PT - JOURNAL ARTICLE AU - Feng Jin AU - Zengyi Chang TI - Discovery of a shortened version of SecA (SecA<sup>N</sup>) that conceivably functions as a protein-conducting channel AID - 10.1101/121335 DP - 2017 Jan 01 TA - bioRxiv PG - 121335 4099 - http://biorxiv.org/content/early/2017/05/25/121335.short 4100 - http://biorxiv.org/content/early/2017/05/25/121335.full AB - During biogenesis, nascent polypeptides of many proteins have to be translocated across biological membranes, relying on specific protein-conducting channels. It remains a great challenge to unequivocally identify the specific channel proteins for transl ocating particular client proteins in living cells. In Gram-negative bacteria, proteins destined to the inner membrane, periplasmic compartment or outer membrane are all initially synthesized in the cytoplasm and have to be translocated across the inner (i.e., the cytoplasmic) membrane. The prevailing perception is that all these translocations are accomplished by using the same SecYEG translocon. Nevertheless, this perception, formed largely based on genetic and in vitro studies, has yet been proved by direct in vivo analysis. Here, mainly via in vivo protein photocrosslinking analysis mediated by genetically incorporated unnatural amino acid pBpA, we revealed that, strikingly, the nascent β-barrel outer membrane proteins (OMPs) are translocated across the inner membrane by using SecAN (a shortened form of SecA), instead of SecYEG, as the translocon. This conclusion is made on the basis of our following observations. First, although direct interaction between the nascent periplasmic proteins and SecY, but not between nascent OMPs and SecY, was observed in living cells. Second, the processing of precursors of periplasmic proteins, but not OMPs, are severely reduced in a SecY-defective strain. Third, a shortened form of SecA, lacking its C-terminal region, thus designated as SecAN, although does not interact with any nascent periplasmic proteins, directly interact with both nascent OMPs and the periplasmic region of the BamA protein, a key component for the assembly of OMPs. Fourth, SecAN is present wholly in the membrane fraction and as homo-oligomers. Fifth, an OmpF-SecAN-BamA ternary complex (in addition to the OmpF-SecAN binary complex) could be clearly detected by our dual photocrosslinking analysis in living cells. An updated model for how proteins are translocated across the inner membrane of Gram-negative bacterial cells is proposed based on our new findings reported here.