Figure 1.

The prRBCS transit peptide directs the specific binding and import of prRBCS-GST into chloroplasts. A, Schematic representation of constructs used in this study. The red rectangles represent the transit peptides, and the blue rectangles represent the mature protein regions or the passenger proteins. The numbers above each construct indicate the positions of Cys residues, with the first residue of the mature protein designated as +1 and residues in the transit peptide designated with negative numbers. The AtToc159G recombinant protein construct, composed of AtToc159 residues 727 to 1,092 with a C-terminal His₆ tag, also is depicted. All constructs are drawn to scale. a.a., Amino acids. B, Binding and import of prRBCS-GST into isolated chloroplasts. GST (lane 1) and prRBCS-GST (lane 2) were purified from the soluble fraction of E. coli. Two different amounts were incubated with ATP-depleted chloroplasts in the presence of 0.1 and 3 mm ATP for the binding and import reactions, respectively. Samples from the import reaction were further treated with 0.2 mg mL⁻¹ thermolysin. Samples were analyzed by SDS-PAGE and immunoblotting with anti-GST antibody.