21ENGBIO - Converting a cellular dustbin into a protein storing organelle

植物养活世界。人类和动物食用的大多数蛋白质（直接或间接）来自植物，特别是种子。植物种子蛋白质在分泌途径中合成，分泌途径是包括内质网、高尔基复合体、内体区室和液泡的细胞膜系统。植物种子细胞将蛋白质储存在内质网、液泡或两者中。蛋白质贮藏泡（PSV）是双子叶植物（如豆科植物）的首选贮藏部位，也是支撑种子消耗的主要营养库。植物也越来越多地被用作生产高价值药用蛋白质的宿主，包括最近的COVID-19疫苗。主要的挑战是确保植物产生的蛋白质稳定地积累和储存。在非种子细胞中，液泡通常提供了一个恶劣的环境，在大多数植物组织中发挥降解（裂解）功能（类似于动物细胞中的溶酶体）。早期尝试将单个种子蛋白质或高价值蛋白质（如抗体）表达到转基因叶细胞的液泡中，导致蛋白质随时间降解。在这个项目中，我们打算测试一个starteg，把裂解空泡变成蛋白质储存空泡。在过去的5年中，我们研究了PSV是如何在种子成熟过程中形成的：在种子胚细胞中，有一个单一的溶胞，在成熟过程中充满了储存蛋白，然后分裂形成多个，蛋白质负载的PSV。最近，我们发现种子贮藏蛋白在液泡内形成一个单独的液相，产生密集的液滴;这些液滴与液泡膜相互作用，使其弯曲，形成芽;芽最终分离形成许多PSV。因此，我们假设蛋白质液滴形成的物理过程，称为液-液相分离（LLPS），是从裂解液泡转变为储存液泡的关键驱动因素。这是非常令人兴奋的：如果我们的假设是正确的，我们将能够诱导这一过程，将植物非种子组织（如叶）和非植物生物体（如酵母）中的裂解液泡转化为储存蛋白质的细胞器。在这个项目中，我们将测试我们的假设，通过表达一组种子贮藏蛋白，和其他蛋白质，这是已知的经历LLPS，在植物叶片和酵母。因此，我们将能够确定哪些蛋白质是最适合触发溶解到储存液泡转换。因此，我们将提供原理证明和蓝图，将细胞“垃圾箱”重新利用为高价值蛋白质内容物的蛋白质储存室。

Plants feed the world. Most proteins eaten by humans and animals come (either directly or indirectly) from plants, in particular seeds. Plant seed proteins are synthesised in the secretory pathway - a system of cellular membranes that comprises the endoplasmic reticulum, the Golgi complex, endosomal compartments, and the vacuole. Plant seed cells store proteins in the endoplasmic reticulum, the vacuole, or both. The protein storage vacuole (PSV) is the preferred storage site in dicotyledonous plants such as legumes and is the main nutritional repository underpinning seed consumption. Plants are also increasingly used as hosts to produce high value pharmaceutical proteins, including, recently, a vaccine against COVID-19. The main challenge is to ensure that plant-produced proteins are accumulated and stored stably. In non-seed cells, the vacuole normally provides a harsh environment, serving a degradative (lytic) function in most plant tissues (akin to the lysosome in animal cells). Early attempts to express individual seed proteins, or high-value proteins such as antibodies, into the vacuoles of transgenic leaf cells led to the proteins to be degraded over time. In this project we propose to test a starteg to turn lytic vacuoles into protein storage vacuoles. In the last 5 years we have studied how PSV are formed during seed maturation: in seed embryo cells, there is a single lytic vacuole, which during maturation becomes filled with storage proteins and then divides to form multiple, protein-laden PSV. Very recently we have discovered that the seed storage proteins form a separate liquid phase inside the vacuole, which creates dense droplets; these droplets interact with the membrane of the vacuole and cause it to bend, forming buds; the buds eventually separate to form many PSV. We therefore hypothesise that this physical process of protein droplet formation, called liquid-liquid phase separation (LLPS), is the key driver of the transition from a lytic vacuole to a storage vacuole. This is very exciting: if our hypothesis is correct, we will be able to induce this process to convert lytic vacuoles, both in plant non-seed tissues (such as leaves), and in non-plant organisms, such as yeast, into protein-storing organelles. In this project we will test our hypothesis by expressing a panel of seed storage proteins, and other proteins which are known to undergo LLPS, in plant leaves and in yeast. We will therefore be able to establish which proteins are the most suitable for triggering the lytic-to-storage vacuole conversions. Therefore we will provide both the proof of principle and the blueprint for re-purposing cellular 'dustbins' into protein-storing compartments for high value protein contents.