Award details

Functional analysis of ER and Golgi subdomains

Reference	BB/K007181/1
Principal Investigator / Supervisor	Dr Jurgen Denecke
Co-Investigators / Co-Supervisors
Institution	University of Leeds
Department	Ctr for Plant Sciences
Funding type	Research
Value (£)	382,092
Status	Completed
Type	Research Grant
Start date	30/09/2013
End date	29/09/2016
Duration	36 months

Abstract

Transport of proteins between organelles of the plant secretory pathway is controlled by complex cascades of cyclic reactions involving conditional conformational changes and interactions between proteins and other bio-molecules. Using innovative in vivo protein transport assays my team has discovered two novel compartments of the secretory pathway: 1) The plant ER import site (ERIS) was found to be physically separated from ER export sites and associated Golgi bodies (daSilva et al., 2004, Plant Cell 16, 1753-1771) and may help to explain how cargo and receptors segregate in the ER. 2) A specialized subpopulation of Golgi bodies was discovered (see case for support) that specifically contains high levels of a membrane spanning protein that resembles the HDEL-receptor ERD2 but is differentiated by an additional transmembrane domain at the N-terminus (Hadlington and Denecke, Curr Opin Plant Biol 3, 461-468). These two new compartments enrich the roadmap of the known transport steps and their further analysis is essential to understand the true complexity of the pathway in plants. This project will deliver specific knowledge on the sorting motifs and transport route required to mediate segregation of transport machinery to ERIS domains of the ER, their biogenesis and the transport cycle of ERD2. ERIS function is one of the last missing links in the retrograde transport route of the pathway and we will identify new proteins that take part in the process of COPI vesicle fusion with the ER. In addition, we will complete the knowledge base on anterograde ERD2 transport and unravel the biological meaning of the plant-unique ERD2-related protein family that specifically labels only a subset of Golgi bodies. These observations stand out internationally, and will explore completely new avenues of fundamental research and plant biotechnology allowing us to ultimately design artificial protein sorting receptors and storage organelles for biotechnology.

Summary

Understanding how proteins are sorted to the right place in a living cell is comparable to the task of the Royal Mail to first sort letters and packages by destination and secondly to deliver them correctly at minimal cost and in a reasonable timeframe. The Golgi apparatus is a sorting station of the secretory pathway from which proteins can be sorted in at least three directions, to the cell surface (secreted proteins), to the vacuole or the endoplasmic reticulum (ER). The ER can be compared to the manufacturing site for a range of products that will be shipped first to the Golgi for subsequent sorting and delivery. To stay in business, manufacturing sites require efficient transport routes to distribution centres. This also means that vehicles should not travel when they are not properly loaded, and they should be doing something useful when they return, such as bringing some mail back. This is not an easy task, and the same is true for the sorting of proteins in living cells. However, cells are very efficient at organising cost-effective supply chains and have developed very efficient transport processes in which no step is perfect but little is left to chance due to efficient back-up mechanisms. This includes the recycling of essential machinery so that they can be engaged in multiple transport reactions during their life-span. Numerous protein sorting signals (address labels) have been described in the last 20 years and in many cases receptor molecules (lorry drivers) have been identified that bind to the sorting signals and package them into transport vesicles (the lorries). It is however much less clear how the sorting receptors know how to find their way in the cell. In other words how exactly do the lorry drivers reach their right destination, who gives them their instructions and who sends them back for new jobs when they have delivered their cargo? In the cell, sorting receptors must not only bind to ligands in one compartment, they must also transport them to another compartment, release them there, and return back to the original compartment to select new proteins again. We would now like to use our recent discoveries to shed light on the complete process of ER retention in plants. Many proteins exported from the ER are meant to be returned, just like the wooden pallets are not discarded in modern logistic firms but they are in fact recycled so that they can be used again to be packed with new cargo. One problem is that there are many more proteins to be recycled than receptors available. Receptors have to bind their cargo very efficiently in the Golgi and preferentially release them in the ER in a place where they will stay for a longer time, whilst the receptors move back to the Golgi using a frequent flyer ticket. This is why we find it exciting that ER import sites and ER export sites are physically separated from each other and we can now start to explore how this is achieved. We have also discovered that plants contain two types of Golgi bodies. This is very exciting, because it suggests that instead of one basic distribution centre the plant uses at least two. It was always believed that the Golgi apparatus is a single organelle, so our finding is of major importance. The new Golgi type can be seen with fluorescently tagged receptors (lorry drivers) which represent a new class found in plants, algae and some unicellular organisms but is absent in animals and yeasts. We must first learn how this new class of receptors reaches the new class of Golgi bodies, and next we would like to see what happens if we eliminate this class of receptors and see why plants use these receptors. Finally, we need to identify more proteins residing in the new compartments that we have discovered to gain a better understanding of protein sorting to the plant vacuoles, which host the vast majority of edible protein on earth. Plant proteins constitute a major food source and must be harnessed as well as we can.

Impact Summary

Plant Biotechnology is in urgent need for new breakthroughs which demonstrate measurable consumer benefits in the use of plants to manufacture molecules of added value. The public is still extremely sceptical regarding any form of research leading to genetic manipulation of plants, and it is therefore of paramount importance to discover new strategies to harness the potential of plants to produce real consumer values. Constructive engineering to obtain gain of function in the secretory pathway is crucially dependent on a complete understanding of all the organelles and sub-organelles that exist in the pathway, as well as the signals/mechanisms that allow receptors to complete a full transport cycle. This project will yield a critical mass of data to design of custom made receptor molecules that can boost ER retention of plant cells without affecting the overall yield. It will pave the way towards next generation pathway engineering strategies and the beginning of true synthetic biology for bio-manufacturing procedures involving the plant secretory pathway as a green factory. Prof. Denecke (Leeds) has extensive experience with IP and filing patents, supplemented by significant experience and passion for process technology and process innovation. The University of Leeds is priviledged to maintain knowledge/technology transfer offices to maximise the potential for exploitable IP and to facilitate industrial links. When plant molecular biology was at its infancy J. Denecke became a pioneer in plant cell transfection and transient expression systems. He developed the first quantitative secretion assays for plant cells, introduced a variety of cargo molecules for the analysis of protein sorting signals, including transport time-courses, dose response analysis and multiple cargo assays. Leading scientists worldwide have adopted the system and the laboratory hosts regular visitors worldwide to acquire the necessary skills for their own research goals. J. Denecke was thefirst to demonstrate with proper proteins that secretion to the cell surface is the default pathway and that soluble proteins can be secreted without carrying active sorting signals. He spearheaded new experimental strategies to co-express cargo molecules of the secretory pathway with effector molecules to specifically interfere with house-keeping transport reactions. The main principle is to monitor early responses of a complex system by rapid transient expression systems in which one parameter is the sole variable. This allows researchers to identify primary responses and not long-term indirect effects. Since 2003, access to state of the art confocal laser scanning microscopes at Leeds has introduced live fluorescence microscopy as a key-technique which has profoundly transformed the research. Results have shed light on fundamental biological questions and were published in high impact journals at regular intervals, including 20 publications in The Plant Cell, the leading journal in the field, and three filed patents on the use of pathway engineering in biotechnology. Whilst at York and later in Leeds, his research group has organised international training workshops on transient expression technology. Researchers leaving the Denecke lab have often published well above average and the lab has a strong track-record in launching young scientists to become research leaders themselves as over 60% of the PhD students and postdoctoral researchers from Denecke's team now work in permanent academic environments running their own research teams, including a number of assistant professors (Luis daSilva, Brazil; Peter Pimpl, Germany; Nathalie Leborgne-Castel, France). Other researchers work for the government (Belinda Phillipson, CSL York) or industry (Phil Taylor, Monsanto; Jane Hadlington, Qiagen).

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Plant Science
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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