Architecture of the active post-translational Sec translocon.

In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel (PCC). In the post-translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre-opened state. Here, we present a cryo-EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N-terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C-terminus of Sec63. Together, we obtained a near-complete and integrated model of the active Sec complex.

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.

The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent - hydrophobic ß-barrel Outer-Membrane Proteins (OMPs) - are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the ß-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL) - an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane 'insertase' YidC - contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.