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。由于纤维素的丰富，它代表了一种巨大的可再生资源，用于制造生物燃料、生化物质和生物材料，这些材料释放的有害温室气体要少得多。棉纤维几乎是纯纤维素，然而，为了更好地利用纤维素，我们需要能够利用锁在植物细胞壁中的大量纤维素。纤维素是由糖链结合在一起形成高度不溶性的纤维素微纤维。虽然这些微原纤维完全由葡萄糖构成，但由于微原纤维的结构使它们难以消化，因此很难释放葡萄糖。使纤维素更容易被消化的一种方法是通过降低纤维素微纤维的水平或组织，并加入更高比例的组织不太好的纤维素，即无定形纤维素，从而使糖更容易被获取。大型纤维素合酶复合物（细胞内制造纤维素的蛋白质复合物）进出细胞表面的运动和控制细胞表面复合物数量是控制纤维素结晶度的重要因素。最近的一项突破表明，木质生物质可以通过去除聚合物木质素来制造坚固、轻便和灵活的材料。虽然这种“灵活、可塑”的木材保留了一些额外的基质，但大部分由纤维素组成，这在很大程度上决定了它的结构特性。虽然大多数植物的微原纤维具有相似数量的葡萄糖链，但在低等植物，特别是藻类中发现的纤维素差异很大。微原纤维可以更大，形状各异。制造这些新型纤维素微原纤维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原纤维需要其他蛋白质。识别这些额外蛋白质的一种方法是使用一种称为“接近标记”的技术。顾名思义，这种技术使用诱饵蛋白质来标记附近的其他蛋白质。我们使用在纤维素合成的各个方面都必不可少的蛋白质作为诱饵，这些诱饵将一个小分子，在我们的例子中是生物素，转移到附近的蛋白质上。然后我们就能确定被标记蛋白质的身份。这些信息可以用来清楚地看到哪些蛋白质彼此接近，并识别纤维素合成各个方面所需的大多数蛋白质，如果不是全部的话。这个建议有两个部分。在第一部分中，我们将使用接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可能纯粹偶然需要。在第二部分中，我们建议展示一些我们已经通过接近标记确定的蛋白质的重要性。特别是，我们将确定特定的蛋白质在协调纤维素的合成与其他基质多糖的沉积中是否重要；确定大型纤维素合酶复合物是如何被引导到细胞表面的适当部位的；并确定纤维素合成酶复合体的活性或制造纤维素所需的其他组分是否受到磷酸基团的调节，以及蛋白质磷酸化过程是否对纤维素的合成在生长过程中以及对环境信号的响应中受到调节很重要。

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.