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

植物叶绿体蛋白质稳定性N-端规则体内测试的定量报告系统？Klaas van Wijk，康奈尔大学简短摘要渴望应用程序1834636第1部分：项目的非技术描述。植物和藻类不同于其他有机体，如人类和细菌，因为它们的细胞中有一种额外的细胞器，即叶绿体。这些叶绿体含有数千种不同的蛋白质，它们完成包括光合作用在内的许多关键功能。叶绿体还包含几种名为蛋白酶的酶系统，一旦不再需要叶绿体蛋白质，它们就会选择性地降解它们。该项目旨在建立一个实验系统，该系统将允许确定蛋白质中导致它们被蛋白酶去除的信号。更好地了解蛋白质的稳定性规律对于作物的改良具有重要意义，也有助于更好地设计产生具有药用价值的产品和作物生物强化的叶绿体蛋白质。该项目还将用于教育和培训STEM学生和博士后，包括代表性不足群体的成员。第2部分：项目的技术说明。蛋白质氨基末端易于修饰，在细菌、真核生物中是蛋白质稳定性的主要决定因素，也许在叶绿体和非光合体中也是如此。N-末端在蛋白质稳定性中的作用在N-末端规则中被概念化，该规则规定，当某些氨基酸暴露在蛋白质的N-末端时，充当降解的触发物(退化)。基于几种类型的间接实验证据，以及进化论证，我们假设叶绿体中一定存在N-端规则，但这一假设尚未得到证实。在叶绿体中检测N-端规则的主要障碍是一个合适的在体定量报告系统。这项急切的提议旨在基于为其他生物系统开发的新的定量工具和技术的组合来生成这样一个系统。然后，这项拟议的研究将使用该系统直接测试拟南芥叶绿体中可能的N-降解子以及叶绿体ClpS1和CLP蛋白酶和伴侣系统的参与。因此，这项工作可能对叶绿体蛋白稳定和细胞器中蛋白质稳定性N端规则的进化适应做出变革性的贡献。确定这样的N-端规则将对叶绿体蛋白质稳定性具有很高的预测性。该奖项反映了NSF的法定使命，并已通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.