期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2010
卷号：107
期号：25
页码：11289-11294
DOI：10.1073/pnas.1002408107
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 [α]-isoprene to unsaturated trans- or cis- [α]-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 Man5GlcNAc2-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.
