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

根据现有的知识，大肠杆菌通过两种主要途径将新合成的蛋白质转运到膜内和膜外。在翻译过程中，通过核糖体结合信号识别颗粒（SRP），协同翻译易位主要发生在内膜蛋白（imp）的早期。随后的膜插入是由停靠在膜嵌入转座子上的核糖体的蛋白质合成驱动的。相反，外膜蛋白（OMPs）和质周蛋白（PPs）跨膜的翻译后易位不依赖于核糖体对接，而是由经空腔对接的SecA驱动。对于一些蛋白质，依赖seca的易位由伴侣蛋白SecB进一步促进。最近来自我们和其他实验室的证据表明，关于细菌蛋白质分泌和膜插入的经典观点的某些方面必须进行修订。首先，我们发现SecA直接与核糖体结合，共同翻译选择底物。这意味着将易位模式明确地划分为共同易位模式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，其他研究表明SecA是将一部分imp插入膜中所必需的，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同与翻译后易位途径的因子网络，提供对新生链的共翻译选择的全面理解。我们将首先通过SeRP识别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与新生底物结合，以及新生链中的哪些分子特征介导结合（例如序列基序、疏水拉伸、折叠和链长）。其次，通过定量比较SRP、SecA、SecB和TF的相互作用特征以及突变细胞中其他SeRP研究，我们将探索这些因素作为一个功能网络的协调程度。目的是阐明功能拮抗和冗余的原则，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翻译核糖体的共翻译底物选择和膜靶向相协调。该研究将扩展我们对核糖体蛋白质分选的理解，并揭示共翻译作用因子网络如何支持大肠杆菌中新合成蛋白质的有效易位。

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.