Cotranslational membrane protein biogenesis by an EMC-bound translocon

Secretory and membrane protein biogenesis occurs at ribosome-bound translocons in the endoplasmic reticulum (ER) membrane. Translocons consist of the Sec61 protein-conducting channel and various maturation factors including chaperones, modification enzymes and membrane insertases, which assemble dynamically in response to the biogenesis needs of a nascent polypeptide. In human cells, nascent multipass membrane proteins utilize the ER membrane protein complex (EMC) and the multipass translocon (MPT), but how these factors are deployed during biogenesis is unknown. Here we combine selective ribosome profiling, cryo-electron tomography and site-specific photocrosslinking to reveal the substrate range, timing and mechanism of EMC recruitment. We show that multipass proteins initially engage the MPT for insertion and chaperoning, with EMC being recruited when substrates begin to exceed the cytosolic or intramembrane capacity of MPT. Substrate-engaged EMC is observed at the interface between the MPT's protected membrane cavity and the bulk ER membrane, where it is poised for insertion, chaperoning and assembly. These results show how EMC and MPT cooperate at the ribosome-bound translocon to facilitate multipass membrane protein biogenesis.