Fig. 2. Insertion of Toc34 and Toc34C++ into chloroplasts under various conditions. (A) [³H]-Leucine-labelled Toc34 was incubated for 5 minutes with chloroplasts (lanes 2-9) then treated with thermolysin (Thr, lanes 3, 5, 7 and 9). Before incubation, chloroplasts were incubated for 10 minutes with 10 µg of purified preSSU (pSSU, lanes 4 and 5) or 10 mM spermine (spm, lanes 6 and 7). Lane 1 shows 10% translation product (TP). (B) [³H]-Leucine-labelled translation product minus RNA (lanes 2 and 3), or Toc34C++ (lanes 5 and 6), was used for insertion into chloroplasts, which was followed by thermolysin treatment (Thr, lanes 3 and 6). Lanes 1 and 4 show 10% translation product. A model of the orientation of the wt protein and the charge mutant C++ is presented between A and B. (C) [³H]-Leucine-labelled Toc34 or [³⁵S]-methionine-labelled precursor of AOX (10% translation product in lane 1) were incubated with mitochondria for 15 minutes (lanes 2-4); mitochondria were then treated with 120 µg/ml thermolysin (lane 3, Th) or 100 mM Na₂CO₃ (lane 4, M) for 30 minutes at 4°C. Thermolysin activity was stopped by the addition of EDTA (lane 3) and membranes were isolated by centrifugation at 200,000 g (lane 3).