Mapping protein-protein interactions in the Escherichia coli Twin Arginine Translocase
type: CreativeWork
description: The Twin Arginine Translocase (Tat) system is a membrane-bound transport system present in plants and bacteria that has the remarkable ability to export fully folded proteins across a lipid bilayer, powered by the protonmotive force. In Escherichia coli the Tat system is composed of only three essential membrane proteins, the homologous TatA and TatB proteins, and the highly hydrophobic TatC core subunit. The transport mechanism involves a TatBC recognition complex which binds substrates through their signal peptides, with TatA being recruited to form an oligomeric structure that facilitates protein transport. Current models suggest that TatA oligomers locally disrupt membrane lipids allowing the substrate to move from the cytoplasm to the periplasm. Here, biochemical methods were used to further understand interactions between these components. Affinity purification experiments in detergent solution, using a His-tagged TatC variant, resulted in co-purification of TatB along with a small amount of associated TatA. The amount of co-purifying TatA was notably increased when TatB was absent, suggesting that the proteins may compete for the same binding site on TatC. Disulphide crosslinking experiments performed in vivo identified a binding site for TatA at the sixth transmembrane helix of TatC occupied in the resting state, indicating that TatA interacts constitutively with the TatBC recognition complex. Further crosslinking experiments found that the protonmotive force was required for TatA to occupy this constitutive site, explaining the poor yields of TatA co-purifying with TatC in detergent solution. In response to increased substrate flux through the Tat pathway, disulphide crosslinking demonstrated that TatA no longer occupied its constitutive site, instead binding at the fifth transmembrane helix of TatC, a site previously shown to be occupied by TatB. This supports the proposition that TatA and TatB occupy this site differentially, with TatB potentially acting …
abstract: The Twin Arginine Translocase (Tat) system is a membrane-bound transport system present in plants and bacteria that has the remarkable ability to export fully folded proteins across a lipid bilayer, powered by the protonmotive force. In Escherichia coli the Tat system is composed of only three essential membrane proteins, the homologous TatA and TatB proteins, and the highly hydrophobic TatC core subunit. The transport mechanism involves a TatBC recognition complex which binds substrates through their signal peptides, with TatA being recruited to form an oligomeric structure that facilitates protein transport. Current models suggest that TatA oligomers locally disrupt membrane lipids allowing the substrate to move from the cytoplasm to the periplasm. Here, biochemical methods were used to further understand interactions between these components. Affinity purification experiments in detergent solution, using a His-tagged TatC variant, resulted in co-purification of TatB along with a small amount of associated TatA. The amount of co-purifying TatA was notably increased when TatB was absent, suggesting that the proteins may compete for the same binding site on TatC. Disulphide crosslinking experiments performed in vivo identified a binding site for TatA at the sixth transmembrane helix of TatC occupied in the resting state, indicating that TatA interacts constitutively with the TatBC recognition complex. Further crosslinking experiments found that the protonmotive force was required for TatA to occupy this constitutive site, explaining the poor yields of TatA co-purifying with TatC in detergent solution. In response to increased substrate flux through the Tat pathway, disulphide crosslinking demonstrated that TatA no longer occupied its constitutive site, instead binding at the fifth transmembrane helix of TatC, a site previously shown to be occupied by TatB. This supports the proposition that TatA and TatB occupy this site differentially, with TatB potentially acting …
author: Moore, Kristoffer
language: eng
date published: 2016
date modified: 2026-07-24T02:08:19Z
provider: University of Dundee
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Dundee