FIGURE 3.

Cross-linking of an RR precursor to translocase-embedded TatA. A, immunoblot analysis of the TatABC contents of INV that had been prepared from cells overproducing, besides TatBC, wild-type TatA (Tat⁺-INV, here denominated TatA(wt)BC) or the L10Bpa variant of TatA (TatA(L10)BC)). TatA(L10)ΔBC INV were obtained from a strain overexpressing selectively the TatA variant and therefore containing only the low amounts of chromosomally expressed TatBC. The amounts of vesicles applied to SDS-PAGE and immunoblot analysis were normalized with respect to their adsorption at 280 nm (54). B, no cross-linking to TatA in the presence of substoichiometric amounts of TatBC. For experimental details, see the legend for Fig. 1. UV-dependent adducts to pSufI are labeled with triangles. C, pSufI was synthesized in vitro in the presence of the indicated INV. To assay translocation of pSufI into these vesicles, one aliquot of each reaction was treated with proteinase K (PK) to visualize the vesicle-protected, i.e. translocated fractions of the precursor and mature form of SufI (pSufI and mSufI, respectively). In the absence of high amounts of TatBC, translocation is negligible. D, pSufI was synthesized in vitro in the presence of the indicated INV. Samples were UV-irradiated as indicated and either directly TCA-precipitated or first treated with Na₂CO₃ and separated into soluble (S) and pelletable (P) material.