Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion

Abstract

The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria)1. In eukaryotic cells the PCC is located in the membrane of the endoplasmic reticulum where it can bind to translating ribosomes for co-translational protein transport. The Sec complex consists of three subunits (Sec61α, β and γ) and provides an aqueous environment for the translocation of hydrophilic peptides as well as a lateral opening in the Sec61α subunit that has been proposed to act as a gate for the membrane partitioning of hydrophobic domains2. A plug helix and a so-called pore ring are believed to seal the PCC against ion flow and are proposed to rearrange for accommodation of translocating peptides2,3. Several crystal and cryo-electron microscopy structures revealed different conformations of closed and partially open Sec61 and SecY complexes2,4,5,6,7,8. However, in none of these samples has the translocation state been unambiguously defined biochemically. Here we present cryo-electron microscopy structures of ribosome-bound Sec61 complexes engaged in translocation or membrane insertion of nascent peptides. Our data show that a hydrophilic peptide can translocate through the Sec complex with an essentially closed lateral gate and an only slightly rearranged central channel. Membrane insertion of a hydrophobic domain seems to occur with the Sec complex opening the proposed lateral gate while rearranging the plug to maintain an ion permeability barrier. Taken together, we provide a structural model for the basic activities of the Sec61 complex as a protein-conducting channel.

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Figure 1: Generation and cryo-electron microscopy structures of translocating and inserting ribosome–Sec61 complexes.
Figure 2: Models for idle and translocating Sec61.
Figure 3: Model for the membrane inserting Sec61.
Figure 4: Conformational transitions of Sec61 during co-translational protein translocation and membrane insertion.

Accession codes

Accessions

EMBL/GenBank/DDBJ

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Cryo-electron microscopy maps for the idle 80S–Sec61 complex, the LepT–RNC–Sec61 complex and the LepM–RNC–Sec61 complex have been deposited in the EMDataBank with accession codes EMD-2510, EMD-2511 and EMD-2512. The respective coordinates for electron-microscopy-based models are deposited in the Protein Data Bank (4CG7, 4CG5 and 4CG6 for the Sec61 complex and 3J5Z, 3J60, 3J62, 3J61 for the updated model of the wheat germ ribosome).

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Acknowledgements

We thank C. Ungewickell for assistance with cryo-electron microscopy, E. van der Sluis for discussions, S. Funes for help with reagent preparation, B. Dobberstein for endoplasmic reticulum membranes, F. Förster and S. Pfeffer for performing mass spectrometry analysis. This work was supported by grants of the German Research Council (SFB594 to R.B. and B.B., SFB646 to T.B. and R.B., GRK1721 to R.B.). R.B. acknowledges support by the Center for Integrated Protein Science and the European Research Council (Advanced Grant CRYOTRANSLATION).

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Contributions

M.G., T.B. and R.B. designed the study. M.G. established protocols for generation of translocation and insertion intermediates, processed and interpreted the cryo-electron microscopy structures, M.G and T.B built molecular models for the Sec61 complex and prepared all figures. B.B. prepared SRP and assisted in preparing wheat germ translation extract and PKRM. J.-P.A. assisted in data processing, C.B.-G. built a refined model of the wheat germ ribosome. O.B. performed cryo-electron microscopy data collection, M.G., T.B. and R.B. interpreted results and wrote the paper.

Corresponding author

Correspondence to Roland Beckmann.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Western blot and mass spectrometry analysis of the purified LepM sample.

Protein content and western blot analysis of the LepM-purification of LepM-containing RNC–Sec61 complexes for flow through (Ft, left) and elution (E, right) fractions. a, Blot probing for the haemagglutinin-tagged nascent chain of LepM. Glycosylated peptidyl-tRNA (Gly-PtRNA, *), unglycosylated peptidyl-tRNA (ut), glycosylated free peptide (gp), unglycosylated free peptide (up) and degradation bands (deg) are indicated. b, Blot probing for the Sec61α protein (*). c, Same as a, but with a mock purification using streptavidin beads pre-saturated with desthiobiotin to determine the level of nonspecific binding to the beads. d, Ribosomal proteins and subunits of the Sec61 complex identified in the sample by mass spectrometry. Ribosomal proteins of the large subunit are in grey, of the small subunit yellow and subunits of the Sec61 complex are highlighted in red.

Extended Data Figure 2 Cryo-electron microscopy reconstructions of ribosome–PCC complexes and resolution curves.

Cryo-electron microscopy maps of the idle 80S–Sec61 complex (left), the LepT–RNC–Sec61 complex (middle) and the LepM–RNC–Sec61 complex (right) at 6.9 Å, 7.4 Å and 7.8 Å, respectively, according to the Fourier shell correlation at cutoff 0.5.

Extended Data Figure 3 Fourier shell correlation curves between experimental maps and molecular models.

Left, Fourier shell correlation curves between the updated model for the wheat germ ribosome with the empty 80S–Sec61 map, the LepT–RNC–Sec61 map and the LepM–RNC–Sec61 map. Right, Fourier shell correlation curves between the models for empty, LepT- and LepM-engaged Sec61 and respective densities, which were cut out using a soft mask.

Extended Data Figure 4 Model for mammalian (Canis familiaris) Sec61 complexes bound to the wheat germ (Triticum aestivum) ribosome.

a, Left panel, fitting of the idle 80S–Sec61 complex. For ribosomal proteins and RNA a refined model of the wheat germ 80S ribosome45 was used. Right panel, isolated density for the Sec61 complex without the mixed detergent/lipid micelle. Views are: front view focusing on the lateral gate (TM2 and TM7), cytoplasmic view and side view focusing on the plug. b, As in a for the translocating LepT–RNC–Sec61 complex. c, As in a for the inserting LepM–RNC–Sec61 complex. The extra transmembrane segment belonging to the variable region of LepM is indicated (green; TM).

Extended Data Figure 5 Comparison of Sec61 models with available crystal structures and cryo-electron microscopy models.

a, Model for the idle Sec61 complex. b, Idle Sec61 compared to Methanococcus jannaschii crystal structure of SecYEβ2. c, Idle Sec61 compared to Sec61 in-vitro-reconstituted with a translating ribosome with a type II signal-anchor sequence7. d, Model for the LepT-engaged Sec61 complex. e, LepT–Sec61 compared to Thermotoga maritima crystal structure of SecYEG bound to SecA4. f, LepT–Sec61 compared to Sec61 as in c. g, Model for the LepM-engaged Sec61 complex. h, Model for Escherichia coli SecYE embedded in native membrane environment (nanodiscs) bound to a translating ribosome and a signal-anchor sequence8. i, LepM–Sec61 compared to Pyrococcus furiosus crystal structure of SecYE6. In all comparison images colours for idle and engaged Sec61 are as in a and TM10 is shown in magenta. For better clarity TM10 is shown in pink and the plug in pale green for the referenced Sec complexes we compare with our structures.

Extended Data Figure 6 Model for a peptide translocating through LepT–Sec61.

a, Cytoplasmic view and b, side view focusing on the plug and TM10. A poly-alanine model peptide (green) has been placed, showing that in principle there is enough space between plug, TM5 and TM10 to accommodate an extended translocating nascent chain. The path of the model peptide through the Sec61 complex has been arbitrarily placed and alternative routes, especially above and below the central pore ring, are possible. NC, nascent chain.

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Gogala, M., Becker, T., Beatrix, B. et al. Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion. Nature 506, 107–110 (2014). https://doi.org/10.1038/nature12950

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