自噬与核糖体的关系

KellyandBedwell

FIGURE8.ModelforselectiveturnoveroftranslationandmRNAde-cayfactorsduringnitrogenstarvationbytheautophagyandproteasomalpathways.(A)Nitrogenexcessconditions.(B)Nitrogenstarvationcon-ditions.Seetextfordetails.

(Ossareh-Nazarietal.2010).EngagementoftheUbp3pcom-plexleadstothetransportofDcp2pandPop2ptotheprotea-some,wheretheirdegradationoccurs(step3).Similarly,theUbp3pcomplexrecruitseRF3andeIF4GIintoearlyauto-phagosomes,leadingtotheirsubsequentdegradationbyautophagy(step4).TheselectivedegradationofDcp2p,Pop2p,eRF3,andeIF4GImaygraduallybecomelimitingasprocessingand/orturnoverdepletesUbp3p(step5)duringprolongednitrogenstarvation.

Theubiquitin–proteasomesystemwaspreviouslyshowntobedispensableforbothribophagyandautophagyinyeast(Kricketal.2010;Ossareh-Nazarietal.2010).However,ourresultssuggestthattheubiquitin–proteasomesystemmedi-atestheregulateddegradationofselectedproteinsduringnitrogenstarvation.Wealsofoundthatproteasomaldegra-dationofDcp2pandPop2pisdependentonTOR1inhibi-tion,suggestingthattheTOR1pathwayregulatesabranchoftheubiquitin–proteasomesystem(Fig.5).PreviousresultshavealsosuggestedinterplaybetweentheproteasomeandtheTOR1signalingpathway.Chanetal.(2000)foundthatsomeproteasomemutantsarehypersensitivetorapamycin.Furthermore,rapamycinadditionpromotesproteasomeac-tivation,whileproteasomeinhibitiondecreasesTORfunc-tioninmammaliancells(Jinetal.2009;Koetal.2011).Theseresultsprovidestrongevidenceofcrosstalkbetweentheubiquitin–proteasomeandTOR1pathways,andthatTOR1activityinverselycorrelateswithproteasomefunctionundersomeconditions.

Apreviousstudyfoundthatproteasomeactivityplaysanimportantroleinmaintainingaminoacidavailabilityduringtheearlystagesofacuteaminoacidstarvationinmammaliancells,whiletheautophagysystemprovidesaminoacidsatlatertimes(VabulasandHartl2005).Here,weobservedasimilartemporalsequenceofeventsforselectiveproteindeg-906

RNA,Vol.21,No.5

radationfollowingnitrogenstarvationinyeast.Dcp2pandPop2pwererapidlyandselectivelydepletedbytheprotea-some,whileeIF4GI,eRF3,andPat1pweredegradedmoreslowlybyautophagy.OthertranslationandmRNAturnoverfactorsweredegradedbyautophagyatevenslowerratesthatwerecomparabletotherateofribosometurnoverbyautoph-agy.Finally,somefactorswerecompletelystableoverthesametimeperiod.Theselective,temporallycontrollednatureofthisprocesssuggeststhattheturnoveroftheseproteinsisnotabulkprocessthatsimplyoccurstoprovideaminoacidstothestarvingcell.Instead,therapidlossofDcp2pandPop2pmayincreasethestabilityofaspecificsubsetofmRNAs,aspreviouslyshownforstrainslackingtheRNAturnoverfactorsXrn1pandDcp1p(Heetal.2003).Similarly,depletionofeIF4GIandeRF3couldreprogramthetranslationmachinerytofacilitatethesynthesisofproteinsrequiredforsurvivalduringnitrogenstarvationconditions.Furtherstudieswillberequiredtotestthesepredictions.MATERIALSANDMETHODS

Nitrogenstarvationassays

Cellsweregrowntologphaseinsyntheticdextrose(SD)mediaanddilutedovernight.Whenculturesreachedacelldensityof0.5A600units/mL,5A600unitsofcellswereharvestedandflashfrozenasthetimezerosample.Therestoftheculturewascentrifuged,washedwithnitrogenstarvationmedia(SDmediumwithoutaminoacidsornitrogen)(Difco)andresuspendedinthesamevolumeofnitrogenstarvationmedia.Cellsweresubsequentlyharvestedattheindicatedtimes.Sampleswereflashfrozenandkeptat 80°CuntiltheywereprocessedforWesternblotting.Insomeexperimentscellswerewashedinnitrogenstarvationmediawith100μMMG132(CaymenChemical)andresuspendedinthesame.Inotherexperi-ments,cellsweregrowntolog,atime=0timepointwastaken,then200nMrapamycin(LCLaboratories)wasaddedandaliquotstakenattheindicatedtime-points.

Westernblottingandantibodies

ToharvestproteinsforWesternblots,5A600unitsofcellswereadd-edto100%trichloroaceticacid(TCA)toachieveafinalTCAcon-centrationof5%.Cellswereincubatedonicefor30min,harvestedbycentrifugation,washedfourtimeswith100%acetone,anddriedundervacuum.Cellswerethenresuspendedin100μLofsodiumdodecylsulfate(SDS)boilingbuffer(1%SDS,50mMTris,pH7.5,1mMEDTA),andlysedbyagitationwithglassbeads(fourcycles,1mineach)with1minonicebetweeneachcycle.ProteinconcentrationsweredeterminedbytheLowrymethod(Lowryetal.1951).Fifteentotwenty-fivemicrogramsofproteinwasloadedintoeachlaneofan8%SDSpolyacrylamidegel.ProteinwasthentransferredtoanImmobilon-Ptransfermembrane(Millipore)us-ingaGenieElectrophoreticTransfersystem(IdeaScientific).Themembraneswereblockedin0.3%Tween20–phosphate-bufferedsalinebuffercontaining5%nonfatmilk.Membraneswereincubat-edinthesamebufferwiththeindicatedantibodiesorantiserumattheirrecommendedconcentrationsfor2hatroom

temperature.