FIGURE 1.

Backsliding of a translocation intermediate after ATP depletion. A, proOmpA (pOA) derivatives with cysteines at positions 175 and 202 or 164 and 202 were translocated into urea-washed inverted membrane vesicles (IMVs) under oxidative conditions for 15 min at 37 °C. After formation of a translocation intermediate (lanes 2 and 11), the samples in lanes 7 and 16 were further incubated with DTT and ATP (Chase). The other samples were depleted of ATP by addition of hexokinase and glucose (HK/Gl), placed at 37 °C, and aliquots were taken at the times indicated (lanes 3–6 and 12–15). The samples in lanes 8 and 17 received HK/Gl at the beginning of the translocation reaction. Lanes 1 and 10 demonstrate full translocation under reducing conditions. Aliquots of samples 2 and 11 were proteolyzed in the presence of Triton X-100 (TX-100)(lanes 9 and 18). All samples were treated with proteinase K, separated by SDS-PAGE, and analyzed by autoradiography. B, as in A, but with proteoliposomes (PL) containing purified E. coli SecY complex. C, quantification of backsliding from A and B. The intensities were normalized to that of the protected fragment at t = 0, and the background was subtracted. The mean and S.D. of three independent experiments are shown.