Figure 2.

Golgi-dependent trafficking of phaseolin in Arabidopsis protoplasts. A, Western-blot analysis of phaseolin under various conditions. Arabidopsis protoplasts were transformed with phaseolin alone or together with AtSar1[H74L]. To inhibit glycosylation, tunicamycin (10 μg mL^–1) was added to the incubation medium right after transformation and the protoplasts were incubated for 24 h. Protein extracts were prepared from the protoplasts and were treated with endo H. The migration patterns of phaseolin in these protein extracts were analyzed by western-blot analysis using an anti-phaseolin antibody. Bands I and II indicate glycosylated and unglycosylated phaseolin, respectively. Arrowheads indicate fragmented forms of phaseolin. B, Effect of AtSar1[H74L] on the secretion of phaseolin Δ418. Protoplasts were transformed with phaseolin Δ418 alone or together with AtSar1[H74L]. Protein extracts were prepared from the protoplasts (Cell) and medium (Med) and were analyzed for the presence of phaseolin Δ418 with the antiphaseolin antibody. C, The effect of BFA on the destiny of phaseolin. Protoplasts derived from leaf cells of Arabidopsis were transformed with phaseolin and were incubated in the presence (+BFA) or absence (–BFA) of BFA for 24 h. Protein extracts were prepared and analyzed for phaseolin by western-blot analysis. Arrow and arrowheads indicate intact and three fragmented forms of phaseolin, respectively. D, Localization of phaseolin in the presence of AtSar1[H74L]. Protoplasts were transformed with phaseolin and AtSar1[H74L] or phaseolin Δ418 and AtSar1[H74L] and localization of phaseolin proteins was detected with antiphaseolin antibody in fixed protoplasts. Bar = 20 μm. E, Localization of phaseolin in the presence of BFA. Arabidopsis protoplasts transformed with phaseolin or sialyltransferase (ST):green fluorescent protein (GFP) plus binding protein (BiP):red fluorescent protein (RFP) were incubated in the presence (+BFA) or absence (–BFA) of BFA for 6 to 24 h, and localization of phaseolin was detected by immunohistochemistry using the antiphaseolin antibody followed by FITC-labeled anti-rabbit IgG antibody. ST:GFP and BiP:RFP were detected directly by GFP and RFP signals, respectively, from intact protoplasts. Arrowheads indicate ST:GFP localized at the Golgi apparatus. CH, Chloroplasts. DsRed was used to generated BiP:RFP. Bar = 20 μm. F, Quantification of localization patterns. The number of protoplasts was counted based on the pattern shown in E (a and c) for phaseolin. More than 50 protoplasts were scored at each time and at least three independent experiments were performed. Disc and ER indicate the patterns shown in E (a and c, respectively).