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Published November 17, 2020 | Supplemental Material + Published
Journal Article Open

A ribosome-associated chaperone enables substrate triage in a cotranslational protein targeting complex


Protein biogenesis is essential in all cells and initiates when a nascent polypeptide emerges from the ribosome exit tunnel, where multiple ribosome-associated protein biogenesis factors (RPBs) direct nascent proteins to distinct fates. How distinct RPBs spatiotemporally coordinate with one another to affect accurate protein biogenesis is an emerging question. Here, we address this question by studying the role of a cotranslational chaperone, nascent polypeptide-associated complex (NAC), in regulating substrate selection by signal recognition particle (SRP), a universally conserved protein targeting machine. We show that mammalian SRP and SRP receptors (SR) are insufficient to generate the biologically required specificity for protein targeting to the endoplasmic reticulum. NAC co-binds with and remodels the conformational landscape of SRP on the ribosome to regulate its interaction kinetics with SR, thereby reducing the nonspecific targeting of signalless ribosomes and pre-emptive targeting of ribosomes with short nascent chains. Mathematical modeling demonstrates that the NAC-induced regulations of SRP activity are essential for the fidelity of cotranslational protein targeting. Our work establishes a molecular model for how NAC acts as a triage factor to prevent protein mislocalization, and demonstrates how the macromolecular crowding of RPBs at the ribosome exit site enhances the fidelity of substrate selection into individual protein biogenesis pathways.

Additional Information

© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 06 May 2020; Accepted 20 October 2020; Published 17 November 2020. We thank J. W. Chin for sharing PyltRNA/RS plasmid, D. Baltimore for sharing EMCV IRES sequence, A. Hoelz for sharing PreScission protease, R. M. Voorhees for advice on RRL in vitro translation and RNC purification, R. L. Gonzalez Jr for advice on the single-molecule TIRF setup, and members of the Shan lab for discussions and advice on this work. This work was supported by National Institutes of Health grant R01 GM078024, R35 GM136321, NSF grant MCB-1929452, and the Gordon and Betty Moore Foundation through grant GBMF2939 to S.-o.S. Data availability: Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. Structural data associated with Fig. 1a are available in the Protein Data Bank under accession code 4UG0 (ribosome)78; and in Electron Microscopy Database under accession codes: EMD-3037 (SRP)6; EMD-4938 (NAC)23; EMD-6105 (RAC)79; and EMD-0202 (NatA/E)80. Source data are provided with this paper. Code availability: The scripts for modeling of cotranslational protein targeting are available at GitHub: https://github.com/emc2emc2/2020_cotranslational_targeting. Author Contributions: H.-H.H. and S.-o.S. conceived and designed the study. H.-H.H., J.H.L., and S.C. expressed and purified proteins and RNAs. H.-H.H. performed experiments, analyzed data, and performed simulations. H.-H.H. and S.-o.S. prepared the figures and wrote and edited the manuscript. S.-o.S. supervised the project and secured funding. Corresponding author: Correspondence to Shu-ou Shan. The authors declare no competing interests. Peer review information: Nature Communications thanks all the anonymous reviewers for their contributions to the peer review of this work. Peer review reports are available.

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