自噬与核糖体的关系

Selectiveproteindepletionduringnitrogenstarvation

TABLE1.StrainsandplasmidsusedStrainYDB0646YDB0686YDB0679YDB0680YDB0681YDB0682YDB0683YDB0687YDB0688YDB0665YDB0689YDB0695YDB0694MHY501MHY659YDB0717YH129/wYH129/1YDB0708YDB0709YWO607YWO612yJC750

Description

MATaleu2-3,112his3-11,15trp1-1ura3-1ade1-14pop2::TRP1PSI–

MATaleu2-3,112his3-11,15trp1-1ura3-1ade1-14pop2::TRP1PSI–atg7Δ::HIS3pDB1227MATaleu2-3,112his3-11,15trp1-1ura3-1ade1-14pop2::TRP1PSI–ubp3Δ::LEU2pDB1227MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;atg7Δ::TRP1DCP2-HA::HIS3MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1DCP2-HA::TRP1

MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;ubp3Δ::HIS3DCP2HA::TRP1MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;ubp3Δ::HIS3PAT1-HA::TRP1MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;UBP3-HA3::TRP1MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1PAT1-HA::TRP1MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;atg7Δ::TRP1

MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;UBP3-HA3::TRP1;atg7Δ::LEU2MATapre1-1pre2-2ura3leu2-3,112his3-11,15CansGal+DCP2-HA::HIS3MATaura3leu2-3,112his3-11,15CansGal+DCP2-HA::HIS3MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1

MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1;ubp3Δ::HIS3MATαhis3-Δ200;leu2-3,112;ura3-52;lys2-801;trp1-1eIF4GI-HA::TRP1MATaura3leu2-3,112his3-1,15canSGal+

MATαura3leu2-3,112his3-1,15canSGal+pre1-1

MATaura3leu2-3,112his3-1,15canSGal+ubp3Δ::HIS3

MATαura3leu2-3,112his3-1,15canSGal+pre1-1ubp3Δ::HIS3MATaura3leu2-3,112his3-11,15CansGal+

MATapre1-1pre2-2ura3leu2-3,112his3-11,15CansGal+MATa,ura3,leu2,his3,met15,LSM1-HA::HIS3

ReferenceKeelingetal.(2006)ThisstudyThisstudyThisstudyThisstudyThisstudyThisstudyThisstudyThisstudyThisstudyThisstudyThisstudy

Ameriketal.(2000)Ameriketal.(2000)ThisstudyDieterWolfDieterWolfThisstudyThisstudyDieterWolfDieterWolfJeffColler

Whenasecondaryantibodywasrequired,eitherRabbitanti-mouse(MPBiomedical,55436)orRabbitanti-rat(Abcam,ab6703)serawereused.Boundantibodiesweredetectedusing[125I]-proteinA(PerkinElmer,NEX146L100UC),andresultswerevisualizedusingaStormPhosphorImager(GEHealthcare).AmousemonoclonalantibodytoS.cerevisiaePgk1p(22C5D8;Invitrogen,cat.No.459250)wasusedasaninternalcontrol.RabbitantiserumtoeIF4GIwasagiftfromMichaelAltmann(Bersetetal.1998).RabbitantiseratoS.cerevisiaeeEF3,eEF1A,eEF2weregiftsfromTerryKinzy,andrabbitantiseratoeIF2α,eIF5B,andeIF5AweregiftsfromTomDever.RabbitantiserumtoTom70p(Mas70p)waspublishedpreviously(Kohetal.2001),aswererabbitantiseratoeRF3oreRF1(Salas-MarcoandBedwell2004).Mousemonoclo-nalantibodiestoS.cerevisiaeRpl3p(ScRPL3)wereobtainedfromtheDevelopmentalStudiesHybridomaBank.HAantibodies(Covance,MMS-101R)wereusedtodetectHA-taggedversionsofLsm1p,Pat1p,Dcp2p,Pop2p,andUbp3p.Pab1pantibodiesareavailablecommercially(EnCorBiotech,MCA-1G1).Insomecases,nonspecificbandswereconsistentlyobservedinWesternblotsusingHAantibodies.Whenthesewerenearproteinsofinterest(e.g.,Dcp2porUbp3p),thecorrectbandwasconfirmedbyitsabsenceinextractsfromthesamestrainlackingtheHA-taggedprotein.

at30°Cwithshaking.After15mina150×chasemix(1mg/mLme-thionine,1mg/mLcysteine,15%yeastextract)wasaddedto1×finalconcentrationandcellswereincubatedat30°Cfor2mininashak-ingwaterbath.Cellswerethenrapidlyharvestedandwashed.OnetubewaswashedinSDmediumwithoutmethionine,whiletheoth-erwaswashedinSDmediumwithoutnitrogenoraminoacids.Cellswerespundownoncemoreandresuspendedinthesamewashme-dium.Time-pointsweretakenattheindicatedtimesandflashfro-zen.Sampleswereincubatedwith5%TCAandwashedtwicewithice-coldacetone.Thesampleswerethendried,resuspendedin50μLSDSboilingbuffer,disruptedbyglassbeadlysis,andsubjectedtoimmunoprecipitationaspreviouslydescribed(Bedwelletal.1987).ArabbitantiserumtoTom70p(Mas70p)wasusedasanin-ternalcontrolforimmunoprecipitationsamples(Kohetal.2001).Thefinalsamplesweresubjectedto8%SDS-PAGE,andthegelwasdriedontofilterpaper.Radiolabeledproteinsweredetectedus-ingaStormPhosphorImager(GEHealthcare).

Polysomeprofiles

Polysomeprofileswereconductedasdescribed(Landryetal.2009).Briefly,strainsweregrowninSDmediumtoacelldensityof0.5A600units/mL.Cycloheximidewasaddedtoafinalconcentrationof0.1mg/mL,andcellswereharvestedbycentrifugation(6000rpm,4minat4°C)andwashedtwicewithlysisbuffer(20mMTris–HClatpH8.0,140mMKCl1.5mMMgCl2,0.5mMDTT,1%TritonX-100,0.1mg/mLcycloheximide,1mg/mLheparin).Aftercentrifugation(7000rpmfor4min,4°C),pelletswereresuspendedinlysisbufferandcellswerelysedbyglassbeadbeating.Lysateswereclearedbycentrifugationat9500rpmfor5minat4°Candlayeredovera20%–50%sucrosegradient(containing20mMTris–HClatpH8.0,140mMKCl,5mMMgCl2,0.5mMDTT,0.1mg/mL

Pulse-chaseexperiments

Tomeasureproteinhalf-life,16A600unitsofcellsweregrowntolog(0.5A600)insyntheticdextrose(SD)mediumovernightat30°C.Theculturewasthensplitintotwoequalportions.CellswerespundownandresuspendedinthesameprewarmedmediatoanOD600of4OD/mLandincubatedat30°Cfor5mininashakingwaterbath.EasyTag[35S]proteinlabelingmix(PerkinElmer)wasaddedtoeachtubeat200μCi/mLfinalvolumeandincubatedfor15min

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