1. Rft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane.
- Author
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Chen, Shuai, Pei, Cai-Xia, Xu, Si, Li, Hanjie, liu, Yi-Shi, Wang, Yicheng, Jin, Cheng, Dean, Neta, and Gao, Xiao-Dong
- Subjects
ENDOPLASMIC reticulum ,ASPARAGINE ,PHENOTYPES ,LIPIDS ,TRANSLOCATOR proteins - Abstract
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 purified Rft1 catalyzes the translocation of M5GN2-PP-Dol across the lipid bilayer. These data, combined with in vitro results demonstrating substrate selectivity and rft1∆ phenotypes, confirm the molecular identity of Rft1 as the M5GN2-PP-Dol ER flippase. Whether Rft1 plays a role during M5GN2-PP-Dol translocation has been controversial for over two decades. In this work, a reconstituted in vitro assay demonstrates that purified Rft1 is sufficient to flip M5GN2-PP-Dol across the lipid bilayer. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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