File Download

There are no files associated with this item.

  Links for fulltext
     (May Require Subscription)
Supplementary

Article: Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum

TitleDistinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum
Authors
Issue Date2008
PublisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistry
Citation
Biochemistry, 2008, v. 47 n. 30, p. 7937-7946 How to Cite?
AbstractTransbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc 3Man 9GlcNAc 2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc 3Man 9GlcNAc 2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man 5GlcNAc 2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at ∼4 S. Man 7GlcNAc 2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25°C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity ∼5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447-450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase. © 2008 American Chemical Society.
Persistent Identifierhttp://hdl.handle.net/10722/188677
ISSN
2015 Impact Factor: 2.876
2015 SCImago Journal Rankings: 1.769
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorSanyal, Sen_US
dc.contributor.authorFrank, CGen_US
dc.contributor.authorMenon, AKen_US
dc.date.accessioned2013-09-03T04:12:43Z-
dc.date.available2013-09-03T04:12:43Z-
dc.date.issued2008en_US
dc.identifier.citationBiochemistry, 2008, v. 47 n. 30, p. 7937-7946en_US
dc.identifier.issn0006-2960en_US
dc.identifier.urihttp://hdl.handle.net/10722/188677-
dc.description.abstractTransbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc 3Man 9GlcNAc 2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc 3Man 9GlcNAc 2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man 5GlcNAc 2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at ∼4 S. Man 7GlcNAc 2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25°C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity ∼5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447-450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase. © 2008 American Chemical Society.en_US
dc.languageengen_US
dc.publisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistryen_US
dc.relation.ispartofBiochemistryen_US
dc.subject.meshBiological Transporten_US
dc.subject.meshEndoplasmic Reticulum - Metabolismen_US
dc.subject.meshGlycerophospholipids - Metabolismen_US
dc.subject.meshLiposomes - Metabolismen_US
dc.subject.meshMicrosomes - Metabolismen_US
dc.subject.meshModels, Biologicalen_US
dc.subject.meshOligosaccharides - Chemistry - Metabolismen_US
dc.subject.meshPhospholipid Transfer Proteins - Metabolismen_US
dc.subject.meshProteolipids - Metabolismen_US
dc.subject.meshSaccharomyces Cerevisiae Proteins - Metabolismen_US
dc.titleDistinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulumen_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.doi10.1021/bi800723nen_US
dc.identifier.pmid18597486-
dc.identifier.scopuseid_2-s2.0-48249088868en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-48249088868&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume47en_US
dc.identifier.issue30en_US
dc.identifier.spage7937en_US
dc.identifier.epage7946en_US
dc.identifier.isiWOS:000257860100019-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridSanyal, S=16069600000en_US
dc.identifier.scopusauthoridFrank, CG=24481396300en_US
dc.identifier.scopusauthoridMenon, AK=7202324192en_US

Export via OAI-PMH Interface in XML Formats


OR


Export to Other Non-XML Formats