The universally conserved Sec translocon and the YidC/Oxa1-type insertases mediate biogenesis of -helical membrane proteins, but the molecular mechanism of their cooperation haves remained mysterious despite the decades of extensive research. Recent structural analysis of the eukaryotic systemchallenged the long-standing paradigm of the nascent helices emerging via the lateral gate of the translocon, as an alternative “back-of-Sec” route was identified, thus raising the question about the evolutionary conservation of the insertion mechanism. Here, we combine cryogenic electron microscopy with cell-free protein synthesis to visualize folding intermediates of the polytopic membrane protein NuoK, the subunit K of NADH-quinone oxidoreductase, in lipid-based nanodiscs containing either SecYEG alone or both SecYEG and YidC. We describe the most complete model of the ribosome-bound SecYEG in the lipid bilayer, though no nascent chain was found at the lateral gate. Instead, we report recruitment of YidC to the back of the translocon at the late stage of NuoK
insertion, and visualize the nascent chain taking a novel route from the ribosome into the lipid membrane at the SecY-SecE-YidC interface. The essential C-terminus of SecY hinders the nascent chain to access the lateral gate and routes it instead to the alternative insertion pathway. The experimental model of the insertase complex allowed us to identify a salt bridge stabilizing SecY-YidC complex, validated by tests in vivo. Our data provide the first detailed insight on the elusive complex in the physiologically relevant environment, highlight the importance of the nascent chain for the dynamic SecYEG-YidC assembly, and prove the evolutionary conservation of the “back-of-Sec” insertion route.

Max Busch (HHU Düsseldorf), Cristian Rosales Hernandez (LMU Munich), Michael Kamel (HHU Düsseldorf & Uni Osnabrück), Yulia Schaumkessel (HHU Düsseldorf), Thomas Becker (LMU Munich), Roland Beckmann (LMU Munich) , Alexej Kedrov (HHU Düsseldorf)