Abstract
The dynamics of G protein-mediated signal transduction depend on the two-dimensional diffusion of membrane-bound G proteins and receptors, which has been suggested to be rate-limiting for vertebrate phototransduction, a highly amplified G protein-coupled signaling pathway. Using fluorescence recovery after photobleaching (FRAP), we measured the diffusion of the G protein transducin alpha-subunit (Galpha(t)) and the G protein-coupled receptor rhodopsin on disk membranes of living rod photoreceptors from transgenic Xenopus laevis. Treatment with either methyl-beta-cyclodextrin or filipin III to disrupt cholesterol-containing lipid microdomains dramatically accelerated diffusion of Galpha(t) in its GTP-bound state and of the rhodopsin-Galphabetagamma(t) complex but not of rhodopsin or inactive GDP-bound Galphabetagamma. These results imply an activity-dependent sequestration of G proteins into cholesterol-dependent lipid microdomains, which limits diffusion and exclude the majority of free rhodopsin and the free G protein heterotrimer. Our data offer a novel demonstration of lipid microdomains in the internal membranes of living sensory neurons.
Highlights
Two-dimensional diffusion of membrane proteins is central to G protein-mediated signal transduction
Roles for lipid microdomains, such as rafts, in the regulation of membrane protein dynamics have been implicated in G protein signaling and other signaling pathways [2], but current evidence is not definitive, and it is unknown whether lipid microdomains play a role in phototransduction
Characterization of Purified G protein transducin ␣-subunit (G␣t)-enhanced green fluorescent protein (EGFP)—To examine the dynamic properties of transducin in intact photoreceptor cells, we produced a fusion of G␣t with EGFP for measurements by fluorescence recovery after photobleaching (FRAP)
Summary
Buffers—Standard buffers contained (in mM): buffer A (Ringer’s solution), HEPES 5, pH 7.7, NaCl 110, CaCl2 2.0, KCl 2.5, MgCl2 1.2, saturated with 95% O2, 5% CO2; buffer B, HEPES 10, pH 7.2, MgCl2 3.0, arginine 105, glutamic acid 105, EGTA 1.0, DTT 1.0; buffer C (Marc’s modified Ringer’s solution), HEPES 5, pH 7.5, NaCl 100, CaCl2 2.0, KCl 2.0, MgCl2 1.0; buffer D, sucrose 250, KCl 75, spermidine trihydrochloride 0.5, spermine tetrahydrochloride 0.2, pH 7.4; buffer E, Tris 5.0, pH 7.2, MgCl2 0.5, DTT 2; buffer F, MOPS 20, pH 7.5, NaCl 50, MgCl2 2.0, EDTA 0.1, DTT 2; buffer G, HEPES 50, pH 7.5, EDTA 1.0, EGTA 3.0, MgCl2 5.0, DTT 1, GDP 0.1, phenylmethylsulfonyl fluoride; buffer H, Tris 20, pH 7.5, MgCl2 5, EDTA 0.1, DTT 2, GDP 0.1, and phenylmethylsulfonyl fluoride ϳ20 mg/liter; buffer I, MOPS 20, pH 7.5, NaCl 50, MgCl2 2, EDTA 0.1, DTT 2; buffer J, Tris 25, glycine 192, 0.1% (w/v) SDS, pH 8.3. Characterization of Purified G␣t-EGFP—G␣t-EGFP expressed in Sf9 cells was purified by anion exchange chromatography, reconstituted with G␥t purified from bovine rod outer segments, and assayed along with purified bovine G␣␥t for [35S]GTP␥S uptake in the presence of photoisomerized rhodopsin in urea-washed rod disk membranes. Preparation of Samples for FRAP Measurements—To measure the diffusion coefficients of EGFP-rhodopsin and EGFPG␣t in rod photoreceptor cells, Xenopus tadpoles at about stage 45 were anesthetized in 0.01% 3-aminobenzoic acid ethyl ester and sacrificed, and eyes were dissected in control Ringer’s solution. A single photoreceptor cell with EGFP fluorescence was selected and located to the center of a field, which was usually 29.6 ϫ 29.6 m or 256 ϫ 256 pixels; 256 gray level images were collected in the xy plane. To correct for fading of fluorescence due to taking images, the intensity of the non-bleached area on photoreceptor cells over time was fitted using the first order decay Equation 1. Several scans were always needed prior to the beginning of the FRAP experiments to locate the cell, find the correct focus position, and center it in the image field
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