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1、JournalofCellularBiochemistry35129-136(1987)MolecularBiologyofIntracellularProteinSortingandOrganelleAssembly1-8ExpressionofCoronavirusElandRotavirusVPIOMembraneProteinsFromSyntheticRNAJohnArmstrong,MalcolmMcCrae,andAlanColmanDepartmentofBiologicalSciences,UniversityofWarw
2、ick,Coventry,CV47AL,EnglandSomevirusesacquiretheirenvelopesbybuddingthroughinternalmembranesoftheirhostcell.WehaveexpressedtheclonedcDNAforglycoproteinsfromtwosuchviruses,theElproteinofcoronavirus,whichbudsintheGolgiregion,andVPlOproteinofrotavirus,whichassemblesintheendop
3、lasmicreticulum.Mes-sengerRNAwaspreparedfrombothcDNAsbyusingSP6polymeraseandeithertranslatedinvitroorinjectedintoculturedCVlcellsorXenopusoocytes.InCVlcells,theElproteinwaslocalisedtotheGolgiregionandVPlOproteintotheendoplasmicreticulum.InXenopusoocytes,theElproteinacquire
4、dpost-transla-tionalmodificationsindistinguishablefromthesialylated,O-linkedsugarsfoundonviralprotein,whiletheVPlOproteinacquiredendoglycosidase-H-sensitiveN-linkedsugars,consistentwiththeirlocalisationtotheGolgicomplexandendo-plasmicreticulum,respectively.Thusthetwoprotei
5、nsprovidemodelswithwhichtostudytargetingtoeachoftheseintracellularcompartments.WhentheRNAswereexpressedinmatured,meioticoocytes,theVPlOproteinwasmodifiedasbefore,buttheElproteinwasprocessedtoamuchlesserextentthanininterphaseoocytes,consistentwithacessationofvesiculartransp
6、ortduringcelldivision.Keywords:expression,CoronavirusEl,RotavirusVP10,membraneproteins,syntheticRNAIntheeukaryoticcell,theroughendoplasmicreticulumisthesiteofsynthesisforbothsecretoryproteinsandintegralproteinsoftheplasmamembrane.Toreachthecellsurface,bothtypesofproteinmus
7、ttraversetheGolgicomplex.Duringthistransportprocess,aseriesofpost-translationalmodificationsmayoccur.Sincethemodifyingenzymesarelocalisedalongthesecretorypathway,theendoplasmicretic-ulumandGolgicomplexconstituteaseriesofmembrane-limitedcompartments,eachapparentlywithadisti
8、nctcomplementofproteins[reviewedin1-41.Howaretheseproteinsconfinedtotheirappropriatedesti
