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

Alice Verchère,Andrew Cowton,Robert Häner,Monika Rauch,Aurelio Jenni,Johannes Graumann,Peter Bütikofer,Anant K. Menon

doi:10.1038/s41598-020-80956-0

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.

Highlights

The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol
M5-DLO scramblase activity is due to a single protein or unique protein complex, present as a homogeneous entity in the ER, we developed a mass spectrometry-based activity correlation profiling approach[27,28] to identify scramblase candidates from a crude mixture of detergent-solubilized ER membrane proteins (Fig. 2)
The underlying concept involves resolving the proteins in the crude mixture into a number of fractions using any separation technique of choice, measuring the scramblase activity of each fraction to generate an activity profile and, in parallel, using mass spectrometry to determine the relative abundance of individual proteins across the fractions (Fig. 2a)

Summary

Introduction

The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol. Asparagine (N)-linked protein glycosylation is found in all domains of life[1,2,3,4,5] In eukaryotes, it takes place in the lumen of the endoplasmic reticulum (ER) via oligosaccharyltransferase (OST)-mediated en bloc transfer of a pre-synthesized oligosaccharide (Glucose3Mannose9N-acetylglucosamine2 (Glc3Man9GlcNAc2) in yeast and humans) to specific asparagine residues in newly translocated p roteins[6,7]. The ER membrane protein Rft[1] was proposed as the M5-DLO scramblase almost two decades a go[18,19], but subsequent work showed that whereas Rft[1] is clearly important for N-glycosylation, it appears to have no direct role in translocating M5-DLO across the ER m embrane[16,20,21,22,23].

Methods

Results

Conclusion