Figure 5

Inhibition of intracellular trafficking of diverse cargo proteins. A Vacuolar localization of sporamin:GFP, a fusion protein between sporamin and green fluorescent protein (GFP). Protoplasts isolated from TRV and TRV:NAG leaves were transformed with the sporamin:GFP construct. Fluorescent and bright field images are shown. Red fluorescence indicates chlorophyll autofluorescence. Scale bars: 10 μm. B Localization of invertase:GFP. Because invertase:GFP is a secretory protein, its fluorescence was not readily detected within protoplasts of TRV control . Scale bars: 10 μm. C Localization of GKX. GKX is a chimeric ER membrane marker [27]. Scale bars: 10 μm. D Localization of GFP control in the cytosol and the nucleus of TRV protoplasts. Scale bars:10 μm. E Trafficking assay of sporamin:GFP. Protein extracts prepared from protoplasts transformed with the sporamin:GFP construct were analyzed by western blotting using anti-GFP antibody. Mock indicates untransformed TRV protoplasts. F Trafficking assay of invertase:GFP. Protein extracts prepared from protoplasts (P) and medium (M) were analyzed by western blotting using anti-GFP antibody. G Endo H resistance of GKX glycans. Protein extracts from protoplasts transformed with the GKX construct were treated with endo H and analyzed by western blotting using anti-GFP antibody. S and R indicate endo H-sensitive GKX proteins (ER form) and endo H-resistant proteins (Golgi form), respectively.