunit without its constitutive ␥-subunit partner sufficiently stresses the cellular biosynthetic and/or chaperone machinery to induce cell death.

Photoreceptor cells have a remarkable capacity to adapt the sensitivity and speed of their responses to ever changing conditions of ambient illumination. Recent studies have revealed that a major contributor to this adaptation is the phenomenon of light-driven translocation of key signaling proteins into and out of the photoreceptor outer segment, the cellular compartment where phototransduction takes place. So far, only two such proteins, transducin and arrestin, have been established to be involved in this mechanism. To investigate the extent of this phenomenon we examined additional photoreceptor proteins that might undergo light-driven translocation, focusing on three Ca 2؉ -binding proteins, recoverin and guanylate cyclase activating proteins 1 (GCAP1) and GCAP2. The changes in the subcellular distribution of each protein were assessed quantitatively using a recently developed technique combining serial tangential sectioning of mouse retinas with Western blot analysis of the proteins in the individual sections. Our major finding is that light causes a significant reduction of recoverin in rod outer segments, accompanied by its redistribution toward rod synaptic terminals. In both cases the majority of recoverin was found in rod inner segments, with ϳ12% present in the outer segments in the dark and less than 2% remaining in that compartment in the light. We suggest that recoverin translocation is adaptive because it may reduce the inhibitory constraint that recoverin imposes on rhodopsin kinase, an enzyme responsible for quenching the photoexcited rhodopsin during the photoresponse. To the contrary, no translocation of rhodopsin kinase itself or either GCAP was identified.