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Fig. 4. FRET data of the anchor constructs indicate that G_q and G_q localize to different microdomains. (A) Subcellular localization of G_q-anchor constructs imaged by confocal microscopy revealed predominant plasma membrane labeling. Bar, 10 µm. (B) Schematic representation of constructs of heterotrimeric G_q protein anchor constructs. Abbreviations as in Fig. 3. (C) The FRET vectors show E_max values of G_q-anchor construct (blue and yellow column headings highlight mCFP- and mCit-labeled constructs, respectively) and microdomain markers (rows) pairs. The E_max 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 N_qC-mCit /mCFP-tK (bold italics) versus N_GAP-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 G_q-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 G_q (bottom) that were used for the two G_q-anchor constructs reveal a more expanded stretch of amino acids with basic side chains (red) on one side of the G_q 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).

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