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Rft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane

Author

Listed:
  • Shuai Chen

    (Jiangnan University
    Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Cai-Xia Pei

    (Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Si Xu

    (Jiangnan University)

  • Hanjie Li

    (Jiangnan University)

  • Yi-Shi liu

    (Jiangnan University)

  • Yicheng Wang

    (Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Cheng Jin

    (Chinese Academy of Sciences)

  • Neta Dean

    (Stony Brook University, Stony Brook)

  • Xiao-Dong Gao

    (Jiangnan University
    Chinese Academy of Sciences
    Chinese Academy of Sciences)

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.

Suggested Citation

  • Shuai Chen & Cai-Xia Pei & Si Xu & Hanjie Li & Yi-Shi liu & Yicheng Wang & Cheng Jin & Neta Dean & Xiao-Dong Gao, 2024. "Rft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane," Nature Communications, Nature, vol. 15(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-48999-3
    DOI: 10.1038/s41467-024-48999-3
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    References listed on IDEAS

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    1. Jonne Helenius & Davis T. W. Ng & Cristina L. Marolda & Peter Walter & Miguel A. Valvano & Markus Aebi, 2002. "Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein," Nature, Nature, vol. 415(6870), pages 447-450, January.
    2. Christian Hirsch & Robert Gauss & Sabine C. Horn & Oliver Neuber & Thomas Sommer, 2009. "The ubiquitylation machinery of the endoplasmic reticulum," Nature, Nature, vol. 458(7237), pages 453-460, March.
    3. Sheng-Tao Li & Tian-Tian Lu & Xin-Xin Xu & Yi Ding & Zijie Li & Toshihiko Kitajima & Neta Dean & Ning Wang & Xiao-Dong Gao, 2019. "Reconstitution of the lipid-linked oligosaccharide pathway for assembly of high-mannose N-glycans," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
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