Structural insights into TRAP association with ribosome-Sec61 complex, and translocon inhibition by a CADA derivative

Abstract

AbstractDuring co-translational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the ER membrane. Our cryo-EM structure of ribosome-Sec61 shows binding of an ordered heterotetrameric TRranslocon-Associated Protein (TRAP) complex, in which TRAP-γ is anchored at two adjacent positions of 28S rRNA and interacts with ribosomal protein L38 and Sec61α/γ. Four transmembrane helices (TMHs) of TRAP-γ cluster with one C-terminal helix of each α, β, and δ subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP–α/β/δ core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the CADA derivative CK147 as a translocon inhibitor. A structure of ribosome-Sec61-CK147 reveals CK147 binding the channel and interacting with the plug helix from the lumenal side. The CK147-resistance mutations surround the inhibitor. These structures help in understanding the TRAP functions and provide a new Sec61 site for designing translocon inhibitors.Short SummaryCryo-EM structures reveal TRAP binding to ribosome-Sec61 complex, and CK147 inhibiting Sec61 by arresting the plug helix inside the channel.