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

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; Rauch, Monika; Häner, Robert and 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:

13 Jun 2021 03:03

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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