Investigation of SecY protein‐translocation channel in action using a novel in vivo tool (LB198)

Many proteins are targeted by signal sequences to a conserved protein‐conducting channel, formed by the prokaryotic SecY or eukaryotic Sec61 complex, and are translocated across the membrane. The channel forms an hourglass‐shaped pore with a constriction in the middle across the membrane. The channel also has a lateral gate that can open toward the lipid phase. During initiation of translocation, a ribosome‐nascent chain complex binds to the SecY/Sec61 channel, resulting in insertion of the nascent chain. To study the SecY channel engaged in translocation, we developed a novel method generating defined translocation intermediate complexes in E. coli cells. Using this tool, we addressed how the channel is opened by a ribosome‐bound nascent chain and transports large polypeptides and yet prevents passage of small molecules. We determined structures of inactive and active ribosome‐channel complexes with cryo‐electron microscopy. These structures demonstrate that the nascent chain opens the channel through mostly rigid body movements of the two halves of SecY. The polypeptide inserts as a loop into the channel, with the hydrophobic region of the signal sequence intercalated into the open lateral gate. Biochemical analysis shows that the pore residues of SecY form a gasket‐like seal around the translocating polypeptide chain, preventing the permeation of small molecules. This work was supported by HHMI and NIH.