Stereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippase.
Mannose-phosphate-dolichol (MPD) is a multifunctional glycolipid that is synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and used on the opposite side of the membrane in the ER lumen as a mannose donor for protein N-glycosylation, glycosylphosphatidylinositol-anchoring, and C- and O-mannosylation. For this, it must be translocated, i.e., flipped, across the ER membrane. The molecular identity of the MPD translocator (MPD flippase) is not known. Here we show that MPD-flippase activity can be reconstituted in large unilamellar proteoliposomes prepared from phosphatidylcholine and Triton X-100-solubilized rat liver ER-membrane proteins. Using carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl NO(+) as a topological probe to selectively oxidize MPD molecules in the outer leaflet of the reconstituted vesicles, we demonstrate rapid, protein-dependent, ATP-independent transbilayer translocation of MPD from the inner to the outer leaflet. MPD flipping is highly specific. A stereoisomer of MPD was weakly translocated (> 10-fold lower rate) compared with natural MPD. Competition experiments with water-soluble isoprenyl monophosphates showed that MPD flippase recognizes the dolichol chain of MPD, preferring a saturated alpha-isoprene to unsaturated trans- or cis- alpha-isoprene units. Chromatography of the detergent-solubilized ER protein mixture prior to reconstitution indicated that MPD flippase (i) is not a Con A-binding glycoprotein and (ii) can be resolved from the oligosaccharide-diphosphate dolichol flippase that translocates Man(5)GlcNAc(2)-PP-dolichol, a lipid intermediate of N-glycosylation. These data provide a mechanistic framework for understanding MPD flipping, as well as a biochemical basis for identifying MPD flippase.
Department of Biochemistry, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA.
Sumana
S
Sanyal
Anant K
AK
Menon
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002845:Chromatography / Q000379:methods; D003902:Detergents / Q000494:pharmacology; D004287:Dolichol Monophosphate Mannose / Q000737:chemistry*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D008051:Lipid Bilayers / Q000737:chemistry*; D008099:Liver / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010710:Phosphates / Q000737:chemistry; D049294:Phospholipid Transfer Proteins / Q000737:chemistry / Q000502:physiology*; D021381:Protein Transport; D051381:Rats; D012685:Sepharose / Q000737:chemistry
D003902:Detergents; D008051:Lipid Bilayers; D010710:Phosphates; D049294:Phospholipid Transfer Proteins; D004287:Dolichol Monophosphate Mannose; D000255:Adenosine Triphosphate; D017830:Octoxynol; D012685:Sepharose