Verchère, Alice; Cowton, Andrew; Jenni, Aurelio; Rauch, Monika; Häner, Robert; Graumann, Johannes; Bütikofer, Peter; Menon, Anant K. (2021). Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry. Scientific reports, 11(1), p. 1411. Springer Nature 10.1038/s41598-020-80956-0
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The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic
reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol. Remarkably, whereas
glycosylation occurs in the ER lumen, the initial steps of Glc3Man9GlcNAc2-PP-dolichol synthesis
generate the lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic side of
the ER. Glycolipid assembly is completed only after M5-DLO is translocated to the luminal side.
The membrane protein (M5-DLO scramblase) that mediates M5-DLO translocation across the ER
membrane has not been identifed, despite its importance for N-glycosylation. Building on our ability
to recapitulate scramblase activity in proteoliposomes reconstituted with a crude mixture of ER
membrane proteins, we developed a mass spectrometry-based ’activity correlation profling’ approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Data curation prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins is necessary for M5-DLO scramblase activity. Our results instead strongly suggest that M5-DLO scramblase activity is due to a protein, or protein complex, whose activity is regulated at the level of quaternary structure.
Item Type: |
Journal Article (Original Article) |
|---|---|
Division/Institute: |
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine 08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP) |
Graduate School: |
Graduate School for Cellular and Biomedical Sciences (GCB) |
UniBE Contributor: |
Cowton, Andrew Robert, Jenni, Aurelio Leandro, Rauch, Monika, Häner, Robert, Bütikofer, Peter |
Subjects: |
500 Science > 570 Life sciences; biology 600 Technology > 610 Medicine & health 500 Science > 540 Chemistry |
ISSN: |
2045-2322 |
Publisher: |
Springer Nature |
Language: |
English |
Submitter: |
Robert Häner |
Date Deposited: |
10 Jun 2021 12:12 |
Last Modified: |
02 Mar 2023 23:34 |
Publisher DOI: |
10.1038/s41598-020-80956-0 |
PubMed ID: |
33446867 |
BORIS DOI: |
10.7892/boris.152791 |
URI: |
https://boris.unibe.ch/id/eprint/152791 |
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