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

Reference	BB/W012162/1
Principal Investigator / Supervisor	Professor Lorenzo Frigerio
Co-Investigators / Co-Supervisors	Dr Fabrizio Alberti, Professor Halim Kusumaatmaja, Dr Joseph McKenna
Institution	University of Warwick
Department	School of Life Sciences
Funding type	Research
Value (£)	99,735
Status	Current
Type	Research Grant
Start date	15/02/2023
End date	14/02/2024
Duration	12 months

Abstract

Protein storage vacuoles (PSV) are unique plant cell organelles, mostly found in seeds, which accumulate seed storage proteins (SSP). PSV therefore are key to human and animal nutrition. PSV are functionally very different to the vacuoles found in non-seed tissues, which have a degradative, lysosome-like function. Over the last 5 years we asked what made PSV a safe haven for proteins and studied PSV biogenesis in the model plant Arabidopsis. We found that PSV in seed embryo cells arise from early vacuoles that are lytic, like every other plant vacuole. As SSP are sorted to those vacuoles, they undergo liquid-liquid phase separation (LLPS) and form droplets in the vacuolar lumen. LLPS is likely triggered by the intrinsically disordered regions and prion-like domains present on many SSP. LLPS has two consequences: 1. it likely shelters SSP from degradation by vacuolar proteases, which are excluded from the SSP droplets; 2. SSP droplets wet the tonoplast. We have very recently found that particular wetting conditions induce tonoplast curvature and the formation of buds, which eventually result in the observed, numerous, SSP-filled PSV in mature seed cells. We therefore hypothesise that the expression of phase-separating SSP is both necessary and sufficient to re-engineer a lytic vacuole into a storage vacuole. In this project we test this hypothesis by transiently expressing combinations of SSP with different LLPS properties, and other proteins which are well known to induce LLPS, in the lytic vacuoles of leaf cells. We also propose to re-engineer the yeast (lytic) vacuole into a PSV-like structure, by sorting different phase-separating proteins to the yeast vacuole. We will also use these experiments, in conjunctions with biophysical measurements, to correlate the behaviour of individual (or combinations of proteins) with the remodelling of vacuoles, therefore providing a blueprint for re-engineering lytic compartments in different biological systems.

Summary

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.

Committee	Not funded via Committee
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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