Eukaryotic oligosaccharyltransferase (OST) is a multi-subunit protein complex embedded in the membrane of the endoplasmic reticulum (ER). It catalyzes the en bloc transfer of a high-mannose oligosaccharide from a dolichol-pyrophosphate carrier onto asparagines located in glycosylation sequons (sequence N-X-S/T) of secretory proteins. Using single-particle cryo-electron microscopy, we determined the structure of a fungal octa-subunit OST complex reconstituted in lipidic nanodiscs at 3.31Å resolution. It revealed the arrangement of the eight subunits and suggested that eukaryotic OST complexes have a conserved architecture. The activity of reconstituted Saccharomyces cerevisiae OST was assessed using an in vitro glycosylation assay with synthetic LLO and peptide substrates. Using the crystal structure of a bacterial homolog of the catalytic STT3 subunit (the PglB protein of Campylobacter lari) as a reference, features critical for the catalytic activity and substrate recognition on STT3 could be identified.
The structure not only revealed ordered lipid molecules, but also a large N-glycan attached to a conserved Asn residue within STT3. This glycan lines a cavity that may serve as the glycan-binding pocket for the donor LLO substrate. By docking the structure of yeast OST into previously determined tomography maps, insight into distinct features of OST complexes either involved in co-translocational N-glycosylation (OST complexes associated with the translocon) or in post-translocational N-glycosylation (standalone OST complexes containing a subunit with redox chaperone) could be gained. Our results not only reveal the architecture of the OST complex and suggests roles for the non-catalytic subunits, they also suggest mechanisms by which eukaryotic OST complexes stimulate N-glycosylation over folding of large numbers of secretory proteins.