Conductivity of the Bacterial Translocon Involved in Translocation at the Single Molecule Level
Sprache des Vortragstitels:
Englisch
Original Tagungtitel:
Linz Winter Workshop
Sprache des Tagungstitel:
Englisch
Original Kurzfassung:
Most of membrane and secretory proteins use the bacterial translocon SecYEG for membrane insertion or translocation. In the resting state translocon is sealed by a hydrophobic ring of six isoleucines in its middle and a short helix, the so-called plug (1) and hence is not conductive to ions. Insertion of a polypeptide chain dislocates the plug. According to the current hypothesis translocon stays sealed for ions during translocation by engulfing the chain by the ring of isoleucines in a gasket-like manner (2). To test this hypothesis, we monitored the ion conductivity of the stalled translocation complex consisting of SecYEG and a translocation intermediate which couldn?t be fully translocated because of the folded domain on the C-terminus. This complex possessed ion channel activity (3). Physiological values of the membrane potential ?? reduced the conductivity, suggesting that the translocation intermediate either moved out of the lateral gate into the lipid phase or slid back into the donating compartment, so that the plug and pore ring could again seal the channel. Here we prevented the intermediate from either backsliding or exiting through the lateral gate by (i) stalling a highly charged helix, the voltage sensor domain S4 of the potassium channel KvAP in the SecYEG pore and (ii) locking it between the ribosome on one side of the membrane and calmodulin on the other side. ?? was unable to gate this complex. We observed several conductivity levels, suggesting that the S4 helix was free to sample between the aqueous environment of the pore and the lipid interior. This observation (i) confirms the general view of how the translocon distinguishes membrane proteins from secretory proteins and (ii) disproves the hypothesis that the pore ring acts to maintain the membrane barrier in an active translocon.