Residue-by-residue analysis of cotranslational membrane protein integration in vivo
- Stockholm University, Sweden
- University of Ljubljana, Slovenia
- California Institute of Technology, United States
Abstract
We follow the cotranslational biosynthesis of three multi-spanning E. coli inner membrane proteins in vivo using high-resolution Force Profile Analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation, and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.
Data availability
All fFL values measured in this study are included as Figures Source Data.
Article and author information
Author details
Thomas F Miller III
Funding
Knut och Alice Wallenbergs Stiftelse (2017.0323)
- Gunnar von Heijne
Novo Nordisk Fonden (NNF18OC0032828)
- Gunnar von Heijne
Vetenskapsrådet (NNF18OC0032828)
- Gunnar von Heijne
National Institutes of Health (R01GM125063)
- Thomas F Miller III
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Randy Schekman, University of California Berkeley, United States
Publication history
- Received: October 23, 2020
- Accepted: February 5, 2021
- Accepted Manuscript published: February 8, 2021 (version 1)
- Version of Record published: February 16, 2021 (version 2)
Copyright
© 2021, Nicolaus et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Residue-by-residue analysis of cotranslational membrane protein integration in vivo
eLife 10:e64302.
https://doi.org/10.7554/eLife.64302
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