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 (Gα(t1)), mutant Gα(t1) that is predicted to be N-acylated and S-palmitoylated (Gα(t1)A3C), and mutant Gα(t1) uncoupled from light-activated rhodopsin (Gα(t1)-Ctα(s)), were examined by EGFP-fluorescence imaging and fluorescence recovery after photobleaching (FRAP). Similar to Gα(t1), Gα(t1)A3C and Gα(t1)-Ctα(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 Gα(t1) consistent with the diffusion of the separated Gα(t1)GTP and Gβ(1)γ(1) on the membrane surface. Unexpectedly, the kinetics of longitudinal diffusion were comparable for Gα(t1)GTP with a single lipid anchor and heterotrimeric Gα(t1)β(1)γ(1) or Gα(t1)-Ctα(s)β(1)γ(1) with two lipid modifications. This contrasted the lack of the longitudinal diffusion of the Gα(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 Gα(t1)β(1)γ(1).
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.
Vasily
V
Kerov
Nikolai O
NO
Artemyev
D000818:Animals; D030801:Animals, Genetically Modified; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D008027:Light; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000166:cytology / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D015293:Transducin / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin; D015293:Transducin