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

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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.