Accurate Molecular Decision Making during Protein Biogenesis

项目摘要：蛋白质生物合成过程中的精确分子决策 准确的蛋白质生物合成对于产生和维持一个功能性的蛋白质结构是必不可少的。 蛋白质组我们的长期目标是了解不同的分子机制， 细胞中的蛋白质生物合成途径准确地选择新生蛋白质底物， 它们的正确折叠、定位和成熟。两个主要组成部分定义了我们的研究 在这个周期里， 首先，我们将结合生物化学、生物物理学和体内实验， 从核糖体中出现的新生蛋白质被选择的机制， 核糖体相关蛋白生物合成因子（RPB）在拥挤的空间中进行加工， 核糖体隧道出口这些研究将包括一种新的共翻译膜蛋白 SecA介导的靶向途径，介导N-末端甲硫氨酸切除的酶， 细菌中的新生蛋白质，以及细菌中SRP介导的共翻译蛋白质靶向， 哺乳动物系统除了研究的生化和生物物理机制， 单独的蛋白质生物合成途径，我们也将阐明如何每个这些因素 在进行翻译期间，与其他RPB在空间和时间上进行协调， 协调重塑了各个途径的效率和保真度。 其次，我们将破译的机制，其中聚集倾向膜蛋白是 在它们的翻译后过程中被有效地保护并容易地引导到靶膜 面向.这些研究将使用两种膜蛋白生物合成途径作为模型： 不依赖ATP的伴侣蛋白cpSRP 43，它使我们能够在生物物理分辨率下破译， 小分子伴侣有效保护多通道的分子机制 膜蛋白客户端，并实现其客户端相互作用的时空调节， 缺乏ATP酶循环或辅伴侣;（ii）尾锚定蛋白（GET）的引导进入 途径，它提供了一个出色的系统来破译多组分Hsp 70- 辅伴侣级联保护，漏斗，并分流新生的膜蛋白，在他们的 定向投放。对GET途径的研究也将使我们能够深入了解 细胞中类似分子伴侣网络的设计和组织原理。 所提出的实验不仅会产生高分辨率的理解 单个蛋白质的生物合成途径，而且还建立有价值的工具，试剂，探索 其他蛋白质生物合成机制的作用。最重要的是，这项研究将产生 重要的概念框架，以了解新生蛋白质是如何准确选择的 在拥挤的胞质环境中进入适当的生物合成途径。

Project Summary: Accurate Molecular Decision Making during Protein Biogenesis Accurate protein biogenesis is essential for the generation and maintenance of a functional proteome. Our long term goal is to understand the molecular mechanisms by which diverse protein biogenesis pathways in the cell accurately select nascent protein substrates and ensure their correct folding, localization, and maturation. Two major components define our research program in this grant cycle. First, we will use a combination of biochemical, biophysical, and in vivo experiments to decipher the mechanisms by which nascent proteins emerging from the ribosome are selected and processed by ribosome-associated protein biogenesis factors (RPBs) in the crowded space of the ribosome tunnel exit. These studies will include a new co-translational membrane protein targeting pathway mediated by SecA, enzymes mediating N-terminal methionine excision on nascent proteins in bacteria, and co-translational protein targeting mediated by SRP in the mammalian system. In addition to studying the biochemical and biophysical mechanisms of the individual protein biogenesis pathways, we will also elucidate how each of these factors coordinates with other RPBs in space and time during ongoing translation, and how this coordination reshapes the efficiency and fidelity of the individual pathways. Second, we will decipher the mechanisms by which aggregation-prone membrane proteins are effectively protected and facilely guided to the target membrane during their post-translational targeting. These studies will use two membrane protein biogenesis pathways as models: (i) an ATP-independent chaperone cpSRP43, which allows us to decipher, at biophysical resolution, the molecular mechanisms by which a small chaperone effectively protects multi-pass membrane protein clients and achieves spatiotemporal regulation of its client interactions in the absence of ATPase cycles or cochaperones; (ii) the guided-entry of tail-anchored proteins (GET) pathway, which provides an excellent system to decipher how a multi-component Hsp70- cochaperone cascade protects, funnels, and triages nascent membrane proteins during their targeted delivery. Investigation of the GET pathway will also allow us to gain insights into the design and organizational principles of analogous chaperone networks in the cell. The proposed experiments will not only generate high resolution understandings of the individual protein biogenesis pathways, but also establish valuable tools, reagents to explore the action of other protein biogenesis machineries. Most importantly, this research will generate important conceptual frameworks to understand how nascent proteins are accurately selected into their appropriate biogenesis pathways in the crowded cytosolic environment.