自噬与核糖体的关系

FIGURE2.ThestabilitiesofeRF3andeIF4GIaredecreasedfollowingtheonsetofnitrogenstarvation.Pulse-chasemeasurementsofproteinhalf-lifewerecarriedoutfor(A)eRF3and(B)eIF4GI.Measurementswerecarriedouttwiceforeachproteinandconditionwithsimilar

results.

TheabundanceofDcp2pandPop2pisrapidlyreducedinaproteasome-dependentmannerfollowingtheonsetofnitrogenstarvation

WealsoexaminedtheabundanceofDcp2pandPop2pfol-lowingtheonsetofnitrogenstarvation.Dcp2pisthecatalyticsubunitoftheDcp2p/Dcp1pdecappingcomplex,whilePop2p(alsoknownasCaf1p)isacomponentoftheCcr4p–Pop2pdeadenylasecomplex.Wefoundthatthesteady-statelevelofbothproteinswasreducedinarapidmannerfollowingtheonsetofnitrogenstarvation(Fig.3A,B),withtheirlevelsdecreasingbyfour-tofivefoldwithin2h.Intriguingly,theirdegradationwasnotblockedintheatg7Δstrain,indicatingthattheirdepletionwasnotduetodegradationbyautophagy.SinceautophagydidnotmediatethedecreasedabundanceofDcp2pandPop2p,wenextaskediftheproteasomewasre-sponsible.Wefoundthatadditionoftheproteasomeinhib-itorMG132totheculturemediumpartiallystabilizedbothproteinsduringnitrogenstarvation(Fig.3C,D).TheseresultssuggestthatbothDcp2pandPop2pbecomesubstratesforproteasomaldegradationfollowingtheonsetofnitrogenstar-vation.Toconfirmtheroleoftheproteasome,weexaminedthelevelsoftheseproteinsinapre1-1/pre2-2strain,whichcarriesmutationsintwoofthethreecatalyticsubunitsof

Selectiveproteindepletionduringnitrogenstarvation

the20Sproteasome(Heinemeyeretal.1991,1993).WefoundthatbothDcp2pandPop2pwerepartiallystabilizedinapre1-1/pre2-2strainwhengrownat30°C(asemi-restric-tivetemperature)(Fig.3E,F).TheseresultsconfirmthattherapiddecreaseinabundanceofDcp2pandPop2pfollowingtheonsetofnitrogenstarvationisatleastpartiallymediatedbytheproteasome.

ToconfirmthattheDcp2pandPop2pproteinsaresubjecttodirectproteasomaldegradationundertheseconditions,

we

FIGURE3.Dcp2pandPop2parerapidlydegradedbytheproteasomefollowingtheonsetofnitrogenstarvation.(A)WesternblotsofDcp2pandPop2pabundanceinwild-typeandatg7Δstrainsharvestedatthein-dicatedtimesfollowingtheonsetofnitrogenstarvation( N).(B)QuantitationofWesternblotsfrompanelA.(C)WesternblotsandquantitationofDcp2pinawild-typestrainharvestedatvarioustimesfollowingashifttonitrogenstarvation( N).(D)WesternblotsandquantitationofPop2pinawild-typestrainharvestedatvarioustimesfollowingashifttonitrogenstarvation( N).InpanelsCandD,MG132(100µM)wasaddedattheonsetofnitrogenstarvationasindi-cated.(E)WesternblotsandquantitationofDcp2pinwild-typeandpre1-1,pre2-2strainsharvestedatvarioustimesfollowingashifttoni-trogenstarvation( N).(F)WesternblotsandquantitationofPop2pinwild-typeandpre1-1,pre2-2strainsharvestedatvarioustimesfollowingashifttonitrogenstarvation( N).TheexperimentsshowninpanelsEandFwerecarriedoutat30°C.ForDcp2pblots,thearrowindicatestheDcp2pband,whilethesmallstarindicatesanonspecificbandrecog-nizedbytheHAantibody.Forquantitation,proteinabundancewasnormalizedtoPgk1pfromthesameextractasaninternalcontrol.Allexperimentswerecarriedouttwoormoretimeswithsimilarresults.Bargraphsareplottedasmean±standarddeviation.

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