Mutations in basic cytosolic loops of SecYEG abolish its stimulatory effects. (A) Stimulated GTP hydrolysis by the RNC-bound SRP and FtsY in the absence (open circles) and presence (closed circles) of 12 µM mtSecYEG. The data were fit to Eq. 3. The broken line is the data for wild-type SecYEG from Fig. 2 A. (B) Effect of SecYEG charge reversal mutants on GTP hydrolysis from the RNC–SRP–FtsY complex. Solid black, R255E/R256E/R357 (mtSecYEG); red, R357E; blue, R255E/R256E. The broken line is the data for wild-type SecYEG from Fig. 2 B. (C) Equilibrium titration of the early complex in the presence of 24 µM mtSecYEG (closed circles). The data were fit to Eq. 1 and gave a Kd value of 162 ± 4 nM. Titration in the presence of DDM (solid line) and wild type SecYEG (broken line) are from Fig. 1. (D) Effect of mtSecYEG on the stability of the early complex. Kd values were determined as described in C. The data with wild-type SecYEG (broken line) are from Fig. 1 C. (E) Equilibrium titration of the early targeting complex formed by FtsY-NG in the presence of 0.05% DDM (black) or 24 µM SecYEG (red). The data were fit to Eq. 1 in the Materials and methods, and Kd values are given in F. (F) Summary of the effects of SecYEG on the stability of the early targeting complex formed with full-length FtsY and FtsY-NG. The data with full-length FtsY are from Fig. 1. A, C, D, and E show representative data from two to three replicates. Error bars in B and F are standard deviations from two to three experiments.
