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

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.

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei

“…Parental cells (SmOx P9) showed a strong radiolabeled GPEET band, as well as a weak band corresponding to EP procyclin ( Fig. 3 A ), and as expected ( 41 ), no labeled procyclins were detected in a control sample of T. brucei procyclic forms that lack TbGPI13, the enzyme that adds phosphoethanolamine to the third mannose of the GPI anchor. Interestingly, TbGPI2-KO cells showed a radiolabeled band with a lower apparent mass than GPEET procyclin in parental cells.…”

“…1 A ). Slower growth of GPI-deficient procyclic cells has been reported previously in some instances, for example, after knocking out TbGPI13 or TbGPI10 ( 24 , 41 ), but not TbGPI12 ( 25 ). Growth was restored by expressing an ectopic copy of TbGPI2 (TbGPI2-HA, bearing a C-terminal 3x HA tag) in the TbGPI2-KO parasites ( Fig.…”

Towards understanding the extensive diversity of protein N‐glycan structures in eukaryotes

“…A GlcNAc and five Man residues then are sequentially added from UDP‐GlcNAc and GDP‐Man by the successive actions of multi‐enzymatic complexes ALG13/14 and the three mannosyltransferases ALG1 (β(1,4)‐mannosyltransferase), ALG2 (α(1,3)/(1,6)‐mannosyltransferase) and ALG11 (α(1,2)‐mannosyltransferase) (Gao, Nishikawa & Dean, 2004 ) to yield the dolichol pyrophosphate heptasaccharide Man 5 GlcNAc 2 ‐P‐P‐Dol. At this stage, the LLO undergoes a trans bilayer translocation across the ER membrane, requiring the activity of a flippase (Alaimo et al ., 2006 ; Rush, 2016 ; Verchère et al ., 2021 ). In humans, deficiency of the proposed flippase RFT1 leads to a severe decrease in N ‐glycosylation site occupancy on newly synthesized glycoproteins (Vleugels et al ., 2009 ).…”

Systematic in silico discovery of novel solute carrier-like proteins from proteomes

“…However, these molecules have at least 12-14 sugar moieties, so given their size, it is unlikely that a single transporter could catalyze their flipping. Later studies refuted the scramblase concept and suggested instead that RFT1 could serve as an accessory protein to a flippase, but would not act as a flippase itself [107][108][109].…”