Genetic and molecular characterization of the regulation of chloroplast protein import in Arabidopsis thaliana

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Plastids are a group of specialised organelles widely distributed through plants and algae. The most prototypical member of the group is undoubtedly the chloroplast, which is the site of photosynthesis and many other essential biosynthetic processes. Plastids derive from ancestral cyanobacteria through an endosymbiotic event, and as a result retain a eubacterial-type genome. Within the course of evolution, over 90% of the ancestral protein-coding genes have been transferred into the nuclear genome. The encoded plastid-bound proteins must therefore be transferred back into plastids after synthesis, and this is facilitated by a dedicated import apparatus (the TOC/TIC complexes). Over last few years a proteolytic system called the Ubiquitin-Proteasome System (UPS) has been recognized to target TOC proteins for degradation by tagging with ubiquitin. By regulating the import pathway, the plastid proteome can be altered to assist the acclimation under particular environment or developmental stage. Recently, a chloroplast-associated protein degradation (CHLORAD) system has been identified. In CHLORAD, SP1 (SUPPRESSOR OF ppi1 LOCUS 1) acts as the E3 ligase, SP2 (SUPPRESSOR OF ppi1 LOCUS 2) functions as an exit channel, and the ATPase Cdc48 provides the motor force for protein extraction. One important question is: how is the cytosolic Cdc48 tethered to chloroplasts? PUX10, which belongs to a UBX-domain-containing protein family functioning as Cdc48 cofactors, was identified to interact with Cdc48, SP1, SP2 and TOC proteins on the chloroplast membrane. I will carry on dissecting its functional link with CHLORAD by analysing its topology, the roles of its two specific domains (UBX and UBA), and its physiological significance in plastid biogenesis. In parallel, a larger scale genetic screen for suppressor mutations of ppi1 had identified a new suppressor of ppi1 locus called sp3. Preliminary characterization suggests that SP3 associates with a CUL4-based E3 ligase complex acting as a substrate receptor. To build on this, I aim to understand: what the substrates of SP3 are; the interaction between SP3 and TOC components; and what physiological roles SP3 has in chloroplast biogenesis and stress tolerance.The proposed project falls into the remit of cross-research council and BBSRC priority areas. As plastids contribute massively to crop productivity and quality, the knowledge gained from this project will facilitate our capability to sustainably enhance agriculture production and battle the issues relating to food, nutrition and health. Furthermore, understanding the predicted role of TOC degradation in responding to abiotic stress may contribute to coping with global food security and living with environment change.