The integration of tail anchored membrane proteins by the twin-arginine translocase

All bacteria, whether they are 'friendly' or otherwise, have one thing in common. In order to colonize their niches they need to communicate with the outside world. They achieve this by secreting protein molecules that allow them firstly to detect and then manipulate their environment. Bacteria are surrounded by one or more membranes and a rigid wall, which together form a protective barrier. Secreting proteins into the environment requires that these molecules are able to pass through the membrane barrier. In order to achieve this, bacteria have transporters located in the membrane that allow the passage of proteins to the outside. Understanding how these protein transporters work is critical if we wish to control this process to prevent disease, or engineer microbes to decontaminate toxic environments.

We study a protein transporter called the Tat system, that is conserved in almost all bacteria and in plant chloroplasts, and we use E. coli as a convenient model system in which to study these processes. The Tat system plays a very important role in the physiology of many different bacteria and it is essential for photosynthesis in plants. Proteins are made up of long, linear chains of amino acids which fold up after they are made. Proteins are only functional once they have folded into their final 3-dimensional structure. Proteins that are secreted are functional outside the bacterial cell. The Tat system is unusual because unlike most other protein transporters it only transports proteins after they have already folded. Proteins that are destined to be secreted by the Tat system have a special signature sequence of amino acids, termed a 'twin arginine signal' at their start. This signal targets the protein to the Tat machinery that is embedded in the membrane, and facilitates its secretion. The Tat machinery itself is made up of three components - TatA, TatB and TatC. The TatA, TatB and TatC components initially form a 1:1:1 complex with each other and this complex is responsible for recognizing each of the different proteins that are targeted for secretion by Tat system, by interacting with the twin arginine signal. After the signal has been bound by TatABC this triggers additional copies of TatA component to assemble into a ring-like structure, which can then allow transport of the protein. After the protein has been transported the TatA ring disassembles ready for another round of secretion.

Some of the Tat substrate proteins are unusual because they contain a membrane interaction domain at their C-terminus that anchors them into the membrane. In E. coli these membrane-anchored Tat substrates are essential for the bacterium to adapt to different growth conditions, and to survive during infection of mammals. We are interested in understanding how the Tat pathway is able to integrate these proteins into the membrane. We have isolated mutants in the Tat system that cannot integrate these proteins, instead secreting them completely across the membrane. We want to understand why these mutants are defective in integrating the Tat substrate proteins. This work will help us to understand how the Tat machinery recognises membrane proteins, and may also help us to understand the mechanism by which the Tat machinery disassembles once transport is complete.

The Tat system of bacteria and chloroplasts carries out the unusual, and mechanistically challenging, task of moving folded proteins across biological membranes. Substrates of the Tat transport system are responsible for a wide range of cellular processes in bacteria and are essential for plant photosynthesis. 20% of the Tat substrates in Escherichia coli are anchored to the periplasmic side of the membrane by a C-terminal transmembrane helix ('C-tail'). We have developed a genetic screen to identify substitutions in the Tat translocase that are defective in the integration of C-tails. This application will build on our findings to:

Probe properties of C-tails that influence integration by the Tat pathway;

Utilise this and a related genetic screen to identify Tat variants defective in C-tail recognition and integration;

Undertake biochemical analysis of C-tail integration by the Tat translocase and;

Investigate whether the mechanism of C-tail integration is related to protein folding quality control.

Taken together this work is expected to lead to fundamental advances in our understanding of the integration of membrane proteins by the Tat system, and to shed light on the mechanism of translocase disassembly and quality control.

Beneficiaries of this research include:

i) Biotech companies which produce proteins of therapeutic and industrial relevance. The work described here may offer the potential for the isolation of Tat translocases that can export both folded and unfolded proteins. Bacterial protein secretion systems are utilised as an aid to downstream processing of protein products of therapeutic and industrial utility. Although much prior industrial usage has focused on the Sec pathway, a number of recombinant proteins are recalcitrant to export by Sec, especially products that require cytoplasmic posttranslational modification or folding. The Newcastle University Faculty of Medical Sciences Enterprise Team has a wealth of industrial contacts and will help maximise the impact of all findings of commercial value. As and when appropriate, results will be peer-reviewed and published.

ii) Members of the wider academic community. The primary mechanism for communication of this research will be through publication in peer review international journals. Open access publishing options will be used where available. We will liaise at the time of publication with the Newcastle University and BBSRC Press offices to ensure publicity of results of interest to the general public. Our results will also be made available on our regularly updated web site. Note also that the Tat system is now featured in mainstream cell biology text books such as Molecular Biology of the Cell and so our data will potentially impact on future editions of standard texts. Strains and other resources will be made available as appropriate.

iii) The staff employed on this project. Newcastle University takes training of early career researchers seriously, thereby ensuring a successful contribution to the knowledge-led economy of UK Plc. The PDRA will be encouraged to be innovative in their work. There will be opportunities for them to train undergraduate, postgraduate and visiting scientists. They will be given multiple opportunities to present their findings at major research conferences, facilitating their career development through the acquisition and refining of key presentational and networking skills. Furthermore, the appointed PDRA will have access to training in transferable/generic skills through the professional development schemes. In line with the Concordat 2009, the PDRA will be actively encouraged to undertake at least 5 days training in personal professional development per annum. In addition, Newcastle University has an annual appraisal scheme to actively facilitate the career development of staff, including PDRAs and PIs.

iv) The general public. It is important that members of the general public are aware and supportive of how tax payers' money is spent on scientific research. Therefore as part of our work on this project, we will engage with local communities, through face-to-face discussion of our work and family focussed scientific event days. The applicant is an experienced science communicator, having participated in many outreach events to schools and the general public. We will work together with colleagues in the Faculty of Medical Sciences to develop a microbiology outreach activity that will showcase the work in this project alongside other microbiology research in the Faculty. The staff employed on this project will fully participate in this event.