biostudies-literatureChen SNational Natural Science Foundation of China (National Science Foundation of China)5157https://www.ebi.ac.uk/biostudies/studies/S-EPMC11182771biostudies-literatureUnknown15(1)The eukaryotic asparagine (N)-linked glycan is pre-assembled as a fourteen-sugar oligosaccharide on a lipid carrier in the endoplasmic reticulum (ER). Seven sugars are first added to dolichol pyrophosphate (PP-Dol) on the cytoplasmic face of the ER, generating Man5GlcNAc2-PP-Dol (M5GN2-PP-Dol). M5GN2-PP-Dol is then flipped across the bilayer into the lumen by an ER translocator. Genetic studies identified Rft1 as the M5GN2-PP-Dol flippase in vivo but are at odds with biochemical data suggesting Rft1 is dispensable for flipping in vitro. Thus, the question of whether Rft1 plays a direct or an indirect role during M5GN2-PP-Dol translocation has been controversial for over two decades. We describe a completely reconstituted in vitro assay for M5GN2-PP-Dol translocation and demonstrate that puNature communicationsRft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane.PMC111827713227134231971216Li HPei CXJin CGao XDXu SDean NChen SWang YLiu YSfalseRft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane.The eukaryotic asparagine (N)-linked glycan is pre-assembled as a fourteen-sugar oligosaccharide on a lipid carrier in the endoplasmic reticulum (ER). Seven sugars are first added to dolichol pyrophosphate (PP-Dol) on the cytoplasmic face of the ER, generating Man5GlcNAc2-PP-Dol (M5GN2-PP-Dol). M5GN2-PP-Dol is then flipped across the bilayer into the lumen by an ER translocator. Genetic studies identified Rft1 as the M5GN2-PP-Dol flippase in vivo but are at odds with biochemical data suggesting Rft1 is dispensable for flipping in vitro. Thus, the question of whether Rft1 plays a direct or an indirect role during M5GN2-PP-Dol translocation has been controversial for over two decades. We describe a completely reconstituted in vitro assay for M5GN2-PP-Dol translocation and demonstrate that pu2024-01-01T00:00:00Z2024 Jun2025-04-04T12:47:47.804Z2025-04-04T12:47:47.804ZS-EPMC111827713888634010.1038/s41467-024-48999-3