Figure 3.

A Tat precursor interacts transiently with TatB before its translocation. (A) The two indicated Bpa variants of pSufI were synthesized in the presence of INV, one-half of each reaction containing the uncoupler CCCP at 0.1 mM, the other half only the solvent DMSO. Samples were either digested with Proteinase K (PK) to visualize protease-resistant (i.e., translocated precursor and mature forms of SufI) or otherwise irradiated with UV light. The labeled photo-adducts are those identified in Figure 2. Whereas CCCP completely blocks translocation of pSufI into INVs and also abolishes cross-linking to TatA, the TatB adducts of pSufI were not affected by the uncoupler. (B) After synthesis of pSufI in the presence of INV, vesicles were collected by centrifugation, resuspended, and incubated in the absence or presence of an ATP regenerating system (cf. Materials and Methods). The ATP-dependent increase in translocation is paralleled by a loss of cross-linking to TatB.