FIGURE 4.

PMF drives translocation of I₂₈ into mature OmpA. A, [³⁵S]proOmpA translocation was carried out in the presence of D209N SecA (60 μg/ml) as described in Fig. 3A, D209N SecA, for 15 min at 37 °C, followed by the addition of WT SecA (60 μg/ml). The time after WT SecA addition is indicated. At 20 min, PMF was imposed by adding 5 mm succinate. At each indicated time, an aliquot (25 μl) was withdrawn and chilled on ice to stop the reaction, followed by proteinase K (1 mg/ml) digestion. For the 15- and 20-min samples, the same amount (25 μl) was mixed with the same amount of a urea (8 m) solution, followed by incubation on ice for 5 min before proteinase K digestion (15* and 20*, respectively). The positions of proOmpA (p) and mature OmpA (m), together with those of intermediates (I₂₈, I₂₆ and I₁₈) are indicated by arrows. The specific band after urea treatment (asterisk) and that at the gel front are also indicated. B, the translocated bands detected in A were quantitated and plotted against time after WT SecA addition. The numbers of Met and Cys were considered to be as in Fig. 3B.