Network analysis of co-translationally acting factors mediating membrane targeting of proteins in E. coli

介导大肠杆菌中蛋白质膜靶向的共翻译作用因子的网络分析

• 批准号: 407215628
• 负责人: Dr. Günter Kramer
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/407215628?language=en
• 关键词: Network; analysis; co; translationally; acting

According to established knowledge, E. coli employs two main pathways to translocate newly synthesized proteins into and across the membrane. Co-translational translocation of mostly inner membrane proteins (IMPs) initiates early during translation by ribosome binding of the signal recognition particle (SRP). Subsequent membrane insertion is driven by protein synthesis of ribosomes docked to the membrane-embedded translocon. In contrast, post-translational translocation of outer membrane proteins (OMPs) and periplasmic proteins (PPs) across the membrane is independent of ribosome docking and driven by translocon-docked SecA. For some proteins SecA-dependent translocation is further facilitated by the chaperone SecB. Recent evidence from us and other labs implies that some aspects of the classical view on bacterial protein secretion and membrane insertion must be revised. First, we find that SecA directly binds to ribosomes to co-translationally select its substrates. This implies that the clear separation into co- and post-translational translocation modes may be incorrect. Second, we recently found that SecA and SecB also engage ribosomes synthesizing IMPs, and others showed that SecA is required for the insertion of a subset of IMPs into the membrane, suggesting there is significant overlap between both pathways.The proposed project aims at providing a comprehensive understanding of the co-translational selection of nascent chains by the network of factors that triages proteins to the co- versus post-translational translocation pathways. We will first identify the nascent chain interactomes of SecA and SecB by SeRP and determine when during synthesis each factor engages nascent substrates and which molecular features in nascent chains mediate binding (e.g. sequence motifs, hydrophobic stretches, folds and chain lengths). Second, by quantitatively comparing interaction profiles of SRP, SecA, SecB and TF and additional SeRP studies in mutant cells, we will explore to what extent these factors are orchestrated as a functional network. The aim is to elucidate the principles of functional antagonism and redundancy employed to maximize the specificity and efficiency of substrate selection. Third, we will study whether the kinetics of protein synthesis is coordinated with co-translational substrate selection and membrane targeting of the translating ribosome. The study will extend our understanding of protein sorting at the ribosome and reveal how the network of co-translationally acting factors supports efficient translocation of newly synthesized proteins in E. coli.

根据已知的知识，E.大肠杆菌利用两种主要途径将新合成的蛋白质转运到膜内和穿过膜。大多数内膜蛋白（IMP）的共翻译易位在翻译早期通过信号识别颗粒（SRP）的核糖体结合启动。随后的膜插入由对接到膜嵌入的易位子的核糖体的蛋白质合成驱动。相反，外膜蛋白（OMPs）和周质蛋白（PPs）跨膜的翻译后易位是独立的核糖体对接和驱动的translocon-docked SecA。对于某些蛋白质，SecA依赖性易位进一步由分子伴侣SecB促进。我们和其他实验室的最新证据表明，必须修改有关细菌蛋白质分泌和膜插入的经典观点的某些方面。首先，我们发现SecA直接与核糖体结合，共同选择其底物。这意味着明确分为共翻译和翻译后易位模式可能是不正确的。其次，我们最近发现SecA和SecB也参与核糖体合成IMP，其他人表明SecA是将IMP子集插入膜所必需的，这表明两种途径之间存在显著的重叠。拟议的项目旨在通过将蛋白质分类到共与后的因子网络，提供对新生链的共翻译选择的全面理解。翻译易位途径。我们将首先通过SeRP鉴定SecA和SecB的新生链相互作用组，并确定在合成过程中每个因子何时参与新生底物，以及新生链中的哪些分子特征介导结合（例如序列基序，疏水延伸，折叠和链长）。其次，通过定量比较SRP，SecA，SecB和TF的相互作用概况和突变细胞中的其他SeRP研究，我们将探索这些因素在多大程度上被编排为一个功能网络。目的是阐明功能性拮抗和冗余的原则，以最大限度地提高底物选择的特异性和效率。第三，我们将研究蛋白质合成的动力学是否与共翻译底物选择和翻译核糖体的膜靶向相协调。这项研究将扩展我们对蛋白质在核糖体上分选的理解，并揭示协同作用因子网络如何支持新合成蛋白质在大肠杆菌中的有效转运。杆菌