Probing the mechanism of protein export by the bacterial Tat transport system
Lead Research Organisation:
University of Dundee
Department Name: College of Life Sciences
Abstract
All bacteria produce proteins that operate on the outside of the bacterial cell. A good example of this is 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.
The Tat system is found in many different bacteria, including those which cause human diseases such as Mycobacterium tuberculosis (which causes tuberculosis) and Salmonella (which causes food poisoning). Scientists have shown for many different pathogenic bacteria that the Tat system is essential for them to cause disease, and they are becoming increasingly interested in developing drugs that prevent the Tat system from working.
In the bacterium E. coli, the Tat transporter is a large protein complex made up of three types of proteins, named TatA, TatB and TatC. These proteins are organised into two different sub-complexes. There is a sub-complex made up of equal amounts of TatB and TatC. This sub-complex plays a very important role in recognising the proteins that are going to be transported across the membrane. The other sub-complex is made up of many copies of the TatA protein. This forms the channel in the membrane through which the proteins are transported.
In order to understand how the Tat machinery works, and ultimately to design new drugs to stop it working, we need to know how the different proteins (or subunits) in the sub-complexes are arranged. This application is aimed at understanding how the TatBC sub-complex is organised. Some of the questions we want to answer in this project are (i) where are the interfaces between the TatB and TatC proteins located? (ii) How do the interfaces change to allow dynamic movement of TatB and TatC during the transport of proteins through the Tat pathway? Finally we know that during the transport of proteins through the Tat system the TatA and TatBC subcomplexes come together to transport proteins. We believe that when this happens TatA might bind to TatC in the same way that TatB does, and we would like to test this by carrying out similar experiments.
The Tat system is found in many different bacteria, including those which cause human diseases such as Mycobacterium tuberculosis (which causes tuberculosis) and Salmonella (which causes food poisoning). Scientists have shown for many different pathogenic bacteria that the Tat system is essential for them to cause disease, and they are becoming increasingly interested in developing drugs that prevent the Tat system from working.
In the bacterium E. coli, the Tat transporter is a large protein complex made up of three types of proteins, named TatA, TatB and TatC. These proteins are organised into two different sub-complexes. There is a sub-complex made up of equal amounts of TatB and TatC. This sub-complex plays a very important role in recognising the proteins that are going to be transported across the membrane. The other sub-complex is made up of many copies of the TatA protein. This forms the channel in the membrane through which the proteins are transported.
In order to understand how the Tat machinery works, and ultimately to design new drugs to stop it working, we need to know how the different proteins (or subunits) in the sub-complexes are arranged. This application is aimed at understanding how the TatBC sub-complex is organised. Some of the questions we want to answer in this project are (i) where are the interfaces between the TatB and TatC proteins located? (ii) How do the interfaces change to allow dynamic movement of TatB and TatC during the transport of proteins through the Tat pathway? Finally we know that during the transport of proteins through the Tat system the TatA and TatBC subcomplexes come together to transport proteins. We believe that when this happens TatA might bind to TatC in the same way that TatB does, and we would like to test this by carrying out similar experiments.
Technical Summary
The Tat system is a highly unusual protein export pathway that transports folded proteins across the cytoplasmic membranes of bacteria. It is required for the virulence of many important human pathogens including Pseudomonas aeruginosa , Legionella pneumophila and Escherichia coli 0157, and it is essential for viability of pathogenic Mycobacteria. The Tat system therefore represents a novel target for development of antibacterial compounds, particularly since it is absent in animal cells.
Proteins substrates are targeted to the Tat system by means of N-terminal signal peptides that contain a conserved twin arginine motif. In E. coli TatA, TatB and TatC are the essential components of the Tat protein export machinery and they interact in a dynamic manner to form the membrane-located protein complexes necessary for substrate transport.
TatB and TatC form an equimolar complex that has previously been purified. This complex binds Tat substrates, probably solely through their twin arginine signal peptides. No high resolution structural information is available for the TatBC complex and there is little information about how it is organised. We have used cysteine mapping experiments to identify a patch on the transmembrane helix of TatB that specifically interacts with one of the six transmembrane helices in TatC. We wish to build on these observations, taking both in vivo and in vitro approaches to find out (i) whether the disulphide cross-linking is sensitive to different stages of the Tat protein transport cycle, the presence of substrate or signal peptide and dissipation of the protonmotive force. (ii) To identify whether a second patch on TatB contacts other regions of TatC and (iii) to ascertain whether TatA can bind in a similar manner to TatC.
Taken together the experiments will reveal important molecular details about the contact sites between Tat components and the conformational changes that occur during protein transport.
Proteins substrates are targeted to the Tat system by means of N-terminal signal peptides that contain a conserved twin arginine motif. In E. coli TatA, TatB and TatC are the essential components of the Tat protein export machinery and they interact in a dynamic manner to form the membrane-located protein complexes necessary for substrate transport.
TatB and TatC form an equimolar complex that has previously been purified. This complex binds Tat substrates, probably solely through their twin arginine signal peptides. No high resolution structural information is available for the TatBC complex and there is little information about how it is organised. We have used cysteine mapping experiments to identify a patch on the transmembrane helix of TatB that specifically interacts with one of the six transmembrane helices in TatC. We wish to build on these observations, taking both in vivo and in vitro approaches to find out (i) whether the disulphide cross-linking is sensitive to different stages of the Tat protein transport cycle, the presence of substrate or signal peptide and dissipation of the protonmotive force. (ii) To identify whether a second patch on TatB contacts other regions of TatC and (iii) to ascertain whether TatA can bind in a similar manner to TatC.
Taken together the experiments will reveal important molecular details about the contact sites between Tat components and the conformational changes that occur during protein transport.