Figure 11.

A substrate‐induced TatC self‐crosslink in transmembrane helix 5 of TatC.

A. Whole cells of strain DADE (ΔtatABCD ΔtatE) harbouring pTat101 co‐producing TatA, TatB and the single cysteine variant M205C of TatC, alone or with additional plasmid pQE80‐CueO were cultured as described in Experimental Procedures and subjected to oxidising (O) or reducing (R) conditions, or left untreated (control; C). Samples (50 μg of membrane protein) were resolved by SDS–PAGE (12% acrylamide), and TatC monomers and dimers visualised by western blotting using an anti‐TatC antibody.

B. Cells of the same strain harbouring pTat101 co‐producing TatA, TatB and the single cysteine variant M205C of TatC together with the indicated additional TatC substitutions alone (top panel) or with pQE80‐CueO (middle and bottom panels) were treated as in part A and samples (50 μg of membrane protein) were resolved by SDS–PAGE (12% acrylamide), and blotted with anti‐TatC (middle panel) or anti‐his antibody (bottom panel). Note that the fast migrating band detected on the anti‐his blot for the oxidised sample most probably represents a crosslinked form of CueO containing an intramolecular disulphide.