The bacterial twin-arginine translocation (Tat) pathway has the unique ability to export pre-folded proteins across the cytoplasmic membrane. Its name came from the almost invariant twin-arginine motif in the signal peptide of Tat substrates. Escherichia coli, a Gram-negative bacterium, is typically used to understand Tat function in bacteria. Until now, evidence has shown that TatA, TatB and TatC comprise the minimally functional unit, moreover, a quality control system exists to monitor the assembly of cofactors and the correctly folding state of proteins which avoids the futile export and initiates the degradation of rejected molecules. The research presented in this thesis sought to gain insight into the quality control mechanism of the Tat pathway in E. coli, and also study the relationship between transport and maturation of substrates. In the first place, a novel Tat substrate, YedY, was used to analyse the nature and variety of proofreading functions operating in conjunction with the Tat pathway. The single substitutions in three predicted ligands for the YedY molybdopterin centre led to complete inhibition of export and variable degradation of mutated YedY forms, indicating an effective proofreading activity. Circular dichroism spectroscopy and inductively coupled plasma mass spectrometry of purified proteins demonstrated the change of secondary structures between YedY and mutated variants, and also indicated the content of Mo in molybdopterin cofactor within proteins. The data suggested that the three mutated forms failed to correctly assemble cofactor which resulted in rejection by the Tat export pathway on the basis of the different changes of secondary structures. Further analysis shows that none of the known export chaperones for molybdenum cofactor-containing Tat substrates is required for YedY biogenesis; export is unaffected in cells lacking DmsD and TorD. In the second place, maturation of pre-YedY was blocked when an Ala>Leu substitution was made at the -1 position of the signal peptide, and a membrane-bound precursor form accumulated in the membrane. However, the mature domain had been transferred to the periplasm. The accumulation did not block transport of other Tat substrates, indicating the precursor exited from the translocation channel and integrated into the membrane bilayer. Since the precursor was not detected in the periplasm, it was suggested that the precursor has undergone lateral transfer into the bilayer during translocation. These results are discussed in relation to the overall mechanism of translocation and proofreading by the Tat pathway in E. coli.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:560105 |
| Date | January 2011 |
| Creators | Ren, Chao |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/45794/ |
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