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

纤维素是一种非常丰富的聚合物，存在于所有植物细胞的细胞壁中。由于其丰富性，纤维素是一种巨大的可再生资源，可用于制造生物燃料，生物化学品和生物材料，这些材料涉及更少的有害温室气体排放。棉纤维几乎是纯纤维素，然而，为了更好地利用纤维素，我们需要能够使用锁定在植物细胞壁中的大量纤维素。纤维素由结合在一起以形成高度不溶性纤维素微纤维的糖链组成。虽然这些微纤维仅由糖葡萄糖组成，但由于微纤维结构使其难以消化，因此难以释放葡萄糖。使纤维素更容易消化的一种方法是通过降低纤维素微纤维的水平或组织，并掺入更高比例的组织较差的纤维素（称为无定形纤维素），使糖更容易获得。大的纤维素合成酶复合物（在细胞中制造纤维素的蛋白质复合物）向细胞表面和从细胞表面的移动以及控制细胞表面处的复合物的数量是控制纤维素结晶度的重要因素。最近的一项突破已经证明了如何通过去除聚合物木质素来利用木质生物质制造坚固，轻质和灵活的材料。虽然这种“灵活，可塑”的木材保留了一些额外的基质，但大多数是由纤维素组成的，这在很大程度上决定了其结构特性。虽然大多数植物的微纤维具有相似数量的葡萄糖链，但在低等植物中发现的纤维素，特别是藻类，差异很大。微纤维可以大得多，形状也不同。制造这些新型纤维素微纤丝的植物材料有可能产生具有更有用结构特性的全新一代新型生物材料。我们目前还不能做到这一点，因为我们对制造纤维素的酶复合物是如何组装和运输到细胞表面的了解还不够，组装一个大的酶复合物并将其运输到细胞表面以制造纤维素微纤维需要其他蛋白质。鉴定这些额外蛋白质的方法之一是使用称为“邻近标记”的技术。顾名思义，这种技术使用诱饵蛋白来标记附近的其他蛋白质。我们使用已知在纤维素合成的不同方面必不可少的蛋白质作为诱饵，这些诱饵将一个小分子，在我们的情况下是生物素，转移到附近的蛋白质上。然后，我们能够确定标记的蛋白质的身份。这些信息可以用来清楚地看到哪些蛋白质彼此接近，并确定大多数，如果不是全部，纤维素合成的所有方面所需的蛋白质。在第一部分中，我们将使用邻近标记来识别三种不同细胞类型中的纤维素合成蛋白。研究不同系统中的纤维素合成使我们能够区分在所有条件下制造纤维素所需的核心成分和更多的外围蛋白质，这些蛋白质仅在某些条件下需要，或者可能纯粹是偶然在附近需要。在第二部分中，我们建议证明我们已经通过邻近标记确定的一些蛋白质的重要性。特别是，我们将确定特定的蛋白质是否在协调纤维素的合成与其他基质多糖的沉积中是重要的;确定大的纤维素合成酶复合物如何被引导到细胞表面的适当部分;并确定纤维素合成酶复合物或制备纤维素所需的其它组分的活性是否通过加入磷酸基团来调节，以及这种蛋白质磷酸化对于在生长期间以及响应于环境信号如何调节纤维素的合成是重要的。

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.