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- Publisher Website: 10.1073/pnas.1002408107
- Scopus: eid_2-s2.0-77954947798
- PMID: 20534553
- WOS: WOS:000279058000027
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Article: Stereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippase
Title | Stereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippase |
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Authors | |
Keywords | Dolichol-Linked-Oligosaccharide N-Glycosylation |
Issue Date | 2010 |
Publisher | National Academy of Sciences. The Journal's web site is located at http://www.pnas.org |
Citation | Proceedings Of The National Academy Of Sciences Of The United States Of America, 2010, v. 107 n. 25, p. 11289-11294 How to Cite? |
Abstract | 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- tetramethyl-piperidine 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 Man 5GlcNAc 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. |
Persistent Identifier | http://hdl.handle.net/10722/188681 |
ISSN | 2023 Impact Factor: 9.4 2023 SCImago Journal Rankings: 3.737 |
ISI Accession Number ID | |
References |
DC Field | Value | Language |
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dc.contributor.author | Sanyal, S | en_US |
dc.contributor.author | Menon, AK | en_US |
dc.date.accessioned | 2013-09-03T04:12:44Z | - |
dc.date.available | 2013-09-03T04:12:44Z | - |
dc.date.issued | 2010 | en_US |
dc.identifier.citation | Proceedings Of The National Academy Of Sciences Of The United States Of America, 2010, v. 107 n. 25, p. 11289-11294 | en_US |
dc.identifier.issn | 0027-8424 | en_US |
dc.identifier.uri | http://hdl.handle.net/10722/188681 | - |
dc.description.abstract | 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- tetramethyl-piperidine 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 Man 5GlcNAc 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. | en_US |
dc.language | eng | en_US |
dc.publisher | National Academy of Sciences. The Journal's web site is located at http://www.pnas.org | en_US |
dc.relation.ispartof | Proceedings of the National Academy of Sciences of the United States of America | en_US |
dc.subject | Dolichol-Linked-Oligosaccharide | en_US |
dc.subject | N-Glycosylation | en_US |
dc.title | Stereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippase | en_US |
dc.type | Article | en_US |
dc.identifier.email | Sanyal, S: sumana@wi.mit.edu | en_US |
dc.identifier.authority | Sanyal, S=rp01794 | en_US |
dc.description.nature | link_to_subscribed_fulltext | en_US |
dc.identifier.doi | 10.1073/pnas.1002408107 | en_US |
dc.identifier.pmid | 20534553 | - |
dc.identifier.scopus | eid_2-s2.0-77954947798 | en_US |
dc.relation.references | http://www.scopus.com/mlt/select.url?eid=2-s2.0-77954947798&selection=ref&src=s&origin=recordpage | en_US |
dc.identifier.volume | 107 | en_US |
dc.identifier.issue | 25 | en_US |
dc.identifier.spage | 11289 | en_US |
dc.identifier.epage | 11294 | en_US |
dc.identifier.isi | WOS:000279058000027 | - |
dc.publisher.place | United States | en_US |
dc.identifier.scopusauthorid | Sanyal, S=16069600000 | en_US |
dc.identifier.scopusauthorid | Menon, AK=7202324192 | en_US |
dc.identifier.issnl | 0027-8424 | - |