Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli.

Johann Habersetzer,Jon Cherry,Tracy Palmer,Phillip J Stansfeld,Kristoffer Moore,Grant Buchanan

doi:10.1098/rsob.170091

Johann Habersetzer, Jon Cherry + Show 4 more

Open Access

https://doi.org/10.1098/rsob.170091

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Journal: Open biology	Publication Date: Aug 1, 2017
Citations: 26	License type: cc-by

Affiliation: University of Dundee, University of Oxford

Abstract

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

Highlights

The twin-arginine protein transport (Tat) pathway operates in parallel with the general secretory (Sec) pathway to export proteins across the cytoplasmic membrane of bacteria and archaea, and the thylakoid membrane of plant chloroplasts
Experiments where that cross-links between Tha4 (TatA) or TatB were present individually as the sole TatA/B family protein indicated that each of these proteins was capable of occupying both sites
Locking the Tat system into the resting state through the introduction of substitutions in TatC that prevent signal peptide binding demonstrated that TatB occupies the polar cluster site under these circumstances, as proposed previously [15], with TatA occupying the newly identified site

Summary

Introduction

The twin-arginine protein transport (Tat) pathway operates in parallel with the general secretory (Sec) pathway to export proteins across the cytoplasmic membrane of bacteria and archaea, and the thylakoid membrane of plant chloroplasts. Tat substrates have N-terminal signal peptides containing a conserved twin-arginine motif and are transported across the membrane in a folded state driven by the protonmotive force [1 –3]. The Tat machinery comprises membrane proteins from the TatA and TatC families. Most Gram-negative bacteria, and plant thylakoids, have two functionally distinguishable TatA paralogues (TatA and TatB in bacteria) that have distinct roles in Tat transport Tat transport is initiated by binding of the signal peptide of a Tat substrate to the Tat(A)BC receptor complex. This complex, which contains several copies of TatB and TatC, is multivalent and appears to function as an obligate oligomer [10,11,12,13]. The Tat(A)BC complex is stable and can interact with substrates in the absence of TatA [13,14], it is likely that in vivo some TatA constitutively associates with this complex, most likely in an equimolar ratio with TatB and TatC [10,15,16,17]

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