Award details

Coordinating different protein translocation machineries during assembly of a membrane protein

Reference	BB/L000768/1
Principal Investigator / Supervisor	Professor Tracy Palmer
Co-Investigators / Co-Supervisors
Institution	University of Dundee
Department	School of Life Sciences
Funding type	Research
Value (£)	406,287
Status	Completed
Type	Research Grant
Start date	01/04/2014
End date	31/07/2017
Duration	40 months

Abstract

The Rieske iron-sulphur protein from actinobacteria is unusually long and has three transmembrane domains (TMD). We have recently shown that two distinct protein transport machineries participate in the biogenesis of the TMD of this protein - the Sec pathway, along with YidC, integrates the first two TMD whilst the Tat system is required for the integration of TMD3 and the translocation of the globular domain across the membrane. The precise features of the protein that coordinate its release from the Sec machinery following integration of TMD2 and recognition of a membrane-tethered twin-arginine signal sequence by the Tat pathway are not clear, but reside in the cytoplasmic loop region between TMD2 and TMD3. Building on our prior studies we now aim to: 1. Dissect the features of the actinobacterial Rieske protein that result in its release from the Sec pathway and facilitate recognition by the Tat pathway. We will undertake site-directed and random mutagenesis approaches to isolate mutations that (i) prevent release of the polypepide from the Sec pathway and (ii) prevent recognition of the tethered signal sequence by the Tat pathway. Results will be confirmed biochemically. 2. Exploit the membrane anchor of the actinobacterial Rieske protein as a tool to explore the mechanism by which twin arginine signal peptides are translocated by the Tat pathway. Specifically we will use topological labelling studies of the 3 TMD of the Rieske protein to assess whether the TatC protein can translocate signal sequences and larger polypeptides across the membrane. 3. Characterise new examples of dual targeted membrane proteins that we have recently discovered. We will confirm that these are novel examples of dual targeted proteins and build on this to probe features of these novel proteins that permit dual recognition.

Summary

All cells are surrounded by lipid membranes. However, in order to allow the passage of important metabolites and other substances biological membranes contain proteins. Proteins located in the lipid membrane are unusual because they are mainly hydrophobic (water-hating) unlike other proteins which are hydrophilic (water-loving). This presents a problem for the cell because the hydrophobic membrane proteins must be inserted into the membrane from the aqueous cytoplasm where they are made. If greasy membrane proteins are allowed to accumulate in the cytoplasm they aggregate, causing cell stress and ultimately cell death. Therefore membrane proteins are usually made co-translationally. This means that the hydrophobic segments of proteins are never exposed to the cytoplasm but are effectively threaded into the membrane as they are synthesised by the ribosome. A transmembrane channel in a specialised membrane protein complex called Sec receives the unfolded hydrophobic segments and releases them sideways into the membrane where they interact with lipids and sometimes with another protein called YidC. Once in the lipid bilayer the membrane protein will fold into its final, active conformation. We recently reported a very important exception whilst examining the mechanism of membrane insertion of the Rieske protein from Streptomyces coelicolor. Rieske proteins are found in almost all organisms and are very important because they help to transfer electrons during respiration or photosynthesis. The electron transfer properties of the Rieske protein are due to the presence of an iron sulphur cluster, which is an iron ion, held in place by inorganic sulphur, and ultimately co-ordinated in a non-covalent manner to cysteine residues in the protein. Normally Rieske proteins in bacteria are synthesised in the cytoplasm, they bind their iron-sulphur cluster, attain their fully folded conformation and are moved across the lipid bilayer through a large channel called Tat which is big enough for folded proteins to pass through. The Rieske protein is guided to the Tat channel by a short, moderately hydrophobic signal sequence at its N-terminus which ultimately anchors the Rieske protein into the membrane, with its large folded domain containing the iron sulphur cluster facing the opposite side. We noted that the Rieske protein from Streptomyces coelicolor and closely related bacteria was much larger and more hydrophobic than expected and anticipated that this might alter the route of membrane insertion. Indeed we were able to show that the first half of the protein required Sec for its membrane integration, but that the remaining hydrophobic sequence and the folded iron sulphur cluster-containing domain required the Tat pathway. This was unexpected because the Sec machinery does not normally release the ribosome during co-translational insertion of membrane proteins, but must do so in this case to allow the remainder of the protein to be synthesised at the cytoplasmic side of the membrane, the iron sulphur cluster to be bound and the protein to fold. This project aims to build on our previous discovery to understand just how the Sec and Tat machineries coordinate to assemble a single membrane protein. To this end we will screen for mutants in which the Sec pathway can no longer release the Rieske protein. We will use similar approaches to select mutants that can no longer allow the protein to be recognised by the Tat pathway. Although our initial studies identified the Rieske protein as a dual targeted membrane protein, we have now analysed the genome sequences of many more bacteria and archaea and have identified new candidates for dual targeting. We will also analyse two of these novel membrane proteins and confirm the route/s by which they are targeted to the membrane. We anticipate that our results will firmly establish that dual targeting is a common feature found in many bacteria and archaea.

Impact Summary

Beneficiaries of this research include: i) Biotech companies interested in protein secretion, surface display, anti-infectives. Such companies will benefit from the proposed research programme since it will generate new knowledge on novel pathways for surface display, membrane protein assembly and proteins essential for bacterial virulence (for example the Rieske protein; the Tat pathway, both of which are essential in some bacteria including Mycobacterium tuberculosis, the causative agent of tuberculosis). We will 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. The PDRA will also be encouraged to design and supervise undergraduate projects and to become involved in science communication. 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 has teamed up with the Dundee Science Centre to organize a 2-day event in May 2010 and March 2012 entitled "Magnificent Microbes", in which the applicant fully participated. This event attracted teachers, children, the general public and the local media to learn about the research going on in the Division of Molecular Microbiology and educated, inspired and entertained the public about microbiology research. We will repeat an improved version event in 2014 and 2016 which would be during the course of the project.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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