EAGER: Quantitative reporter systems for in vivo testing of an N-end rule for protein stability in plant chloroplasts

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Quantitative reporter systems for in vivo testing of an N-end rule for protein stability in plant chloroplasts ? Klaas van Wijk, Cornell University Short abstract EAGER application 1834636Part 1: A nontechnical description of the project. Plants and algae are different from other organisms, such as humans and bacteria, in that they have an extra type of organelle in their cells, namely chloroplasts. These chloroplasts contain several thousand different proteins that fulfill many critical functions, including photosynthesis. The chloroplast also contains several enzymatic systems, named proteases, which selectively degrade chloroplast proteins once they are no longer needed. This project aims to build an experimental system that will allow to determine the signals within proteins that lead to their removal by proteases. A better understanding of the stability rules of proteins is important for improvement of crops and also will help in a better design of chloroplast proteins that generate products of pharmaceutical value and biofortification of crops. The project will also serve to educate and train students and postdocs in STEM, including members of underrepresented groups.Part 2: A technical description of the project. Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts and non-photosynthetic plastids. The role of the N-terminus in protein stability is conceptualized in the N-end rule, which states that certain amino acids, when exposed at the N-terminus of a protein, act as triggers (degrons) for degradation. Based on several types of indirect experimental evidence, as well as evolutionary arguments, we postulate that an N-end rule in plastids must exist, however that has not been proven. The main impediment for testing for an N-end rule in chloroplasts is a suitable in vivo quantitative reporter system. This EAGER proposal aims to generate such a system based on a combination of novel quantitative tools and techniques developed for other biological systems. The proposed research will then use this system to directly test possible N-degrons in chloroplasts and the involvement of chloroplast ClpS1 and the Clp protease and chaperone system in Arabidopsis. Therefore, this work could make a transformative contribution to chloroplast proteostasis and the evolutionary adaptation of the N-end rule for protein stability in organelles. Identification of such an N-end rule would be highly predictive for chloroplast protein stability.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.