Substrate-transporter complexes in the twin-arginine protein transport system

Lead Research Organisation: University of Oxford
Department Name: Biochemistry


Some proteins operate on the outside of the bacterial cell, for example the toxins produced by bacterial pathogens. Since all proteins are made inside the bacterium the extracellular proteins must be moved out of the cell across the normally impermeable cell membrane. This task is carried out by machines termed protein transporters that are located in the cell membrane. One type of transporter moves unfolded proteins, threading them across the membrane like string through the eye of a needle. By contrast, a second type of transporter, which we term the Tat system, moves folded proteins across the membrane. This is much more challenging than threading and so it is thought that the Tat system operates by an unusual mechanism. The Tat system is required for many bacterial processes including energy generation, cell division, pathogenesis, and the nitrogen-fixing symbiosis of soil bacteria with plants. The Tat protein transport system is not only found in bacteria but is also present in the chloroplasts of plants where it is essential to form and maintain the proteins required to carry out photosynthesis. The heart of the Tat system is a complex formed by two proteins called TatB and TatC. This complex sits in the cell membrane and recognises and binds the proteins that are to be transported. It then binds another protein called TatA to form the active transporter. This project aims to characterise the complexes formed between the Tat components and the transported protein in more detail with the aim of increasing our understanding of the unusual mechanism of the Tat system. The Tat system is a possible drug target because it is required for bacterial pathogenesis but is not found in humans. It is also of biotechnological interest because it could be utilised to secrete useful protein products.

Technical Summary

The Tat system of bacteria and chloroplasts carries out the unusual, and mechanistically challenging, task of moving folded proteins across biological membranes. The membrane proteins TatA, TatB and TatC are the essential components of the Tat pathway. During the Tat translocation cycle they are believed to form a series of dynamically changing complexes with the substrate protein. This proposal builds on our success in developing methods to trap, purify in biochemical quantitites, and structurally characterize a transport intermediate involving TatB, TatC, and substrate. A series of experiments will probe Tat mechanism by: [a] Attempting to trap and purify analogues of the fully assembled translocation site containing substrate and TatA, TatB, and TatC. [b] Testing whether the structural re-arrangement of the TatBC complex upon substrate binding that we have identified by electron microscopy (i) requires the transmembrane proton electrochemical gradient (ii) involves protomer loss from the complex. [c] Testing whether the transmembrane proton electrochemical gradient causes the TatBC complex to bind substrate more tightly. [d] Exploiting our discovery of a stable TatAC-substrate complex to (i) investigate the nature of the interactions of TatA with TatC and with substrate and (ii) to investigate the different functional roles of the homologous TatA and TatB proteins.
Description The twin arginine (Tat) protein transport system is involved in moving folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Tat system is of potential use as pathway to produce proteins of industrial or pharmacological utility.
A membrane bound receptor protein complex containing the proteins TatB and TatC recognises `signal peptides' on proteins that will be transported by the Tat system. Our work characterised the interactions between the signal peptide and TatBC using a range of biochemical and biophysical techniques.
We identified two modes of signal peptide binding which differ in their binding strength. We found that there was one high affinity binding site in each TatBC complex but multiple low affinity binding sites. This implies a functional asymmetry with the TatBC complex which we infer to be of mechanistic importance. We found that the TatB protein is essential to the formation of the high affinity binding site. These results have significant implications for the mechanism of the Tat transporter.
Exploitation Route Demonstrates that techniques used to analyse protein-protein interactions between water-soluble proteins can be adapted to the analysis of much larger, multi-subunit, membrane protein complexes.
Sectors Pharmaceuticals and Medical Biotechnology

Description Substrate-receptor interactions in the Tat protein transport pathway
Amount £460,972 (GBP)
Funding ID MR/K000721/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2013 
End 01/2016
Description School science demonstrations 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Engagement of children with science.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013