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 firstTM(TM1) oriented to the outside (type I) or the inside (typeII) of the cell. Here, we studyTM1 topogenesis during ongoing translation in a bacterial in vitro system, applying real-timeFRETand protease protection assays. We find thatTM1 of the type I protein LepB reaches the translocon immediately upon emerging from the ribosome. In contrast, the typeIIprotein EmrD requires a longer nascent chain beforeTM1 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 ofTM1 and negative charges in the tunnel wall. EarlyTM1 inversion is abrogated by charge reversal at the N-terminus. Kinetic analysis also shows that co-translational membrane insertion ofTM1 is intrinsically rapid and rate-limited by translation. Thus, the ribosome has an important role in membrane protein topogenesis.