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. Author manuscript; available in PMC: 2012 Jan 1.
Published in final edited form as: Mol Cell Neurosci. 2010 Oct 31;46(1):340–346. doi: 10.1016/j.mcn.2010.10.006

Diffusion and light-dependent compartmentalization of transducin

Vasily Kerov 1, Nikolai O Artemyev 1,2
PMCID: PMC3018668  NIHMSID: NIHMS250490  PMID: 21044685

Abstract

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 (Gαt1), mutant Gαt1 that is predicted to be N-acylated and S-palmitoylated (Gαt1A3C), and mutant Gαt1 uncoupled from light-activated rhodopsin (Gαt1-Ctαs), were examined by EGFP-fluorescence imaging and Fluorescence Recovery After Photobleaching (FRAP). Similarly to Gαt1, Gαt1A3C and Gαt1-Ctαs 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 oderate acceleration of the lateral diffusion for the activated Gαt1 consistent with the diffusion of the separated Gαt1GTP and Gβ1γ1 on the membrane surface. Unexpectedly, the kinetics of longitudinal diffusion were comparable for Gαt1GTP with a single lipid anchor and heterotrimeric Gαt1β1γ1 or Gαt1-Ctαsβ1γ1 with two lipid modifications. This contrasted he lack f the longitudinal diffusion of the Gαt1A3C mutant apparently caused by its stable two-lipid attachment to the membrane and suggests the existence of a mechanism that facilitates axial diffusion of Gαt1β1γ1.

1. Introduction

The principal components of the vertebrate phototransduction cascade, rhodopsin, transducin (Gt1), and cGMP phosphodiesterase-6 (PDE6), reside on the disc membranes within a specialized outer segment (OS) compartment of rod photoreceptor cells. Photoexcited rhodopsin (R*) stimulates exchange of GTP for GDP on the α-subunit of heterotrimeric Gt1 (Gαt1β1γ1) causing Gαt1GTP to dissociate from Gβ1γ1 and R* and activate PDE6 (Burns and Arshavsky, 2005; Fu and Yau, 2007; Lamb and Pugh, 2006). The diffusion controlled encounter between the phototransduction proteins at the membrane surface is thought to control the kinetics of photoresponse (Calvert et al., 2001; Pugh and Lamb, 1993). In addition to lateral diffusion along the plane of the disc membranes, Gαt1GTP, Gβ1γ1, and heterotrimeric Gt1 can diffuse longitudinally (along the long axis of the OS). The axial diffusion or inter-disc protein transfer apparently underlies light-induced translocation of Gαt1GTP and Gβ1γ1 from the OS to the inner segment (IS) (Artemyev, 2008; Calvert et al., 2006; Nair et al., 2005; Rosenzweig et al., 2007; Slepak and Hurley, 2008; Sokolov et al., 2002; Wang et al., 2008). Furthermore, longitudinal diffusion may serve as a mechanism for the IS→OS transport of translocated or newly synthesized Gt1 in the dark. The N-terminal acylation of Gαt1 and farnesylation of Gγ1 provide for a relatively weak interaction of the individual subunits with the disc membrane, whereas the dual lipidation of Gαt1β1γ1 promotes a comparatively stable membrane association (Bigay et al., 1994; Kosloff et al., 2008). According to the diffusion model of light-dependent translocation of transducin, the activation of Gt1 by R* facilitates dissociation of Gαt1GTP and Gβ1γ1 subunits from the membrane allowing them to diffuse to the IS (Artemyev, 2008; Burns and Arshavsky, 2005; Calvert et al., 2006; Slepak and Hurley, 2008). For the protein translocation to occur, the Gt1 activation rate must exceed the capacity of the GTPase activating protein (GAP) complex to inactivate Gαt1GTP (Kerov et al., 2005; Lobanova et al., 2007). Inactivation of Gαt1GTP with subsequent formation of the heterotrimer leads to re-association of Gt1 with the membrane and prevents diffusion. The simple diffusion model predicts that the longitudinal diffusion of activated transducin is faster than that of the heterotrimeric Gt1. However, a recent investigation of transducin diffusion in rods of transgenic Xenopus laevis yielded surprising findings. The activated Gαt1GTP was found to diffuse laterally and longitudinally significantly slower than Gαt1β1γ1 (Wang et al., 2008). It is not unexpected that Gαt1 would diffuse slower as a part of the activated Gαt1/PDE6 complex, particularly since the diffusion of PDE6 in ROS is remarkably slow (Muradov et al., 2009). Yet, the slower mobility was exhibited by the entire pool of activated Gαt1 well in excess to rod PDE6 (Wang et al., 2008). The proposed underlying mechanism is sequestration of Gαt1GTP by lipid microdomains (Wang et al., 2008). Although the reported slow rate of longitudinal diffusion of activated Gαt1 is sufficient to explain the light-dependent redistribution of transducin, the study underscores the lack of complete understanding of the translocation phenomenon (Wang et al., 2008).

To probe transducin targeting and translocation mechanisms, we have generated transgenic Xenopus laevis expressing human EGFP-Gαt1 and its mutants in rod photoreceptors. Localization and diffusion of mutant transducins were examined by EGFP-fluorescence imaging and Fluorescence Recovery After Photobleaching (FRAP). The A3C mutation was introduced into EGFP-Gαt1 to create an artificial palmitoylation site. By analogy with Gαi, the A3C mutant is expected to be both N-acylated at Gly2 and palmitoylated at Cys3, thereby enhancing its affinity for the membrane (Chen and Manning, 2001; Wedegaertner et al., 1995). A Gαt1 mutant with the replacement of the C-terminal 11 residues by the corresponding residues of Gαs (Gαt1-Ctαs) was produced to investigate the effects of rhodopsin/transducin interactions on the light-dependent compartmentalization and diffusion of transducin. The C-terminus of Gαt1 is a major rhodopsin recognition site (Bourne, 1997; Oldham et al., 2006; Scheerer et al., 2008) that could potentially serve as a signal motif for targeting of transducin to the OS. Biochemical studies indicated that the Gαt1-Ctαs mutant is not activated by R* (Natochin et al., 2001). Our analysis revealed an increase rather than decrease in the lateral D value of the activated Gαt consistent with the diffusion of the separated Gαt1GTP and Gβ1γ1 on the membrane surface. Interestingly, the axial diffusion kinetics were equivalent for Gαt1GTP with a single lipid anchor and heterotrimeric Gt1 with two lipid modifications. In contrast, the Gαt1A3C mutant failed to diffuse axially, apparently due to its strong two-lipid attachment to the membrane.

2. Materials and methods

2.1. Generation of transgenic tadpoles

The transgene construct was prepared using a modified pXOP(−508/+41)EGFP vector (Mani et al., 2001; Muradov et al., 2009). The EGFP sequence was inserted into the helical domain of Gαt1 between Met115 and Pro116 via a 6-residue linker sequence (SGGGGS) (Fig. 1) (Wang et al., 2008). The cDNA sequence of human Gαt was obtained from the Missouri cDNA Resource Center. The Gαt1-115 sequence was PCR-amplified using a 5′-primer with an NcoI site and a 3′-primer with the N-terminal sequence of EGFP added to Gαt specific sequence. The Gαt1116-350 sequence was PCR-amplified using a 5′-primer with the C-terminal sequence of EGFP added to Gαt1 specific sequence, and a 3′-primer containing an XbaI site. The two resulting PCR products were used as primers in the PCR reaction with pXOP(−508/+41)EGFP as a template. The Gαt1 construct with the internally-fused EGFP sequence was subcloned into the pXOP vector using NcoI/XbaI sites to produce pXOP(−508/+41)EGFP-Gαt1. The SalI site was introduced in front of the promoter region for linearization of the plasmid and transgenesis by restriction enzyme mediated integration (REMI) (Kroll and Amaya, 1996). The pXOP(−508/+41)EGFP-Gαt1 template was amplified with primers coding the A3C mutation or the 11 C-terminal amino acids of Gαs to generate transgenic Gαt1A3C and Gαt1-Ctαs tadpoles. The sequences of the final constructs were confirmed by direct sequencing. Preparation of sperm nuclei and egg extract, and REMI were performed as previously described (Kroll and Amaya, 1996). Transgenic tadpoles were identified 7–8 days post-injection by visual examination for EGFP fluorescence in the eye using a dissecting microscope equipped with a GFP filter.

Fig. 1. Expression of EGFP-Gαt1 and mutants in retinas of transgenic X. laevis.

Fig. 1

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(A). Map of the transgene. L – linker. The Sal I site was used to linearize the transgene plasmid for transgenesis. (B) A piece of retina of a transgenic tadpole expressing EGFP- Gαt1. EGFP-fluorescence/DIC overlay. Bar – 20 μm. (C) Samples of retinal extracts from tadpoles expressing EGFP-fused Gαt1 (1), Gαt1A3C (2), and Gαt1-Ctαs (3) (equivalent to 2 retinas at about stage 50 X. laevis tadpole with an average fluorescence intensity in the eye) were immunoblotted with rabbit anti-GFP antibodies (Invitrogen) (left) or with anti-Gαt1 antibodies K-20 (Santa Cruz Biotech) (right). Arrow indicates frog Gαt1.

2.2. Live cell imaging

X. laevis tadpoles at about stage 50 were dark-adapted overnight and anesthetized with 0.1% tricaine methylsulphonate. Eyes were enucleated, retinas extracted and put into 60 μl oxygenated Ringer’s solution on a microscope glass under dim red light. Retinas were dissected into ~0.2×0.2 mm pieces using two 30G needles. For light adaptation, tadpoles were exposed to light in Petri dishes (~800 lux, 40 min) prior to anesthesia, and the retina pieces were prepared under room light as described above. EGFP-fluorescence was examined using a Zeiss LSM 510 confocal microscope. Mean fluorescence intensities in the OS and IS of light-adapted EGFP-Gαt1 and EGFP-Gαt1A3C rods were quantified using Z-stack projection images and ImageJ. The Z-stack projection images were obtained from 1 μm Z-sections covering an entire diameter of a single rod (~7–8 μm). Gαt1 and EGFP-Gαt1A3C rods with average and similar fluorescence were selected and analyzed with the same microscope settings.

2.3 Immunofluorescence

X. laevis tadpoles were dark-adapted for at least 12 hrs, anesthetized in 0.1% tricaine methylsulphonate, and dissected under dim red light. The heads were fixed in 4% paraformaldehyde in PBS at 22°C for 4 hours in full darkness. For light adaptation, tadpoles in Petri dishes were placed under white fluorescent bulbs (~800 lux, 40 min), followed by anesthesia and dissection. After fixation, the eyeballs were submersed in a 30% sucrose solution in PBS for 12 hrs at 4°C and then embedded in tissue freezing medium (TBS) and frozen on dry ice. Radial sectioning (10 μm) of the retina was performed using a cryomicrotome Microm HM 505E. Retinal cryosections were air-dried and kept at −80°C until use. Before staining, sections were incubated in 0.1% Triton/PBS for 30 min followed by incubation with 5% BSA in PBS for another 30 min. Labeling with rabbit anti-rod Gαt antibody K-20 (1:1000)(Santa Cruz Biotech, Santa Cruz, CA) was performed in 0.1% Triton/PBS containing 5% BSA for 3 hours at 25°C. Following 2-hr incubation with goat anti-rabbit AlexaFluor 555 secondary antibodies (Invitrogen) (1:1000), the sections were visualized using a Zeiss LSM 510 confocal microscope.

2.4. Transducin extraction

After overnight dark-adaptation of transgenic X. laevis, tadpole eyeballs (~20) were isolated under room light (~1000 lux), homogenized with pestle and sonicated in 250 μl of 20 mM Tris (pH=7.0) buffer containing 150 mM NaCl, 2 mM MgSO4, complete protease inhibitor mixture (Roche) and 10 μM GTPγS (buffer A). The homogenate was exposed to 2000 lux light for 5 minutes, spun (2500g, 1 min) to remove cell debris and nuclei, and centrifuged for 1 hour at 100000g. The supernatant was collected as soluble fraction S. The pellet was rinsed with 200 μl of buffer A, resuspended in 120 μl of buffer A, and centrifuged (1 hour, 100000g). The procedure was repeated using buffer A without GTPγS, and the pellet was dissolved in 100 μl 1% SDS (membrane fraction M). All procedures were performed on ice or at +4°C.

2.5. Analysis of transducin diffusion

The lateral and longitudinal (axial) diffusion of transducin were assessed by measuring Fluorescence Recovery After Photobleaching (FRAP) in living Xenopus rods as previously described (Wang et al., 2008) with minor modifications (Muradov et al., 2009). A typical recording involved imaging of a transgenic rod cell attached to a small piece of retina at 25°C in Ringer’s buffer (5 mM HEPES, pH 7.7, 110 mM NaCl, 2 mM CaCl2, 2.5 mM KCl, 1.2 mM MgCl2). To study diffusion of Gαt1β1γ1, rhodopsin in transgenic rods was converted to opsin by pre-incubation with 10 mM hydroxylamine under room light conditions. Diffusion of Gαt1GTP was monitored using Ringer’s buffer oxygenated with 95% O2/5% CO2 and containing 10 mM glucose. The membrane permeabilization with α-toxin, used previously to study diffusion of Gαt1GTPγS (Wang et al., 2008), caused an apparent loss of native rod morphology and was excluded from our analysis. Nucleotide depletion with 6 mM 2-deoxyglucose and 10 mM sodium azide was used to accumulate the Gt-R* complex (Wang et al., 2008). Diffusion coefficients were determined according to Wang et al. (2008) using GraphPad Prizm 4. Groups of measurements were compared with two-tailed unpaired t test.

2.6. Western Blotting

Tadpole retinas were homogenized in 5% SDS and boiled for 5 min. After centrifuging at 16 000 g for 5 minutes at 22°C supernatant was collected. The protein concentration was determined by a detergent-compatible method (DC Protein Assay, Bio-Rad, 500–0114). Samples of retinal homogenates after centrifugation (16 000 g, 5 min) were subjected to SDS-PAGE in 10% gels, electrotransferred onto nitrocellulose membranes, and probed with antibodies against EGFP (A-11122, Invitrogen) and rod transducin (K-20, Santa Cruz Biotech) (both at 1:1000 dilution). The antibody-antigen complexes were detected using anti-rabbit antibodies conjugated to horseradish peroxidase (Sigma) and ECL reagent (Amersham Pharmacia Biotech.).

3. Results

3.1. Expression, localization, and translocation of EGFP-Gαt1 and mutants in transgenic rods

The EGFP-fusion protein of human Gαt1, and the A3C and Gαt1-Ctαs mutants were expressed in rods of transgenic X. laevis under control of the Xenopus opsin promoter XOP (Mani et al., 2001) (Fig. 1A). The site for insertion of EGFP in the helical domain of Gαt1 between Met115 and Pro116 was selected based on an earlier report that showed complete functionality of the similarly fused Gαq (Hughes et al., 2001). The intact properties of the EGFP-fusion protein of bovine Gαt1 with the analogous insertion site have been confirmed (Wang et al., 2008). As it has been shown for other XOP-directed transgenes, Gαt1 transgene expression varied between retinas from different tadpoles and also between rods within the same retina (Moritz at al., 2001; Wang et al., 2008). Western blot analysis indicated that on average the levels of EGFP-Gαt1, A3C, and Gαt1-Ctαs were less than 10% of the endogenous frog Gαt1 (Fig. 1B). Thus, levels of EGFP-Gαt1 and mutants in the majority of rods were significantly lower than the endogenous Gαt1 level. EGFP-fluorescence imaging of the retina of transgenic tadpoles showed that all three EGFP-fusion proteins, Gαt1, A3C, and Gαt1-Ctαs, were properly targeted to the OS in the dark-adapted tadpoles, suggesting intact protein transport (Fig. 2). Following light adaptation of transgenic EGFP-Gαt1 tadpoles (800 lux, 40 min), a fraction of EGFP-Gαt1 translocated from the OS to the IS (Fig. 2). A similar degree of translocation (IOS/IIS=2.3±0.2, n=7) of native frog Gαt1 under these illumination conditions was observed in WT tadpoles using immunofluorescence analysis with Gαt1 specific antibodies K-20 (not shown), suggesting that the EGFP insert did not alter transducin translocation. In comparison to the frog Gαt1, light-induced translocation of Gαt1 in mouse rods is more extensive (Kerov et al., 2005; Lobanova et al., 2007; Rosenzweig et al., 2007). The quantitative difference of Gαt1 translocation in frog and mouse rods appears to be species-specific.

Fig. 2. Localization of G αt1, Gαt1A3C, and Gαt1-Ctαs in dark- and light-adapted rods.

Fig. 2

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(A) EGFP-fluorescence in living rods expressing EGFP-fusion proteins of Gαt, Gαt1A3C, and Gαt-GαsCt. Transgenic tadpoles were dark adapted overnight (D). After dark adaptation, some tadpoles were exposed to light (800 lux/40 min) in a Petri dish (L). Bar -10 μm. (B) Mean fluorescence intensities in the OS (red line) and IS (yellow line) of light-adapted rods were quantified using Z-stack projection images and ImageJ. The ratio of the intensities (IOS/IIS) (mean±SE) was determined for control Gαt1 rods (2 tadpoles, 8 rods) and Gαt1A3C rods (2 tadpoles, 8 rods) (p=0.003).

EGFP-Gαt1-Ctαs (hereafter EGFP is omitted) did not translocate from the OS to the IS in response to light (Fig. 2A). This observation is in agreement with the lack of Gαt1-Ctαs activation by R* and the requirement of Gt activation for its translocation (Artemyev, 2008; Calvert et al., 2006; Slepak and Hurley, 2008). In comparison to Gαt1, the translocation of the Gαt1A3C mutant was clearly impaired. Only a very weak EGFP signal was detectable in the IS of the majority of rods after light adaptation (Fig. 2AB). Control immunofluorescence staining of the Gαt1A3C transgenic retina demonstrated that light-dependent translocation of endogenous Gαt1 in rods expressing Gαt1A3C was unaffected (Fig. 3). The Gαt1A3C phenotype is consistent with palmitoylation of the majority of Gαt1A3C molecules. To further examine the extent of palmitoylation of Gαt1A3C, we performed analysis of transducin partitioning between the soluble GTPγS extract and membrane-bound fraction. In control X. laevis, the bulk of endogeneous frog Gαt1 and transgenic Gαt1 was found in the GTPγS extract with only ~15% of transducin remaining in the membrane fraction (Fig. 4). In contrast, about 50% of Gαt1A3C remained in the membrane fraction following extraction with GTPγS, whereas the membrane-bound fraction of frog Gαt1 was similar to that in control X. laevis (Fig. 4). Since palmitoylated Gαt1A3C may partially be extracted from the membrane, the results of the extraction experiments indicate that ~50% or more of the mutant Gαt1 is palmitoylated.

Fig. 3. Light-dependent translocation of total transducin in EGFP-Gαt1A3C rods.

Fig. 3

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Localization of total Gαt1 (red, anti-Gαt1 K-20 immunofluorescence) and EGFP-Gαt1A3C (green, EGFP-fluorescence) in the dark (D) and after light exposure (800 lux, 40 min) (L). OS – outer segment, IS – inner segment. Bar -5 μm.

Fig. 4. Membrane association of Gαt1A3C.

Fig. 4

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Soluble GTPγS-extracted (S) and membrane (M) fractions were obtained from control EGFP-Gαt1 and EGFP-Gαt1A3C tadpoles as described in Materials and Methods. The fractions were analyzed by Western blotting with anti-Gαt1 antibodies K-20 (Santa Cruz Biotech) (right). Open and filled arrows indicate EGFP-fused and frog Gαt1, respectively.

3.2. Lateral diffusion of Gαt1 and mutants in transgenic rods

The mobilities of Gαt1, Gαt1A3C, and Gαt-GαsCt, in various states of activation were assessed by FRAP (Fig. 5, Table 1). To validate the FRAP protocol, we first measured lateral diffusion of rhodopsin using the transgenic rhodopsin-EGFP X. laevis model described previously (Jin et al., 2003). The D value for rhodopsin-EGFP was 0.10 μm2/s (or 0.28 μm2/s after the correction for the slowing effect of incisures) (not shown) (Govardovskii et al., 2009; Poo and Cone, 1974). This value is comparable to D values obtained previously for rhodopsin-EGFP with a similar FRAP analysis (Wang et al., 2008) and for native rhodopsin with alternative biophysical techniques (Govardovskii et al., 2009; Poo and Cone, 1974; Wey et al., 1981).

Fig. 5. Representative measurement of lateral diffusion of EGFP-Gαt1β1γ1.

Fig. 5

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(A). Pre- and post-bleach images of a transgenic ROS at different time points. The retina was pre-incubated in Ringer’s buffer containing 10 mM hydroxylamine under ambient light conditions to monitor diffusion of Gαt1β1γ1. 150 images (256×256 pixels) were collected at 196 msec/frame with no intervals between the scans. (B) Intensity integrated along y-axis for each x-axis pixel of the selected box region from the first post-bleach image. The plot is used to determine the width of a bleach region with a Gaussian profile and the depth of bleach according to the protocol of Wang et al. (2008). (C) Intensities of the bleached region during the experimental time course corrected for background and fading (black) and the fitting curve to a one-dimensional diffusion equation 3 in Wang et al. (2008).

Table 1.

Lateral and longitudinal diffusion coefficients of EGFP-Gαt1 and its mutants in different activation states

Lateral:
State Gαt1 Gαt1A3C Gαt1-Ctαs
Gαt1GTP 0.145±0.013 (6)* 0.114±0.008 (11) 0.120±0.017 (8)
Gαt1β1γ1/R* 0.065±0.008 (7) 0.076±0.004 (5) 0.125±0.007 (6)
Gαt1β1γ1 0.105±0.007 (8) - 0.119±0.018 (6)
Longitudinal:
State Gαt1 Gαt1A3C Gαt1-Ctαs
Gαt1GTP 0.0039±0.0004 (6) <0.001, F>0.8(6)** 0.0051±0.0013 (7)
Gαt1β1γ1/R* 0.0018±0.0004 (4) <0.001, F>0.8 (5) 0.0039±0.0011 (9)
Gαt1β1γ1 0.0039±0.0003 (6) - 0.0046±0.0025 (6)
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*

mean±standard error (N). Data are not adjusted for the effect of incisures

**

F - immobile fraction

To study diffusion of Gαt1β1γ1, rhodopsin in transgenic rods was converted to opsin by pre-incubation with 10 mM hydroxylamine similarly as described (Wang et al., 2008). The lateral diffusion coefficient for Gαt1β1γ1 was 0.105±0.007 μm2/s. Diffusion of Gαt1GTP monitored using an oxygenated Ringer’s buffer containing 10 mM glucose (Wang et al., 2008) was moderately accelerated compared to Gαt1β1γ1 (D=0.145±0.013 μm2/s) (p=0.013). A significant decrease in the mobility of Gαt1β1γ1 was observed upon its sequestration by R* under nucleotide-depleting conditions (0.065±0.008 μm2/s) (p=0.0023). In comparison to Gαt1GTP, the lateral diffusion of the activated Gαt1A3C mutant was modestly slower (p=0.048) (Table I). The diffusion of the heterotrimeric A3C was not investigated since hydroxylamine promotes hydrolysis of palmitoyl moieties. Interestingly, the kinetics of the lateral diffusion of Gαt1-Ctαs were similar under all tested conditions suggesting that Gαt1-Ctαs was not sequestered or activated by R* (Table I). The calculated immobile fractions of transducin and mutants for lateral diffusion in all of the examined states (Gαt1β1γ1, Gαt1GTP, and Gαt1β1γ1/R*) were negligible (<1%).

3.3. Longitudinal diffusion of Gαt1 and mutants in transgenic rods

The longitudinal diffusion of Gαt1, Gαt1A3C, and Gαt1-GαsCt was probed by bleaching a stripe perpendicular to the long axis of the transgenic ROS (Fig. 6). The longitudinal diffusion of Gαt1β1γ1 was determined to be markedly slower than its lateral diffusion (Table I). Surprisingly, the axial D value for the activated Gαt1GTP was analogous to that for the heterotrimeric protein. Sequestration of Gαt1β1γ1 by R* decreased D for longitudinal diffusion by more than 2-fold (p=0.0027). The A3C mutation essentially blocked the longitudinal diffusion of the activated state or the R*-bound transducin (Table I). The interdisc transfer rates of Gαt1-Ctαs were highly variable and not significantly different across the three experimental conditions. The axial diffusion rates of Gαt1-Ctαs were generally comparable to those of Gαt1GTP and Gαt1β1γ1 (Table I).

Fig. 6. Representative measurement of longitudinal diffusion of EGFP-Gαt1β1γ1.

Fig. 6

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(A). Pre- and post-bleach images of a transgenic ROS at different time points. The retina was pre-incubated in Ringer’s buffer containing 10 mM hydroxylamine to monitor diffusion of Gαt1β1γ1. 150 images were collected with a 2-sec interval between the scans. (B) Intensities of the bleach region during the experimental time course corrected for background and fading (black), and the fitting curve to one-dimensional diffusion equation 3 in Wang et al. (2008).

4. Discussion

Biochemical studies have shown that the activation of Gt1 by R* loosens its interaction with the disc membranes and causes solubilization of Gαt1GTP in vitro (Bigay et al., 1994; Kosloff et al., 2008). Considering the high density of disc membranes in the OS, it remains unclear whether Gαt1GTP partitions to the cytosolic fraction in vivo. Because the viscosity of the membrane is much greater than the viscosity of cytoplasm, diffusion of a protein with a lipid anchor on a surface of a membrane is dictated by the embedded anchor (Saffman and Delbruck, 1975). If Gαt1GTP dissociates from the membrane, its lateral diffusion coefficient would be increased by more than an order of magnitude as suggested by comparison of the diffusion coefficients for transducin and EGFP in the ROS (Wang et al., 2008). If, however, Gαt1GTP dissociates from Gβ1γ1 but stays on the membrane, the embedded radius would be reduced by ~2 fold and only a ~10–15% increase in the diffusion coefficient is then predicted from the theoretical calculations of Saffman and Delbruck (1975). Our data show a moderate ~40% increase in the lateral diffusion rate of Gαt1GTP in comparison to Gαt1β1γ1. We interpret these results as an indication that Gαt1GTP remains largely associated with the membrane, but its lifetime on the membrane is reduced compared to Gαt1β1γ1.

The lateral diffusion coefficients of Gαt1GTP and the R*-Gαtβ1γ1 complex obtained in this study are in general agreement with the previous results (Wang et al., 2008). However, the lateral and longitudinal D values of Gαt1β1γ1 reported by Wang et al. (2008) are ~3.5 and ~10-fold greater, respectively, than the values obtained here. One potentially significant difference between the experimental protocols is that we analyzed only rods that are still attached to small pieces of retina. To test this possibility, we also analyzed diffusion of Gαt1β1γ1 in dissociated rods. We found that Gαt1β1γ1 diffuses laterally ~1.5 times faster (D=0.16±0.02 μm2/s) and longitudinally ~2.5 times faster (D= 0.0095±0.0025 μm2/s) in comparison to the diffusion in attached rods. Apparently, dissociated rods have reduced viability, especially after the treatment with hydroxylamine, and this in part accounts for the discrepancy. The diffusion analysis of Wang et al. (2010) presumes bleaching through the full width of the ROS. In this regard, Calvert et al. (2010) pointed out that with the bleaching at a single focus level, Wang et al. (2010) might have overestimated the diffusion rates due to radial diffusion of unbleached molecules from out-of-focus regions into the scan plane. We imaged the 3-dimensional bleached volume under our experimental settings using EGFP immobilized in 40% polyacrylamide gel (Suppl. Fig. 1). This analysis indicated a relatively uniform bleaching in the axial direction within at least ±3 μm from the focal plane. Thus, the bleached volume spanned the entire width of the rods used in this study.

Generally, the observed diffusion coefficients for Gαt1GTP and Gt, corrected for the effect of incisures in frog discs membrane, are lower than previously assumed (Pugh and Lamb, 1993) or theoretically predicted (Saffman and Delbruck, 1975; Wang et al., 2008). This may indicate that transducin diffusion is retarded by protein-protein interactions, macromolecular crowding, and/or association with more viscous lipid microdomains. A recent study indicated that the mobility of a soluble photoactivatable GFP in the frog ROS is much slower than predicted (Calvert et al., 2010). According to the analysis of Pugh and Lamb (1993) (equation 3), the D values for Gt (0.105 μm2/s) (Table 1) and R* (0.14 μm2/s) (Wang et al., 2008) would result in the encounter rate of ~900 s−1 between a single R* and Gt in frog ROS. Even without the correction for incisures, this rate is well above the actual activation rate of 120–150 Gt s−1 per R* estimated in amphibian rods (Leskov et al., 2000). Thus, the apparently slow diffusion of transducin is sufficient to account for its known R*-dependent activation rate.

The D values for longitudinal diffusion of Gt and Gαt1GTP were 27 and 37 fold smaller than corresponding D for the lateral diffusion. Unexpectedly, the interdisc transfer kinetics of Gt and Gαt1GTP were equivalent despite a lower membrane affinity of Gαt1GTP. It appears that the longitudinal diffusion of Gt and Gαt1GTP is limited by tortuosity rather than by the protein dissociation from the discs. The tortuosity factor of the frog OS due to obstruction of axial diffusion by stacked membrane discs is ~20 or even ~40 if the incisures are inaccessible for diffusion (Calvert et al., 2010). Although the longitudinal diffusion of Gαt1GTP is rather slow, it might be adequate to explain the light-dependent translocation of transducin in frogs rods, which appears to be less efficient than in mouse rods. Transducin transport in the dark in the IS→OS direction is significantly slower than its light-dependent translocation. In terms of kinetics, this process may well be accounted for by the axial diffusion.

The equivalence of the rates of longitudinal diffusion of Gt and Gαt1GTP raises important questions. What determines compartmentalization of Gt to the OS and prevents Gt from leaking out to the IS in the dark? Ciliary proteins such as centrins can potentially block diffusion of Gt in the OS→IS direction (Giessl et al., 2006). Another possibility is that Gt is targeted and retained in the OS in the dark due to its affinity for the membranes and/or its interactions with rhodopsin. The Gαt1-Ctαs mutant transgenic Xenopus model was generated to probe the effects of rhodopsin/transducin interactions on the light-dependent compartmentalization and diffusion of transducin. The C-termini of Gα subunits are the major interaction sites of heterotrimeric G-proteins with activated GPCRs. The C-terminus of Gαt1 is a major rhodopsin recognition site that is required for the Gt activation by R* (Burns and Arshavsky, 2005; Lamb and Pugh, 2006). It could potentially serve as a signal motif for targeting transducin to the OS. The C-terminus of Gαt1 also weakly interacts with opsin (Scheerer et al., 2008) and may even interact with the ground “dark” state of rhodopsin as suggested by studies using plasmon-waveguide resonance spectroscopy (Alves et al., 2005) and computational analyses (Fanelli and Dell’Orco, 2005). Two main observations have been made using the Gαt1-Ctαs model. Correct localization of Gαt1-Ctαs to the OS demonstrates the Gαt1 C-terminus is not involved in the targeting of Gt (Fig. 2). The failure of Gαt1-Ctαs to translocate to IS during light adaptation indicates that Gαt1-Ctαs·β1γ1 is not activated by R* and confirms that this activation is necessary for Gt translocation to the IS. The analysis of Gαt1-Ctαs diffusion is consistent with the impaired interaction with R*. The kinetics of lateral diffusion of Gαt1-Ctαs were similar under conditions favoring formation of Gαt1β1γ1, Gαt1GTP or the R*-Gαt1β1γ1 complex. Furthermore, the Gαt1-Ctαs model presents an important control for the analysis of longitudinal diffusion of Gt. It allowed exclusion of transducin activation by opsin as a source of the axial diffusion of Gt. Gt is activated by opsin with 10−3 R* efficiency (Cornwall and Fain, 1994; Melia et al., 1997), but this still may potentially influence the slow longitudinal FRAP kinetics. Indeed, the D value for the axial diffusion of Gαt1-Ctαs·β1γ1 was comparable to that of Gαt1β1γ1, demonstrating the interdisc transfer for the heterotrimer uncoupled from R* activation.

If longitudinal diffusion of transducin underlies its light-dependent translocation to the IS, a correlation must exist between the two phenomena. Blocking or slowing of the axial diffusion of Gαt1 would then lead to the block or attenuation of the protein translocation. We sought to manipulate the membrane affinity of Gαt1 by introducing an artificial palmitoylation site at the N-terminus with the A3C mutation. Several lines of evidence suggest that a dominant fraction of the Gαt1A3C mutant in frog rods was palmitoylated. The Gαt1A3C mutant was properly targeted to the OS in the dark, but its light-dependent translocation to the IS was markedly impaired (Fig. 2). A large fraction of Gαt1A3C remained membrane-bound following extraction of retinal membranes with GTPγS. The kinetics of the lateral diffusion of Gαt1A3C were slower than those of Gαt1 with one lipid anchor, and similar to the kinetics of Gαtβ1γ1 with two lipid modifications. Lastly, the A3C mutation halted the longitudinal diffusion of transducin with immobile fraction exceeding 80%. Apparently, two lipids, N-acyl and S-palmitoyl, cause stable attachment of Gαt1A3C to the membrane. The lack of axial diffusion of Gαt1A3C differs from the ability of Gαt1β1γ1 to diffuse longitudinally. We hypothesize that the longitudinal diffusion of Gαtβ1γ1 in the OS is facilitated by sequestration of one of the two lipid anchors via interactions with phosducin, PrBP/d, UNC119, or another unknown binding partner (Norton et al., 2004; Sokolov et al., 2004; Zhang et al., 2010). Our analysis supports the essential role of the lipid modifications of transducin in the control of the protein mobility and compartmentalization.

Supplementary Material

01

Acknowledgments

This work was supported by the National Institutes of Health Grant RO1 EY-12682 to N.O.A.

Abbreviations

Gt1

rod G protein transducin

Gαt1

rod transducin α-subunit

PDE6

hotoreceptor phosphodiesterase-6

R*

photoexcited rhodopsin

FRAP

Fluorescence Recovery After Photobleaching

ROS (OS)

rod outer segment(s)

RIS (IS)

rod inner segment(s)

GTPγS

guanosine 5′-O-(3-thiotriphosphate)

Footnotes

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References

  1. Alves ID, Salgado GFJ, Salamon Z, Brown MF, Tollin G, Hruby VJ. Phosphatidylethanolamine enhances rhodopsin photoactivation and transducin binding in a solid supported lipid bilayer as determined using plasmon-waveguide resonance spectroscopy. Biophys J. 2005;88:198–210. doi: 10.1529/biophysj.104.046722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Artemyev NO. Light-dependent compartmentalization of transducin in rod photoreceptors. Mol Neurobiol. 2008;37:44–51. doi: 10.1007/s12035-008-8015-2. [DOI] [PubMed] [Google Scholar]
  3. Bigay J, Faurobert E, Franco M, Chabre M. Roles of lipid modifications of transducin subunits in their GDP-dependent association and membrane binding. Biochemistry. 1994;33:14081–14090. doi: 10.1021/bi00251a017. [DOI] [PubMed] [Google Scholar]
  4. Bourne HR. How receptors talk to trimeric G proteins. Curr Opin Cell Biol. 1997;9:134–142. doi: 10.1016/s0955-0674(97)80054-3. [DOI] [PubMed] [Google Scholar]
  5. Burns ME, Arshavsky VY. Beyond counting photons: trials and trends in vertebrate visual transduction. Neuron. 2005;48:387–401. doi: 10.1016/j.neuron.2005.10.014. [DOI] [PubMed] [Google Scholar]
  6. Calvert PD, Govardovskii VI, Krasnoperova N, Anderson RE, Lem J, Makino CL. Membrane protein diffusion sets the speed of rod phototransduction. Nature. 2001;411:90–94. doi: 10.1038/35075083. [DOI] [PubMed] [Google Scholar]
  7. Calvert PD, Strissel KJ, Schiesser WE, Pugh EN, Jr, Arshavsky VY. Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends Cell Biol. 2006;16:560–568. doi: 10.1016/j.tcb.2006.09.001. [DOI] [PubMed] [Google Scholar]
  8. Calvert PD, Schiesser WE, Pugh EN., Jr Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium. J Gen Physiol. 2010;135:173–196. doi: 10.1085/jgp.200910322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen CA, Manning DR. Regulation of G proteins by covalent modification. Oncogene. 2001;20:1643–1652. doi: 10.1038/sj.onc.1204185. [DOI] [PubMed] [Google Scholar]
  10. Cornwall MC, Fain GL. Bleached pigment activates transduction in isolated rods of the salamander retina. J Physiol. 1994;480:261–279. doi: 10.1113/jphysiol.1994.sp020358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fanelli F, Dell’Orco D. Rhodopsin activation follows precoupling with transducin: inferences from computational analysis. Biochemistry. 2005;44:14695–14700. doi: 10.1021/bi051537y. [DOI] [PubMed] [Google Scholar]
  12. Fu Y, Yau KW. Phototransduction in mouse rods and cones. Pflugers Arch. 2007;454:805–819. doi: 10.1007/s00424-006-0194-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Giessl A, Trojan P, Rausch S, Pulvermüller A, Wolfrum U. Centrins, gatekeepers for the light-dependent translocation of transducin through the photoreceptor cell connecting cilium. Vision Res. 2006;46:4502–4509. doi: 10.1016/j.visres.2006.07.029. [DOI] [PubMed] [Google Scholar]
  14. Govardovskii VI, Korenyak DA, Shukolyukov SA, Zueva LV. Lateral diffusion of rhodopsin in photoreceptor membrane: a reappraisal. Mol Vis. 2009;15:1717–1729. [PMC free article] [PubMed] [Google Scholar]
  15. Hughes TE, Zhang H, Logothetis DE, Berlot CH. Visualization of a functional Gαq-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4−. J Biol Chem. 2001;276:4227–4235. doi: 10.1074/jbc.M007608200. [DOI] [PubMed] [Google Scholar]
  16. Jin S, McKee TD, Oprian DD. An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis. FEBS Lett. 2003;542:142–146. doi: 10.1016/s0014-5793(03)00368-5. [DOI] [PubMed] [Google Scholar]
  17. Kerov V, Chen D, Moussaif M, Chen YJ, Chen CK, Artemyev NO. Transducin activation state controls its light-dependent translocation in rod photoreceptors. J Biol Chem. 2005;280:41069–41076. doi: 10.1074/jbc.M508849200. [DOI] [PubMed] [Google Scholar]
  18. Kosloff M, Alexov E, Arshavsky VY, Honig B. Electrostatic and lipid-anchor contributions to the interaction of transducin with membranes: Mechanistic implications for activation and translocation. J Biol Chem. 2008;283:31197–31207. doi: 10.1074/jbc.M803799200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kroll KL, Amaya E. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development. 1996;122:3173–3183. doi: 10.1242/dev.122.10.3173. [DOI] [PubMed] [Google Scholar]
  20. Lamb TD, Pugh EN., Jr Phototransduction, dark adaptation, and rhodopsin regeneration. Invest Ophthalmol Vis Sci. 2006;47:5137–5152. doi: 10.1167/iovs.06-0849. [DOI] [PubMed] [Google Scholar]
  21. Leskov IB, Klenchin VA, Handy JW, Whitlock GG, Govardovskii VI, Bownds MD, Lamb TD, Pugh EN, Jr, Arshavsky VY. The gain of rod phototransduction: reconciliation of biochemical and electrophysiological measurements. Neuron. 2000;27:525–537. doi: 10.1016/s0896-6273(00)00063-5. [DOI] [PubMed] [Google Scholar]
  22. Lobanova ES, Finkelstein S, Song H, Tsang SH, Chen CK, Sokolov M, Skiba NP, Arshavsky VY. Transducin translocation in rods is triggered by saturation of the GTPase-activating complex. J Neurosci. 2007;27:1151–1160. doi: 10.1523/JNEUROSCI.5010-06.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mani SS, Batni S, Whitaker L, Chen S, Engbretson G, Knox BE. Xenopus rhodopsin promoter. Identification of immediate upstream sequences necessary for high level, rod-specific transcription. J Biol Chem. 2001;276:36557–36565. doi: 10.1074/jbc.M101685200. [DOI] [PubMed] [Google Scholar]
  24. Melia TJ, Cowan CW, Angleson JK, Wensel TG. A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin. Biophys J. 1997;73:3182–3191. doi: 10.1016/S0006-3495(97)78344-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moritz OL, Tam BM, Papermaster DS, Nakayama T. A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern. J Biol Chem. 2001;276:28242–28251. doi: 10.1074/jbc.M101476200. [DOI] [PubMed] [Google Scholar]
  26. Muradov H, Boyd KK, Haeri M, Kerov V, Knox BE, Artemyev NO. Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods. J Biol Chem. 2009;284:32662–32669. doi: 10.1074/jbc.M109.049916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nair KS, Hanson SM, Mendez A, Gurevich EV, Kennedy MJ, Shestopalov VI, Vishnivetskiy SA, Chen J, Hurley JB, Gurevich VV, Slepak VZ. Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions. Neuron. 2005;46:555–567. doi: 10.1016/j.neuron.2005.03.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Natochin M, Moussaif M, Artemyev NO. Probing the mechanism of rhodopsin-catalyzed transducin activation. J Neurochem. 2001;77:202–210. doi: 10.1046/j.1471-4159.2001.t01-1-00221.x. [DOI] [PubMed] [Google Scholar]
  29. Norton AW, Hosier S, Terew JM, Li N, Dhingra A, Vardi N, Baehr W, Cote RH. Evaluation of the 17-kDa prenyl-binding protein as a regulatory protein for phototransduction in retinal photoreceptors. J Biol Chem. 2005;280:1248–1256. doi: 10.1074/jbc.M410475200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Oldham WM, Van Eps N, Preininger AM, Hubbell WL, Hamm HE. Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins Nat. Struct Mol Biol. 2006;213:772–777. doi: 10.1038/nsmb1129. [DOI] [PubMed] [Google Scholar]
  31. Poo M, Cone RA. Lateral diffusion of rhodopsin in the photoreceptor membrane. Nature. 1974;247:438–441. doi: 10.1038/247438a0. [DOI] [PubMed] [Google Scholar]
  32. Pugh EN, Jr, Lamb TD. Amplification and kinetics of the activation steps in phototransduction. Biochim Biophys Acta. 1993;1141:111–149. doi: 10.1016/0005-2728(93)90038-h. [DOI] [PubMed] [Google Scholar]
  33. Rosenzweig DH, Nair KS, Wei J, Wang Q, Garwin G, Saari JC, Chen CK, Smrcka AV, Swaroop A, Lem J, Hurley JB, Slepak VZ. Light-driven translocation of signaling proteins in vertebrate photoreceptors. J Neurosci. 2007;27:5484–5494. doi: 10.1523/JNEUROSCI.1421-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Saffman PG, Delbruck M. Brownian motion in biological membranes. Proc Natl Acad Sci U S A. 1975;72:3111–3113. doi: 10.1073/pnas.72.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krauss N, Choe HW, Hofmann KP, Ernst OP. Crystal structure of opsin in its G-protein-interacting conformation. Nature. 2008;455:497–502. doi: 10.1038/nature07330. [DOI] [PubMed] [Google Scholar]
  36. Slepak VZ, Hurley JB. Mechanism of light-induced translocation of arrestin and transducin in photoreceptors: interaction-restricted diffusion. IUBMB Life. 2008;60:2–9. doi: 10.1002/iub.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sokolov M, Lyubarsky AL, Strissel KJ, Savchenko AB, Govardovskii VI, Pugh EN, Jr, Arshavsky VY. Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation. Neuron. 2002;34:95–106. doi: 10.1016/s0896-6273(02)00636-0. [DOI] [PubMed] [Google Scholar]
  38. Sokolov M, Strissel KJ, Leskov IB, Michaud NA, Govardovskii VI, Arshavsky VY. Phosducin facilitates light-driven transducin translocation in rod photoreceptors. Evidence from the phosducin knockout mouse. J Biol Chem. 2004;279:19149–19156. doi: 10.1074/jbc.M311058200. [DOI] [PubMed] [Google Scholar]
  39. Wang Q, Zhang X, Zhang L, He F, Zhang G, Jamrich M, Wensel TG. Activation-dependent hindrance of photoreceptor G protein diffusion by lipid microdomains. J Biol Chem. 2008;283:30015–30024. doi: 10.1074/jbc.M803953200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wedegaertner PB, Wilson PT, Bourne HR. Lipid modifications of trimeric G proteins. J Biol Chem. 1995;270:503–506. doi: 10.1074/jbc.270.2.503. [DOI] [PubMed] [Google Scholar]
  41. Wey CL, Cone RA, Edidin MA. An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis. Biophys J. 1981;33:225–232. doi: 10.1016/S0006-3495(81)84883-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zhang H, Constantine R, Davis M, Inana G, Jorgensen EM, Baehr W. Unc119/RG4 regulates G protein trafficking in sensory neurons by recognizing the acylated Gα N-Terminus. ARVO Annual Meeting. 2010 (abstract) [Google Scholar]

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