Exploiting a cellulose synthase interactome to understand assembly and trafficking of the plant cellulose synthase complex

Cellulose is a very abundant polymer in the wall that surrounds all plant cells. As a result of its abundance, cellulose represents a massive renewable resource for making biofuels, biochemicals and biomaterials that involve much smaller releases of harmful greenhouse gases. Cotton fibres are almost pure cellulose, however, to better exploit cellulose, we need to be able to use the huge quantities of cellulose locked up in plant cell walls. Cellulose is composed of chains of sugars bound together to form a highly insoluble cellulose microfibril. Although these microfibrils are made solely of the sugar glucose, it is hard to release the glucose as the microfibril structure makes them hard to digest. One way of making cellulose more readily digestible is to allow the sugars to be accessed more easily, by reducing the level or organisation of the cellulose microfibril and incorporate a higher proportion of less well organised cellulose known as amorphous cellulose. Movement of the large cellulose synthase complex, the protein complex which makes cellulose in cells, to and from the cell surface and controlling the number of complexes at the cell surface are important factors that control cellulose crystallinity.
A recent breakthrough has demonstrated how woody biomass can be utilised to make strong, light and flexible materials by removing the polymer lignin. While this "flexible, mouldable" wood retains some additional matrix, the majority is composed of cellulose that largely determines its structural properties. While the microfibrils of most plants have similar numbers of glucose chains, the cellulose found in lower plants, particularly algae, vary enormously. Microfibrils can be much larger and vary in shape. Plant material making these novels cellulose microfibrils has the potential to generate an entirely new generation of novel biomaterials with even more useful structural properties. We are not currently able to do this because we do not understand enough about how the enzyme complexes that makes cellulose are assembled and transported to the cell surface.
Assembling a large enzyme complex and transporting it the cell surface to make cellulose microfibrils requires other proteins. On way of identifying these additional proteins is to use a technique known as "proximity labelling". As the name suggests this technique uses a bait protein to label other nearby proteins. We use proteins known to be essential in different aspects of cellulose synthesis as bait and these baits transfer a small molecule, biotin in our case, onto nearby proteins. We are then able to determine the identity of the labelled proteins. This information can be used to clearly see which proteins are close to each other and identify most, if not all, of the proteins required for all aspects of cellulose synthesis.
There are two parts to this proposal. In the first, part we will use proximity labelling to identify cellulose synthesis proteins in three different cell types. Studying cellulose synthesis in different systems allows us to distinguish core components required to make cellulose under all conditions from more peripheral proteins, those that are only required under certain conditions or maybe nearby purely by chance. In the second part, we propose to demonstrate the importance of some of the proteins we have already identified by proximity labelling. In particular, we will identify if particular proteins are important in coordinating the synthesis of cellulose with the deposition of other matrix polysaccharides; identify how the large cellulose synthase complex is guided to the appropriate part of the cell surface; and determine if the activity of the cellulose synthase complex or other components required to make cellulose are regulated by the addition of phosphate groups and whether this process of protein phosphorylation is important for how the synthesis of cellulose is regulated during growth and in response to environmental signals.

Cellulose is a very abundant biopolymer with huge potential for use as renewable feedstock for production of biofuels and novel biomaterials. There are enormous opportunities if we were able to alter the basic cellulose microfibril in higher plants either to increase its digestibility or to generate entirely new cellulose microfibril structures by exploiting the variation in plant microfibril structure exhibited by algal species. One of the obstacles to achieving these aims is our lack of understanding of the mechanism of cellulose synthase complex assembly and trafficking. While there has been some progress in identifying individual components involved in trafficking of the complex a comprehensive analysis is lacking. In this proposal, we will use 11 different bait proteins together with 4 different control proteins tagged with TurboID, a recently developed improved version of the BirA enzyme that exhibits excellent biotinylation in plants. We have already used this tag to identify a robust network covering cellulose synthesis in the primary cell wall, composed of 411 interactions between 265 proteins. As part of this proposal, we will also develop a similar interactome for cellulose synthesis in the secondary cell walls. A comparison of primary and secondary wall cellulose synthesis interactomes will allow us to distinguish a core set of proteins and interactions required for cellulose synthesis under all conditions, from other proteins that may interact only at certain growth stages or under specific environmental conditions. To demonstrate the utility of the dataset, we will analyse 3 small families of proteins from our primary wall interactome and demonstrate: their importance to the regulation of cellulose synthesis by external cues; how cellulose synthase complexes are targeted to certain parts in the plasma membrane; and how cellulose synthesis is coordinated with the deposition of other matrix polysaccharides to make a functional secondary cell wall.