Membrane protein insertion and quality control by the bacterial holo-translocon and FtsH chaperone/protease complex

All cells are surrounded by membranes that act as a barrier. Proteins embedded in the membrane are required for the transport of nutrients and information (signals) across this barrier. Translocation systems are required to transport proteins into the membrane or across the membrane to the cellular location where they can fulfil their tasks. Translocation systems recognise the specific proteins to be translocated via signals embedded in the sequence of amino acids from which they are constructed.
The Sec translocation system is well studied and conserved from the bacterium Escherichia coli to humans highlighting its importance. Findings about the mechanism of the SecYEG translocon from bacterial cells can thus inform us how similar systems work in our own bodies. SecYEG is a membrane protein complex that comprises 3 subunits. It contains a central channel for protein translocation through the membrane. The channel can also open up on the side to allow the lateral passage of proteins into the membrane.
Additional accessory proteins were identified in bacteria to help translocating and folding the membrane proteins and to assemble them into larger complexes. Despite the fact that these proteins are essential for cell survival, much less is known about how these proteins work. This is mostly due to the fact that they form a large assembly with the SecYEG translocon and it was difficult to produce this higher-order complex for experimental studies. By using new technology we have succeeded in generating the large holo-translocon complex and suggest here to study how the accessory proteins in the holo-translocon help to fold up membrane proteins that emerge from the ribosome, which is the protein factory of the cell. To this end, we will directly look at these molecular machines consisting of membrane-protein synthesizing ribosomes and active holo-translocon machinery by state-of-the-art electron microscopy and image processing. Due to recent developments in hard and software, this technology now enables to obtain unprecedented high-resolution structures and thus insights into the molecular interplay of translocation proteins with the synthesizing ribosome and with the translocated, to be folded membrane protein.
Proteins that are not correctly folded are recognized by the cellular quality control system. This system first attempts to fold up the protein with the help of energy, and if unsuccessful, degrades the mis-folded protein. Two components of the bacterial membrane are thought to have a key role: YidC and the FtsH-HflKC complex. Both are also present as homologous proteins in a human cellular organelle, the mitochondrion. How they work and recognize the unfolded protein is unknown today. We will elucidate here how these machines work together to ensure the proper folding of membrane proteins and to remove unfolded proteins that could be detrimental to the cell. We will use purified components to biochemically dissect the interplay and the mechanism of the folding/degradation machine, and we will use electron microscopy to visualize at high resolution the relevant complexes which we identify in this work.
Our studies will thus provide essential new insights into the poorly understood process of membrane protein genesis, folding, and concomitant quality control.

HTL is active in protein secretion and membrane protein integration. We determined the architecture of HTL in an integrated approach combining cryo-EM, SANS, biochemistry and proteomics. We observed intriguing conformational changes in the SecD periplasmic domain in HTL supporting an active role of SecD during substrate translocation. We discovered a lipid-filled cavity in HTL which could serve as a protected folding space for membrane proteins, strikingly mirroring assisted folding of newly synthesized proteins in the cytoplasm. Importantly, SANS indicated that HTL is a dynamic machine, capable of flexibly adapting to accommodate variable sizes of translocating proteins.
We propose now to study the actively translocating HTL in complex with the ribosome. We will isolate native ribosomal assemblies from cell membranes and affinity-purify them. The structure of the ribosome-HTL complexes will be determined by cryo-EM at high resolution.
We will further explore membrane protein biogenesis and quality control by producing the FtsH-HflKC chaperone/protease complex and study its interactions with YidC and HTL. We will use pull-down, crosslinking and in vitro translocation experiments to study protein-protein interactions and interactions with the substrate. Further putative interaction partners will be identified by mass spectrometry. We will determine the stoichiometry of the relevant complexes by size-exclusion chromatography, blue native gel electrophoresis and/or analytical ultracentrifugation. Stable supercomplexes between FtsH-HflKC and YidC or HTL, if confirmed, will be studied by cryo-EM.
Our work seeks to address the fundamental question of how bacterial membrane proteins are inserted into the membrane, folded and assembled to complexes and how aberrant, misfolded membrane proteins are detected and eliminated. What is the role and interplay of HTL, YidC and FtsH-HflKC in these processes, and what is the structural basis of the underlying mechanisms?

There is an urgent need for novel antibiotics. Interestingly, it has been shown that the FtsH protease complex reinforces the bactericidal effects of antibiotics that inhibit translation elongation, e.g. tetracycline and chloramphenicol. This leads to stalling of co-translational translocation. Such 'jammed' translocation machines are recognized by FtsH - our proposed work should reveal how - and subsequently degraded, a suicidal reaction. Unfortunately, cells have developed mechanisms to counteract: the Cpx envelope stress response induces YccA production, an inhibitor of FtsH. The HTL, FtsH-HflKC and YccA are thus interesting targets for antibiotics. Notably, YccA has a human homologue Bax Inihibitor-1, an anti-apoptotic protein that acts on the tumour suppressor Bax. Bax contributes to apoptosis upon prolonged stress in protein secretion in the endoplasmic reticulum.
I. Collinson (Co-I) is involved in a collaboration with the Dundee Drug Discovery unit who seek to target the bacterial Sec machinery as a target for novel antibiotics. Several compounds have been identified that inhibit the secretion activity of the core-SecY complex. The holo-complex also presents a good target for potential antibiotics. Therefore, new information gained on the mechanism of membrane protein insertion and quality control will be fed into the existing drug discovery platform that may then lead to the development of new and improved drug discovery strategies.
Applications of this work are identified from within the department (through regular discussion with our Impact lead and industrial liaisons) as well as by continuing discussions with our Research and Enterprise Department. Any outcomes of this work that are exploitable, notably in terms of intellectual property or knowledge transfer to the private sector, are handled by the highly experienced team within RED; who engage closely with funders when appropriate. The ACEMBL system, which was specifically developed to enable production of multiprotein complexes such as HTL, has been patented by our previous employer (EMBL) and was successfully commercialized (by Geneva Biotech SARL). We anticipate that similar technological advances may arise from the present project to the benefit of the wider academic and industrial R&D community.

This project includes considerable opportunity to train researchers involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration with public communication and outreach programmes and the extensive network of University schemes to benefit the training and development of research staff, an area where University of Bristol is very active.

Former research members of my laboratory now work for F. Hoffmann-La Roche Ltd (Basel), AstraZeneca (Cambridge) and as university lecturer (Maitre de conferences, University of Grenoble). Thus, the environment provided by my own lab as well as the University as a whole is highly conducive to career development of our staff beyond academic, basic science research and thus will contribute to the economic development of the nation. Our projects are also very data intensive, and the management and analysis of such large (multi-terabyte) datasets is applicable to many areas of professional life.

This work will lead to significant image data (2D and 3D) that is readily used in both public understanding of science and artistic arenas. Through our public engagement plans, entering competitions, and other outreach activities, this work therefore will contribute to local exhibitions and to communicate science to the public.