Co‐translational insertion and topogenesis of bacterial membrane proteins monitored in real time

Integral membrane proteins insert into the bacterial inner membrane co‐translationally via the translocon. Transmembrane (TM) segments of nascent proteins adopt their native topological arrangement with the N‐terminus of the first TM (TM1) oriented to the outside (type I) or the inside (type II) of the cell. Here, we study TM1 topogenesis during ongoing translation in a bacterial in vitro system, applying real‐time FRET and protease protection assays. We find that TM1 of the type I protein LepB reaches the translocon immediately upon emerging from the ribosome. In contrast, the type II protein EmrD requires a longer nascent chain before TM1 reaches the translocon and adopts its topology by looping inside the ribosomal peptide exit tunnel. Looping presumably is mediated by interactions between positive charges at the N‐terminus of TM1 and negative charges in the tunnel wall. Early TM1 inversion is abrogated by charge reversal at the N‐terminus. Kinetic analysis also shows that co‐translational membrane insertion of TM1 is intrinsically rapid and rate‐limited by translation. Thus, the ribosome has an important role in membrane protein topogenesis.