Figure 3.

CmTic110 is imported into pea chloroplasts. (a) In vitro translated [³⁵S]Met-CmTic110 (Tr, lane 1) was treated with thermolysin directly (lane 2) or incubated with isolated pea chloroplasts (Cpt, 100 μg chlorophyll) under import conditions for 30 min. After import, a small portion of the chloroplasts (10 μg chlorophyll) were centrifuged through a 40% Percoll cushion to re-isolate intact chloroplasts (lane 3). The rest of the chloroplasts were digested with thermolysin, and then recovered through a 40% Percoll cushion (lane 4). Some of the thermolysin-treated chloroplasts (81 μg chlorophyll) were further lysed hypotonically and separated into membrane (M, lane 5) and soluble (S, lane 6) fractions by centrifugation. Samples were analyzed by SDS-PAGE, Coomassie blue staining and fluorography. Twenty micrograms of proteins were loaded in lanes 3–6. The arrow indicates the full-length CmTic110 and the bracket marks the three imported and process CmTic110 inside chloroplasts. LS, large subunit of ribulose biphosphate carboxylase in the stroma, which serves as a marker for the soluble fraction; CAB, chlorophyll a/b-binding protein of the thylakoid membrane, which serves as a marker for the membrane fraction. (b) Import of CmTic110D363x. The experimental conditions and lane designations are the same as (a), except lane 5 is the soluble fraction and lane 6 is the membrane fraction.