YidC from Escherichia coli forms an ion-conducting pore upon activation by ribosomes.
The universally conserved protein YidC aids the insertion and folding of transmembrane polypeptides independently or as a part of the SecYEG translocon complex. In the former scenario, the lipid-exposed YidC surface equipped with a highly conserved positively charged arginine is thought to facilitate membrane insertion of the nascent chain by providing a countercharge for the acidic residues at the polypeptides N-terminal region. Here we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon binding a ribosome or ribosome-nascent chain complex (RNC). This pore is closed in the absence of ribosomes. As this pore is not visible in the published monomeric YidC structure, we used AlphaFold to construct the model of a parallel YidC dimer. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, and single-molecule fluorescence microscopy. In the dimeric model, the conserved positively charged arginine and many residues interacting with nascent chains point into the putative pore. This result suggests the possibility of an alternative mode of YidC-assisted membrane protein insertion.