Figure 3.

Import reactions were initiated by the addition of chloroplasts to 1× IB (final concentration in the reaction was 0.33 mg/mL chlorophyll) containing radiolabeled prOE17, 5 mm MgCl₂ and 5 mm ATP. A, Reactions incubated in the absence of both inhibitors; B, reactions incubated with azide alone; C, reactions incubated with nigericin alone; and D, reactions incubated with both inhibitors. Lanes 1, Intact chloroplasts; lanes 2, washed thylakoids (containing contaminating envelope membranes); lanes 3, supernatant fraction after envelope lysis of sample in lanes 2 containing stromal components and envelope membranes; and lanes 4, protease-treated thylakoids. The reactions were treated as in Figure 1. After this incubation, the samples were placed back in the light at 25°C for an additional 15 min. The chloroplast envelopes in lanes 2 and 4 were osmotically lysed in lysis buffer (see Methods) containing 2 μm nigericin, whereas the samples in lanes 1 were maintained in isotonic buffer (1× IB with 2 μm nigericin). Reactions were incubated on ice in the dark for 5 min. The osmoticum was then restored to samples in lanes 2 and 4 with the addition of 2× IB containing 2 μm nigericin (an equivalent volume of 1× IB with 2 μm nigericin was added to the samples in lanes 1). Samples in lanes 4 were protease treated with thermolysin (200 μg/mL with 5 mm CaCl₂); samples in lanes 1 and 2 received 5 mm CaCl₂ only. After inactivating thermolysin with the addition of 25 mm EDTA, membranes were pelleted and the supernatant collected from the samples in lanes 2. This supernatant was precipitated with 1.5 m PCA and loaded in lane 3. The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p) and mature (m) proteins are noted.