Back to overview

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
Show all

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

Type of Open Access Publisher (Gold Open Access)


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.