Figure 4.

Dominant negative point substituted variants of TatC interact with TatB and TatA. Crude membrane fractions of the E . coli strain DADE (Δtat ABCD ΔtatE) harbouring pREP4 (Zamenhof and Villarejo, 1972) and over‐producing TatA, TatB and hexa‐histidine‐tagged wild‐type or amino‐acid substituted TatC, as indicated were solubilised with digitionin and the TatC‐his protein purified using nickel‐charged beads as described in Experimental Procedures. In each case a sample that was loaded onto the beads (load) along with the sample that was eluted from the beads (elute) were separated by SDS–PAGE (12% acrylamide), electroblotted and immunoreactive bands were detected with either anti‐his, anti‐TatB or anti‐TatA antisera. As a control, solubilised membranes from strain DADE/pREP4 overproducing TatA, TatB and a non‐tagged variant of TatC (lanes labelled No his) were used to show that there was no unspecific binding of TatA and TatB to the beads. Five microlitres of sample was loaded in each lane.