FIGURE 4.

Forward translocation after reduction of the disulfide-bonded loop. A, a translocation intermediate of radiolabeled proOmpA (pOA) with cysteines at positions 175 and 202 was generated with proteoliposomes as in Fig. 1B, and an aliquot was immediately digested with proteinase K (lane 2). 10 mm DTT and excess unlabeled pOA were added, and samples were taken at the times indicated and digested with proteinase K (lanes 3–6). An aliquot of sample 2 was proteolyzed in the presence of Triton X-100 (TX-100)(lane 8). Lane 1 demonstrates translocation under reducing conditions. Lane 7 had hexokinase and glucose (HK/Gl) present from the onset of translocation. The sample in lane 9 received excess unlabeled pOA at the beginning of the translocation reaction. B, quantification of translocation assays performed as in A. For the disappearance of the intermediate, the intensities were normalized to that observed at t = 0. For the appearance of the full-length species, intensities were normalized with respect to the plateau level at t = 8 min.