FIGURE 5.

Translocation arrest of TorA-PhoA requires a functional targeting to the Tat translocase. A, oxidized TorA-PhoA containing the wild-type RR motif as well as its KR and KK variants were analyzed for complete and partial translocation into Tat⁺-INV. B, effect of DCCD (0.5 mm) and CCCP (0.1 mm) on complete and partial translocation of SufI and oxidized TorA-PhoA into Tat⁺-INV. Stock solutions of both drugs were prepared in DMSO, which was added in equivalent amounts to control reactions. C, in contrast to CCCP, DCCD interferes with binding of oxidized TorA-PhoA to Tat⁺-INV. For experimental details see Fig. 2. D, oxidized TorA-PhoA was synthesized in the presence of Tat⁺-INV (control). INV were reisolated by centrifugation at 100,000 × g for 30 min at 4 °C and resuspended in compensating buffer (35) containing 3.2% (w/v) polyethylene glycol 6000, 2 mm DTT, 5 mm GSSG, and either no further additives or an ATP-regenerating system, and incubated for 25 min at 37 °C before PK treatment.