自噬与核糖体的关系

FIGURE5.TOR1inhibitionbyrapamycininducesproteasomaldegra-dationofDcp2pandPop2p.(A)WesternblotsandquantitationofDcp2pabundancefollowingtheadditionof200nMrapamycin(+rap)toawild-typestrain.(B)WesternblotsandquantitationofPop2pabundancefollowingtheadditionof200nMrapamycin(+rap)toawild-typestrain.ForpanelsAandB,MG132(100µM)wasaddedwhereindicated.(C)WesternblotsandquantitationofDcp2pabundancefollowingtheadditionof200nMrapamycin(+rap)towild-typeandtor1-1strains.(D)Westernblotsandquantita-tionofPop2pabundancefollowingtheadditionof200nMrapamycin(+rap)towild-typeandtor1-1strains.ForDcp2pblots,thearrowindi-catestheDcp2pband,whilethesmallstarindicatesanonspecificbandrecognizedbytheHAantibody.Forquantitation,proteinabundancewasnormalizedtoPgk1pfromthesameextractasaninternalcontrol.Allexperimentswerecarriedouttwoormoretimeswithsimilarresults.Bargraphsareplottedasmean±standard

deviation.

decreasedduringnitrogenstarvation(Fig.6A),andthedecreaseoffull-lengthUbp3pcoincidedwiththeappearanceofafragmentthatwas～30kDasmaller.SinceourUbp3pconstructcarriedacarboxy-terminalHAepitopetag,thisin-dicatedthatthesmallerspecieslackedtheamino-terminalre-gionthatincludestheBre5pbindingsite(Lietal.2007).WefoundthatUbp3pturnoverwasreducedintheatg7Δstrain(Fig.6B),aswellasfollowingtheadditionofMG132(Fig.6C).Together,theseresultsindicatethatboththeautophagyandproteasomalpathwaysparticipateinUbp3pdepletionduringnitrogenstarvation.

SinceourresultssuggestthatUbp3pparticipatesintheproteasomaldegradationofDcp2pandPop2p,wetestedforageneticinteractionbetweentheubp3Δmutationandthepre1-1mutationofthe20Sproteasome.SerialdilutionsofWT,ubp3Δ,pre1-1,andubp3Δ/pre1-1strainswereplated

Selectiveproteindepletionduringnitrogenstarvation

onYPDplatesintheabsenceorpresenceof1nMrapamy-cin.ColonysizeandcellviabilityofthesestrainsweresimilaronYPDplatesgrownat30°C(Fig.6D,leftpanel).WhenYPDplateswereincubatedat37°C,wefoundthatthecolonysizeofthepre1-1strainwasreduced,whiletheubp3Δ/pre1-1doublemutantgrewmoreslowlyandexhibitedpoorerviabilitythaneitherofthesinglemutants(Fig.6D,centerpanel).Whenthesestrainsweretestedonplatescontaining1nMrapamycinat30°C,thepre1-1strainexhibitedonlyaslightgrowthdefect(Fig.6D,rightpanel).Theubp3Δstrainexhibitedbothreducedviabilityandslowgrowth,indicat-inganenhancedsensitivitytoTOR1inhibitionasobservedpreviously(Kraftetal.2008;Ossareh-Nazarietal.2010).Finally,theubp3Δ/pre1-1doublemutantexhibitedbothslowgrowthandagreaterlossofviabilitythantheubp3Δstrain.Theseresultsdemonstratethatrapamycinsensitivityoftheubp3Δstrainisexacerbatedbythepre1-1mutation,furtherimplicatingTOR1intheregulationofproteasomal

function.

FIGURE6.Ubp3pisdegradedduringnitrogenstarvation.(A)WesternblotsandquantitationofUbp3pfollowingtheonsetofnitrogenstarva-tion( N)inawild-typestrain.eRF3isincludedasacontrol.(B)WesternblotsandquantitationofUbp3pfollowingtheonsetofnitro-genstarvation( N)inwild-typeandatg7Δstrains.(C)WesternblotsandquantitationofUbp3pfollowingtheonsetofnitrogenstarvation( N)inawild-typestrain.MG132(100µM)wasaddedwhereindicat-ed.ForpanelsA–C,thearrowindicatesthelocationoffull-lengthUbp3p.ThelargestarisastableUbp3pdegradationproduct,whilethesmallstarindicatesanonspecificbandrecognizedbytheHAanti-body.(D)Evidenceofageneticinteractionbetweentheubp3Δandpre1-1proteasomalmutations.Wild-type,ubp3Δ,pre1-1,andpre1-1/ubp3ΔcellswereadjustedtoacelldensityofoneAYPDplateswith1ng/mLrapamycin600unit/mLandspot-tedonYPDplates,orusingfivefoldserialdilutionsattheindicatedtemperatures.Allexperimentswerecar-riedouttwoormoretimeswithsimilarresults.Bargraphsareplottedasmean±standarddeviation.

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