Substrate-induced assembly and functional mechanism of the membrane protein insertase SecYEG-YidC

The Sec translocon and the YidC/Oxa1-type insertases universally mediate biogenesis of α-helical membrane proteins, but the molecular basis of their cooperation has remained disputed. Recent discovery of multi-subunit insertases assembled at the back of the translocon in fungi and higher eukaryotes has raised questions about the architecture and mechanism of the putative bacterial ortholog SecYEG-YidC. Here, we combine cryogenic electron microscopy with cell-free protein synthesis to visualize biogenesis of the SecYEG/YidC-dependent multipass membrane protein NuoK. The nascent chain of NuoK does not enter the lateral gate of SecYEG but instead crosses the translocon towards its back side, where YidC is recruited in the nascent substrate-dependent manner. The SecY-YidC interface promotes folding of the transmembrane helices before insertion, consistent with thermodynamic principles of membrane protein folding. YidC forms extensive contacts with the nascent chain, suggesting its key role in the insertion event. These findings provide mechanistic insight into membrane protein insertases, support evolutionary conservation of a gate-independent insertion route, and expand current models of membrane protein biogenesis.