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First published online August 9, 2007
doi: 10.1242/10.1242/jcs.001404

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A FRET map of membrane anchors suggests distinct microdomains of heterotrimeric G proteins

Ecole Polytechnique Fédérale de Lausanne (EPFL), Institut des Sciences et Ingénierie Chimiques, CH-1015 Lausanne, Switzerland

View larger version (39K): [in this window] [in a new window]   Fig. 1. Analysis of FRET on the cell membrane demonstrates the distinct levels of co-clustering of microdomain markers. (A) Illustration of our reductionistic approach to study activation-state-dependent microdomain localization of heterotrimeric G proteins. (Top) Activation followed by dissociation of the heterotrimer leads to a separation of G and G subunits. (Bottom) Only the lipid anchors of inactive heterotrimer and active G were fused to fluorescent proteins, and the microdomain localization of these G-protein-anchor constructs was studied in relation to microdomain markers (see B and C) using FRET between mCFP as a donor and mCit as an acceptor. (B) Schematic representation of microdomain markers with their lipid anchors. P, palmitoyl; G, geranylgeranyl; F, farnesyl; pb, polybasic sequence. Source refers to the proteins from which targeting sequences were derived. (C) Subcellular localization of microdomain markers imaged by confocal microscopy. All constructs were predominantly localized to the plasma membrane, with minor labeling of internal membranes or, in the case of the Rac1-derived construct, nuclear labeling. Only the mCit constructs are representatively shown. Bars, 10 µm. (D) By plotting the FRET efficiency (E) against the normalized acceptor surface concentration (cA) at a constant donor mole fraction (xD=0.50±0.17), we obtained information about the clustering of donor and acceptor fluorophores. A random distribution of fluorophores (Wolber and Hudson, 1979) cannot describe our data (left, hashed curve). We found that the FRET efficiencies increased after a cA offset towards a plateau value Emax (indicated by solid horizontal line in the left plot). We therefore adapted the double exponential function of Wolber and Hudson (Wolber and Hudson, 1979), further taking the cA offset and the maximum efficiency Emax into account. This lead to fits which described all of our FRET data adequately (from left to right, 2: 22.6, 25.3 and 9.7). Each datapoint was calculated on a single cell. Representative examples of indicated FRET pairs are shown. (E) The Emax matrix of microdomain-marker FRET pairs. FRET values of mCFP-tH/mCit-tH and mCFP-tK/mCit-tK are significantly higher (bold) than those of tH-polybasic pairs (P<0.001 or P<0.05, respectively, 2-tailed Student's t-test). No significant differences were found for consistently high mCFP-polybasic/mCit-polybasic sequence pairs (bold). Emax values are given in percent ± s.d.; n, number of independent experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Sensitized acceptor emission FRET imaging confirms the distinct FRET levels on the plasma membrane. Left to right: FRET-channel images, FRET-efficiency images and dependencies of the FRET efficiency of indicated FRET pairs on the acceptor intensity, corresponding to approximately 500-2000 acceptors/µm2. Each datapoint was calculated on one region of interest on plasma membranes with donor mole fractions of xD=0.5±0.17 were analysed. Note that for mCFP-tH/mCit-tH the FRET originating from putative internal membranes show similar values as that originating from the plasma membrane. Consistent with the negligible FRET from soluble fluorophores at these concentrations (supplementary material Fig. S2E), FRET from the nucleus is close to zero for mCFP-tR/mCit-tR. Bars, 5 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 3. The Gi/o protein anchor construct FRET vector is used to characterize Gi/o and Gi/o microlocalizations. (A) Schematic representation of heterotrimeric Gi/o protein anchor constructs. M, myristoyl; P, palmitoyl; G, geranylgeranyl; FP, fluorescent protein (i.e. either mCFP or mCit). Source refers to the proteins from which targeting sequences were derived. (B) The subcellular localization of Gi/o-anchor constructs was imaged by confocal microscopy. Bar, 10 µm. (C) The FRET vectors show the Emax values of pairs of Gi/o anchor constructs and microdomain markers. Column headings give names of G-protein-anchor constructs, row headings give co-expressed microdomain markers. The Emax values are given in % ± s.d.; n, number of independent experiments. The value for Ni2C-mCit/mCFP-tH is significantly larger than the Gi/o-anchor construct/FP-tH pairs (P<0.01, 2-tailed Student's t-test), whereas the value for Ni2C-mCit/mCFP-tR is significantly smaller than that for the Gi/o-anchor construct/FP-tR pairs (P<0.001, 2-tailed Student's t-test). (D) Plots of the FRET efficiency (E) against the normalized acceptor surface concentration (cA) at a constant donor mole fraction of indicated FRET pairs with fitted curves as described (from left to right, 2: 3.1 and 6.2).

View larger version (38K): [in this window] [in a new window]   Fig. 4. FRET data of the anchor constructs indicate that Gq and Gq localize to different microdomains. (A) Subcellular localization of Gq-anchor constructs imaged by confocal microscopy revealed predominant plasma membrane labeling. Bar, 10 µm. (B) Schematic representation of constructs of heterotrimeric Gq protein anchor constructs. Abbreviations as in Fig. 3. (C) The FRET vectors show Emax values of Gq-anchor construct (blue and yellow column headings highlight mCFP- and mCit-labeled constructs, respectively) and microdomain markers (rows) pairs. The Emax values are given in percent with standard deviations and number of independent experiments, n, in brackets. Differences between bold and regular typed values in one row are significant (P<0.01, 2-tailed Student's t-test), however, the P value of NqC-mCit /mCFP-tK (bold italics) versus NGAP-43-mCFP/mCFP-tK is, P<0.05. (D) Plots of the FRET efficiency (E) against the normalized acceptor surface concentration (cA) at a constant donor mole fraction of indicated FRET pairs with fitted curves as described (from left to right, 2: 11.0; 10.3). (E) Since the crystal structures of heterotrimeric G-protein subunits show a -helical conformation for their N-terminus (Sprang, 1997), we compared the GAP-43- and Gq-derived targeting sequences in helical wheel projections. These show that the first 20 amino acids of GAP-43 (top) and the first 41 amino acids of Gq (bottom) that were used for the two Gq-anchor constructs reveal a more expanded stretch of amino acids with basic side chains (red) on one side of the Gq helix compared with the GAP-43 helix. These amphipathic helices might modify their microlocalization, which is primarily directed by palmitoylation (sites are encircled in orange; green, acidic side chains; blue, polar side chains).

View larger version (30K): [in this window] [in a new window]   Fig. 5. (A) The direct comparison of G-protein-anchor FRET pairs suggests that Gq and Gi/o share the same microdomain, whereas Gi/o and Gq do not. The Emax values are given in % ± s.d.; n, number of independent experiments. Emax values of heterotrimer construct FRET pairs are not significantly different (dark grey), whereas the values of G construct FRET pairs Ni2-mCFP/Ni2-mCit and NGAP-43-mCFP/Ni2-mCit (light grey) are significantly different (P<0.05, 2-tailed Student's t-test). Blue and yellow column headings highlight mCFP- and mCit-labeled constructs, respectively. (B) The FRET map of heterotrimeric G-protein microdomains based on the results of their membrane anchors. This scheme is based on the Emax relationships, where a high Emax value relates to a large overlap of the microdomains or a higher probability of the respective molecules to co-cluster. As an example, the arrow shows how the Gi/o subunit displaces after activation. Its starting microdomain has a lesser `proximity' to the tR-microdomain-marker (low FRET), than the destination microdomain of active Gi/o (high FRET). Thus, activation results in an increase of FRET for the FRET pair Gi/o-construct/tR (Fig. 6C).

View larger version (54K): [in this window] [in a new window]   Fig. 6. FRET experiments with full-length heterotrimeric-G-protein constructs confirm displacement of the G subunit into another microdomain after activation, as predicted by the FRET vectors of the anchor constructs. (A) Schematic representation of fluorescent full-length heterotrimeric-G-protein constructs. We fused the fluorescent protein (FP) to the N-terminus of the G subunits and targeted these fusion constructs using the targeting sequences of the G-protein-anchor constructs. (B) Confocal imaging confirmed that all full-length G constructs are predominantly localized to the plasma membrane. Bars, 10 µm. (C) FRET changes were calculated after stimulating cells with AlF4- (30 µM, 40 minutes, 22°C). Fluorescence of cells co-expressing indicated full-length G-protein constructs, microdomain markers and in addition unlabelled G12, was measured in a sensitive spectrofluorometer. Numbers at the bars give the average FRET change in percent with standard deviations and number of independent experiments, n, in brackets.

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