Abstract
Protein biogenesis at the endoplasmic reticulum requires translocons comprising the Sec61 protein-conducting channel and several dynamically associated accessory factors. Here we used transcriptome-wide selective ribosome profiling in human cells to monitor cotranslational interactions of accessory factors for N-glycosylation (the OST-A complex) and multipass membrane protein synthesis (the GEL, PAT and BOS complexes). OST-A was preferentially recruited to open Sec61 channels engaged in polypeptide translocation; conversely, GEL, PAT and BOS were recruited synchronously to closed Sec61 channels and stabilized by newly inserted transmembrane domains. Translocon composition changed repeatedly and reversibly during the synthesis of topologically complex multipass membrane proteins. These data establish the molecular logic that underlies substrate-driven translocon remodeling, events that are crucial for the efficient biogenesis of secretory and membrane proteins.