AbstractThe Tat protein export system transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. In Escherichia coli, the Tat system is composed of the TatA, TatB and TatC proteins. TatB and TatC assemble into a multimeric receptor complex that recognises and binds the substrate, before the TatA protomers cluster at the TatBC complex to facilitate substrate transport.
A genetic screen was devised to explore the oligomeric state of TatC, reasoning that the isolation of dominant negative TatC variants that inactivate the Tat system in the presence of a functional copy of wild type TatC would provide strong evidence TatC is an obligate oligomer. Single dominant negative TatC substitutions were isolated that were located in the first and second periplasmic loops of TatC. These substitutions did not prevent TatC from interacting with TatB, TatA, itself or with a Tat substrate. Blue Native PAGE analysis showed that the TatC variants were unable to form the 440 kDa TatBC complex. Surprisingly, the substitutions did not prevent TatC:TatC self-interactions in the periplasmic regions, detected by disulphide cross-linking, but they did abolish a substrate-induced interaction at the fifth transmembrane helix of TatC. Fluorescence microscopy experiments revealed that the dominant negative TatC variants prevented the polymerisation of TatA-YFP in vivo. These results show that TatC possesses at least two interaction interfaces and imply that the periplasmic loops are critical for the transition between substrate binding and TatA polymerisation.
Accessibility of single cysteine substitutions in TatC was probed by PEG-Mal labelling in intact cells. TatB was shown to be important for the proper insertion of TatC into the membrane. The absence of TatA led to accessibility changes in the vicinity of the fifth transmembrane domain of TatC, where both TatA and TatB are known to dock. This suggests that TatA and TatB may share an overlapping binding site.
|Date of Award
|Biotechnology and Biological Sciences Research Council
|Tracy Palmer (Supervisor)
- Escherichia coli