Translocon remodeling during protein synthesis at the ER

Protein biogenesis at the endoplasmic reticulum (ER) is coordinated by the Sec61 translocon whose dynamic subunit composition is essential for maintaining a functional proteome. How nascent secretory and membrane proteins recruit accessory factors to the translocon, and what controls the interplay between these factors during synthesis, is poorly understood. Here we use selective ribosome profiling to systematically define the cotranslational interactions of accessory factors for N-glycosylation (OST-A complex) and multipass membrane protein synthesis (GEL, PAT and BOS complexes). OST-A is actively recruited during translocation of long lumenal segments through an open Sec61 channel. This occurs independently of N-glycosylation acceptor sites, and is disfavored when Sec61 is closed. By contrast, the GEL, PAT and BOS complexes sample ribosomes docked at a closed Sec61 channel. Their recruitment is highly synchronized, and stabilized by newly inserted transmembrane domains (TMDs). Conversely, GEL, PAT and BOS binding is disfavored during translocation of long segments through open Sec61, or synthesis of long cytosolic segments. Analysis of large, topologically complex multipass proteins reveals that translocon composition can change repeatedly and reversibly during synthesis of a single polypeptide. These data establish a simple molecular logic underlying translocon remodeling during biogenesis of secretory and membrane proteins. Overall design: Selective ribosome profiling of HEK293 cells expressing near-endogenous levels of Flag-tagged OST48, RPN2, TMCO1, CCDC47, and Nicalin. Both the total membrane ("input") and affinity-purified fractions ("IP") were collected for profiling.