Diffusion and light-dependent compartmentalization of transducin

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-α subunit (Gαt1), mutant Gαt1 that is predicted to be N-acylated and S-palmitoylated (Gαt1A3C), and mutant Gαt1 uncoupled from light-activated rhodopsin (Gαt1-Ctαs), were examined by EGFP-fluorescence imaging and fluorescence recovery after photobleaching (FRAP). Similar to Gαt1, Gαt1A3C and Gαt1-Ctαs 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 Gαt1 consistent with the diffusion of the separated Gαt1GTP and Gβ1γ1 on the membrane surface. Unexpectedly, the kinetics of longitudinal diffusion were comparable for Gαt1GTP with a single lipid anchor and heterotrimeric Gαt1β1γ1 or Gαt1-Ctαsβ1γ1 with two lipid modifications. This contrasted the lack of the longitudinal diffusion of the Gαt1A3C mutant apparently caused by its stable two lipid attachment to the membrane and suggests the existence of a mechanism that facilitates axial diffusion of Gαt1β1γ1.