FIG. 8.
Retrograde PM-Golgi trafficking of Gαq depends on Gαq palmitoylation by Golgi apparatus-resident DHHC3 and -7. (A) HeLa cells were transfected with Gαq-GFP (green and white) and GalT-mCherry (red) expression vectors with or without Gβ1γ2. The Golgi region (white circle) marked by GalT-mCherry was bleached, and then fluorescent recovery of Gαq-GFP was monitored by acquiring images every 10 s. Fluorescence intensities were plotted in graphs (right). (B) HeLa cells expressing Gαq-GFP (green and white) and GalT-mCherry (red) were treated with or without 2-BP for 30 min before FRAP analysis. The region (white circle) identified with GalT-mCherry was bleached, and then fluorescent recovery of Gαq-GFP was monitored. Treatment with 2-BP inhibited the recovery of fluorescence. (C) Knockdown (KD) of DHHC3 and -7 inhibited the Golgi and PM targeting of Gαq-GFP, resulting in the diffuse cytoplasmic localization. (D) Palmitoylation-deficient Gαq (CS)-GFP showed similar cytoplasmic distribution and rapid recovery both around the GalT-positive region (CS region 1) and at cytoplasmic region (CS region 2). The recovery of palmitoylation deficient Gαq (CS)-GFP was as rapid as that of DHHC3 and -7 knocked down cells in panel C. Scale bar, 10 μm. Error bars show ±SD (n = 5). WT, wild type.