Probing the Ion Permeability of the Bacterial Translocon with a Locked Translocation Intermediate at the Single Molecule Level
Sprache des Vortragstitels:
2. 61th Biophysical Society Meeting 2017
Sprache des Tagungstitel:
The bacterial translocon SecYEG is responsible for membrane insertion or the translocation of most of membrane and secretory proteins. The idle translocon is sealed to ions by a hydrophobic ring of six isoleucines in its middle and a short helix, the so-called plug (1). Insertion of a polypeptide chain dislocates the plug. By engulfing the chain in a gasket-like manner, the ring is thought to keep the barrier to ions (2). When testing this hypothesis, we found that the complex of SecYEG and a stalled translocation intermediate possesses 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.