自噬与核糖体的关系

KellyandBedwell

FIGURE1.AsubsetoftranslationandRNAturnoverfactorsarerap-idlydegradedbyautophagyfollowingtheonsetofnitrogenstarvation.(A)WesternblotsofRpl3pandPgk1p,andanRNAgelofrRNAfromcellsharvestedatvarioustimesfollowingtheonsetofnitrogenstarvation.QuantitationofRpl3p(center)andrRNA(right)abundanceisalsoshown.(B)WesternblotsoftranslationandRNAturnoverfactorsfromawild-typestrainwhoseabundancedidnotdecreasefollowingashifttonitrogenstarvation( N).(C)WesternblotsoftranslationandRNAturnoverfactorsinwild-typeandatg7Δstrainsthatshowedamodestdecreaseinabundancefollowingashifttonitrogenstarvation( N).(D)WesternblotsoftranslationandRNAturnoverfactorsinwild-typeandatg7Δstrainsthatshowedasignificantdecreaseinabun-dancefollowingashifttonitrogenstarvation( N).Ashifttoglucosestarvation( Glu)fortheindicatedtimeswasalsoexamined.(E)QuantitationofWesternblotsfromDforproteinsshowingthelargestchangesinabundanceafterexposuretonitrogenstarvation( N)fortheindicatedtimes(comparedwiththeribophagycontrol,Rpl3p).(F)WesternblotsofeRF3andeIF4GIinanatg19Δstrainafterexposuretonitrogenstarvation( N)fortheindicatedtimes.Quantitationisshowntotheright.ProteinabundancewasnormalizedtoPgk1pfromthesameextractasaninternalcontrol.Allexperimentswerecarriedouttwoormoretimeswithsimilarresults.Bargraphsareplottedasmean±standarddeviation.

theabundanceoftheselatterproteinsuponnitrogenstarva-tion,weutilizedanatg7Δstrain.Atg7pshareshomologywithE1activatingenzymesandmediatestheconjugationofAtg12pwithAtg5p,andAtg8pwithphosphatidylethanol-amine.TheabsenceofAtg7pblocksautophagosomeforma-900

RNA,Vol.21,No.5

tion,anessentialearlystepofautophagy(Fengetal.2014).Wefoundthatthedecreaseintheabundanceofthesepro-teins(aswellastheappearanceofadegradationproductforeIF5A)wasblockedintheatg7Δstraininamannersim-ilartoRpl3p(Fig.1C).Theseresultssuggestthatthereducedabundanceoftheseproteinsisduetodegradationbytheautophagypathway.

Interestingly,wefoundthattheabundanceofthreeotherproteins,thetranslationfactorseIF4GIandeRF3,aswellasthedecappingenhancerPat1p,wasreducedmuchmorerap-idlythanRpl3p,withdecreasesoffour-tofivefoldwithin4hofnitrogenstarvation(Fig.1D,E).Thedecreasedabundanceoftheseproteinswasalsomediatedprimarilybyautophagy,sincetheywerestabilizedinanatg7Δstrainunderthesameconditions.However,theinabilityoftheatg7Δmutationtocompletelyblockthedecreaseoftheseproteinsmaysuggestthatanothermechanism,suchasreducedsynthesis,mayalsocontributetotheirdecrease.Thedegradationoftheseproteinsisspecificfornitrogenstarvation,sincetheonsetofglucosestarvationdidnotinduceeIF4GIdegradationaspreviouslyreported(Gelperinetal.2002)oreRF3degrada-tion(Fig.1D).TheseresultsdemonstratethattheabundanceofasubsetoftranslationandmRNAturnoverfactorsisrap-idlyreducedfollowingtheonsetofnitrogenstarvation,andmuchofthisdecreaseisdependentupontheautophagypathway.

Thecytoplasmtovacuole(Cvt)pathwayisaspecializedformofautophagythatdeliversatleasttwohydrolases,α-mannosidase(Ams1)andaminopeptidaseI(Ape1),tothevacuole.DuringassemblyoftheCvtcomplex,Atg19pservesasacargoreceptor(Lynch-DayandKlionsky2010).TotestwhethertheCvtpathwayalsoparticipatesinthepref-erentialdegradationofeIF4GIandeRF3duringnitrogenstarvation,weexaminedtheeffectofanatg19Δmutationontheirdepletion.Wefoundthatthedecreasedabundanceofbothproteinswasunaffectedbytheatg19Δmutation(Fig.1F),indicatingthattheCvtpathwayisnotresponsiblefortheirturnover.

ToconfirmthatthereducedabundanceofeRF3andeIF4GIwasmediated(atleastinpart)byproteindegradation,wecarriedoutmetabolicpulse-chaseexperimentsusingan-tibodiestotheseproteins(Fig.2).Twoidenticalcultureswerelabeledfor15minwith[35S]-methionineinnitrogenrichconditions.Achaseperiodwastheninitiatedbytheadditionofexcessunlabeledmethionineandcellsfrombothcultureswererapidlyharvested.Subsequently,oneculturewasresus-pendedinnitrogenrichmedium,whiletheotherwasshiftedtonitrogen-freemedium.Aliquotsofeachwereharvestedattimepointsthereaftertoquantitatetheamountof[35S]-labeledeRF3andeIF4GIremaining.Wefoundthatthehalf-lifeofbothproteinsdecreasedsignificantlyfollowingtheonsetofnitrogenstarvation(Fig.2A,B).Theseresultsconfirmthatanincreaseintherateofdegradationbyauto-phagyplaysasignificantroleinthereducedabundanceofeRF3andeIF4GIfollowingtheonsetofnitrogen

starvation.