Residue-by-residue analysis of cotranslational membrane protein integration in vivo.

Felix Nicolaus,Salmo Mohammed Abdullahi,Ane Metola,Gunnar von Heijne,Thomas F Miller III,Daphne Mermans,Amanda Liljenström,Ajda Krč,Matthew Zimmer

doi:10.7554/elife.64302

Felix Nicolaus, Salmo Mohammed Abdullahi + Show 7 more

Open Access

https://doi.org/10.7554/elife.64302

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Abstract

We follow the cotranslational biosynthesis of three multispanning Escherichia 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.

Highlights

Most integral membrane proteins are cotranslationally integrated into their target membrane with the help of translocons such as bacterial SecYEG and YidC, and the eukaryotic Sec61 and EMC complexes (Rapoport et al, 2017; Chitwood et al, 2018)
In constructs where the transmembrane helix (TMH) engages in an interaction that generates a strong enough pulling force F on the nascent chain at the point when the ribosome reaches the last codon of the arrest peptide (AP), pausing will be prevented and mostly full-length protein will be produced during a short pulse with [35S]-Met (Figure 1b, middle)
We have previously shown that a model TMH composed of Ala and Leu residues generates a peak in an force profile (FP) recorded with the SecM(Ec) AP that reaches half-maximal amplitude (Nstart) when the N-terminal end of the TMH is ~45 residues away from the polypeptide transferase center (PTC) (Ismail et al, 2012), and a recent real-time FRET study of cotranslational membrane integration found that the N-terminal end of the first TMH in a protein reaches the vicinity of the SecYEG translocon when it is 40–50 residues away from the PTC (Mercier et al, 2020)

Summary

Introduction

Most integral membrane proteins are cotranslationally integrated into their target membrane with the help of translocons such as bacterial SecYEG and YidC, and the eukaryotic Sec and EMC complexes (Rapoport et al, 2017; Chitwood et al, 2018). While the energetics of translocon-mediated integration of a transmembrane a-helix (TMH) is reasonably well understood (Hessa et al, 2007), the actual integration process is not, other than in general terms. We have applied FPA and coarse-grained molecular dynamics (CGMD) simulations to follow the cotranslational membrane integration of three multispanning Escherichia coli inner membrane proteins of increasing complexity (EmrE, GlpG, BtuC), providing the first residue-by-residue data on membrane protein integration in vivo

Results

Discussion

Materials and methods