Figure 6.

Pro residues in the TP are crucial for efficient import of an inner envelope membrane protein AtTic110 into chloroplasts. A, Sequences of AtTic110[1-80] and its P/A substitution mutant. P/A substitutions are indicated in red. B, Localization of reporter proteins. Protoplasts were observed 12 h after transformation. Green, red, and yellow signals represent GFP, chlorophyll autofluorescence, and overlap between green and red signals, respectively. Scale bar = 20 μm. C, Western-blot analysis. Protein extracts from protoplasts transformed with the indicated constructs were analyzed by western blotting using anti-GFP antibody. Pre, precursor form; Pro, processed form. D, Chloroplast isolation. Chloroplasts were isolated and analyzed as described in Figure 1D. E, Thermolysin sensitivity of precursors. Protoplasts transformed with the indicated constructs were gently lysed and treated with thermolysin. Subsequently, protein extracts were analyzed by western blotting using anti-GFP, anti-Toc159, and anti-Tic110 antibodies. Pre, precursor form; Pro, processed form.