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

细胞内蛋白酶具有许多功能，并且必须在识别其底物时具有高度选择性。本项目主要研究拟南芥中质体定位的Clp蛋白酶的功能和底物。ClpPR蛋白酶是含有10种不同ClpPR蛋白的双环状复合物，其中大多数是胚胎发生或幼苗发育所必需的。附着在桶上的是功能未知的小ClpS1，2蛋白质，是光合生物所特有的。质体中的clp底物和底物识别机制尚不清楚。目标是：i）通过完成ClpPR突变体的遗传分析来确定哪些ClpPR蛋白对于胚胎发生或幼苗发育是必需的。将进一步测试ClpR家族内可能的功能冗余，ii）使用clpr2 - 1突变体进行靶向生物化学实验以理解ClpPR复合物在质体基因表达和蛋白质稳态中的作用，iii）通过纯化的Clp复合物的生物化学分析来确定ClpPRS复合物的不对称结构的组成，iv）鉴定Clp蛋白酶底物，更广泛的影响：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蛋白水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，目的是改善质体作为过量生产具有例如营养或药用价值的外源蛋白质（例如可食用疫苗）的位点。该研究将提供本科生、研究生和博士后水平的植物分子遗传学和生物化学方面的多学科培训。 PI和co-PI还将参加高中教师的课程开发研讨会，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.