title: 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 Glc 3Man 9GlcNAc 2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc 3Man 9GlcNAc 2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man 5GlcNAc 2-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 approximately 4 S. Man 7GlcNAc 2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25 degrees C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity approximately 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.
authors.affiliation: Department of Biochemistry, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA.
authors.forename: Sumana
authors.initials: S
authors.lastname: Sanyal
authors.forename: Christian G
authors.initials: CG
authors.lastname: Frank
authors.forename: Anant K
authors.initials: AK
authors.lastname: Menon
mesh_terms: D001692:Biological Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D020404:Glycerophospholipids / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D020404:Glycerophospholipids; D008081:Liposomes; D009844:Oligosaccharides; D049294:Phospholipid Transfer Proteins; D011510:Proteolipids; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes