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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.

SUBMITTER: Sanyal S

PROVIDER: S-EPMC2646664 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum.

Sanyal Sumana S Frank Christian G CG Menon Anant K AK

Biochemistry 20080703 30

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 cy ...[more]

PMID: 18597486

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Project description:The endoplasmic reticulum (ER) is the subcellular site where proteins following the secretory pathway acquire their proper tertiary and, in certain cases, quaternary structures. Species that are not yet properly folded are prevented from exit to the Golgi apparatus and, if permanently misfolded, are transported to the cytosol, where they are degraded in the proteasomes. This review deals with a mechanism, applicable to proteins that are N-glycosylated in the ER, by which the quality control of folding is performed. Protein-linked monoglucosylated glycans, formed by glucosidase I- and glucosidase II-dependent partial deglucosylation of the oligosaccharides transferred from dolichol diphosphate derivatives in N-glycosylation (Glc(3)Man(9)GlcNAc(2)), mediate glycoprotein recognition by two ER-resident lectins, membrane-bound calnexin (CNX) and its soluble homologue, calreticulin (CRT). A still not yet fully confirmed interaction between the lectins and the protein moieties of folding glycoproteins may occur after lectin recognition of monoglucosylated structures. Further deglucosylation of glycans by glucosidase II, and perhaps also by a change in CNX/CRT and/or in the substrate glycoprotein conformation, liberates the glycoproteins from their CNX/CRT anchors. Glycans may be then reglucosylated by the UDP-Glc:glycoprotein glucosyltransferase (GT), and thus be recognized again by CNX/CRT, but only when linked to not yet properly folded protein moieties, as this enzyme behaves as a sensor of glycoprotein conformation. Deglucosylation/reglucosylation cycles catalysed by the opposing activities of glucosidase II and GT only stop when proper folding is achieved. The interaction between CNX/CRT and a monoglucosylated glycan is one of the alternative mechanisms by which cells retain not yet properly folded glycoproteins in the ER; in addition, it enhances folding efficiency by preventing protein aggregation and thus allowing intervention of classical chaperones and other folding-assisting proteins. There is evidence suggesting that both glycoprotein glucosylation and mannose removal, respectively mediated by GT and ER mannosidase I, might be involved in cell recognition of permanently misfolded glycoproteins bound for proteasome degradation.

Dataset Information

Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum.

Publications

Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum.

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