Probing the mechanism of the Tat protein transport system at the single complex level in whole bacterial cells

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. In this project we want to increase our understanding of how the Tat system works. We have added labels to the various proteins involved in the Tat pathway which make them appear as coloured spots when viewed in a special type of microscope. We can even see the spots in whole bacterial cells. By studying the behaviour of the spots we can learn about how the Tat components function in living cells. 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 Tat protein transport system functions to export folded proteins across the bacterial cytoplasmic membrane. TatA, TatB and TatC are the essential components of the bacterial Tat protein export pathway and interact in a dynamic manner to form the membrane-located protein complexes necessary for substrate transport. We will use strains that express fluorescently-tagged Tat components at wild-type levels in conjunction with advanced imaging techniques to study individual Tat components under physiologically relevant conditions in whole bacterial cells.


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Alcock F (2013) Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system. in Proceedings of the National Academy of Sciences of the United States of America

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Leake MC (2008) Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging. in Proceedings of the National Academy of Sciences of the United States of America

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Tarry MJ (2009) Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system. in Proceedings of the National Academy of Sciences of the United States of America

Description Protein transporters move proteins across cell membranes during cell synthesis. Usually the proteins are transported in an unfolded state. However, the Tat protein transporter found in bacteria and chloroplasts moves folded proteins. The Tat system is of potential use as pathway to produce proteins of industrial or pharmacological utility.
In this study we probed the mechanism of the unusual Tat protein transport system by labelling the protein components of the system with fluorescent tags and watching their behaviour in living bacterial cells. This allowed us for the first time to directly seen the complexes. We developed methods to determine the number of proteins in a complex using the stepwise bleaching of the fluorescent labels by a laser light source. Using this approach we were able to show that the main transporter component, TatA, is present in the cell membrane in two pools, either dispersed as n = 4 protomers, or in ring-shaped, variable-stoichiometry, higher oligomer complexes (nmean=25) built from TatA tetramers. We were able to demonstrate that formation of the higher order TatA oligomers required the other Tat pathway components.
Exploitation Route Our demonstration that photobleaching methods could be used to determine the stoichiometry of mobile fluorophore-labelled complexes has inspired other to use this approach.
Sectors Pharmaceuticals and Medical Biotechnology

Description No non-academic applications to date.
Description In vitro analysis of Tat protein transport using single molecule fluorescence methods
Amount £686,310 (GBP)
Funding ID BB/H018050/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2010 
End 12/2013