自噬与核糖体的关系

roleintheattenuationofautophagy(Yuetal.2010;ShinandHuh2011).

Untilrecently,autophagywasthoughttofacilitaterandomdegradationofcytoplasmicmacromoleculesandorganelles.However,theinvolvementofubiquitinasaspecificityfactorforselectiveautophagyisemerging,andrecentevidencesug-geststhatcrosstalkexistsbetweenproteasome-mediateddeg-radationandselectiveautophagy(Kraftetal.2010).Manyexamplesofselectiveautophagyhavebeenreported.Oneofthese,ribophagy,specificallydegradesribosomesuponnitro-genstarvation.Both40Sand60Sribosomalsubunitsarede-gradedbyribophagy,butonly60SribosomalsubunitdecayrequirestheactivityofthedeubiquitinaseUbp3p(Kraftetal.2008).Kraftetal.(2008)foundthatRpl25pandother60Sribosomalsubunitproteinswereenrichedwithubiquitinconjugatesinaubp3Δstrainduringnitrogenstarvation,sug-gestingthatdeubiquitinationofribosomalproteinsmayfacilitatedegradationof60Ssubunitsduringnitrogenstarvation.Inaddition,Ubp3phasbeenshowntophysicallyinteractwithCdc48p,akeycomponentoftheubiquitin–proteasomesystem.Cdc48pbindstomultiplecofactors,in-cludingthedeubiquitinaseUbp3pandtheubiquitinligaseUfd3p(RumpfandJentsch2006;DargemontandOssareh-Nazari2012).Theinteractionofthesedistinctclassesofubiq-uitin-processingenzymeswithCdc48pisthoughttomakeita“decisionplatform”forsubstrateubiquitination.Finally,theribosome-associatedE3ubiquitinligaseLtn1p/Rkr1pwasrecentlyfoundtoantagonizeUbp3pactivityandactasaninhibitorofribophagyduringgrowthinnutrient-richconditions(Ossareh-Nazarietal.2014).

Besidesribosomes,asmallsubsetofcytoplasmicproteins,includingeIF4GI,havebeenshowntoundergoselectivedeg-radationduringnitrogenstarvationinSaccharomycescerevi-siae(Bersetetal.1998;Gelperinetal.2002;OnoderaandOhsumi2004;Shimobayashietal.2010).eIF4GIisalsode-gradeduponrapamycinaddition,whileothertranslationini-tiationfactorssuchaseIF4EandeIF4Aremainstableundertheseconditionsinbothmammalianandyeastcells(Bersetetal.1998;PowersandWalter1999;Kuruvillaetal.2001;Ramirez-Valleetal.2008).TheselectivedegradationofeIF4GIduringnitrogenstarvationisinterestingfromthegeneregulationstandpointsincedepletionofeIF4GIinmammaliancellsbyshRNAknockdownnotonlydecreasesthetranslationofmRNAsinvolvedincellproliferation,butalsopromotestheinductionofautophagy(Ramirez-Valleetal.2008).DeletionoftheyeastTIF4631geneencodingeIF4GIimpairsglobaltranslationinitiationratesandcellgrowth(Clarksonetal.2010).TheseresultssuggestthattheabundanceofeIF4GIbroadlyinfluencesthetranslationofmanyclassesofmRNAs.

GiventhepreviousfindingsthatribosomesandeIF4GIundergoselectivedegradationduringnitrogenstarvation,weexaminedthefateof14translationandmRNAdecayfac-torsundertheseconditions.Wefoundthattwotranslationfactors,eRF3andeIF4GI,arerapidlydegradedbyautophagy

Selectiveproteindepletionduringnitrogenstarvation

inamannerthatrequirestheribophagydeubiquitinaseUbp3p.Furthermore,twomRNAturnoverfactors,Dcp2pandPop2p,weredepletedduringnitrogenstarvationatafasterratethaneRF3oreIF4GI.ThedecreasedabundanceofDcp2pandPop2pwasUbp3p-dependent,butalsore-quiredtheproteasomepathwayinamannerthatwasre-pressedbyTOR1duringnutrient-richconditions.WealsofoundthatUbp3pitselfisdepletedduringnitrogenstarva-tion.Takentogether,ourdatashowthatUbp3pmediatesthedepletionofasubsetoftranslationandRNAturnoverfactorsbyboththeproteasomeandautophagypathwaysdur-ingnitrogenstarvation.RESULTS

AsubsetoftranslationandRNAturnoverfactorsaredegradedbyautophagyduringnitrogenstarvation

Previousstudieshaveshownthatnitrogenstarvationcausestheselectivedepletionofribosomesthroughaprocesscalledribophagy(Kraftetal.2008;Ossareh-Nazarietal.2010).Toinitiallyexploreribophagyinourstrains,wemonitoredtheabundanceoftheendogenouslargeribosomalsubunitpro-teinRpl3p,aswellasthe18Sand25SrRNAsduringnitrogenstarvation.WefoundthattheinitialabundanceofRpl3pandbothrRNAswasreducedbytwofoldwithin24hoftheonsetofnitrogenstarvation(Fig.1A);nofurtherdecreasewasob-servedafter48hofstarvation.Thus,ribophagydecreasedri-bosomecontentbyuptotwofoldinourstrainsfollowingtheonsetofnitrogenstarvation.Thereafter,ribosomeabundancereequilibratedatanewsteady-statelevel.

Thisdecreaseinribosomecontentuponnitrogenstarva-tionledustoinquireaboutthefateofotherproteinsassoci-atedwiththepost-transcriptionalcontrolofgeneexpression.Todothis,weexaminedthefateofninetranslationfactorsandfiveRNAdecayfactorsfollowingtheonsetofnitrogenstarvation.Thechoiceoftheseproteinswasprimarilybasedontheavailabilityofspecificantibodiesorepitope-taggedex-pressionclonesofeach.ChangesintheabundanceoftheseproteinswerecomparedwiththeribophagycontrolproteinRpl3p,whoseabundanceroutinelydecreased10%–25%inthefirst6hfollowingnitrogendepletion.Thecytosolicpro-teinPgk1pandthemitochondrialproteinTom70pwereusedascontrolsandfoundtobestableundertheconditionsused(Fig.1B–D).

Wefoundthattheabundanceofthreetranslationfactors(eIF2α,eIF5B,andeEF3)andtwoRNAturnoverfactors(Lsm1pandTpa1p)didnotchangeappreciablyduringthefirst6hofnitrogenstarvation(Fig.1B).Fiveotherproteins(thetranslationfactorseIF5A,eEF1A,eEF2,eRF1,andthepoly(A)bindingproteinPab1p)exhibitedmodest(20%–40%)decreasesinabundancefollowingtheonsetofnitrogenstarvation,roughlysimilartotheribophagymarkerRpl3p(Fig.1C).Toexaminetheroleofautophagyinreducing

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