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Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Verchère Alice, Cowton Andrew, Jenni Aurelio, Rauch Monika, Häner Robert, Graumann Johannes, Bütikofer Peter, Menon Anant K.,
Project Molecular identification of lipid transporters for protein glycosylation
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Original article (peer-reviewed)

Journal Scientific Reports
Volume (Issue) 11(1)
Page(s) 1411 - 1411
Title of proceedings Scientific Reports
DOI 10.1038/s41598-020-80956-0

Open Access

URL http://doi.org/10.1038/s41598-020-80956-0
Type of Open Access Publisher (Gold Open Access)

Abstract

Abstract The oligosaccharide required for asparagine (N) -linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc 3 Man 9 GlcNAc 2 -PP-dolichol. Remarkably, whereas glycosylation occurs in the ER lumen, the initial steps of Glc 3 Man 9 GlcNAc 2 -PP-dolichol synthesis generate the lipid intermediate Man 5 GlcNAc 2 -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 identified, 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 profiling' 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.
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