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V. Kerov, N. O. Artemyev; Compartmentalization and Diffusion of Mutant Transducins in Transgenic Xenopus laevis Rods. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2991.
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© ARVO (1962-2015); The Authors (2016-present)
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.
Transgenic X. laevis have been produced that express EGFP-fused human rod transducin- subunit (Gt1) or mutant Gt1 subunits which are predicted to be S-palmitoylated (G2A/A3C), N-acylated and S-palmitoylated (A3C), or uncoupled from light-activated rhodopsin (uncGt1). Expression, localization and light-dependent translocation of mutant Gt1 subunits were analyzed by Western blotting and EGFP-fluorescence. Lateral and longitudinal diffusion of mutant transducins were investigated using Fluorescence Recovery After Photobleaching (FRAP).
EGFP-Gt1 and the mutant proteins were expressed in Xenopus rods at average levels below 5% of the endogenous frog Gt1. The A3C and uncGt1 mutants were correctly targeted to the rod outer segments (OS) similarly to EGFP-Gt1, whereas G2A/A3C was mislocalized to the inner segments (IS). The uncGt1 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 Gt1.
Correct N-terminal lipid modification of Gt1, 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.
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