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The Protein:Protein Interaction Landscape of A. thaliana Chloroplasts

Equipped with a photosynthetic apparatus that uses the energy of solar radiation to fuel formation of organic compounds, chloroplasts are the metabolic factories of mature leaf cells. The first steps of energy conversion are catalyzed by a machinery of protein complexes, which can dynamically interact with each other for optimizing biochemical processes under changing environmental conditions. Chloroplast protein-protein-interactions can be studied by complexome profiling, which relies on the mild solubilization of protein complexes, their separation under native conditions, and - finally - their identification by LC-MS. To get deeper insights into the organization of high molecular mass protein complexes and their involvement in chloroplast adaption to changing environmental conditions, an improved complexome profiling protocol is reported here. By first applying a MS-cleavable cross- linker, labile protein complexes are stabilized during the isolation procedure and their separation on large pore blue native (BN) polyacrylamide gels. The influence of different detergents and cross-linker concentration was systematically tested. Optimized conditions were finally used to investigate plants acclimated to four different light intensities. In total, more than 800 mass spectrometry runs were performed, yielding thousands of protein abundance profiles along large-pore BN gels. The potential of the workflow is demonstrated by the identification of components of the PSII repair machinery and their dynamic behavior in dependence to different light intensities, as well as by the discovery of a ~8 MDa RNA-polymerase complex.

Impressum & Datenschutz   |   Institute of Plant Genetics   |   Leibniz Universität Hannover