Role of Flippases in Protein Glycosylation in the Endoplasmic Reticulum

Abstract

Glycosylation is essential to the synthesis, folding, and function of glycoproteins in eukaryotes. Proteins are co- and posttranslationally modified by a variety of glycans in the endoplasmic reticulum (ER); modifications include C- and O-mannosylation, N-glycosylation, and the addition of glycosylphosphatidylinositol membrane anchors. Protein glycosylation in the ER of eukaryotes involves enzymatic steps on both the cytosolic and lumenal surfaces of the ER membrane. The glycans are first assembled as precursor glycolipids, on the cytosolic surface of the ER, which are tethered to the membrane by attachment to a long-chain polyisoprenyl phosphate (dolichol) containing a reduced α-isoprene. The lipid-anchored building blocks then migrate transversely (flip) across the ER membrane to the lumenal surface, where final assembly of the glycan is completed. This strategy allows the cell to export high-energy biosynthetic intermediates as lipid-bound glycans, while constraining the glycosyl donors to the site of assembly on the membrane surface. This review focuses on the flippases that participate in protein glycosylation in the ER.

Keywords: congenital disorder of glycosylation; dolichol; flippase; glycosylation.

Figure 1

Topological model for DLO synthesis in the ER. M5-DLO (Step 1), Man-P-Dol (Step 2), and Glc-P-Dol (Step 3) are synthesized on the cytosolic surface of the ER membrane from appropriate sugar-nucleotide donors and Dol-P and diffuse transversely to the lumenal monolayer where they participate in the synthesis of full-length G3-DLO. The polar head groups of the dolichol-linked intermediates do not spontaneously cross the ER membrane but are escorted across the highly hydrophobic lipid bilayer by a specialized class of membrane transporters referred to as flippases, numbered arbitrarily as indicated (Flippase 1: M5-DLO Flippase; Flippase 2: Man-P-Dol Flippase; Flippase 3: Glc-P-Dol Flippase; and Flippase 4: Dol-P Flippase). The G3 oligosaccharide is transferred to protein nascent chains releasing a molecule of Dol-P-P, which is dephosphorylated by DolPP1; Dol-P is then returned to the cytosolic monolayer by Flippase 4 (Step 4) for utilization in further rounds of DLO synthesis.

Figure 2

Man-P-Dol acts as mannosyl donor for the synthesis of M9-DLO, GPI anchors, and C- and O-mannosylated proteins. Man-P-Dol is synthesized on the cytosolic monolayer of the ER membrane from GDP-Man and Dol-P and flips (Flippase 2) to the lumenal monolayer where it functions as mannosyl donor for four mannosyltransferases in the DLO pathway and three mannosyltransferases in GPI anchor assembly. At least seven O-mannosyltransferases have been characterized in yeasts and mammals. The number of C-mannosyltransferases remains unknown. Following mannosyl transfer, Dol-P is released and returned to the cytosolic monolayer by Flippase 4.

Figure 3

Transport of ³²P-Cit into the lumen of intact rat liver ER vesicles. Panel A, open circle (○), ³²P-Cit was incubated with sealed rat liver ER vesicles at 22°C for the indicated periods of time. Following incubation, the transport reactions were diluted with 0.5 mL ice-cold buffer (10 mM Tris-CL, pH 7.4, 0.25 M sucrose) and filtered through a Millipore 0.45 µm HA filter. The filters were washed with an additional 10 mL ice-cold buffer and assayed for radioactivity, as described earlier. Panel A, closed circle (●), sealed rat liver ER vesicles were pre-incubated with ³²P-Cit for 10 minutes and diluted with 0.5 mL, 10 mM Tris-Cl, pH 7.4, and 0.25 M sucrose all at 22°C. At the indicated times, the diluted reactions were filtered through a 0.45 µm HA filter, washed with an additional 10 mL ice-cold buffer, and assayed for radioactivity. Panel B, large unilamellar synthetic phospholipid vesicles were reconstituted from octyl glucoside soluble phospholipids in the presence of ³²P-Cit, as described elsewhere., The vesicles containing ³²P-Cit entrapped in the lumen were incubated with 10 mM Tris-Cl, pH 7.4, and 0.25 M sucrose at 22°C for the indicated periods of time and chromatographed on a 30 mL column of Sephadex G-50 equilibrated in 10 mM Tris-Cl, pH 7.4, and 0.25 M sucrose. Fractions of 1 mL were collected and monitored for radioactivity by scintillation spectrometry. The percentage of the radioactivity eluting in the void volume of the column is plotted versus time of incubation.