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Published October 2019 | Supplemental Material + Accepted Version
Journal Article Open

The molecular mechanism of cotranslational membrane protein recognition and targeting by SecA

Abstract

Cotranslational protein targeting is a conserved process for membrane protein biogenesis. In Escherichia coli, the essential ATPase SecA was found to cotranslationally target a subset of nascent membrane proteins to the SecYEG translocase at the plasma membrane. The molecular mechanism of this pathway remains unclear. Here we use biochemical and cryoelectron microscopy analyses to show that the amino-terminal amphipathic helix of SecA and the ribosomal protein uL23 form a composite binding site for the transmembrane domain (TMD) on the nascent protein. This binding mode further enables recognition of charged residues flanking the nascent TMD and thus explains the specificity of SecA recognition. Finally, we show that membrane-embedded SecYEG promotes handover of the translating ribosome from SecA to the translocase via a concerted mechanism. Our work provides a molecular description of the SecA-mediated cotranslational targeting pathway and demonstrates an unprecedented role of the ribosome in shielding nascent TMDs.

Additional Information

© 2019 Springer Nature Limited. Received 04 May 2019; Accepted 12 August 2019; Published 30 September 2019. Data Availability: Cryo-EM maps are deposited in the electron microscopy databank (EMDB) with accession codes EMD-10073 (RNCRodZ–SecA) and EMD-10074 (SecA, local refinement), and model coordinates are deposited in the worldwide PDB with accession code PDB 6S0K. Other data are available from corresponding authors upon reasonable request. We thank A. McDowall and H. Wang for assistance with negative stain electron microscopy data collection, J. Rothman for sharing plasmid ApoE422k, D. Boehringer and A. Scaiola for the support with EM data collection and processing, M. Leibundgut and M. Saurer for the support with model building and members of the Shan and Ban groups for discussions and comments on the manuscript. We also thank T. Miller, M. Zimmer and F. Huber for helpful discussions. Cryo-EM data was collected at the Scientific Center for Optical and Electron Microscopy at the ETH Zurich (ScopeM). We gratefully acknowledge the support of NVIDIA Corporation for the Titan Xp GPU used in this research through a GPU Grant program awarded to A.J.; M.J. was supported by the internal research grant of the ETH to N.B. (ETH-40 16-2). This work was supported by National Institutes of Health grant GM107368A and the Gordon and Betty Moore Foundation through grant GBMF2939 to S.S. and by the Swiss National Science Foundation (SNSF) (grant number 310030B_163478), National Center of Excellence in Research (NCCR) RNA & Disease Program of the SNSF (grant number 51NF40_141735) to N.B. Author Contributions: S.W. and S.S. conceived the project. S.W. performed most of the biochemical experiments and analyzed data. A.J. acquired cryo-electron microscopy data and performed reconstructions and model building. M.J. performed initial sample preparation for cryo-EM analysis. C.Y. measured the association rate constant of SecA binding to RNCRodZ. N.B. and S.S. supervised the structural and biochemical experiments, respectively. All authors interpreted the data and contributed to the final versions of the manuscript. The authors declare no competing interests.

Attached Files

Accepted Version - nihms-1537248.pdf

Supplemental Material - 41594_2019_297_Fig10_ESM.jpg

Supplemental Material - 41594_2019_297_Fig11_ESM.jpg

Supplemental Material - 41594_2019_297_Fig12_ESM.jpg

Supplemental Material - 41594_2019_297_Fig13_ESM.jpg

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Supplemental Material - 41594_2019_297_Fig15_ESM.jpg

Supplemental Material - 41594_2019_297_Fig8_ESM.jpg

Supplemental Material - 41594_2019_297_Fig9_ESM.jpg

Supplemental Material - 41594_2019_297_MOESM1_ESM.pdf

Supplemental Material - 41594_2019_297_MOESM2_ESM.pdf

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Additional details

Created:
August 22, 2023
Modified:
October 18, 2023