Compartmentalization and Diffusion of Mutant Transducins in Transgenic Xenopus laevis Rods

Abstract

Purpose: : Transgenic Xenopus laevis models were designed to probe the roles of transducin/rhodopsin interactions and lipid modifications in transducin targeting and membrane mobility in rod photoreceptors.

Methods: : Transgenic X. laevis have been produced that express EGFP-fused human rod transducin- subunit (G_t1) or mutant G_t1 subunits which are predicted to be S-palmitoylated (G2A/A3C), N-acylated and S-palmitoylated (A3C), or uncoupled from light-activated rhodopsin (uncG_t1). Expression, localization and light-dependent translocation of mutant G_t1 subunits were analyzed by Western blotting and EGFP-fluorescence. Lateral and longitudinal diffusion of mutant transducins were investigated using Fluorescence Recovery After Photobleaching (FRAP).

Results: : EGFP-G_t1 and the mutant proteins were expressed in Xenopus rods at average levels below 5% of the endogenous frog G_t1. The A3C and uncG_t1 mutants were correctly targeted to the rod outer segments (OS) similarly to EGFP-G_t1, whereas G2A/A3C was mislocalized to the inner segments (IS). The uncG_t1 failed to translocate from the OS to the IS in response to light, while light-dependent translocation of A3C was markedly impaired. The FRAP analysis indicated significant effects of the mutations on the lateral and longitudinal diffusion of G_t1.

Conclusions: : Correct N-terminal lipid modification of G_t1, but not its interaction with rhodopsin, is required for proper targeting of transducin to the OS in the dark. Lipid modification of transducin- controls both diffusion and light-dependent translocation of the protein.

Keywords: photoreceptors • signal transduction