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Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein

Nature volume 415, pages 447–450 (2002)Cite this article

Abstract

N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER1. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells2,3,4. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive2,3,4. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.

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Figure 1: Biosynthesis of the N-linked oligosaccharides requires enzymatic reactions on both sides of the ER membrane.
Figure 2: RFT1 repression reduces N-linked glycosylation by altering the synthesis of lipid-linked oligosaccharides.
Figure 3: RFT1 repression results in a distinct protein glycosylation defect.
Figure 4: Overexpression of RFT1 improves N-linked glycosylation in a Δ alg11 strain.
Figure 5: Overexpression of RFT1 improves growth of Δ alg11 (YG1365) and Δ alg3 Δ alg11 (YG1363) strains.

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Acknowledgements

We thank C. J. Waechter for his contributions. We also thank F. Reggiori and A. Conzelmann, and T. Immervoll and M. Gentzsch for providing antibodies against Gas1 protein and chitinase, respectively. P. Orlean provided the dpm1-6 strain. This work was supported by grants from the Swiss National Science Foundation to M.A., the National Institutes of Health to P.W. and D.T.W.N., and the Canadian Institutes of Health Research to M.A.V. P.W. is an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Institute of Microbiology, Swiss Federal Institute of Technology, Zürich, Zürich, CH-8092, Switzerland

    Jonne Helenius & Markus Aebi

  2. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Davis T. W. Ng

  3. Department of Microbiology and Immunology, University of Western Ontario, London, N6A 5C1, Ontario, Canada

    Cristina L. Marolda & Miguel A. Valvano

  4. Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, 94143-0448, California, USA

    Peter Walter

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Correspondence to Markus Aebi.

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Helenius, J., Ng, D., Marolda, C. et al. Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein. Nature 415, 447–450 (2002). https://doi.org/10.1038/415447a

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