Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry.
Sci Rep
; 11(1): 1411, 2021 01 14.
Article
em En
| MEDLINE
| ID: mdl-33446867
ABSTRACT
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 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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MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Espectrometria de Massas
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Trypanosoma brucei brucei
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Proteínas de Protozoários
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Proteínas de Saccharomyces cerevisiae
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Retículo Endoplasmático
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Hexosiltransferases
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Proteínas de Membrana
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article