Figure 3.

TatA^L9C interacts with TatC^F213C in vivo. (a,e,g,h) Western blot analysis (separated on 12.5% polyacrylamide gels) of whole cells of E. coli strain DADE-P producing the indicated Cys substitutions in TatA and TatC (and wild-type TatB, from plasmid pUNITATCC4) following exposure to 1.8 mM CuP (oxidizing) or 10 mM DTT (reducing) for 1 min. Cross-linked products were visualized by immunoblotting using anti-TatC antibodies. The asterisk in (h) indicates a faint TatAC cross-link. (b) The TatA^L9C–TatC^F213C oxidized (O) and reduced (R) samples from (a) were separately probed with an anti-TatA antibody (note that the TatA monomer that is in large excess has been run off the bottom of the gel). (c,f) Cells of strain DADE harbouring plasmid pTAT101 producing (c) TatA^L9C and wild-type TatB along with either TatC^V212C or TatC^F213C, or (f) TatA^S5C, wild-type TatB and TatC^F213C, as indicated, were incubated with 1.8 mM CuP (O) or 10 mM DTT (R) for 1 min. (d) Structural model of TatA interacting with TatC at the TatA constitutive binding site. The backbone distances between TatA^S5/L9/TatC^F213, TatA^I6/TatC^V212, TatA^A13/TatC^I220 and TatA^V17/TatC^E227 are shown. (i) Cells of strain DADE producing TatA^L9C and wild-type TatB alongside TatC^{F94A,E103A,M205C} or TatC^{F94A,E103A,F213C} (annotated TatC^FEA,M205C or TatC^FEA,F213C, respectively) from pTAT101 were left untreated (C) or incubated with 1.8 mM CuP (O) for 1 min. For (c,d), following quenching, membranes were prepared, samples separated by SDS-PAGE (12.5% polyacrylamide) and immunoblotted using an anti-TatC antibody.