Genetic Analysis of the Escherichia coli Tat Pathway

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DESCRIPTION (provided by applicant): The Twin Arginine Transporter (Tat) is a recently discovered pathway for protein secretion in bacteria and plants. The overall objective of this proposal is to elucidate the mechanism of protein secretion by the Tat pathway using genetic and biochemical means. A unique feature of this application is the development by the PI of novel high throughput screening methodologies that have greatly aided the genetic dissection of the Tat pathway. The proposed studies are focused on the dissection of some of the key early steps in Tat export: Specific Aim 1 will focus on leader peptide binding and the assembly of the translocon complex: 1.1 The analysis of the features of Tat leader peptides that mediate their recognition by the Tat apparatus and also the mechanism by which a subset of Tat leader peptides avoid misrouting into Sec. 1.2 Genetic identification of the TatC region that is responsible for the binding of the Tat consensus motif within the leader peptide. 1.3 The exploitation of a defective leader peptide and its cognizant TatC suppressor that specifically recognizes only that leader (isolated though the studies in 1.2) for the biochemical analysis of changes in Tat protein oligomerization and the events that lead to the formation of the translocon complex. In studies under Specific Aim 2 we will examine the folding quality control feature of Tat. We will seek to: 2.1 Isolate Tat mutations that disable the folding quality control feature of the pathway to permit the export of unfolded polypeptides. 2.2 Examine the role of TatB, and specifically its cytoplasmic domain which our preliminary studies reveal to have chaperone activity and therefore may be responsible for the binding of unfolded mature polypeptides and 2.3 Investigate in detail, the relationship between folding kinetics of substrate proteins and Tat export competence. Overall, the studies proposed in this application will lead to a significant advance in our understanding of the Tat pathway. The revised application is likely to yield significant and completely unique insights into the Tat pathway that, apart from their mechanistic significance, will be of considerable medical benefit, given the established importance of Tat export in bacterial pathogenicity.