Tracking the stepwise movement of a membrane-inserting protein in vivo

Abstract Proper membrane insertion is crucial for the structure and function of membrane proteins in all cells. The YidC insertase plays an essential role in this process, but the molecular mechanism of YidC-mediated insertion remains unknown. Here we track the stepwise movement of Pf3 coat through YidC by obtaining a series of translational arrested intermediates, and investigated them by thiol crosslinking. We show that Pf3 is inserted as a helical hairpin, i.e., the prospective transmembrane segment moves along the YidC greasy slide comprised of TM3 and TM5, while the N-terminal tail transiently folds back into the hydrophilic groove of YidC located in the inner leaflet of the membrane until it is translocated to the periplasm in a subsequent step involving the electrochemical membrane potential. In addition to providing virtual insights about how YidC inserts single spanning membrane proteins, our study also demonstrates a valuable in vivo tracking method that can be applied to study more complicated substrates or other translocases.