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

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.

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.