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

蛋白质氨基末端（即蛋白质中氨基酸线性序列的起点）是蛋白质稳定性的主要决定因素。这在N-degron途径中被概念化，该途径指出某些氨基酸，当在蛋白质的n端异常暴露时，充当蛋白质降解的触发器（degron）。植物叶绿体与原核生物有着古老的关系，它们的N-degron通路涉及细菌样的Clp伴侣-蛋白酶系统，其中n -识别蛋白ClpS1在识别、结合和将N-degron底物传递到Clp伴侣中起着核心作用。这项研究将确定这一重要途径的机制特征。该研究的广泛影响包括其固有的优点，因为植物叶绿体在地球上的能量转导中起着核心作用，该工作可能有助于合理设计针对叶绿体的稳定转基因蛋白，并应用于制药和生物强化产品。其他活动包括跨学科培训，桥接细胞生物学，分子遗传学，定量成像，生物信息学和生物化学。夏季实习将通过我们的nsf赞助REU计划提供。确定叶绿体n -降解途径的分子细节的主要障碍是植物定量报告系统可以测试特定的n -末端。本研究将采用我们新开发的体内报告系统来测定叶绿体中的N-degrons以及叶绿体ClpS1和Clp伴侣-蛋白酶系统的参与情况。独立地，我们的体内ClpC1伴侣捕获方法将确定ClpS1和ClpF对底物选择和传递到Clp伴侣-蛋白酶系统的影响。已鉴定的ClpS1/ClpF依赖底物将被研究其降解。我们还将利用一种独特的转基因报告基因，确定叶绿体谷氨酸t-RNA还原酶的n结构域是如何被ClpS1和ClpF识别为条件底物的。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.