First,wetestedthecapacityofthismutant,namedP6m1,tointeractwiththewild-typeP6byfarproteingelblotassay.
The
The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat
932ThePlantCell
Figure3.SubcellularLocalizationofTruncatedVersionsofEGFP:P6inTobaccoBY-2Cells.
(A)Schematicrepresentationoffull-lengthP6andoftruncatedversionsfusedtotheCterminusofEGFP.P6fragments:thenumberscorrespondtoaminoacidpositionswithintheclonedP6sequence.EGFPisrepresentedbyagreenboxanddeletedversionsofP6byanemptybox,exceptdomainA,whichisyellow(nottoscale).TheotherdomainsofP6arerepresentedasinFigure1.CLSMimages:thenumbersreferto(B).aa,aminoacids.(B)SubcellularlocalizationofEGFP:P6mutants(1to4)intobaccoBY-2cells20hpost-transfectionbybombardment.FluorescenceimageswerecollectedbyCLSMasdescribedinFigure2.Images2and4correspondtothesuperpositionofthe uorescentimageandthecorrespondingdifferentialinterferencecontrastimage.Bars¼10mm.
proteinwasexpressedinE.coli,fractionatedbySDS-PAGE,transferredontoanitrocellulosemembrane,andthenincubatedwith32P-labeledP6.AsshowninFigure6,P6m1nolongerinteractedwithP6,suggestingthattheputativehydrophobicbondsinvolvingLeu14and18arecrucialforformationoftheP6-P6complex(cf.tolaneP6correspondingtothepositivecontrol).WealsofusedP6m1totheCterminusofEGFPandexpresseditinBY-2cells.VisualizationofEGFP:P6m1byCLSMrevealedthatitneverproducedaggregatesbutwasinsteadevenlydistributedthroughoutthecytoplasmandthenucleus(Figure5B,panels5and6)incontrastwithEGFP:P6deletedversionsthatmainlyaccumulatedwithinthenucleus(Figure5B,panels1to4).WealsodeterminedbyAlascanningwhetherotherresiduesofthea-helixareinvolvedintheaggregationofEGFP:P6.InthemutantEGFP:P6m2,theMet,Glu,andAspresidueslocatedbetweenLeuresiduesoftheI1sequenceatpositions15,17,and19,respectively,werereplacedbyAlaresidues,andinmutantEGFP:P6m3,theresiduesoftheEKImotifatpositions11to13wassubstitutedbyanAlatriplet.IncontrastwithEGFP:P6m1,whenexpressedinBY-2cellsbothconstructionswereexclu-sivelyfoundinthecytoplasm;no uorescencecouldbedetectedinthenucleusevenafter48to72hincubationofthetransfectedtobaccocells(Figure5B,panels7to10).However,EGFP:P6m2alsogeneratednumeroussmall uorescentfocithatweresuper-imposedonthediffuse uorescence(Figure5B,panel7;seealsoFigure8C,panel5),whereasEGFP:P6m3wasunabletoformaggregates.
Takentogether,theseresultsdemonstratethattheN-terminala-helixofP6isessentialfortheformationofviroplasmsand,furthermore,thatitsLeuzippermediatestheP6–P6interaction.Variousaminoacidsofthea-helix,notallofwhicharelocatedattheinterfaceofthepredictedcoiled-coilstructure(Figure4B),areimportantfortheaggregationprocess.Ifwerefertoourmodel(Figure4B),GlufromtheEKImotifandLeuatpositions14and18aredirectlyinvolvedintheinteractionbetweenP6molecules,thusexplainingtheinabilityofEGFP:P6m1andEGFP:P6m3toformaggregates.Whethertheotherresidues,inparticularthoselocalizedbetweentheLeuresidues,affectthe
secondary
The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat
CaMVP6IsaNucleocytoplasmicProtein933
Figure4.Fine-ScaleMappingoftheP6–P6InteractionDomain.
(A)SchematicrepresentationofdomainA(aminoacids1to112).TheconservedregioncanbedividedintotwosubdomainsdesignatedA1(aminoacids1to83)andA2(aminoacids84to112).Thetwoinvariantsequences(I1andI2)indomainAareindicatedbysolidlinesandthea-helixbyagraybox.aa,aminoacids.
(B)Top:thesequenceofaminoacids4to31ofP6containsfourtypicalLeuzipperheptadmotifs.Hydrophobicresiduesatheptadpositionsaanddareinbold.TheinvariantsequenceI1andthenearduplicatesequencei1areindicated.LeuresiduessubstitutedinEGFP:P6mbyaGln(atpositions14and16)andbyaHis(atposition18)arespeci ed.Bottom:acomputer-generatedmodelofaparallelcoiled-coilstructureformedbetweentheNterminioftwoP6molecules.Thesidechainsoftheresiduesinpositionsaanddareshown.
(C)GST:A,GST:A1,andGST:A2aswellasGSTandfull-lengthP6wereexpressedinE.coliandsubmittedtofarproteingelblotanalysisusing32P-labeledP6,GST:A,GST:A1,orGST:A2asprobeintheoverlay.Theradioactivecomplexesweredetectedbyautoradiographyafteranexposureof24h.Molecularmassesofmarkerproteinsareindicatedattheleft.
structureoftheNterminusofP6orthecompleteproteinremainsanopenquestion.Indeed,EGFP:P6m2couldstillgivesmallaggregatesbutlostthecapacitytoassembleintolargeperinu-clearviroplasms.
P6IsaNucleocytoplasmicShuttlingProtein
Asmentionedatthebeginning,smallaggregatesofEGFP:P6werefoundinthenucleusofBY-2cells(Figure2,panels4and5)andEGFP:P6DAwaspresentmainlyinthenucleus(Figure3B,panels1and2),althoughthecorrespondingfusionproteinshaveapproximatemolecularmassesof85and75kD,respectively,whicharehigherthanthereportedlimitforpassivediffusion
¨rlichandKutay,1999).We rstsus-acrossnuclearpores(Go