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

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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.