Distinct Flippases Translocate Glycerophospholipids and Oligosaccharide Diphosphate Dolichols across the Endoplasmic Reticulum

Abstract

Transbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc3Man9GlcNAc2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc3Man9GlcNAc2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at ∼4 S. Man7GlcNAc2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25 °C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity ∼5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447−450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase.

Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

“…Thus, glycerophospholipids, glycosylphosphatidylinositol, dolichol-PP-oligosaccharide, dolichol-P-monosaccharides, dolichyl phosphate, and glucosylceramide must all flip across the ER membrane to support cell growth and metabolism. We recently showed that glycerophospholipids, dolichol-PP-oligosaccharide, and dolichyl-phosphate-mannose are transported across the ER by ATP-independent flippases that can be biochemically resolved (21,51). Because it is likely that dolichyl phosphate is flipped by the dolichol-P-monosaccharide flippases (51) and glycosylphosphatidylinositol is translocated by phospholipid/GlcCer flippases (52), we conclude that the ER has four different lipid flippases.…”

“…C, purified Yop1-HA was reconstituted into proteoliposomes. Membrane association of Yop1-HA was demonstrated by flotation in a discontinuous sucrose gradient, as described previously (21), using steps of 750, 750, 1000, 1000, and 750 l of 0, 5, 10, 20, and 30% (w/v) sucrose, respectively. The vesicles floated as a sharp band at the interface between the 10 and 20% sucrose layers.…”

“…The identical behavior of both NBD-PE and NBD-GlcCer flippase activity on hydroxyapatite supports our conclusion that the same flippases are responsible for flipping both lipids. (38,39), but phospholipid flippase activity has been demonstrated in yeast microsomes (19) as well as in proteoliposomes reconstituted with yeast ER membrane proteins (19,21,35). If the rat liver ER phospholipid flippases are able to translocate NBD-GlcCer, we reasoned that the same should be true in the yeast system.…”

“…Approximately 1 ml of TE (ϳ1 mg/ml) was obtained from 400 OD units of cell culture. We previously showed that ice-old Triton X-100 selectively solubilizes ER membrane proteins from crude yeast microsomes (21).…”

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional α1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

“…Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 (11) and another not yet identified protein (12), it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially in an ordered fashion based on differences in the substrate specificity of the various glycosyltransferases (13).…”