Figure 4.

Ionophores impaired the membrane integration of psALB3 and SCY1 but had no effect on TIC40 and FTSH12. Chloroplasts treated without or with 1 μm nigericin and 2 μm valinomycin were incubated with in vitro translated precursor proteins and 5 mm ATP in import buffer (IB) for 20 min in light at 25°C. Intact chloroplasts were lysed and centrifuged to separate SE (St) from membranes (M). Membranes were further extracted with 0.2 m Na₂CO₃, 0.1 m NaOH, or OH⁻/CO₃²⁻ as shown above the gels. An equivalent aliquot of membranes from the SCY1, psALB3, and FTSH12 assays was treated with thermolysin (+T). OE23 localization (i.e. accumulation of iOE23) served as the positive control for the effect of ionophores. DP, Degradation product; i, intermediate protein; m, mature protein; p, precursor protein; P, pellet; S, supernatant; tp, translation product.