自噬与核糖体的关系

KellyandBedwell

againattemptedpulse-chaseexperimentsasdescribedabove.However,wewereunabletodetecteitheroftheseproteinsbyimmunoprecipitation.Therefore,whiledirectproteasomaldegradationofDcp2pandPop2pisthemostdirectinterpre-tationoftheseresults,wecannotyetexcludeanindirectroleoftheproteasomeintheirdecreasedsteady-stateabundancefollowingtheonsetofnitrogenstarvation.Ubp3pisrequiredforselectivedegradationoftranslationandRNAturnoverfactorsbytheautophagyandproteasomalpathwaysduringnitrogenstarvation

ActivationoftheribophagypathwayfollowingtheonsetofnitrogenstarvationrequiresthedeubiquitinaseUbp3panditsassociatedcofactor,Bre5p(Cohenetal.2003;Kraftetal.2010).SinceourresultsshowedthateRF3,eIF4GI,andPat1parerapidlydegradedbyautophagyfollowingtheonsetofnitrogenstarvation,wenextaskedwhethertheirturnoverwasalsoUbp3p-dependent.WefoundthattherapiddecreaseinbotheRF3andeIF4GIabundancewasmoderatedinbothubp3Δandbre5Δstrains(Fig.4A,B),suggestingthatdeubi-quitinaseactivityoftheUbp3p–Bre5pcomplexfacilitatesdegradationoftheseproteins.Incontrast,theubp3Δmuta-tiondidnotalterthedegradationofPat1p.WealsoexaminedwhetherUbp3pisrequiredfortheproteasomaldegradationofDcp2pandPop2pduringnitrogenstarvation.Wefoundthatbothproteinswerestronglystabilizedintheubp3Δstrain(Fig.4A,C).TheseresultsindicatethatUbp3pparticipatesintherapidandselectivereductionofasubsetoftranslationandRNAturnoverfactorsbyboththeautophagyandtheproteasomalpathwaysduringnitrogenstarvation.Proteinsdepletedbytheproteasomeduringnitrogenstarvationarealsoreducedfollowingrapamycinaddition

RapamycinisamacrolideantibioticthatmediatesrapidTOR1inhibition,whichresultsintheinductionofautophagy(NodaandOhsumi1998).SinceourresultsindicatedthatDcp2pandPop2parerapidlydecreasedinaproteasome-dependentmannerfollowingtheonsetofnitrogenstarva-tion,wenextaskedwhetherrapamycinalsoinducedasimilarresponse.WefoundthattheabundanceofDcp2pandPop2palsorapidlydecreaseduponrapamycintreatment(Fig.5A,B).ThisturnoverwasblockedbyMG132,indicatingthattheirdepletionundertheseconditionswasagaindependentuponproteasomefunction.ToconfirmthatTOR1inhibitionwasrequiredforthisdecrease,wetransformedstrainswithaplasmidexpressingtherapamycinresistanttor1-1allele.Thetor1-1mutationpreventsTOR1bindingbytheFKBP-rapa-mycincomplex(SchmelzleandHall2000).Thus,expressionofthismutantalleleshouldpreventdegradationofDcp2pandPop2puponrapamycinadditionifTOR1regulatesthispro-teasomalprocess.Indeed,wefoundthatdegradationof902

RNA,Vol.21,No.5

FIGURE4.TheribophagyfactorUbp3pisrequiredforselectivepro-teindegradationbyboththeautophagyandproteasomalpathwaysdur-ingnitrogenstarvation.(A)WesternblotsofeRF3,eIF4GI,Pat1p,Dcp2p,andPop2pabundanceinwild-type,ubp3Δ,andbre5Δstrainsharvestedattheindicatedtimesfollowingtheonsetofnitrogenstarva-tion( N).(B)QuantitationofresultsfrompanelAforeRF3,eIF4GI,andPat1p.(C)QuantitationofresultsfrompanelAforDcp2pandPop2p.ForDcp2pblots,thearrowindicatestheDcp2pband,whilethesmallstarindicatesanonspecificbandrecognizedbytheHAanti-body.Forquantitation,proteinabundancewasnormalizedtoPgk1pfromthesameextractasaninternalcontrol.Allexperimentswerecar-riedouttwoormoretimeswithsimilarresults.Bargraphsareplottedasmean±standarddeviation.(ND)notdetectable.

bothDcp2pandPop2pwasblockedinstrainsexpressingthetor1-1allele(Fig.5C,D).TheseresultsindicatethatTOR1activityrepressestheproteasomaldepletionofDcp2pandPop2pduringgrowthinnutrient-richconditions,whileTOR1inactivationfollowingnitrogenstarvationorrapamy-cinexposureinducesselective,rapiddepletionofthesepro-teinsbytheproteasome.WenotethatthesmallerdecreaseinPop2pabundanceobservedfollowingrapamycinadditionascomparedwithnitrogenstarvationcouldbeduetothefactthatrapamycinisarelativelyspecificinhibitoroftheTOR1pathway,whilenitrogenstarvationperturbsnotonlytheTOR1pathway,butalsootherphysiologicalprocesses.Ubp3pisdepletedduringnitrogenstarvationandhasageneticinteractionwiththeproteasome

WenextexaminedtheabundanceofUbp3pduringnitrogenstarvation.WefoundthatthelevelofUbp3pis

rapidly