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Article: Stereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippase

TitleStereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippase
Authors
KeywordsDolichol-Linked-Oligosaccharide
N-Glycosylation
Issue Date2010
PublisherNational 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?
AbstractMannose-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 Identifierhttp://hdl.handle.net/10722/188681
ISSN
2015 Impact Factor: 9.423
2015 SCImago Journal Rankings: 6.883
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorSanyal, Sen_US
dc.contributor.authorMenon, AKen_US
dc.date.accessioned2013-09-03T04:12:44Z-
dc.date.available2013-09-03T04:12:44Z-
dc.date.issued2010en_US
dc.identifier.citationProceedings Of The National Academy Of Sciences Of The United States Of America, 2010, v. 107 n. 25, p. 11289-11294en_US
dc.identifier.issn0027-8424en_US
dc.identifier.urihttp://hdl.handle.net/10722/188681-
dc.description.abstractMannose-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.languageengen_US
dc.publisherNational Academy of Sciences. The Journal's web site is located at http://www.pnas.orgen_US
dc.relation.ispartofProceedings of the National Academy of Sciences of the United States of Americaen_US
dc.subjectDolichol-Linked-Oligosaccharideen_US
dc.subjectN-Glycosylationen_US
dc.titleStereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippaseen_US
dc.typeArticleen_US
dc.identifier.emailSanyal, S: sumana@wi.mit.eduen_US
dc.identifier.authoritySanyal, S=rp01794en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1073/pnas.1002408107en_US
dc.identifier.pmid20534553-
dc.identifier.scopuseid_2-s2.0-77954947798en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-77954947798&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume107en_US
dc.identifier.issue25en_US
dc.identifier.spage11289en_US
dc.identifier.epage11294en_US
dc.identifier.isiWOS:000279058000027-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridSanyal, S=16069600000en_US
dc.identifier.scopusauthoridMenon, AK=7202324192en_US

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