The ubiquitous SecY/Sec61–complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent solubilized Sec–complexes have been reported. Here, we present a single–particle cryo–electron microscopy structure of the SecYEG complex in a membrane environment at sub–nanometer resolution, bound to a translating ribosome. Using the SecYEG complex reconstituted in a so–called Nanodisc, we could trace the nascent polypeptide chain from the peptidyl transferase center into the membrane. The reconstruction allowed for the identification of ribosome–lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the PCC. Based on our map and molecular dynamics simulations we present a model of a signal anchor–gated PCC in the membrane.

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The integration of most membrane proteins into the cytoplasmic membrane of bacteria occurs co-translationally. The universally conserved YidC protein mediates this process either individually as a membrane protein insertase, or in concert with the SecY complex. Here, we present a structural model of YidC based on evolutionary co-variation analysis, lipid-versus-protein-exposure and molecular dynamics simulations. The model suggests a distinctive arrangement of the conserved five transmembrane domains and a helical hairpin between transmembrane segment 2 (TM2) and TM3 on the cytoplasmic membrane surface. The model was used for docking into a cryo-electron microscopy reconstruction of a translating YidC-ribosome complex carrying the YidC substrate FOc. This structure reveals how a single copy of YidC interacts with the ribosome at the ribosomal tunnel exit and identifies a site for membrane protein insertion at the YidC protein-lipid interface. Together, these data suggest a mechanism for the co-translational mode of YidC-mediated membrane protein insertion.DOI: http://dx.doi.org/10.7554/eLife.03035.001

The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.Translocase mediates the translocation of precursor proteins (preproteins) across the cytoplasmic membrane of Escherichia coli and other bacteria. The ATPase SecA (1) is the peripheral motor domain of the translocase. The SecYEG complex constitutes a membrane-embedded protein-conducting channel (reviewed in Ref. 2) and a high affinity binding site for SecA (3). When bound to SecYEG, SecA functions as a high affinity membrane receptor for the molecular chaperone SecB with associated preproteins (3). The movement of the preprotein through the protein-conducting channel is driven by multiple cycles of ATP binding and hydrolysis at SecA (4) and the proton motive force.In solution, SecA exist as a homodimer (5) that equilibrates with its monomeric form in a temperature-, salt-and protein concentration-dependent manner (6). Under physiological conditions, the dissociation constant (K D ) for the monomer-dimer equilibrium is ϳ0