Quality control of substrate conformation in the Escherichia coli Twin Arginine protein-targeting pathway

In Escherichia coli, the twin arginine translocase (Tat) is one of the major protein

translocation mechanisms. The Tat system has the ability to transport folded proteins

across the inner membrane. Therefore, it has the ability to discriminate between folding

states. However, it is not well understood how the Tat system senses the folding state of a

substrate. In this study we probed the Tat proofreading mechanism and we investigated

whether Tat substrates in E. coli are translocated by the Tat system due to their rapid

folding kinetics. We demonstrate that the E. coli Tat machinery can process a de-novo designed substrate

(BT6 maquette). Moreover the Tat proofreading mechanism can discriminated between

different folding states of this substrate. This data and the fact that this simple four helix

artificial substrate offers a lot of engineering freedom, suggests that BT6 is an ideal

candidate to study the Tat proofreading mechanism (chapter 3).

In chapter 4, we focussed on the Tat system’s proofreading ability by substituting substrate

surfaces of BT6 maquette. Mutants with substituted surface properties were expressed in

order to understand what Tat senses as folded. Expression assays showed whether the

mutants were accepted or rejected by Tat. We propose that the proofreading system does

not sense a global unfolded state of the substrate but has the ability to sense localised

unfolded regions. Finally, we tested whether Tat substrates fold co- or post-translationally to determine the

speed of the folding kinetics by using an arrest peptide-mediated force measurements assay

(chapter 5). This study was to increase our understanding about the rationale for using the

Tat system.