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Biochemical reconstitution and resolution of lipid flippase activities required for protein glycosylation in the ER
Author(s) -
Me Anant K
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.349.2
Subject(s) - flippase , dolichol , endoplasmic reticulum , glycosylation , biochemistry , chemistry , unfolded protein response , glycoprotein , microbiology and biotechnology , membrane protein , transport protein , biosynthesis , membrane , phosphatidylserine , biology , enzyme , phospholipid
The oligosaccharide donor for protein N‐glycosylation is Glc3Man9GlcNAc2‐PP‐dolichol. This lipid is required in the lumen of the endoplasmic reticulum (ER). However, its biosynthesis is initiated on the cytoplasmic face of the ER necessitating transbilayer movement, or flip‐flop, of three lipids across the ER membrane: the biosynthetic intermediate Man5GlcNAc2‐PP‐dolichol (M5‐DLO), and the sugar donors Man‐P‐dolichol (MPD; required also for glycosylphosphatidylinositol (GPI)‐anchoring, O‐mannosylation and C‐mannosylation in the ER lumen) and Glc‐P‐dolichol. Since flip‐flop of these lipids is intrinsically unfavorable, it is likely facilitated by specific transport proteins, or flippases. The predicted flippases have yet to be identified. We report rapid, ATP‐independent flipping of M5‐DLO and MPD in proteoliposomes generated from detergent‐solubilized ER membrane proteins. We show that the M5‐DLO and MPD flippase activities can be biochemically resolved from each other as well as from the ER glycerophospholipid flippase activity, and are highly specific with respect to the structure of the lipid that they transport. These results provide the first clear indication of at least three specific flippase proteins in the ER and establish the basis for their eventual purification and identification.