Characterisation of the assembled state of the Tat protein transport system

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. Because different folded proteins have different sizes, this means that the Tat system must be able to form channels that can accommodate the different diameters of the folded proteins that it transports. How is this achieved?

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 TatB and TatC components form a 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 TatBC this triggers the 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.

We have been able to isolate mutants that allow the Tat system to transport proteins that have no twin arginine signal. We would like to understand how these mutated Tat systems work - how do they identify proteins to transport? This will help us to understand how the twin arginine signal is able to activate the Tat system. In the long run these mutated Tat systems have the potential to be useful because they may be able to secrete a wide range of different proteins, allowing us to develop them into cell factories to produce and secrete important industrial proteins.

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. The mechanism of Tat transport remains to be elucidated.

The Tat system comprises the three integral proteins TatA, TatB, and TatC. Substrate proteins are recognized by specific signal peptides which bind to sites in TatC and TatB, and signal peptide binding results in the assembly of TatA to form a transmembrane translocation pathway. We have recently identified amino acid substitutions in the TatB component that bypass the requirement for a signal peptide to trigger Tat transport. Our analysis of effects of these substitutions indicates that they promote the assembly of the Tat translocase in the absence of signal peptide interaction. We will build on our findings to:

Characterise the 'assembled' Tat translocase triggered by the TatB amino acid substitutions

Explore the export limitations of the 'assembled' translocase

Isolate suppressors in tatA and tatC that allow signal peptide-independent protein translocation

This work is expected to lead to fundamental advances in our understanding of the assembly of the Tat protein translocase.

Beneficiaries of this research include:

i) Biotech companies which produce proteins of therapeutic and industrial relevance. The work described here offers the potential for the design of variant Tat translocases that can transport substrates in the absence of a Tat targeting sequence (thus overcoming a potentially rate-limiting or problematic targeting step). 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. We have already filed a preliminary patent on our findings and will continue to act to protect any intellectual property and to maximise opportunities for collaborative research or licensing. The Dundee research and innovation team have a wealth of industrial contacts and close links to Scottish Enterprise, 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 University of Dundee 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. The University of Dundee takes training of early career researchers seriously, thereby ensuring a successful contribution to the knowledge-led economy of UK Plc. The appointed 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, the University of Dundee 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. For example, the Division of Molecular Microbiology runs a signature outreach event, 'Magnificent Microbes' over two consecutive days in partnership with the Dundee Science Centre exploring the world of microorganisms. The first day (Friday) is dedicated to Primary 7 pupils from local schools and the second day (Saturday) is open to the general public. This event will run in the spring of 2016 and of 2018, the latter of which would be during the course of the project. The staff employed on this project will fully participate in this event.