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

Abstract

The canonical pathway of N-linked protein glycosylation in yeast and humans involves transfer of the oligosaccharide moiety from the glycolipid Glc3Man9GlcNAc2-PP-dolichol to select asparagine residues in proteins that have been translocated into the lumen of the endoplasmic reticulum (ER). Synthesis of Glc3Man9GlcNAc2-PP-dolichol occurs in two stages, producing first the key intermediate Man5GlcNAc2-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/N-glycosylation scramblase via a mass spectrometry-based 'activity correlation profiling' approach.

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. 3A), 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.…”

“…1A). 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.…”

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

“…1A). 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.…”

“…Thus, loss of TbGPI2 decreases the rate of synthesis of GlcNAc-PI, which may lead to a reduction of the available amount of GPI anchors that can be exported to the cell surface. While procyclic form trypanosomes do not depend on a functioning GPI anchor pathway when cultured in vitro (24, 25), a growth defect is often observed when GPI anchor biosynthesis is disrupted (24, 41). Therefore, any increase of GPI-anchored proteins present on the cell surface is expected to reduce the growth defect and therefore increase the relative fitness.…”

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

“…from yeast cells (BY4741 strain) as previously described 9,47 , except for an additional salt-wash step of the membranes prior to detergent solubilization. Briefly, cells harvested at OD600 ~2 were washed and homogenized using glass beads (0.5 mm) in ice-cold lysis buffer (10 mM HEPES-NaOH pH 7.4, 10 mM MgCl2, 5% (w/v) glycerol, 1 mM dithiothreitol) supplemented with protease inhibitor cocktail.…”