FIGURE 4.

A collapse of the proton-motive force at the membrane vesicles interferes with cross-linking of precursor and TatA. A, pSufI was synthesized in vitro in the presence of Tat⁺-INV. CCCP at a final concentration of 100 μm or alternatively its solvent dimethyl sulfoxide (DMSO) was added during the incubation with INV as indicated. Translocation of pSufI was analyzed as described in the legend for Fig. 3C. B, cross-linking of pSufI and TorA-mCherry to the indicated Bpa variants of TatA in the absence or presence of 100 μm CCCP. UV-dependent adducts to TorA-mCherry and pSufI are labeled with stars and triangles, respectively. C, as in B except that CCCP was titrated from 0 to 200 μm. D, dissipation of the PMF by the combined addition of glucose and hexokinase 10 min before reactions synthesizing pSufI were supplemented with the indicated INV. Samples were either UV-irradiated to obtain adducts to TatA (triangles) or treated with proteinase K (PK) to monitor translocation.