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

Wang, Shuai and Jomaa, Ahmad and Jaskolowski, Mateusz and Yang, Chien-I and Ban, Nenad and Shan, Shu-ou (2019) The molecular mechanism of cotranslational membrane protein recognition and targeting by SecA. Nature Structural & Molecular Biology, 26 (10). pp. 919-929. ISSN 1545-9985. PMCID PMC6858539. https://resolver.caltech.edu/CaltechAUTHORS:20190801-131335661

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[img] PDF (Supplementary Figures 1–8 and Supplementary Notes 1 and 2) - Supplemental Material
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[img] PDF (Reporting Summary) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 1: Controls and additional data to map the interaction of SecA with hydrophobic sequences on nascent protein in the co- versus post-translational mode) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 2: Additional data to map the interaction surface of SecA with nascent chains on RNC and with post-translational substrates) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 3: Image classification and refinement of the structure of the RNCRodZ–SecA complex) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 4: Local resolution and validation of the cryo-EM maps) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 5: Overview of the RodZ TMD binding pocket and the ribosome tunnel region) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 6: Characterization of the samples for fluorescence measurements of RNCRodZ transfer from SecA to SecYEG) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 7: Supporting information for the kinetic simulations, and additional data for chase experiments of the RNCRodZ–SecA complex) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 8: Comparison of the structure from this work with previous structures) - Supplemental Material
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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/s41594-019-0297-8DOIArticle
https://rdcu.be/bSBkUPublisherFree ReadCube access
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858539PubMed CentralArticle
ORCID:
AuthorORCID
Wang, Shuai0000-0002-8920-969X
Jomaa, Ahmad0000-0002-4786-934X
Jaskolowski, Mateusz0000-0001-6618-7048
Yang, Chien-I0000-0001-8606-5013
Ban, Nenad0000-0002-9527-210X
Shan, Shu-ou0000-0002-6526-1733
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.
Funders:
Funding AgencyGrant Number
NVIDIA CorporationUNSPECIFIED
ETH ZurichETH-40 16-2
NIHGM107368A
Gordon and Betty Moore FoundationGBMF2939
Swiss National Science Foundation (SNSF)310030B_163478
Swiss National Science Foundation (SNSF)51NF40_141735
Subject Keywords:Biochemistry; Cell biology; Structural biology
Issue or Number:10
PubMed Central ID:PMC6858539
Record Number:CaltechAUTHORS:20190801-131335661
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190801-131335661
Official Citation:Wang, S., Jomaa, A., Jaskolowski, M. et al. The molecular mechanism of cotranslational membrane protein recognition and targeting by SecA. Nat Struct Mol Biol 26, 919–929 (2019). https://doi.org/10.1038/s41594-019-0297-8
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:97591
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:30 Sep 2019 17:12
Last Modified:21 Apr 2020 22:42

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