Structural basis of regulated N-glycosylation at the secretory translocon

Most human secretory pathway proteins are N-glycosylated by oligosaccharyltransferase complexes as they enter the endoplasmic reticulum. Recent work revealed a substrate-assisted mechanism by which N-glycosylation of the chaperone GRP94 is regulated to control cell surface receptor signaling. Here we report the structure of a GRP94 folding intermediate tethered to a specialized CCDC134-bound translocon. Together with functional analysis, the data reveal how a conserved N-terminal extension in GRP94 inhibits OST-A, and how structural rearrangements within the translocon shield the tethered nascent chain from inappropriate OST-B glycosylation. These interactions depend critically on a hydrophobic CCDC134 groove, which recognizes a non-native conformation of nascent GRP94. These results define a mechanism of regulated N-glycosylation and reveal how the nascent chain customizes translocon subunit composition to facilitate its own biogenesis. Selective ribosome profiling of HEK293 cells expressing near-endogenous levels of 3xFlag-FKBP11 and 3xFlag-CCDC134. Both the solubilized ribosome translocon complexes ("input") and affinity-purified fractions ("IP") were collected for profiling.