The bacterial twin-arginine translocase (Tat) pathway is able to export large folded proteins across the cytoplasmic membrane. It derives its name from the almost invariant twin-arginine motif present in the signal peptide of Tat substrates.\ud Components of the Tat pathway have been identified in many bacteria, and in Escherichia coli one of these essential proteins, TatA, forms large complexes of variable size. The research presented in this thesis sought to gain insight into the Tat pathway in E. coli and the cyanobacterium Synechocystis sp. PCC6803, mainlyfocusing on the TatA component of the E. coli Tat pathway and how its various domains contribute to function and complex formation.\ud Green fluorescent protein tagged with a Tat signal peptide was used to compare the Tat pathway in E. coli and Synechocystis, using biochemical and bioimaging techniques. Substitution of the twin-arginine motif with lysine residues highlighted differences between the specificity of the two systems.\ud The transmembrane (TM) domain and amphipathic helix (APH) of E. coli TatA were analysed using synthetic peptides and the TOXCAT assay. Investigation of the TM domain demonstrated it is α-helical and spontaneously inserts into membranes. It interacts relatively weakly and substitution of a glutamine does not affect these interactions or function of the protein at high expression levels. In contrast, the APH only forms an α-helical secondary structure in high detergent concentration or in the presence of negatively charged lipids, and does not insert into lipid bilayers. Analysis of the unstructured C-terminus of TatA led to the identification of an acidic motif just after the APH. Removal of this motif has a severe effect on function and complex formation of TatA. Removal of the C-terminus does not affect the proofreading abilities of TatA.\ud The research presented here has led to the proposal of a new model for TatA complex formation in E. coli – the weak interactions of the TM domains bring the monomers together, whilst interactions between the acidic motif and other regions stabilise these complexes
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