Selection and Delivery of Substrates to the Essential Clp Protease Complex in Plastids of Arabidopsis Thaliana

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Intellectual Merit:Plastids are essential organelles that are present in every plant cell. Plastids include chloroplasts, the sites of photosynthetic energy generation in plants and algae, amyloplasts, which store starch in roots and tubers, and chromoplasts, which accumulate carotenoids in fruits. Around 14% percent of genes in the plant genome are predicted to encode plastid proteins, underlining the importance of the plastid for plant biology. This project concerns the function and substrates of the essential plastid localized Clp protease machinery in the model plant 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. Intracellular proteases have many functions and must be highly selective in recognizing their substrates. The plastid-localized Clp proteins are clearly important for plastid biogenesis and differentiation, but their mechanism of action in plastids, including Clp substrates and substrate recognition mechanisms, are unknown. The objectives of the project are to identify Clp protease substrates and their degradation signals, to determine the role of ClpS in substrate delivery to the Clp protease system, to determine the function of ClpT1,T2 as a specific plastid component of the Clp system, and to test potential substrates and degradation signals (degrons) identified in objectives 1, 2 and 3 (or in new literature) in vivo. 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 nutritional or other added value. The proposed research will provide multi-disciplinary training in plant molecular genetics and biochemistry at the undergraduate, graduate and post-doctoral levels. The PI will participate in curriculum development workshops for high school teachers to develop genomics-based modules for the classroom.