DETERMINATION OF THE N-DEGRON PATHWAY AND ITS SUBSTRATES IN PLANT CHLOROPLASTS

Protein amino (N) termini (i.e., the start of the linear sequence of amino acids in a protein) are major determinants of protein stability. This is conceptualized in the N-degron pathway which states that certain amino acids, when exposed abnormally at the N-terminus of a protein, act as triggers (degrons) for protein degradation. Plant chloroplasts have an ancient relationship with prokaryotes and their N-degron pathway involves the bacterial-like Clp chaperone-protease system, with the N-recognin ClpS1 playing a central role in recognition, binding, and delivery of N-degron substrates to Clp chaperones. This research will determine the mechanistic features of this important pathway. The Broader impacts of this research include its intrinsic merit as plant chloroplasts play a central role in energy transduction on the planet and the work may facilitate the rational design of stable transgenic proteins targeted to chloroplasts with applications in pharmaceutical and biofortification products. Additional activities include interdisciplinary training, bridging cell biology, molecular genetics, quantitative imaging, bioinformatics, and biochemistry. Summer internships will be offered through our NSF-sponsored REU programs. A main impediment for determining the molecular details of a chloroplast N-degron pathway is an in planta quantitative reporter system that can test specific N-termini of interest. The research will employ our newly developed in vivo reporter system to determine N-degrons in chloroplasts and the involvement of chloroplast ClpS1 and the Clp chaperone-protease system. Independently, our in vivo ClpC1 chaperone trapping approach will determine the impact of ClpS1 and ClpF on substrate selection and delivery to the Clp chaperone-protease system. Identified ClpS1/ClpF dependent substrates will be investigated for their degrons. We will also determine how the N-domain of chloroplast glutamate t-RNA reductase is recognized as a conditional substrate by ClpS1 and ClpF in vivo using a unique transgenic reporter. 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.