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- Publisher Website: 10.1021/bi800723n
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- PMID: 18597486
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Article: Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum
Title | Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum |
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Authors | |
Issue Date | 2008 |
Publisher | American 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? |
Abstract | Transbilayer 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 Identifier | http://hdl.handle.net/10722/188677 |
ISSN | 2021 Impact Factor: 3.321 2020 SCImago Journal Rankings: 1.430 |
ISI Accession Number ID | |
References |
DC Field | Value | Language |
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dc.contributor.author | Sanyal, S | en_US |
dc.contributor.author | Frank, CG | en_US |
dc.contributor.author | Menon, AK | en_US |
dc.date.accessioned | 2013-09-03T04:12:43Z | - |
dc.date.available | 2013-09-03T04:12:43Z | - |
dc.date.issued | 2008 | en_US |
dc.identifier.citation | Biochemistry, 2008, v. 47 n. 30, p. 7937-7946 | en_US |
dc.identifier.issn | 0006-2960 | en_US |
dc.identifier.uri | http://hdl.handle.net/10722/188677 | - |
dc.description.abstract | Transbilayer 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.language | eng | en_US |
dc.publisher | American Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistry | en_US |
dc.relation.ispartof | Biochemistry | en_US |
dc.subject.mesh | Biological Transport | en_US |
dc.subject.mesh | Endoplasmic Reticulum - Metabolism | en_US |
dc.subject.mesh | Glycerophospholipids - Metabolism | en_US |
dc.subject.mesh | Liposomes - Metabolism | en_US |
dc.subject.mesh | Microsomes - Metabolism | en_US |
dc.subject.mesh | Models, Biological | en_US |
dc.subject.mesh | Oligosaccharides - Chemistry - Metabolism | en_US |
dc.subject.mesh | Phospholipid Transfer Proteins - Metabolism | en_US |
dc.subject.mesh | Proteolipids - Metabolism | en_US |
dc.subject.mesh | Saccharomyces Cerevisiae Proteins - Metabolism | en_US |
dc.title | Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum | 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 | 10.1021/bi800723n | en_US |
dc.identifier.pmid | 18597486 | - |
dc.identifier.scopus | eid_2-s2.0-48249088868 | en_US |
dc.relation.references | http://www.scopus.com/mlt/select.url?eid=2-s2.0-48249088868&selection=ref&src=s&origin=recordpage | en_US |
dc.identifier.volume | 47 | en_US |
dc.identifier.issue | 30 | en_US |
dc.identifier.spage | 7937 | en_US |
dc.identifier.epage | 7946 | en_US |
dc.identifier.isi | WOS:000257860100019 | - |
dc.publisher.place | United States | en_US |
dc.identifier.scopusauthorid | Sanyal, S=16069600000 | en_US |
dc.identifier.scopusauthorid | Frank, CG=24481396300 | en_US |
dc.identifier.scopusauthorid | Menon, AK=7202324192 | en_US |
dc.identifier.issnl | 0006-2960 | - |