Journal
BIOCHEMISTRY
Volume 47, Issue 30, Pages 7937-7946Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi800723n
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- NIGMS NIH HHS [GM71041] Funding Source: Medline
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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(3)Man(9)GlcNAc(2)-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc(3)Man(9)GlcNAc(2)-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man(5)GlcNAC(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 similar to 4 S. Man(7)GlcNAc(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 similar to 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.
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