Structure, Function and Substrates of the ClpP/R Proteolytic Machinery in Plastids of Arabidopsis Thaliana

叶绿体和非光合质体中的蛋白质水解知之甚少，但对植物发育和质体功能至关重要。通过生物化学纯化和质谱分析，在根和花瓣的非光合质体以及叶绿体中鉴定了Clp蛋白酶复合物。该复合物含有11种不同的基因产物（ClpP 1，3-6，ClpR 1 -4和ClpS 1 -2）的一个或多个副本，共16个亚基，而植物线粒体含有同源十四聚体ClpP 2复合物。因此，质体的Clp核心复合物是不寻常的复杂性，在细菌和植物线粒体的Clp核心复合物相比。三维同源性建模表明，ClpP/R蛋白很好地配合在一起，在一个四-十聚体环结构，也表明每个亚基的独特贡献。然而，目前尚不清楚为什么质体Clp复合物已经进化到如此复杂，并且对质体Clp机制的底物知之甚少。初步数据表明，大多数（如果不是全部）ClpP和ClpR基因是必需的。 本研究的具体目标是：（1）利用GUS启动子融合技术，研究ClpP和ClpR基因在不同发育阶段的组织特异性表达。(2)描述在自养和异养条件下生长的ClpP和ClpR基因中T-DNA插入系的表型。包括生长发育、超微结构（TEM）、光合活性分析和比较蛋白质组学分析。（3）利用表位标记的转基因植物、交联和质谱技术，确定亲和纯化的Clp复合物中来自不同组织的化学计量学和最近邻。（4）通过与突变的ClpP/R表位标记的转基因互补敲除株系，确定单个ClpP/R质体蛋白的结构/功能关系，并鉴定Clp底物。更广泛的影响：质体中的蛋白质水解知之甚少，但对植物发育也至关重要。该项目将提供深入了解Clp驱动的蛋白水解对植物生长和发育的确切作用。控制质体蛋白水解对于改善叶绿体作为过量生产这些产物的场所将是重要的。这个多学科的项目也将为本科生和研究生在这个项目和植物蛋白质组学和质谱领域提供一个很好的培训基地。该项目还将参加最近为高中生设立的外展计划。

Proteolysis in chloroplasts and non-photosynthetic plastids is poorly understood, but is critical for plant development and plastid function. Clp protease complexes were identified in non-photosynthetic plastids in roots and petals, as well as in chloroplasts, by biochemical purification and mass spectrometry. The complex contains 11 different gene products (ClpP1, 3-6, ClpR1-4 and ClpS1-2) in one or more copies, totaling 16 subunits, whereas plant mitochondria contain homotetradecameric ClpP2 complexes. Thus plastid Clp core complexes are of unusual complexity, as compared to Clp core complexes in bacteria and plant mitochondria. Three-dimensional homology modeling showed that the ClpP/R proteins fit well together in a tetra-decameric ring structure and also suggest unique contributions for each subunit. Yet, it is not clear why the plastid Clp complex has evolved to such complexity and little is known about the substrates of the plastid Clp machinery. Preliminary data suggest that most, if not all, of the ClpP and ClpR genes are essential. The specific objectives of this project are: (1) Determine tissue specific gene expression with promoter GUS fusions for the ClpP and ClpR genes across different developmental stages. (2) Characterize the phenotype of T-DNA insertion lines in ClpP and ClpR genes, grown under autotrophic and heterotrophic conditions. This includes analysis of growth, development, ultra-structure (by TEM) and photosynthetic activity and comparative proteomics analyses (3) Determine stochiometry and nearest neighbors in affinity purified Clp complexes from different tissues, using epitope tagged transgenic plants, crosslinking and mass spectrometry. Compare the experimental information with the generated high resolution 3-D models,(4) Determine structure/function relationships of the individual ClpP/R plastid proteins and identify Clp substrates, through complementation of knockout lines with mutated ClpP/R epitope tagged transgenes. Broader Impacts: Proteolysis in the plastid is poorly understood, but is also critical for plant development. This project will provide insight into the precise role of Clp driven proteolysis for plant growth and development. Controlling plastid proteolysis will be important to improve the chloroplast as the site for overproduction of such products. This multi-disciplinary project will also provide an excellent training ground for undergraduate and graduate students in this project and the area of plant proteomics and mass spectrometry. The project will also participate in a recently established outreach program for high school students.