Abstract
During biogenesis, nascent polypeptides of many proteins have to be translocated across biological membranes by relying on specific protein-conducting channels. It remains a great challenge to unequivocally identify the specific membrane-integrated channel proteins that translocate particular client proteins in living cells. In Gram-negative bacteria, proteins destined to the periplasmic compartment or outer membrane are all synthesized in the cytoplasm and have to be translocated across the inner (i.e., the cytoplasmic) membrane. The currently prevailing perception is that all these transmembrane translocations occur by using the same SecY channel on the inner membrane. Nevertheless, this perception, formed largely based on genetic and in vitro studies, has not yet been proved by direct analysis in living cells. Here, mainly via a systematic in vivo protein photo-crosslinking analyses mediated by a genetically incorporated unnatural amino acid, we revealed that, in contrary to the long-held view, nascent polypeptides of β-barrel OMPs are not translocated across the inner membrane via the SecY channel, but through a shortened version of SecA, designated as SecAN, which exists as a membrane-integrated homo-oligomers. Furthermore, we demonstrated that SecAN is most likely part of the supercomplex that we revealed earlier as one which is responsible for the biogenesis of β-barrel OMPs in living cells and spans the cytoplasm, the inner membrane, the periplasm and the outer membrane.