CaMVP6IsaNucleocytoplasmicProtein935
Inconclusion,ourdataclearlysupporttheideathatP6canenterthenucleusduringviralinfectionandalsoindicatethatP6isanucleocytoplasmicshuttlingprotein.Furthermore,our ndingsalsostronglysuggestthatthesequencedownstreamofdomainAisimplicatedinthenuclearlocalizationofP6,whereastheN-terminalregionofP6mightcontainanuclearexportsignal(NES).
TheNTerminusofP6ContainsanNESThatIsRecognizedbytheCRM-1NuclearExportPathway
ThelatterhypothesisisreinforcedbythefactthatdeletionoftheconservedhydrophobicsequenceI1locatedinsubdomainA1ormutationoftheLeuresiduesatpositions14,16,and18partiallyabolishednuclearexportofP6(Figure5B,panels3to6).Moreover,thisLeu-richsequencebearssomeresemblancetotheNES(EKDTLLIDL)foundintheBR1proteinoftheSquashleafcurlvirus,ageminivirus(WardandLazarowitz,1999),andtotheNESsequenceofseveralknownrapidlyshuttlingnuclearpro-teins,suchasHIVRevprotein(forareview,seePollardandMalim,1998).
ToprovidefurtherevidencethattheaforesaidsequenceisanNES,wedeletedthesequenceI1ormutatedLeuresidues14,16,and18intheEGFP:Afusionprotein(Figure8A)asdescribedabove,andthebehaviorintobaccoBY-2cellsofthemutantswascomparedwiththatofthenonmutatedprotein.TheseexperimentswereperformedwithEGFP:Aratherthanwiththefull-lengthP6becausenuclearaccumulationofEGFP:Amutantswouldbedirectlyrelevanttotheimpairmentoftheexportprocess,whereastheaccumulationofEGFP:P6mutantsinthenucleusdoesnotpermitdiscriminationbetweenanexportdefectandanactiveimportoftheprotein.Indeed,thewild-typefusionproteinEGFP:Awasneverfoundinthenucleus,althoughitisofasizethatshouldpermitittodiffusefreelythroughthenuclearpore(Figure3B,panels3and4).
WhensequenceI1wasremovedfromdomainA,EGFP:ADI1wasequallydistributedinthecytoplasmandthenucleusexceptforthenucleolus(Figure8B,panels1and2),whereasEGFP:Alocalizedexclusivelytothecytoplasmiccompartment(Figure3B,panels3and4).ThesubcellularlocalizationinBY-2cellsofmutantEGFP:Am(Figure8B,panels3and4),inwhichthethreeLeuresiduesofI1werereplacedbypolarresidues,wassimilartothatobservedwithEGFP:ADI1(Figure8B,panels1and2);EGFP:Amwasfoundinboththecytoplasmandinthenucleusbutnotinthenucleolus.TheabsenceofbothEGFP:Amutantsinthenucleolus,incontrastwiththesituationwithEGFP:P6DI1andEGFP:P6pointmutatedversions(Figure5),mightbeduetothefactthattheN-terminalregionofP6isunabletointeractwithribosomalproteins,whereastheEGFP:P6mutantsstillcontainthecorrespondinginteractiondomains(i.e.,mini-TAVandRNAbindingdomainA)(Lehetal.,2000;Parketal.,2001;Bureauetal.,2004).
Alltheseresultssupportamodelinwhich(1)bothEGFP:AandEGFP:Amutantscanenterandexitthenucleusbydiffusion,(2)EGFP:AmoleculesbutneitherEGFP:ADI1norEGFP:Amarerapidlyexportedtothecytoplasm,and(3)thatthesequenceI1functionsasanNESbecausepointmutationsofLeuresiduesinI1impairtheexportofEGFP:A.Thus,theseLeuresidues
appear
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
936ThePlantCell
Figure7.NuclearLocalizationofP6.
Nucleifromhealthy(A)andCaMV-infectedturnipleaves(B)were xedandimmunolabeledwithrabbitanti-P6antibodiesandmouseanti-rabbitIgGcoupledtoAlexa488assecondaryantibodies(image1)andstainedwithpropidiumiodide(image2).Panel3correspondstodifferentialinterferencecontrastimagesandtheright-handimages(panel4)totheirsuperpositionwiththe uorescentandpropidium-stainedimages.Theconfocalimageswerecollectedwithafocaldepthof0.45mm(C).Aseriesofsuchopticalsectionsthroughanucleusisolatedfromaninfectedplant,anti-P6immunolabeled,andstainedwithpropidiumiodide.Bar¼5mm.
tobeessentialresiduesforthenuclearexportofP6asalreadydescribedfornucleocytoplasmicshuttlingproteinspossessingaLeu-richNESandinparticularfortheBR1proteinofSquashleafcurlvirus(WardandLazarowitz,1999).However,wecannottotallyexcludethattheinvariantsequenceI1alsohaspropertiesinvolvedintheretentionoffusionproteininthecytosol.
Therefore,thenuclearexportofP6wasfurtherinvestigated,treatingBY-2cellstransfectedwiththeaforementionedre-combinantplasmidswithleptomycinB,whichspeci callyinhib-itstheCRM-1pathwayinvolvedinthenuclearexportofmanyproteins(Fornerodetal.,1997;Kudoetal.,1998).WhenBY-2cellsexpressingEGFP:Awereincubatedwith100nMlepto-mycinB,6hafterbombardment,the uorescentproteinaccu-mulatedabundantlyinthenucleus(Figure8C,panel2),whereasno uorescencewaspresentinthiscompartmentinuntreatedcontrolcells(Figure8C,panel1),thuscon rmingthatEGFP:Amoleculesareactivelyexportedfromthenucleus.ThefusionproteinEGFP:P6formedlargeaggregatesinthecytoplasmandwasundetectableinthenucleusoftransfectedtobaccocells(Figure8C,panel3),butwhenthelatterweretreatedwithleptomycinB,itwasfoundinboththecytoplasmandinthenucleus(Figure8C,panel4).Thenuclearcompartmentcon-taineddiffuse uorescence,accompaniedasexpectedbymanysmallaggregatesbecauseP6wasabletoself-interact.ThisresultprovesthatP6isactivelytransportedbetweenthenucle-ocytoplasmiccompartments.SimilarexperimentswerealsoperformedwithpointmutatedEGFP:P6versionstodeterminethefunctionalimportanceofdifferentresiduesinthenuclearexportofP6.Asexpected,themutantEGFP:P6m1hadthesamesubcellularlocalizationinBY-2cellstreatedwithleptomycin