Interactions between the TatBC complex and Tat signal peptides during protein transport by the bacterial Tat pathway

Abstract

The Tat and Sec pathways operate in parallel to translocate proteins across the prokaryotic cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Unlike the Sec pathway, which translocates unfolded proteins, substrates of the Tat system are transported in a folded state. In Escherichia coli, the Tat system consists of three membrane proteins, TatA, TatB and TatC. Substrates are targeted to the Tat machinery via N-terminal signal peptides that have a tripartite structure, with a polar n-region, a hydrophobic h-region and a polar c-region. They critically contain an almost invariant twin-arginine (RR) motif within the n-region that is essential to trigger Tat transport. Tat signal peptides interact with the Tat receptor complex that contains multiple copies of TatA, TatB and TatC, probably in 1:1:1 ratio. Substrate binding at the receptor drives recruitment of further TatA molecules to form an oligomer that facilitates substrate transport across the membrane by an unknown mechanism.
In this study, genetic screens have been utilised to gain insight into the role of the Tat signal peptide in protein translocation. Two classes of genetic suppressors have been identified that allow normally inactive substitutions at the signal peptide RR-motif to be recognised by the Tat system, and that can also compensate for inactivating substitutions in the signal peptide binding site on TatC. The first class of the suppressors fell primarily within the transmembrane region of TatB. Biochemical analysis indicated that the suppressors did not act by restoring binding of the substituted signal peptides to the Tat machinery. Instead, they caused conformational change to the receptor complex allowing it to more readily transition to the assembled state. The second class of suppressors were isolated in the signal peptide h-region and conferred increased signal peptide hydrophobicity. These suppressors restored signal peptide binding to the Tat receptor complex. The Tat system was shown to functionally interact with highly hydrophobic signal peptides including two bona fide Sec targeting sequences. This study indicates that Tat signal peptides have dual functionality, to both target substrates to the Tat machinery and to trigger its assembly and that there is unprecedented overlap between Sec and Tat signal peptides.

Date of Award	2017
Original language	English
Awarding Institution	University of Dundee
Supervisor	Tracy Palmer (Supervisor)