Robust Ligand Binding to the Protein Translocation Complex (Secyeg) Requires a Lipid Environment

Abstract

Many secretory proteins pass the membrane barrier via the bacterial SecY complex. SecY is assisted by the motor protein SecA, which is thought to push the polypeptides through the SecY pore with its two-helix finger. This model requires ATP molecules to exchange continuously during translocation. Whether the resulting change in SecA-SecY binding affinity leads to SecA release is unknown. Here we have tested the hypothesis by monitoring SecA binding to the SecY complex using lanthanide resonance energy transfer between a fluorescent label on the tip of its two-helix finger and Tb3+ in a genetically engineered binding pocket on SecY. In the absence of ATP, binding only occurred to SecY molecules that were reconstituted into lipid bilayers, but not to SecY molecules in detergent. In a fraction of these binding events we observed a deep insertion of SecA's two-helix finger into the SecY-channel. These penetrations resulted in relocation of SecY's plug as indicated by measurements of the distance between fingertip and plug. The ‘‘plug’’ is a short helix that is (i) normally located in the center of the channel adjacent to its hydrophobic constriction zone (pore ring) and (ii) blocks the passage of small molecules through the idle SecY complex (1). Observation of ion channel activity in reconstituted planar lipid bilayers confirmed that the plug had moved out of the pore. As previously observed in the case of ribosome nascent chain complexes (2), physiological values of the membrane potential were required to close the channel. We conclude that (i) SecA binding is sufficient to open the protein translocation channel in its lipid environment and (ii) the current transport model needs to be revised to account for SecY's voltage sensitivity.The project was supported by the Austrian Science Fund (P25844, P28213).1. Saparov SM,..,Pohl P (2007) Mol Cell 26:501-509.2. Knyazev DG, ⋯, Pohl P (2014) J Biol Chem 289:24611-24616.