On identifying the eukaryotic N -glycosylation scramblase by activity correlation profiling

The canonical pathway of -linked protein glycosylation in yeast and humans involves transfer of the oligosaccharide moiety from the glycolipid GlcManGlcNAc-PP-dolichol to select asparagine residues in proteins that have been translocated into the lumen of the endoplasmic reticulum (ER). Synthesis of GlcManGlcNAc-PP-dolichol occurs in two stages, producing first the key intermediate ManGlcNAc-PP-dolichol (M5-DLO) on the cytoplasmic face of the ER, followed by translocation of M5-DLO across the ER membrane where the remaining glycosyltransfer reactions occur to complete the structure. The scramblase protein that mediates the translocation of M5-DLO across the ER membrane has not been identified, but activity assays provide compelling evidence that it is an ER membrane protein with exquisite substrate specificity. Here we report on our progress in identifying the M5-DLO/-glycosylation scramblase via a mass spectrometry-based 'activity correlation profiling' approach.