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

• Applicant: Dr. Günter Kramer
• Subject Area: Biochemistry ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 407215628

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.

• DFG Programme: Research Grants