Analysis of ATP-driven translocation activity of reconstituted cross-linked SecA–SecYEG complexes. (A) (Top) steady-state ATPase activity of 0.3 µM SecA, SecA_Y794C, or SecA_A795C in the presence of saturating (1 µM) membrane reconstituted SecY_K268CEG, compared with proteoliposomes containing only the cross-linked complexes (A₇₉₄–Y₂₆₈EG and A₇₉₅–Y₂₆₈EG); in each case with or without 0.7 µM proOmpA. The results are the average of three independent experiments. (Bottom) corresponding levels of translocation activity analyzed by anti-proOmpA immunoblot and compared with wild-type levels of translocation. The results were quantified using ImageJ software; n = 3 (bottom). See Fig. S2, A and B for representative blots and time courses of the translocation assays. (B) As in A but using SecY₁₈₃. See Fig. S2 C for a representative blot. The ATPase results shown are the average of five independent experiments.