Diffusion and light-dependent compartmentalization of transducin are essential for phototransduction and light adaptation of rod photoreceptors. Here, transgenic Xenopus laevis models were designed to probe the roles of transducin/rhodopsin interactions and lipid modifications in transducin compartmentalization, membrane mobility, and light-induced translocation. Localization and diffusion of EGFP-fused rod transducin-alpha subunit (G alpha(t1)), mutant G alpha(t1) that is predicted to be N-acylated and S-palmitoylated (G alpha(t1)A3C), and mutant G alpha(t1) uncoupled from light-activated rhodopsin (G alpha(t1)-Ct alpha(s)), were examined by EGFP-fluorescence imaging and fluorescence recovery after photobleaching (FRAP). Similar to G alpha(t1), G alpha(t1)A3C and G alpha(t1)-Ct alpha(s) were correctly targeted to the rod outer segments in the dark, however the light-dependent translocation of both mutants was markedly impaired. Our analysis revealed a moderate acceleration of the lateral diffusion for the activated G alpha(t1) consistent with the diffusion of the separated G alpha(t1)GTP and G beta(1)gamma(1) on the membrane surface. Unexpectedly, the kinetics of longitudinal diffusion were comparable for G alpha(t1)GTP with a single lipid anchor and heterotrimeric G alpha(t1)beta(1)gamma(1) or G alpha(t1)-Ct alpha(t1)beta(1)gamma(1) with two lipid modifications. This contrasted the lack of the longitudinal diffusion of the G alpha(t1)A3C mutant apparently caused by its stable two lipid attachment to the membrane and suggests the existence of a mechanism that facilitates axial diffusion of G alpha(t1)beta(1)gamma(1). (C) 2010 Elsevier Inc. All rights reserved.
