Function and Organization of the ClpPR Complex in Plastids of Arabidopsis; A Central Protease Essential for Embryogenesis and Seedling Development

胞内蛋白水解酶具有多种功能，在识别其底物时必须具有高度的选择性。本项目涉及拟南芥中必需的叶绿体定位的CLP蛋白水解酶的功能和底物。ClpPR蛋白酶是一个双环复合体，含有10种不同的ClpPR蛋白，其中大部分对胚胎发育或幼苗发育是必需的。桶上附着着小的ClpS1，2蛋白质，功能未知，是光合作用有机体独有的。CLP底物和叶绿体中的底物识别机制尚不清楚。目的是：1)通过完成ClpPR突变体的遗传分析，确定哪些ClpPR蛋白对胚胎发生或幼苗发育是必需的。将进一步测试CLPR家族内可能存在的功能冗余，ii)使用clpr2-1突变体进行有针对性的生化实验，以了解ClpPR复合体在叶绿体基因表达和蛋白质动态平衡中的作用，iii)通过对纯化的CLP复合体进行生化分析来确定ClpPR复合体的不对称结构的组成，iv)确定CLP蛋白酶底物、其降解信号和伴侣递送系统对ClpPR核心复合体的专一性。广泛影响：该项目将有助于确定CLP蛋白酶系统的分子功能和组织，以及其底物和底物识别机制。了解CLP蛋白分解机制和底物识别信号的作用对于控制蛋白质稳定性也是至关重要的，目的是改善叶绿体作为过量生产具有营养或药用价值的外来蛋白质(如可食用疫苗)的场所。这项研究将在本科生、研究生和博士后水平上提供植物分子遗传学和生物化学的多学科培训。PI和共同PI还将参加为高中教师举办的课程开发讲习班，为课堂开发基于基因组学的模块。

Intracellular proteases have many functions and must be highly selective in recognizing their substrates. This project concerns the function and substrates of the essential plastid localized Clp protease machinery in Arabidopsis. The ClpPR protease is a double ring shaped complex containing 10 different ClpPR proteins, most of which are essential for embryogenesis or seedling development. Attached to the barrel are small ClpS1,2 proteins with unknown function and unique to photosynthetic organisms. Clp substrates and substrate recognition mechanisms in plastids are unknown. The objectives are: i) to determine which ClpPR proteins are essential for embryogenesis or seedling development, by completion of genetic analysis of ClpPR mutants. Possible functional redundancy within the ClpR family will be further tested, ii) to carry out targeted biochemical experiments using the clpr2-1 mutant to understand the role of ClpPR complex in plastid gene expression and protein homeostasis, iii) to determine the composition of the asymmetric structure of the ClpPRS complex by biochemical analysis of purified Clp complexes, iv) to identify Clp protease substrates, their degradation signals and specificity of chaperone delivery systems to the ClpPR core complex.Broader impacts: This project will help to determine the molecular function and organization of the Clp protease system, as well as its substrates and substrate recognition mechanisms. Understanding the role of the Clp proteolytic machinery and substrate recognition signals is also critical to control protein stability with the objective to improve the plastid as the site for overproduction of foreign proteins with e.g nutritional or pharmaceutical value (e.g. edible vaccines). The research will provide multi-disciplinary training in plant molecular genetics and biochemistry at the undergraduate, graduate and post-doctoral levels. The PI and co-PI will also participate in curriculum development workshops for high school teachers to develop genomics-based modules for the classroom.