Anterograde and retrograde transport of the plant vacuolar sorting receptor BP80 in vivo

Lead Research Organisation: University of Leeds
Department Name: Ctr for Plant Sciences


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. 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. Numerous protein sorting signals (address labels) have been described in the last 20 years and in many cases receptor molecules (drivers with instructions) have been identified that bind to the sorting signals. However, much less is known about the sorting of the sorting receptors themselves, in other words how do the drivers reach their right destination, and what do they do when they have arrived? In the cell, 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. This process is extremely complex, because it means that receptor should only start transporting when they have bound to the right protein and when the transport carrier has been filled with appropriate cargo. On the other hand, receptors must release the proteins at the right place, and then return with a transport carrier for a new job. This project aims to understand how the plant vacuolar sorting receptor BP80 accomplishes the difficult task of finding its way in the cell and transport proteins to the right place in an efficient manner. We have already started to understand how receptors move forward, but we wish to find out more about these routes and also discover how and when they return from their trips.

Technical Summary

Vacuolar sorting receptors recognise sorting signals but also carry sorting signals themselves. These are thought to be located in the cytosolic tail, but few functional studies have been performed. We have recently established a new assay to simulate vacuolar sorting receptor traffic via in vivo receptor competition and simultaneous live imaging (da Silva et al., 2005; The Plant Cell 17, 132-148). This assay is based on the fact that truncated receptor, in which the ligand binding domain was replaced by green fluorescent protein (GFP-BP80), competes with endogenous receptors for sorting machinery and inhibits sorting of ligands. Competition is quantitatively measured by monitoring increased secretion of vacuolar proteins. This has proven a very useful assay, because it is very specific for BP80 traffic, as shown by numerous controls and the fact that only full length BP80 can complement the semi-dominant behaviour of the competitor. In addition, GFP-BP80 can be directly localised in live cells that express the chimeric construct and thus reveals its steady state levels in the cells. A range of unpublished data illustrate how the assay can be used to study targeting signals of BP80 and are explained in this application. Here it is proposed to use the competition assay using truncated BP80 derivatives as well as the reconstitution assay with full length BP80 to study the sorting signals that control anterograde and retrograde transport of this important receptor molecule. The fast and reliable competition assay is to be used in conjunction with life bio-imaging to monitor the steady state locations of the mutant molecules. In addition to the quantitative work with tobacco protoplasts which will serve to identify mutations with a clear phenotype, receptor traffic is to be studied in transgenic plants (Nicotiana tabacum and Arabidopsis thaliana) in order to verify traffic in tissues within a whole plant context.
Description We found that plant vacuolar sorting receptors carry signals for ER export, endocytosis, early secretory pathway transport to the prevacuole and recycling from there. We also discovered that a novel type of prevacuole exists which is depleted for vacuolar sorting receptors but carries only vacuolar cargo.
Further work on this novel organelle (now termed "late prevacuole" or LPVC) was started and mostly completed during the period of the award but was recently taken to publication stage (a paper submitted to The Plant Cell in February 2017). Essentially, the research suggests that the LPVC discovered in this award is the equivalent of the plant storage vacuole. We found a new receptor that takes a different route to this compartment and we characterised the LPVC biochemically and showed that the density of the LPVC is identical to that of other vacuolar-related organelles in plants, such as the protein storage vacuole (PSV) and dense vesicles (DVs). Both findings are highly significant due to the similarity with sorting pathways in mammalian cells of lysosome-related organelles (eg. melanosomes, synaptic vesicles, platelet dense vesicles), all of which are dependent on AP3, which should therefore be of broad interest to both plant and mammalian research fields.
The work has both fundamental as well as applied impact because it introduces a simple model in which the LPVC represents a functionally independent organelle located at the checkpoint between the routes to protein storage or lytic vacuoles. This model explains and resolves previously conflicting data regarding vacuolar sorting pathways in plants.
Furthermore, we have successfully started to use the information regarding the sorting of this receptor to build designer receptors, a synthetic biology approach to bio-manufacturing. This has enabled us to redirect vacuolar cargo to the plasma membrane or the ER. Further synthetic biology approaches have been tested to help proliferation of LPVCs in non-storage tissues.
Exploitation Route We can engineer high starch crops (potato, cassava, sweet potato) with increased protein content, in order to increase both food and energy output of agricultural land-surfaces.
We can also engineer crops to produce and store high value proteins (vaccines, antibodies medicines, industrial enzymes).
The recent knowledge gained about the density of the LPVC and the ease with which this organelle can be purified will be instrumental in more directed breeding and screening programmes to increase protein content in crops naturally rich in storage proteins (beans, lentils, peas, chickpea, pigeon pea, sorghum, millet, wheat and other cereals)
Sectors Agriculture, Food and Drink,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description The findings were used to describe a novel organelle, the late prevacuole, that could be identical to the so-called dense vesicles and/or storage vacuoles. We are currently using the findings to induce storage vacuoles to boost protein productivity in starch crops which have lytic vacuoles in the tubers. We are also making the first artificial receptors, designed to mediate transport of single heterologous cargo molecules, which is an achievable synthetic biology strategy. Further research started during the award but completed only recently provided evidence that the late prevacuole that we discovered has identical properties compared to dense vesicles and storage vacuoles. For the layman, this means that we have discovered that lytic and storage vacuoles are not two different destinations in plants with different pathways, but that the storage vacuole is an earlier compartment on the route to the lytic vacuole. This insight will make it a lot easier to induce storage vacuoles in plants that naturally do not store high levels of proteins and will have a big impact on food security on Earth. The gained insight also makes the prospect of using plants as green factories for medicines (vaccines, antibodies, hormones, nutraceuticals) much more realistic.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology
Impact Types Cultural,Societal,Economic