Figure 4.

AtRMR1 deletion mutants inhibit the trafficking of phaseolin to the PSV. (A) Inhibition of phaseolin trafficking to the PSV by AtRMR1 deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of the reporter proteins was examined. GFP signals of AALP-GFP and sporamin-GFP were observed from intact protoplasts, whereas phaseolin was detected from fixed protoplasts by immunostaining with antiphaseolin antibody. Un, untransformed protoplasts; CH, chloroplasts. Insets, bright field images of protoplasts. Bars, 20 μm. (B) Quantification of trafficking efficiency. To estimate the trafficking efficiency, transformed protoplasts were counted based on their GFP or immunostaining patterns. More than 100 protoplasts were counted for each transformation in a triplicate experiment. The numbers and error bars indicate the means and SEM, respectively. (C) Western blot analysis of vacuolar trafficking of AALP-GFP and sporamin-GFP. Protein extracts were prepared from protoplasts transformed with the indicated constructs and used for Western blot analysis using anti-HA or anti-GFP antibodies. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA; R6, an empty vector used to balance the amount of plasmid DNA that was introduced into protoplasts. Single arrowhead, precursor; double arrowhead, proteolytically processed form.