Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel 英文参考文献.docVIP

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Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel 英文参考文献.doc

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Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel 英文参考文献

FunctionalReconstitutionintoLiposomesofPurified HumanRhCGAmmoniaChannel IsabelleMouro-Chanteloup1,2,3*,SylvieCochet1,2,3,MohamedChami4,SandrineGenetet1,2,3 ,Nedjma Zidi-Yahiaoui1,2,3,AndreasEngel4,YvesColin1,2,3,OlivierBertrand1,2,3,PierreRipoche1,2,3 1INSERM UMR_S 665, Paris, France, 2Universite′ Paris Diderot-Paris 7, Paris, France, 3Institut National de la Transfusion Sanguine, Paris, France, 4C-CINA, Center for ImagingandNanoanalytics,E.Mu¨llerInstituteforStructuralBiology,Biozentrum,UniversityofBaselMattenstrasse26,Basel,Switzerland Abstract Background:Rhglycoproteins(RhAG,RhBG,RhCG)aremembersoftheAmt/Mep/Rhfamilywhichfacilitatemovementof ammoniumacrossplasmamembranes.ChangesinammoniumtransportactivityfollowingexpressionofRhglycoproteins havebeendescribedindifferentheterologoussystemssuchasyeasts,oocytesandeukaryoticcelllines.However,inthese complexsystems,apotentialcontributionofendogenousproteinstothisfunctioncannotbeexcluded.Todemonstrate that Rh glycoproteins by themselves transport NH3, human RhCG was purified to homogeneity and reconstituted into liposomes,givingnewinsightsintoitschannelfunctionalproperties. Methodology/Principal Findings: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9nmdiameterwithatrimericproteinstructure.Reconstitutionwasperformedwithsphingomyelin,phosphatidylcholine andphosphatidicacidlipidsinthepresenceoftheC12E8detergentwhichwassubsequentlyremovedbyBiobeads.Control ofproteinincorporationwascarriedoutbyfreeze-fractureelectronmicroscopy.Particledensityinliposomeswasafunction oftheLipid/Proteinratio.Whencomparedtoemptyliposomes,ammoniumpermeabilitywasincreasedtwoandthreefold inRhCG-proteoliposomes,dependingontheLipid/Proteinratio(1/300and1/150,respectively).ThisstrongNH3tra