Precursor to cpTatC possesses a stromal targeting transit peptide. (a) Radiolabeled pSSU, pcpTatC, and mature cpTatC (mcpTatC) were each incubated with isolated stroma and 3 mM PMSF with or without 10 mM 1,10-phenanthroline (OP), as shown above the panel, at 25°C, for 30 minutes. Samples were analyzed using SDS-PAGE and fluorography. (b) Scheme illustrating swapping of the pSSU and pcpTatC transit peptides. Numbers above constructs represent the number of amino acids from the translation start site. (c) Radiolabeled pcpTatC, SSUtpcpTatC, pSSU, and cpTatCtpSSU were each incubated with chloroplasts in a protein import assay (Experimental Procedures) for 20 minutes. Chloroplasts were treated with 100 μg/mL thermolysin and then re-purified, washed, lysed, and fractionated. Translation product (Tp) equivalent to 5% of each assay, chloroplasts (Cp), stroma (S), membranes (M), and thermolysin-treated membranes (+T) were analyzed using SDS-PAGE and fluorography. The positions of molecular weight markers are shown at left.