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

叶绿体是一组特殊的细胞器，广泛分布于植物和藻类中。这个群体中最典型的成员无疑是叶绿体，它是光合作用和许多其他基本生物合成过程的场所。质体通过内共生事件从祖先的蓝藻中衍生出来，因此保留了一个真细菌类型的基因组。在进化过程中，90%以上的祖先蛋白质编码基因已经转移到核基因组中。因此，编码的叶绿体结合蛋白必须在合成后转移回叶绿体，这是由专门的输入装置(TOC/TIC复合体)促进的。在过去的几年里，一种被称为泛素-蛋白酶体系统(UPS)的蛋白分解系统被认为是通过标记泛素来靶向降解TOC蛋白的。通过调节输入途径，叶绿体蛋白质组可以被改变，以帮助在特定环境或发育阶段的驯化。最近，一个叶绿体相关蛋白降解(CHLORAD)系统被发现。在CHLORAD中，SP1(ppi1基因座1的抑制子)作为E3连接酶，SP2(ppi1基因座的抑制子2)作为出口通道，ATPase CDC48为蛋白质提取提供动力。一个重要的问题是：胞质CDC48是如何与叶绿体相连的？PUX10是一个含有Ubx结构域的蛋白家族，与叶绿体膜上的CDC48、SP1、SP2和TOC蛋白相互作用，具有CDC48辅因子的功能。我将通过分析它的拓扑结构、它的两个特定结构域(Ubx和Uba)的作用以及它在叶绿体生物发生中的生理意义，来剖析它与ChLORAD的功能联系。与此同时，对ppi1抑制子突变的更大规模的遗传筛查已经确定了ppi1基因座的一个新的抑制子，称为SP3。初步鉴定表明，SP3与以CUL4为基础的E3连接酶复合体结合，作为底物受体。在此基础上，我的目标是了解SP3的底物是什么；SP3和TOC组分之间的相互作用；以及SP3在叶绿体生物发生和逆境耐受性中具有哪些生理作用。由于质体对作物产量和质量有很大贡献，从这个项目中获得的知识将有助于我们可持续地提高农业生产和解决与食品、营养和健康有关的问题。此外，了解TOC降解在应对非生物胁迫中的预测作用，可能有助于应对全球粮食安全和应对环境变化。

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.