Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane1,2. Proteins with highly hydrophobic signal sequences are first recognized by the signal recognition particle (SRP)3,4 and then moved co-translationally through the Sec61 or SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass the SRP and are moved through the channel post-translationally5,6. In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62–Sec63 complex7,8,9, and substrates are moved through the channel by the luminal BiP ATPase9. How the Sec62–Sec63 complex activates the Sec61 channel for post-translational translocation is not known. Here we report the electron cryo-microscopy structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel’s plug domain. Lateral channel opening is triggered by Sec63 interacting both with cytosolic loops in the C-terminal half of Sec61 and transmembrane segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp7010. Our structure shows how the Sec61 channel is activated for post-translational protein translocation.
The coordinates of atomic models of the Sec complex without Sec62 were deposited in the Protein Data Bank with accession code 6ND1. The cryo-EM maps of the Sec complex before and after focused refinement with a mask were deposited with accession code EMDB-0440. All other data are available on reasonable request from the corresponding author.
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We thank Z. Yu, R. Huang, and H.-T. Chou at the HHMI Janelia Cryo-EM Facility for help with microscope operation and data collection, E. Twomey for help with data analysis, R. Gilmore for advice and materials, and S. Shao and M. Catipovic for comments on the manuscript. This work was supported by a Jane Coffin Child fellowship to X.W., and an NIGMS award (R01GM052586) to T.A.R. T.A.R. is a Howard Hughes Medical Institute Investigator.
Nature thanks G. von Heijne and the other anonymous reviewer(s) for their contribution to the peer review of this work.
X.W. designed experiments, purified proteins, collected and analysed electron microscopy data, and built the models. C.C. purified proteins, performed mutagenesis and mutant analysis. T.A.R. supervised the project. T.A.R. and X.W. wrote the manuscript.
Correspondence to Tom A. Rapoport.
Extended Data Fig. 1 Cryo-EM analysis of the Sec complex.
a, The image shows a representative cryo-EM image of Sec-complex particles. Some particles are highlighted with green circles. Four electron microscopy grids were screened and had a similar particle distribution. b, Representative 2D class averages of picked particles collected from images of three grids. c, Image processing workflow for 3D classification and refinement. Shown are views of 3D reconstructions parallel to the membrane, with percentages of the particles in each class indicated. Classes in colour were used for subsequent analysis. d, Euler-angle distribution in two different views. e, Fourier shell correlation (FSC) curve with indicated resolution at FSC = 0.143. f, Local resolution was calculated from the unfiltered half-map and coloured according to the scale on the side. Two different views are shown. g, Map filtered to local resolution using cryoSPARC2. Regions corresponding to the different proteins are coloured as in Fig. 1b.
Extended Data Fig. 2 Examples of the fit of the models into the density map.
a, Density map and model for the indicated segments of Sec63. Interactions 1 and 2 show the interfaces between Sec63 and components of the Sec61 complex. Residues in salmon and pink belong to Sec61 and Sss1, respectively. b, As in a, but for segments of the Sec61 complex. c, As in a, but for the loop of Sec71 interacting with Sec63 and Sec61.
Extended Data Fig. 3 Localization of Sec62 in the Sec complex.
a, Shown is the unsharpened map (grey), low-pass filtered to 8 Å, with models for the components of the Sec complex in ribbon diagram. A homology model for the N-terminal DEP domain of Sec62 (purple) was docked into the map. The acidic C-terminal tail of Sec63 (dotted line on the right panel) wraps around the TM8–TM9 loop of Sec61 and interacts with the cytosolic DEP domain of Sec62. b, Cuts through the middle of the unsharpened map are shown in views from the side and from the cytosol. Density for a transmembrane segment of Sec62 (highlighted by red, dotted ovals) is close to the lateral gate.
Extended Data Fig. 4 The Sec61 channel in the Sec complex has open lateral and closed luminal gates.
Space-filling model of the Sec61 channel in the Sec complex. The left panel shows a side view with the lateral gate in the front. The right panel shows a cut through the space-filling model viewed from the cytosol. Note that the plug domain keeps the luminal gate closed.
Extended Data Fig. 5 Lateral gate changes of the Sec61 channel.
a, The structure of Sec61 in the ribosome-primed state (PDB code 3J7Q) is superimposed on that of the idle channel from M. jannaschii (PDB code 1RH5). Shown are the transmembrane segments as cylinders, viewed from the side (left) and from the cytosol (right). Movements of transmembrane segments are indicated by red arrows. The plug domains are indicated by stars in the right panel. b, As in a, but comparison of the Sec61 channel in the Sec complex with the SecY channel from P. furiosus (PDB code 3MP7). c, The Sec61 channel in the Sec complex is shown together with the signal sequence (SS; in cyan) from a structure of the signal-sequence-opened ribosome-bound Sec61 channel (PDB code 3JC2). The alignment of the Sec61 molecules was done as in Fig. 2c. Note that the lateral gate in the Sec complex is open enough to allow a helix to move through.
Extended Data Fig. 6 Alignment of Sec63 sequences from different species.
Transmembrane segments and the Brl and J domains are shown as black bars above the sequences. The β-sandwich and lasso regions in the Brl domain are indicated as blue lines. Conserved residues are highlighted. Mutated residues involved in the interaction with the Sec61 channel are shown as coloured circles and arrows: interaction 3 at the N terminus in blue; interaction 2 in TM3 in red; interaction 1 in the β-sandwich and lasso segments of the Brl domain in green.
Extended Data Fig. 7 Sec63 expression in wild-type and mutant S. cerevisiae cells.
The strains used in Fig. 3c were analysed for expression of Flag-tagged Sec63. Flag-tagged wild-type or mutant Sec63 was expressed in Sec63Δ S. cerevisiae cells. Equal numbers of cells were analysed by SDS–PAGE, followed by immunoblotting with Flag antibodies. To control for equal loading, the samples were also analysed with antibodies to phosphoglycerate kinase (PGK). The lanes correspond to expression of the following Sec63 proteins: (1) wild-type Sec63; (2) mutations in interface 3 of Sec63 (Y5A/Y7A/D8A); (3) mutations in interface 2 (Y227A/W242A); (4) mutations in interface 2 (Y227A/W242A/W243A); (5) mutations in interface 1 (T444K/S447K/E482A); (6) empty vector. The experiment was repeated twice.