Understanding the TatA channel of the twin-arginine protein translocase

Lead Research Organisation: University of Oxford
Department Name: Biochemistry


Some bacterial proteins operate on the outside of the 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. The Tat protein transport system is also found in plant chloroplasts. One essential component of the Tat system is the protein TatA. Multiple copies of TatA assemble to form a large complex. We have used electron microscopy to visualize the TatA complex. TatA forms a doughnut-shaped structure. This suggests that TatA forms a channel across the cell membrane with proteins being transported through the central hole. A surprising feature of TatA is that individual TatA complexes have different sizes, apparently because they contain different numbers of TatA proteins. It is possible that this size variation allows TatA to pack tightly around the protein that is being transported and thus prevent the co-transport of other molecules. However, the TatA complex may be flexible for a number of other plausible reasons. In this project we want to increase our understanding of how the TatA protein works. In particular we want to learn the significance of the variation in the size of the Tat complexes. The electron microscopy images are too fuzzy to allow us to determine the position or shape of individual proteins within the TatA complex. The organization of the subunits in the complex will instead be probed using methods that determine which parts of the TatA molecule are in contact with other TatA molecules by chemically tying the two proteins together. These links will also be used to determine which parts of the TatA protein have to move relative to other parts of the protein to allow TatA to carry out its function. We will investigate whether the variability in the size of the TatA complexes is linked to tailoring the size of the hole to the substrate by asking whether the size of the ring varies when transporting proteins of differing sizes. We will also test whether individual TatA proteins enter and leave the TatA complex during transport since this is a prediction of the substrate-fitting explanation of the size variations. These and other experiments are expected to give a clearer picture of the operation of TatA. 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 used to secrete useful protein products.

Technical Summary

The membrane proteins TatA, TatB and TatC are the essential components of the Tat protein transport pathway. TatA and TatBC form separate, highly oligomeric, complexes. The TatBC complex functions as a receptor for the substrate protein. TatA is proposed to form the transmembrane protein translocation channel. This suggestion is supported by our recent low resolution structure of the Escherichia coli TatA complex, obtained by negative stain electron microscopy, which shows a variable-diameter ring occluded at one end by a lid structure. This project aims to test, exploit and expand the structural and mechanistic insights arising from our low resolution EM structure of the TatA complex. [1] We have used scanning Cys mutagenesis in combination with oxidant-induced disulfide crosslinking to establish the interfacial contacts between TatA protomers. These experiments will be extended to determine the pattern of local interactions between the TatA protomers by constructing selected TatA variants containing two Cys substitutions. [2] Site-specific disulfide crosslinking will be used to identify conformational changes in TatA that are necessary for Tat transport. TatA variants that exhibit quantitative disulfide crosslinking will be employed and the effect of the crosslinks on Tat transport in vitro will be assessed. [3] We will investigate the mechanistic rationale for the observed variation in size of the TatA complex. It is possible that this size variation allows TatA to pack tightly around the protein that is being transported (Assembly/Disassembly Model). However, it is also possible that TatA protomer interactions are weak for mechanistic reasons and that as a consequence TatA is unstable in detergent solution. We will attempt to discriminate between these two possibilities by: (i) testing whether, as predicted by the Assembly/Dissassembly model, transport of proteins of very different sizes change the size distribution of the TatA complex. This will be assessed by blue native-PAGE. (ii) testing whether, as predicted by the Assembly/Dissassembly model, Tat transport results in subunit exchange between complexes. TatA in growing cells will be labelled with a pulse of [35S]methionine. We will then induce synthesis of an affinity-tagged TatA molecule and determine whether affinity-purified TatA complexes contain radiolabelled protomers. [4] Use single channel recording methods to test whether the TatA complex contains an aqueous, rather than lipid-filled, pore. Proteolysis will be used to remove the lid structure that appears to block access to the pore. [5] The orientation of the TatA amphipathic helix relative to the membrane bilayer is a crucial difference between certain models of TatA structure and mechanism. We will determine the orientation of this helix using Attenuated Total Reflection-Fourier Transform Infrared spectroscopy of aligned membranes.


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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 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.
The TatA protein is the component of the Tat system that allows physical movement of the transported protein through the membrane. To do this it has to polymerize into a large complex. This projected aimed to understand important characteristics of the TatA complex.
We were able to demonstrate that TatA subunits exchange between TatA complexes. This is consistent with the polymerisation model for the mechanism of Tat transport in which TatA complexes need to show dynamic change in the number of proteins in the complex. Molecular details of how the TatA subunits interact in the complex were obtained from spin labelling measurements. These data provide insight into the organisation and mechanism of the Tat system.
Exploitation Route Mechanistic insight into Tat transport may underpin attempts to exploit the Tat system for production of high value recombinant products.
Sectors Pharmaceuticals and Medical Biotechnology

Description Not yet used outside an academic context.
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