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T. G. Wensel, Q. Wang; Activation-Dependent Sequestration of Transducin in Cholesterol-Dependent Microdomains of Disk Membranes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2415. doi: https://doi.org/.
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To determine whether cholesterol regulates the diffusion kinetics of membrane-associated complexes containing the alpha subunit of the phototransduction G protein transducin.
A fusion of EGFP with transducin alpha was expressed in rods of transgenic Xenopus laevis, and diffusion kinetics were monitored by Fluorescence Recovery after Photobleaching (FRAP). Samples were treated with metabolic poisons to stabilize the R*-transducin complex, with hydroxylamine to inactivate R* and stabilize the transducin-GDP heterotrimer, or with glucose or nucleotides to enhance formation of activated transducin alpha. Methyl-beta-cyclodextrin (MBCD) or filipin III was added to disrupt cholesterol-dependent lipid microdomains. S. aureus alpha toxin was used to permeabilize the plasma membranes. Recovery curves were fit to a solution to the diffusion equation that accounts for finite membrane size, and compared to predictions of the Safmann-Delbrück equation for diffusion of membrane proteins.
Diffusion coefficients of photoexcited rhodopsin (R*) and transducin-GDP heterotrimer were consistent with both diffusing in a common bulk lipid phase with relatively low viscosity, as reported previously for R*. Diffusion coefficients of the R*-transducin complex and of transducin alpha-GTP were much slower than expected from the results for R* and transducin-GDP heterotrimer. Addition of MBCD or filipin III greatly accelerated the diffusion of activated transducin alpha-GTP and of the R*-transducin complex, but not of R* or transducin-GDP heterotrimer. The effects of MBCD were blocked by simultaneous addition of cholesterol, which further slowed the diffusion of activated transducin alpha-GTP.
Disk membranes are not homogeneous, but rather contain cholesterol-dependent microdomains with lower effective viscosity than the bulk lipid. Rhodopsin is largely excluded from these microdomains in either its light or dark states, but transducin alpha moves into them (or forms them around itself) upon formation of the R*-transducin complex and of the activated transducin alpha-GTP.
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