Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

Abstract

In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the proteinconducting channel Sec61 and additional complexes involved in nascent chain processing and translocation. As an integral component of the translocon, the oligosaccharyl-transferase complex (OST) catalyses co-translational N-glycosylation, one of the most common protein modifications in eukaryotic cells. Here we use cryoelectron tomography, cryoelectron microscopy single-particle analysis and small interfering RNA-mediated gene silencing to determine the overall structure, oligomeric state and position of OST in the native ER protein translocon of mammalian cells in unprecedented detail. The observed positioning of OST in close proximity to Sec61 provides a basis for understanding how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. The overall spatial organization of the native translocon, as determined here, serves as a reliable framework for further hypothesis-driven studies.

Organization of the mitochondrial translation machinery studied in situ by cryoelectron tomography

“…On the contrary, the distance between the inner mitochondrial membrane and the canonical peptide exit site on the mitoribosome even exceeds that observed between eukaryotic ribosomes and the ER membrane during co-translational protein translocation ( Supplementary Fig. 5) 14 .…”

“…Cryoelectron tomography (CET) is an excellent method for studying the structure of large molecules in their natural context 13 , in particular ribosomal structures and their supramolecular assembly [14][15][16] . Using this approach, we analysed mitoribosome structure and distribution in purified translation-competent mitochondria revealing detailed insights into the organization of the mitochondrial translation machinery and its association with the inner membrane.…”

Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons

“…The mammalian STT3A is found in a complex that associates with the general secretion (Sec) pathway, coordinating cotranslational glycosylation of nascent proteins entering the RER. This macromolecular association is mediated by direct interactions between subunits of the Sec61 translocation machinery and the OTase subunits (7)(8)(9). The STT3B isoform is part of a distinct OTase complex in the RER that is not associated with the Sec translocon and rather glycosylates sites missed by STT3A in a post-translational and/or post-translocational manner (10,11).…”

The Endoplasmic Reticulum-based Acetyltransferases, ATase1 and ATase2, Associate with the Oligosaccharyltransferase to Acetylate Correctly Folded Polypeptides

“…Finally, in humans, mutations that affect the input of acetyl-CoA into the ER lumen are associated with developmental defects (8), as well as a familial form of spastic paraplegia (9). The OST is a multisubunit complex that is structurally positioned in close proximity to the translocon (33). Purification of the OST complex has yielded different subcomplexes with different subunits and different molecular mass (reviewed in Ref.…”