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ChaperoneInteractionsattheRibosomeElkeDeuerling,MartinGamerdinger,andStefanG.
KreftMolecularMicrobiology,DepartmentofBiology,UniversityofKonstanz,78464Konstanz,GermanyCorrespondence:elke.
deuerling@uni-konstanz.
deThecontinuousrefreshmentoftheproteomeiscriticaltomaintainproteinhomeostasisandtoadaptcellstochangingconditions.
Thus,denovoproteinbiogenesisbyribosomesisvitallyimportanttoeverycellularsystem.
Thisprocessisdelicateanderror-proneandrequires,besidescytosolicchaperones,theguidancebyaspecializedsetofmolecularchaperonesthatbindtransientlytothetranslationmachineryandthenascentproteintosupportearlyfoldingeventsandtoregulatecotranslationalproteintransport.
Thesechaperonesincludethebacterialtriggerfactor(TF),thearchaealandeukaryoticnascentpolypeptide-associatedcomplex(NAC),andtheeukaryoticribosome-associatedcomplex(RAC).
Thisreviewfocusesonthestructures,functions,andsubstratesoftheseribosome-associatedchaperonesandhighlightsthemostrecentfindingsabouttheirpotentialmechanismsofaction.
Thelifeofeveryprotein,irrespectiveofitsfunctionororigin,startsbyitsmessengerRNA(mRNA)-templatedtranslationonribo-somes.
Uponsynthesisbyribosomes,theemerg-ingpolypeptidechainsdirectlystarttheirfold-ingprogramintotheuniquethree-dimensional(3D)structuretobecomebiologicallyactive.
However,aboutone-thirdofnewlymadepro-teinsarecotranslationallytransportedtoanoth-erdestinationbeforefolding,forexample,totheendoplasmicreticulum(ER).
Thedenovofold-ingandtransportofproteinsisproblematicbecausehydrophobicresiduesofunfoldedpoly-peptidechainsareaccessible,whichenhancestheprobabilitythatthenewlysynthesizedpro-teinsfollowanunproductiveoffpathwayleadingtomisfoldingandaggregationorprematuredeg-radation(DeuerlingandBukau2004;Hartletal.
2011;Balchinetal.
2016).
Moreover,alargefrac-tionofnewproteinsarecotranslationallymodi-ed,forexample,byamino-terminalacetylationor/andcleavageoftheamino-terminalmethio-nine.
Thus,differentfactorsactcotranslationallyonnascentchainsassuminglyinahighlyspecicandcoordinatedmanner,bothtemporallyandspatially,toensurethefunctionalityofthetrans-latome(Krameretal.
2009;Gamerdinger2016).
Toaccomplishproductivefoldingandtransportandtopreventoffpathways,newlysynthesizedpolypeptidesinteractwithribosome-associatedchaperonesthatpreventinappropriateinter-andintramolecularinteractionsandthuspro-motetransportand/orfoldingintothenativestate.
Ribosome-associatedchaperonesarefoundineverycellbutdiffersignicantlyamongthedifferentkingdomsoflifewithregardtotheirstructureandmechanismofaction.
Whereasprokaryoteshaveonlyoneribosome-associatedchaperone,whichiscalledtriggerfactor(TF),eukaryotesusetwodifferentTF-unrelatedchaperonesystemsattheribosome(Fig.
1),theconservedheterodimericnascentpolypeptide-Editors:RichardI.
Morimoto,F.
UlrichHartl,andJefferyW.
KellyAdditionalPerspectivesonProteinHomeostasisavailableatwww.
cshperspectives.
orgCopyright2019ColdSpringHarborLaboratoryPress;allrightsreservedAdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a0339771onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/Downloadedfromassociatedcomplex(NAC)andaspecializedHsp70/Hsp40dimericchaperonesystemcalledribosome-associatedcomplex(RAC).
RACactsasacofactorforanadditionalribosome-attachedHsp70partnerinyeast(calledSsb)oracytosolicHsp70inhighereukaryotes(DeuerlingandBukau2004;Hartletal.
2011;PreisslerandDeuerling2012).
Recentanalysesofthenascentinteractomeofthesechaperonessuggestthattheyactonalmosteverynewproteinsynthesizedwithonlyafewexceptions(seebelow).
Asubsetofnewlysynthesizedproteinsbindtoothercyto-solicchaperones,forexample,Hsp70orHsp60familymembers,laterduringsynthesisorafterthereleasefromribosomesforfurtherassistanceoftheirdenovofoldingprogram(forreview,seeBuskiewiczetal.
2004;Hartletal.
2011;PreisslerandDeuerling2012).
THERIBOSOMEASPARTNERINCRIMETheribosomeisalargeribonucleoproteinpar-ticleof2.
4MDainbacteriaand4MDaineukaryotes(Steitz2008;Jenneretal.
2012;Klingeetal.
2012).
Itconsistsoftwosubunitsinallorganisms,asmallsubunit(30Sinbacte-ria,40Sineukaryotes)andalargesubunit(50Sinbacteria,60Sineukaryotes).
Fourfunctionalfeaturesareintrinsictoribosomes(Fig.
2A):Therstfeatureisthedecodingcenter,whichen-suresselectionofthecorrectaminoacyl-transferRNA(tRNA)speciedbythecodoninthemRNA.
Thedecodingcenterliesinthesmallsubunitandrecognizesthegeometryofco-don–anticodonbasepairingandstericallydis-criminatesagainstmismatches(SchmeingandRamakrishnan2009).
Thesecondfeaturerepre-sentsthepeptidyl-transferasecenter(PTC),theactivesiteoftheribosomewherepeptidebondformationoccurs.
ThePTCislocatedinacleftwithinthesubunitinterfacewithinthelargeri-bosomalsubunit.
Athirdfeatureistheribosom-altunnelinsidethelargesubunit.
Withalengthof80–100andadiameterof10atitsnarrowestpointand20atitswidestpointitconnectsthePTCwiththeribosomeexitsite50S60S60STriggerfactor30S40SYeastProkaryotesEukaryotesMammals40SMPP11RACRACNACNACSsbHsp70Hsp70L1Ssz1Zuo1Figure1.
Ribosome-bindingchaperones.
Theconceptofribosome-associatedchaperonesthatassistdenovoproteinfoldingisconservedinprokaryotesandeukaryotes,albeitrealizedbydifferenttypesofchaperones.
The30Sor40Sribosomalsubunitisschematicallydrawnindarkgrayandthe50Sor60Ssubunitinmiddleandlightgray,indicatingthatthe50S/60Sisslicedinhalftoillustratetheinteriorwiththeribosomaltunnel.
Thenascentpolypeptide(orange)attachedtoatransferRNA(tRNA)intheP-sitemigratesthroughthetunnelandinteractswithchaperoneswhenitleavestheribosomeattheexitsite.
Inprokaryotes(left),asinglechaperonecalledtriggerfactor([TF],red)bindstransientlytotheribosometowelcomenascentpolypeptides.
Ineukaryotes(right),tworibosome-associatedchaperonesystemsexist:theheterodimericnascentpolypeptide-associatedcomplex([NAC],showninpinkandpurple)andtheribosome-associatedcomplex([RAC],showninyellowandlightgreen),whichconsistsofSsz(lightgreen)andZuotin(Zuo,yellow)inyeastandtheZuo-homologMPP11(yellow)anditscomplexpartnerHsp70L1(lightgreen)inmammals,respectively.
Restrictedtofungi,theHsp70Ssb(darkgreen)additionallybindstoribosomesandactsonnascentpolypeptides,whereasinmammalsRAC(MPP11/Hsp70L1)recruitacytosolicHsp70(darkgreen)tobindtonascentpolypeptides.
E.
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2AdvancedOnlineArticle.
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ThetunnelwallispredominantlycomposedofribosomalRNA(rRNA)andonlyfewribosomalproteins.
TheribosomalproteinsuL4anduL22contributetothetunnelwallandforma10narrowcon-striction30fromthePTC.
ThehighcontentofrRNAgivesthetunnelanoverallelectroneg-ativepotential.
Oncethoughtofasamerelypas-siveconduit,thetunnelisnowacknowledgedtoplayanimportantroleinproteinbiogenesis,forexample,incontextofarrestsequences,whichtriggerPTCinactivationaswellasformingasecludedenvironmentinwhichrstfoldingeventsofthenascentpolypeptidetakeplace,mostlytheformationofα-helicalelements(Wil-sonandBeckmann2011).
Thefactor-bindingplatformcenteredaroundthetunnelexitrepre-sentsthefourthfunctionalfeatureoftheribo-some(Fig.
2B).
ItcomprisesseveralribosomalproteinsandrRNAelementsandconstitutesthebindingsitesforvariousribosome-associatedfactorsthatactonnascentpolypeptidesinclud-ingchaperones,processingenzymes(suchasN-acetyltransferases[NATs])andtargetingfac-tors(e.
g.
,signalrecognitionparticle[SRP]).
Thetunnelexithasadiameterof20andiscomposedofrRNAandfourconservedribo-somalproteins,uL22,uL23,uL24,anduL29(Fig.
2B).
Inaddition,kingdom-specicribo-somalproteinsarealsopresentattheexitsite(e.
g.
,eL19,eL31,eL39inarcheaandeukaryotes;bL17andbL32onlyinbacteria).
Someoftheseribosomalproteinshavebeenshowntoserveasbindingsitesforribosome-associatedfactors.
Inparticular,uL23associateswithmultiplena-scentchain-processingfactors,includingTF,NAC,SRP,andSec61,andwasthereforedubbedthe"universalribosomedockingsite"(Krameretal.
2002;Pooletal.
2002;Wegrzynetal.
2006).
Fromamultitudeofcross-linkingandcryo-electronmicroscopy(cryo-EM)studies,howev-er,amorecomplexpictureemerged,inwhichadditionalribosomalproteins,suchaseL19,eL22,uL22,uL29,eL31,andeL39alsorepresentcrucialcontactsitesforribosome-associatedfac-tors(Pooletal.
2002;Peiskeretal.
2008;Pole-vodaetal.
2008;Pechetal.
2010;Leidigetal.
2013;Zhangetal.
2014;Gumieroetal.
2016;Leeetal.
2016).
Inaddition,ribosome-associatedfactorsalsocontactrRNAelements(Leidig30S/40S50S/60SDCPTCuL23eL19uL29eL39uL22H24eL22eL31TunnelTunnelexitTunnelexitBAFigure2.
Functionalfeaturesofaribosome.
(A)Schematicdepictionofaribosome.
Thedecodingcenter(DC)islocatedinthesmallribosomalsubunit(30Sor40S).
Peptidyl-transferasecenter(PTC),ribosomaltunnel(tunnel),andthetunnelexitarelocatedinthelargeribosomalsubunit(50Sor60S).
Apeptidyl-transferRNA(tRNA)inthePTCwithnascentchain(orange)isincludedtoillustratethepathofanascentchain.
Theconstrictionsitewithintheribosomaltunnelisindicatedbythetwoarrowheads.
(B)Topviewonribosometunnelexit.
Surfacerenderingoftheyeast60Ssubunit(gray)withribosomalproteinsimplicatedincofactorbindingaroundthetunnelexit(whitecircle)highlighted.
eL19(palegreen),eL22(green),uL22(magenta),uL29(marine),eL31(red),eL39(limegreen).
Helix24(H24)ofthe25SribosomalRNA(rRNA)isdepictedinorange.
ChaperoneInteractionsattheRibosomeAdvancedOnlineArticle.
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a0339773onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
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2013;Zhangetal.
2014;Gumieroetal.
2016;Leeetal.
2016).
Theseinteractionsaretypicallybasedonelectrostaticinteractionsbe-tweenpositivelychargedstretcheswithintheas-sociatedfactorsandnegativelychargedRNA.
Ingeneral,ribosomeassociationofindividualfac-torsishighlydynamicandincasesinwhichfactorscompeteforthesameorclosenearbybindingsite(s),bindingoffactorsisoftenmutu-allyexclusive.
Notably,theorderinwhichfac-torsassociatewiththeribosomeandbindthenascentchainisprimarilydeterminedbythechemicalattributesoftheemergingnascentchainitself.
Itislikelythatseveralfactorscanadoptdif-ferentconformationsontheribosomeandhavemorethanonebindingsiteontheribosomede-pendingontheirfunctionalstate.
Forexample,fortheribosome-attachedHsp70Ssbinyeast,differentconformationsandcontactswiththeribosomehavebeensuggestedfortheATP-boundopenandADP-boundclosedconforma-tion(Gumieroetal.
2016).
Clearly,weareonlyatthebeginningofadetailedmechanisticunder-standingandappreciationoftheintricatedy-namicprocessestakingplaceattheribosomeexitsiteandwithintheribosomaltunnel.
STRUCTURES,FUNCTIONS,ANDMECHANISMSOFACTIONOFRIBOSOME-ASSOCIATEDCHAPERONESBacterialTriggerFactorTFisahighlyabundantchaperonefoundinbacteriaandchloroplastsbutnotinthecytosolofeukaryotes.
Itassociatesviaaribosome-bind-ingmotifinitsND(Fig.
3)transientlyina1:1uL23arm1HeadPPlaseTallribosomebindingarm2118149150-GFRxGxxP-43250432360PPlasearm1arm2Ribosome-bindingmotifBACFigure3.
Escherichiacolitriggerfactor(TF).
(A)SchematicrepresentationofthelineardomainorganizationofTF.
Theribosome-bindingdomain("tail")withtheribosome-bindingmotif(residues43–50)isshowninred,thePPIase"head"ingreenand"arm"1and"arm"2inlightblueanddarkblue,respectively.
(B)TFadoptsanextendedthree-dimensionalfold.
(Left)RibbondiagramoftheTFfold.
ColorcodeissimilartoA.
Inpositionsoftheribosome-bindingmotif(residues43–50),thearmsandthePPIaseareindicated.
(Right)SurfacechargedistributionofTFinthesameorientationasintheribbondiagram.
Positivelyandnegativelychargedresiduesareshowninblueandred,respectively.
(C)StructuralmodelofTFboundtotheribosome(differentshadesofgrayasinFig.
1).
ThemaincontactbetweenTF(colorsasinA)involvestheribosome-bindingmotifintheNdomainandtheribosomalproteinuL23(lightgray).
Thenascentpolypeptide(yellow)migratesintotheTFcradleduringsynthesis.
AllTFstructureswerepreparedusingPyMOL(DeLanoScienticLLC)basedonFerbitzetal.
(2004).
E.
Deuerlingetal.
4AdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a033977onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/DownloadedfromratiowithribosomesmainlyviainteractionwiththeribosomalproteinuL23atthetunnelexit(Krameretal.
2002;Ferbitzetal.
2004).
FirstevidencethatTFactsasachaperonefornascentpolypeptideswasprovidedbyexperimentsshowingthatcombinedlossofTFandtheHsp70chaperoneDnaKprovokedsynergisticdefectsindenovoproteinfoldingresultinginglobalproteinaggregationanddecreasedviabil-ityofEscherichiacolicells(Deuerlingetal.
1999;Teteretal.
1999).
TFhasaunique3Dconformation.
Itisanextendeddragon-likestructurewithacentralbodyincludingtwoprotrudingarms,ahead,andatailregion(Fig.
3).
Boundtotheribosomeasmonomerbyitsamino-terminaltail,TFleansovertheribosomalexittunneltherebyexposingitslargeinteriorsurfacespeckledwithmultiplehydrophobicpatchestowardtheexitingnascentchain(Fig.
3B,C;Ferbitzetal.
2004).
Thus,TFisideallypositionedtocaptureemergingchains.
Basedonthecrystalstructure,TFcanaccom-modateentireproteindomainsorevensmallproteins(uptoalengthof130aa)initscentralcavity.
Ribosome-boundTFcanpreventprema-tureandincorrectfoldingofproteinsduringsynthesis.
Forexample,TFretardscotransla-tionalfoldingofrecombinantlyexpressedreyluciferaseinE.
colicells,therebyenhancingthetotalyieldofactiveluciferase(Agasheetal.
2004;Kaiseretal.
2006).
MorerecentdatasuggestthatTFcanreshapeandimprovethefoldingpathwayofaproteinbyprotectingpartiallyfoldedinter-mediates(Singhaletal.
2015;Wrucketal.
2018).
Evenmoreintriguing,TFcanreverseprematurefoldingbyfacilitatingunfoldingofpreformedstructuresinnascentpolypeptides,whichallowsthenascentpeptidetoreentertheproductivefoldingpath(Hoffmannetal.
2012;Mashaghietal.
2013).
AmoredetailedunderstandingoftheTFmechanismofactionwasprovidedbyarecentstudyofSaioandcolleaguesusingsophisticatednuclearmagneticresonance(NMR)techniques(GamerdingerandDeuerling2014;Saioetal.
2014).
Theseinvestigatorsdeterminedthestruc-tureanddynamicsofpuriedTFinteractingwithunfoldedmodelsubstratesinsolution.
TFformsabindingscaffoldwithuptofourdistinctsubstrate-bindingsitesdistributedalongTFs'innersurface(Fig.
3B)withavariableorderofbindingandoccupancy.
Thebindingsitesarecomposedofnonpolarresiduesthatcanformnumeroushydrophobicpocketstobindtohy-drophobicpeptidestretchesof6–10residuesinsubstrateproteins.
Additionally,polarresiduesproximatetothehydrophobic-bindingsitescanbeusedtoformhydrogenbondswiththesub-strateprobablytoenhanceafnityandnavigatebinding.
Importantly,thesebindingsitesshowaexiblelocalarchitectureandtheengagementofindividualresidueswithinthebindingsitesisvariabledependingonthesubstratesegmentboundtoit.
ThishighdegreeofplasticityofTFs'bindingsurfacesexplainshowTFcanservesuchapromiscuousandlargepoolofnascentsubstrates(GamerdingerandDeuerling2014;Saioetal.
2014).
Usingupallofitsbindingsites,TFcandirectlybindupto50amino-acidresi-duesofasubstrate.
ThehydrophobicpeptidestretchesboundbyTFareseparatedbylinkerregionsinbetweenthatremainunboundandmayevenloopoutwardofthecentralcavity.
Perhaps,multipleTFmoleculescanbindsimul-taneouslytoanascentsubstrate,whichenablesTFtoretainalsolargepolypeptidesinanunfold-edstateandprotectthemfromaggregationbyshieldingtheirexposedhydrophobicregions.
ThisinteractionmodeofTFmayevenexerttheforcedrivenbythebindingenergytounfoldmisfoldedpeptidesegmentsoflowthermody-namicstability.
Morethan300differentaggregation-proneproteinspecieswerefoundinTF-andDnaK-decientE.
colicells.
Theidentiedproteinsareinvolvedinmanydifferentcellularprocesses,rangeinsizefrom16kDato140kDa,andarespecicallyenrichedforlarge(>40kDa)multi-domainproteins(Deuerlingetal.
2003).
Selec-tiveribosomeproling(SeRP)providedtherstglobalanalysisofthenascentinteractomeofTF(Ohetal.
2011).
Thistechniquecombinesafn-itypuricationofribosome–TFcomplexesandsubsequentidenticationofthemRNAsegmentthatisbeingreadbyTF-boundribosomes(Beckeretal.
2013).
Thiselegantapproachrevealednewfundamentalfeaturesoftheco-translationalactivityofTF.
TFrecruitmenttoChaperoneInteractionsattheRibosomeAdvancedOnlineArticle.
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As-sumingthat30–40aaofthenascentchainsareburiedintheribosomaltunnel,TFmustbindtoemergingpeptidesonlyoncetheyhaveexposedatleast60–70aaoutsidetheribosome.
Thisini-tialexclusionofTF,whichonlycorrelateswiththelengthofthepolypeptides,providesatimewindowtoallowprocessingenzymestoaccessnascentproteins(Ohetal.
2011).
Theseenzymesarerequired,forexample,toremovetheformyl-moietyandtheinitiatormethioninefromtheaminoterminiofnascentpolypeptidechains.
Thesecondimportantndingfromtheribo-someprolingstudyisthatTFinteractswithallnewlysynthesizedproteins,exceptforthosethatareinsertedintothecytoplasmicmembraneviatheSRPtargetingpathway.
Incontrast,na-scentoutermembraneβ-barrelproteins(Omps)wereamongthestrongestTFinteractorsduringtheirsynthesisonribosomes.
ThissuggeststhatthechaperoneactivityofE.
coliTFisparticularlyimportantforkeepingOmpsinatranslocation-competentconformation,sothattheycanbeefcientlyexportedbytheSecmachinery(Ohetal.
2011).
ItshouldbementionedthatthisndingisinagreementwithpioneeringstudiesbyWicknerandcoworkers,whoin1987initial-lyidentiedE.
coliTFinareconstitutedinvitrotranslocationexperiment,asacytosoliccomponentthatmaintainsproOmpAinatransport-competentconformationfordeliveryintoinside-outmembranevesicles(CrookeandWickner1987).
EukaryoticRibosome-AssociatedSystemsTheNascentPolypeptide-AssociatedComplexTheNACisanevolutionarilyconservedeukary-oticheterodimericproteincomplexcomposedofaα-andβ-subunit,referredtoasαNACandβNAC(Fig.
4A)(Wiedmannetal.
1994).
BothsubunitscontainahomologousNACdomain,whichdimerizebyformingaβ-barrel-likestructurewithahydrophobiccore(Fig.
4A,B)(Liuetal.
2010;Wangetal.
2010).
Besidesthedimerizationdomain,structuralinformationisalsoavailableforthecarboxylterminusofαNAC,whichformsacompactthree-helix-bun-dlemotifcharacteristicforubiquitin-associateddomains(UBAs)(Fig.
4B)(Spreteretal.
2005).
TheremainingpartsofNACincludingtheNDsofbothsubunitsandthecarboxy-terminaldo-mainofβNAChavenotbeenstructurallyre-solvedyet,andmanyregionsinthesedomainsarepredictedtobeintrinsicallydisordered,sug-gestingthatoverallNACshowshighexibility(Pechetal.
2010;Martinetal.
2018).
NACisabundantlyexpressedinequimolarconcentrationrelativetoribosomesandrevers-iblybindsina1:1stoichiometrytoribosomesincloseproximitytotheribosomaltunnelexit(Raueetal.
2007;PreisslerandDeuerling2012).
Cross-linkingdatasuggestthatbothsubunitscaninteractwithnascentchains(Wiedmannetal.
1994),whilespecicallytheamino-termi-nalregionofβNACiscriticalforribosomebind-ing(Wegrzynetal.
2006;Pechetal.
2010).
Stud-iesinyeastshowedthatdeletionoftherst11amino-terminalresiduesormutationofacon-servedpositivelychargedmotifinthecenter(RRKxxKK)abolishesribosomebinding,sug-gestingthattheamino-terminalpartofβNACmakesthemainribosomalcontact(Wegrzynetal.
2006;Pechetal.
2010).
Cross-linkingdatasuggestthatβNACcontactstheribosomeviatheribosomalproteinuL23(Wegrzynetal.
2006);however,otherstudiesalsosuggestaninteractionwitheL31(Pechetal.
2010;Zhangetal.
2012;NyathiandPool2015).
Bothribo-somalproteinsarelocatednexttothenas-centpeptidetunnelexitbutonoppositesides(Fig.
4C),indicatingsomeexibilityofNAConribosomes.
Inaddition,alsotheα-subunitmaycontributetoribosomebindingasarecentstudyshowedacross-linkbetweenαNACandtheribosomalproteinuL29,aneighboringproteinofuL23(Fig.
4C)(NyathiandPool2015).
However,apartfromthesecross-linkingdatanofurtherstructuralinformationisavail-ableyetandtheexactpositioningandstructureofNACattheribosomaltunnelexitremainsobscure.
Theabundanceandthepositionattheribo-somaltunnelexitindicateacentralroleforNACinguardingdenovoproteinsynthesis.
TheE.
Deuerlingetal.
6AdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a033977onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/DownloadedfromdeletionofNACleadstoembryoniclethalityinworms,ies,andmice,demonstratingafundamentalroleofNACintheproteinhomeo-stasisnetwork(DengandBehringer1995;Mar-kesichetal.
2000;Blossetal.
2003).
Despiteitsessentialrole,theinvivofunctionofNACremainedenigmaticforalongtimeandonlyrecentlyimportantfunctionalinsightswereob-tainedinCaenorhabditiselegansshowingaprimaryfunctionofNACinregulatingthecotranslationalproteintransporttotheER(Gamerdingeretal.
2015).
IntheabsenceofNAC,translatingribosomesunspecicallyasso-ciatewiththeSec61transloconintheERmem-braneleadingtothemislocalizationofnascentsubstratestotheER.
Topreventincorrectribo-some–Sec61interactions,ribosomebindingofNACwasfoundtobeessential,indicatingthatNACfunctionsasaregulatorydeviceblockingahigh-afnitySec61-bindingsiteonribosomesnearthetunnelexit.
ThishypothesisisbasedonthefactthatribosomesperseshowaveryhighintrinsicafnitytotheSec61complexinthelownanomolarrangeindependentofwhetherornotasignalsequenceisexposed(Borgeseetal.
1974;JungnickelandRapoport1995).
Thus,tomaintainERtargetingspecicitythebindingofribosomestoSec61mustbeneg-ativelyregulatedbyNAC.
Thisisinagreementwithapreviousinvitrostudyshowingthatpu-riedNACpreventsunspecicbindingofribo-somestoER-derivedmembranes(Molleretal.
1998).
TheactivityofNACopposesthatoftheSRP,whichpromotesthebindingofcorrect,NCuL29eL31TunnelexituL2340S60SNNACdomainUBAdomainNACβNACNACUBAαNACαNAC-RRKKK-2731531101621841321762151ACBβNACFigure4.
Thenascentpolypeptide-associatedcomplex(NAC)ineukaryotes.
(A)SchematicrepresentationofthelineardomainorganizationofhumanαNACandβNAC.
BothsubunitscontainahomologousNACdimeriza-tiondomain.
Aconservedpositivelychargedribosome-bindingmotif(RRKKK)islocatedintheamino-terminaldomainofβNAC.
Aconservedubiquitin-associateddomain(UBA)islocatedattheverycarboxylterminusofαNAC.
(B)CrystalstructureofthehumanNACdimerizationdomain,whichformsacompactβ-barrel-likestructure(blue,βNAC;violet,αNAC,PDB:3MCB).
ThestructureoftheUBAdomain(fromarchaealNAC,PDB:1TR8)consistsofatypicalthree-helix-bundle.
GraydashedlinesindicatepartsofNACthatarenotstructurallyresolvedyet.
(C)Surfacerenderingofyeast60S(gray)and40S(wheat)subunitswithpotentialmajorcontactpointsofNACnearthetunnelexitrevealedbycross-linkingexperiments.
βNACcross-linkstobothuL23andeL31,whereasαNACcross-linkstouL29.
ChaperoneInteractionsattheRibosomeAdvancedOnlineArticle.
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a0339777onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
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org/Downloadedfromsignalsequence-containing,ribosome-nascentchaincomplexes(RNCs)totheERtranslocon(Keenanetal.
2001).
Thisantagonistic"sortandcountersort"interplaybetweenNACandSRPisessentialtoenhanceaccuracyandefciencyofthecellularprotein-sortingmachinery(Gamer-dingeretal.
2015).
ThepivotalphysiologicalroleofNACinERproteintransportisunder-scoredbythefactthatNACdeciencyleadstostronginductionoftheunfoldedproteinre-sponse(UPR)intheERandaccompanyingin-ductionofcellapoptosis,asshowninC.
elegans,zebrash,andhumancells(Hotokezakaetal.
2009;Arsenovicetal.
2012;Murayamaetal.
2015).
Moreover,inadditiontocontrollingtheinherentSec61–ribosome-bindingafnity,NACmaybealsoimportanttoregulatethebindingspecicityofSRPtotranslatingribosomes.
ApreviousinvitrostudyindicatedthatNACisrequiredtopreventSRPtobindtosignalse-quencelessRNCs,indicatingthatNACandSRPuseoverlappingbindingsitesonribosomesandNACinhibitsthelow-afnitybindingofSRPtononsecretoryRNCs(Wiedmannetal.
1994).
Thisndingwaspartiallyreproducedinvivoinyeast,showingthatNACmodulatesSRP-bindingspecicitytosomedegree(delAlamoetal.
2011).
However,theNAC–SRPinterplayremainsobscureandiscontroversiallydis-cussed,asotherinvitrostudiesdidnotndevidenceforanalteredSRP-bindingspecicity(Neuhofetal.
1998;RadenandGilmore1998),and,mostimportantly,becauseoverallSRP-directedERmembranetargetingofRNCsseemsnottobeaffectedinvivointheabsenceofNAC(delAlamoetal.
2011;Gamerdingeretal.
2015).
Inadditiontoitsfunctionasanegativereg-ulatorofERproteintransport,severallinesofevidencealsosuggestafunctionofNACasanATP-independentmolecularchaperone.
Cross-linkingdataindicateadirectbindingofNACtonascentchainsandNACdeletioninyeastandhumancellsleadstoanincreasedubiquitylationofnascentpolypeptidessuggestingthatNACdirectlybindstonascentsubstratestoprotectthemfromprematuredegradation(Wiedmannetal.
1994;Duttleretal.
2013;Wangetal.
2013).
Furthermore,NACisrequiredtopromotegrowthofyeastcellstreatedwiththeprolinean-alogazetidine-2-carboxylicacid(AZC),whichisknowntocorruptthefoldingofnewlysyn-thesizedproteins(Duttleretal.
2013).
NACdeletionalsoexacerbateswidespreadproteinaggregationinyeastcellslackingthemajor,ATP-drivencotranslationalchaperonesystem,theRAC–Ssbsystem(Koplinetal.
2010).
Alto-gether,thesedatasupportaroleforNACasanearlyactingmolecularchaperoneassistingthecotranslationalfoldingofnascentchains.
TheprinciplethatATP-independentribosome-as-sociatedchaperoneshaveacrucialfunctioninthefoldingofmanynewlysynthesizedproteinsiswellestablishedforTFinbacteria,asoutlinedabove.
TFprotectsnascentchainsagainstpre-matureaggregationanddegradationbyusingseveralexiblebindingsitestoshieldhydropho-bicpeptidestretchesinsubstrateproteins.
How-ever,whetherNACactssimilartotheholdasechaperoneTFisspeculative.
Apartfromcross-linkingdatasuggestingthatbothNACsubunitsinteractwithnascentchains,littleisknownaboutNAC'ssubstrate-bindingspecicityandeventheparticularsubstrate-bindingsite(s)ofαNACandβNAChavenotbeenmappedsofar.
Arecentcross-link-massspectrometrystudyindicatedthatNACpredominantlyinteractswithsubstratesviatheexibleamino-terminalregionsofαNACandβNAC;however,whetheracrucialchaperonedomainislocatedinthesedo-mainsisyetunknown(Martinetal.
2018).
Stud-iesinyeastshowedthatNACcanassociatewithpracticallyeveryribosome-attachednascentpolypeptideofacellindicatingthatNACmayserveaverylargepoolofnascentsubstrates(delAlamoetal.
2011).
Incontrasttootherorgan-isms,yeastcellsexpresstwodifferentβNACsub-units,βNACandβ0NAC,thelatterexpressedinloweramounts(Ottetal.
2015).
Interestingly,thecotranslationalsubstratepoolgreatlydif-feredbetweenthetwoNACspeciesthatexistinyeast.
NACcontainingβ0NACpreferentiallyassociateswithproteinsshowinghighintrinsicdisorderandlowerhydrophobicity.
Conversely,NACcontainingtheβ-subunitbindstolongerpolypeptideswithhighhydrophobicityandlow-erintrinsicdisorder(delAlamoetal.
2011).
ThesedifferencesindicatethateachNACsub-E.
Deuerlingetal.
8AdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a033977onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/Downloadedfromunitrecognizesspecicphysicochemicalprop-ertiesofthenascentpolypeptide.
However,β0NACisonlypresentinyeast,suggestingthatitsspecicfunctioniseithernotrequiredoradoptedbycanonicalNACorotherproteinsinhighereukaryotes.
FurtherevidencealsosuggestsafunctionforNACinproteintransporttomitochondria.
KnockdownofNACinducesthemitochondri-alUPRinC.
elegansandcausesmitochondrialdysfunctioninhumancells(Hotokezakaetal.
2009;Gamerdingeretal.
2015).
Moreover,inyeast,NACwasfoundtoenhancetheefciencyofproteinimporttomitochondria(Georgeetal.
1998;FünfschillingandRospert1999).
Inthisrespect,NACpromotestheinteractionofribo-someswiththemitochondrialsurface,suggest-ingthatNACstimulatesimportofmitochondri-alproteinsinacotranslationalmanner(Georgeetal.
2002).
Arecentstudyinyeastshowedthatcotranslationalproteintransporttomitochon-driaismorewidespreadthanpreviouslythought(Williamsetal.
2014).
ThetargetingprocessofRNCsexposingamitochondrialtargetingse-quence(MTS)tothetranslocase(TOMcom-plex)oftheorganelleisnotyetestablished.
However,NACcouldplayamajorrolethereinasitbindstothemitochondrialoutermem-braneproteinOM14inyeast(Lesniketal.
2014).
Throughthisinteraction,translatingri-bosomesgetlocalizedtomitochondriaandbothNACandOM14arerequiredtoenhancepro-teinimportefciency.
Consistentwithamito-chondrialtargetingfunction,β0NACwasfoundtoassociatepreferentiallywithribosomestrans-latingmitochondrialprecursorsinyeast(delAlamoetal.
2011).
Moreover,mitochondrialproteinsinparticulargetmistargetedandmis-localizedtotheERonNACdepletioninC.
elegans(Gamerdingeretal.
2015).
Insum,thesendingssupportafunctionofNACincotranslationalproteintransporttomitochon-dria.
However,thequestionariseshowNAC,whichbroadlybindstoRNCs,canexertdis-criminativetargetingactivitytowardRNCs,ex-posinganMTS.
Moreover,OM14isnotcon-servedandapotentialreceptorforNAConmitochondriainhigherorganismshasnotbeenidentiedyet.
TheRibosome-AssociatedComplexEukaryotesfeatureasecondconservedribo-some-associatedchaperonesystemthatisin-volvedininitialproteinfolding,targeting,andpreventionofaggregationofnewlysynthe-sizedproteins,theRAC.
RACactstogetherwitharibosome-boundHsp70(heat-shock70kDaprotein)inyeast(Ssb)orrecruitscytosolicHsp70tonascentpolypeptidesinmammals(Fig.
1B,C)(PreisslerandDeuerling2012;Zhangetal.
2017).
RACisastableheterodimericcomplexofanHsp40protein(Zuo1inyeast,ZRF1/MPP11inhumans)andadegeneratedATPase-inactiveHsp70protein(Ssz1inyeast,Hsp70L1inhumans)(Fig.
5A,B)(Gautschietal.
2001,2002;Huangetal.
2005;Conzetal.
2007).
MostofourknowledgeofthefunctionandstructureoftheRAC–Hsp70systemisde-rivedfromstudiesinthebaker'syeastSaccharo-mycescerevisiae.
RACstimulatestheATPaseactivityofSsbinyeastandtherebyenhancestheafnityofthisHsp70forunfoldedpolypep-tidesultimatelyassistingdenovoproteinfolding(PreisslerandDeuerling2012;Zhangetal.
2017).
RACitselfseemsnottodirectlycontactthenascentchain(Gautschietal.
2002;Conzetal.
2007),butnonethelessitplaysanimpor-tantroleincoordinatingthesubstrate-bindingspecicityofSsbinyeast(Koplinetal.
2010;Willmundetal.
2013;Dringetal.
2017).
AfunctionalcooperationbetweencomponentsoftheyeastHsp40/Hsp70–chaperonetriadwasrstrevealedbygeneticanalyses.
CellslackingeitherSsborRACorbothdisplayasimilarphe-notype,whichincludessensitivitytohighsaltconcentrations,lowtemperature,andtransla-tioninhibitorydrugssuchasaminoglycosides(Nelsonetal.
1992;Yanetal.
1998;Gautschietal.
2002;Hundleyetal.
2002).
ThersthinttoaroleoftheRAC–Ssbsysteminfoldingofnascentchainsattheribosomecamefromcross-linkingexperimentsestablishingaRAC-dependentinteractionofSsbwithshortnascentchains(Pfundetal.
1998;Gautschietal.
2002;Hundleyetal.
2002).
AninvolvementofRAC–Ssbindenovoproteinfoldingwasfurthercor-roboratedbythefactthattheaminoglycosidesensitivityofracΔssbΔyeastcellscanbepartiallyChaperoneInteractionsattheRibosomeAdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a0339779onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/DownloadedfromZuo1S.
cerevisiaeHumanND11538390411Linker391403Linker380403Linker1512516613144595168184285348433JDZHDNBDSBDβRACRACNBDSBDβNBDSBDβSBDαMD4HB40SCABH44eL31Zuo1uL2260SH24TunnelexitND11509154686161178279346449512549605621JDZHDMD4HBSANTSANTSsz1MPP11Hsp70L1Ssb1Figure5.
Theribosome-associatedHsp70/40systemineukaryotes.
(A)Theyeastribosome-associatedchaperonesystemconsistsoftheHsp40Zuo1,andtheHsp70sSsbandSsz1.
Zuo1andSsz1formthestableheterodimericribosome-associatedcomplex(RAC).
Zuo1bindstotheSsz1nucleotide-binding(NBD)andsubstrate-bindingdomainβ(SBDβ)viaitsamino-terminaldomain(ND).
TheJdomain(JD)ofZuo1isrequiredforstimulationoftheATPaseactivityofitsHsp70partnerSsb.
TheZou1homologydomain(ZHD),thehighlychargedmiddledomain(MD),andthefour-helixbundle(4HB)areinvolvedinribosomebinding.
Ssz1andSsbcontainanamino-terminalNBDandacarboxy-terminalSBD.
Ssz1containsanincompleteSBD,whichconsistsofonlytheSBDβmoiety,whereastheSBDofSsbiscompleteandconsistsofSBDαandSBDβ.
AutonomousribosomebindingofSsbismostlymediatedviaapositivelychargedstretchatthecarboxylterminus.
RACisconservedinmammalswhileSsbhomologsareabsent(andasolubleHsp70isusedinstead).
(B)ThehumanRACcomplexisformedbytheHsp40MPP11andHsp70L1.
MPP11containstwoSANTdomainsatitscarboxylterminus.
(C)Surfacerenderingofyeast60S(gray)and40S(wheat)subunitswithmajorcontactpointswithZuo1highlighted.
ThedottedemptyoutlinecoarselytracestheshapeofZuo1.
Exittunnel(whitecircle),uL22(magenta),uL31(red),helix24(H24)ofthe25SribosomalRNA(rRNA)in60Ssubunit(orange),andhelix44(H44)ofexpansionsegment12of18SrRNAin40Ssubunit(cyan).
S.
cerevisiae,Saccharomycescerevisiae.
E.
Deuerlingetal.
10AdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a033977onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/Downloadedfromsuppressedbyexpressionoftheprokaryoticri-bosome-associatedchaperoneTF(Rauchetal.
2005).
Inaddition,cellsdevoidofSsbshowcaseanincreasedaggregationofnewlysynthesizedproteins,aswellasofribosomalproteinsandribosomebiogenesisfactors(Koplinetal.
2010;Willmundetal.
2013).
ThesendingsindicatethatalossofSsbnotonlynegativelyimpactsonfoldingofcytosolicproteinsbutalsoonribo-somebiogenesis.
Interestingly,theRAC–Hsp70systemisalsoimportantformaintainingtrans-lationaldelity(RakwalskaandRospert2004;Muldoon-JacobsandDinman2006;Leeetal.
2016;Zhangetal.
2017).
Recentstructuralworksignicantlyexpand-edourunderstandingofthefungalRAC–Ssbsystemontheribosome(Leidigetal.
2013;Zhangetal.
2014,2017;Leeetal.
2016).
Ribo-somebindingoftheRACcomplexismediatedsolelybytheZuo1subunit,whichcontactstheribosomalproteineL31closetothetunnelexitviaaconservedchargedregion(Yanetal.
1998;Peiskeretal.
2008).
Moreover,recentcryo-EMstudiesunveiledamuchmoreintricateinterac-tionbetweenZuo1andtheribosome.
Zuo1notonlycontactsthe60Ssubunitbutalsothe40Ssubunitandspans190acrossthesubunits(Fig.
5A,C)(Zhangetal.
2014;Leeetal.
2016).
WhilethecentralZuo1homologydomain(ZHD)bindsthe60Ssubunit(Leidigetal.
2013;Zhangetal.
2014;Kaschneretal.
2015;Leeetal.
2016),afour-helixbundle(4HB)atthecarboxylterminusofZuo1bindstothe40Ssubunit,andthemiddledomain(MD)formsahingebetweenthetwodomains(Zhangetal.
2014;Leeetal.
2016).
TheuniquebindingmodeofRACtotrans-latingribosomeshasseveralmechanisticimpli-cations.
Acommunicationbetweentheribo-somaltunnelandchaperoneactivityatthetunnelexithasbeenanticipatedgiventhatZuo1contactshelix24(H24)ofthe25SrRNAinthe60Ssubunit,andH24inturnisincontactwithribosomalproteinuL22,whichextendsintotheribosomaltunnelandmakescontactwithnascentchainsegmentsattheconstrictionsite.
Conceivably,thenatureofthenascentchaininthetunnelissensedattheconstrictionsitebyuL22andtheinformationmightberelayedviaH24andtheZHDtotheZuo1Jdomain,whichmayinuenceJdomainpositioningwithrespecttoSsbandhenceultimatelyregulateSsbactivityatthetunnelexit(Leeetal.
2016;Zhangetal.
2017).
Moreover,Zuo1interactsviaapositivelychargedsurfacewithhelix44(H44)ofextensionsegment12(ES12)of18SrRNAinthe40Ssub-unit(Zhangetal.
2014;Leeetal.
2016).
BecausetheextendedH44formsthecoreofthedecodingcenter(DC)andmutationswithinH44affecttranslationaldelity(Leeetal.
2016),bindingofZuo1tothetipofH44mightexplainhowRACinuencestranslationaldelity(Leeetal.
2016;Zhangetal.
2017).
AnunusualattributeofRACisthestableinteractionoftheHsp40Zuo1andthedegener-atedHsp70Ssz1,becauseHsp40–Hsp70inter-actionsarenormallytransient(MayerandKityk2015).
Firstbiochemicalinsightsintothesta-bleinteractionofthetwoproteinscamefromhydrogen–deuteriumexchangeexperiments,whichshowedthattheZuo1amino-terminaldomain(ND)bindsstablytoSsz1,makingdi-rectcontactswithboththesubstrate-bindingdomain(SBD)andthenucleotide-bindingdo-main(NBD)ofSsz1(Fiauxetal.
2010).
Therecentcrystalstructureoffull-lengthSsz1incomplexwiththeZuo1ND(bothfromthether-mophilicascomyceteChaetomiumthermophi-lum)fullyconrmedthesendingsandfurtherrevealeduniquefeaturesoftheinteractionofthetwoproteins(Weyeretal.
2017;Zhangetal.
2017).
IncontrasttocanonicalHsp70s,thelink-erbetweenNBDandSBDislongerinSsz1,andisdetachedfromATP-boundNBDallowingZuo1NDtobindthissiteintransinstead.
Inaddition,partsoftheZuo1NDcomplementtheβ-sandwichofSsz1SBDβtherebymimickingSBDβofcanonicalADP-boundHsp70s(despitehavingATPbound).
Overall,theconformationofSsz1intheRACheterodimerlookslikeahybridbetweenADP-andATP-boundHsp70(Weyeretal.
2017).
Ssz1representsinseveralaspectsanatypicalHsp70:First,itbindsATPbutisnotabletohydrolyzeit(atleastatadetectablerate),andATPbindingisnotstrictlyrequiredforitsfunc-tion(Huangetal.
2005;Conzetal.
2007).
Sec-ond,itlackstheliddomainoftheSBDandnoChaperoneInteractionsattheRibosomeAdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a03397711onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/Downloadedfrombindingtoasubstratecouldbedetected(Hund-leyetal.
2002;Huangetal.
2005).
AnSsz1trun-cationvariantlackingtheentireSBDfullycom-plementsgrowthdefectsofanssz1Δstrain(Hundleyetal.
2002;Conzetal.
2007).
Zuo1canonlyefcientlystimulateATPhydrolysisbySsbinthepresenceofSsz1(Huangetal.
2005).
Consequently,itwasproposedthatSsz1'spre-dominantfunctionistofacilitateZuo1'sabilitytofunctionasaJproteinpartnerofSsbontheribosome(Huangetal.
2005).
Itwasalsospec-ulatedthatSsz1mightfulllregulatoryfunc-tionsbyintroducingstructuralrearrangementswithinintheZuo1Jdomain,whichmightstrengthenthecontacttoSsb(Fiauxetal.
2010),andthatSsz1mightplayaroleinre-cruitmentofsubstratesbySsb(Leidigetal.
2013).
Regardless,despitetheimmensepro-gressonthestructuralsite,thefunctionoftheuniqueRACSBDβconformation,aswellasthemechanisticroleofSsz1remainhithertolargelyenigmatic.
Inyeast,twoSsbhomologsexist,Ssb1andSsb2,whichdifferonlyinfouraminoacids(here,collectivelyreferredtoasSsb).
ThestructureofSsbissimilartocanonicalHsp70proteins,ex-ceptthatitcontainsinadditionanuclearexportsequence(NES)atitscarboxylterminus,whichlikelyfacilitatesshuttlingbetweenthenucleusandcytosol(Shulgaetal.
1999).
AsforcanonicalHsp70s,theSsbreactioncycleisdrivenbyco-chaperones(PreisslerandDeuerling2012;MayerandKityk2015).
ATPhydrolysis,whichresultsintightsubstratebinding,isstimulatedbyRAC(Gautschietal.
2002;Hundleyetal.
2002),andnucleotideexchangefactorsforSsbareSse1,Sln1,andFes1(Peiskeretal.
2010).
ThehighlyabundantSsbinteractsindependentofRACwithribosomes,andinwild-typecells50%ofSsbisfoundassociatedwithribosomes(Nelsonetal.
1992;Yanetal.
1998;Peiskeretal.
2010).
Apos-itivelychargedregionclosetotheendofthecarboxy-terminalSBDαisessentialforribosomebinding(Gumieroetal.
2016;Hanebuthetal.
2016).
Likely,thisregionmediatesribosomeas-sociationofSsbviaelectrostaticinteractionswithexpansionsegmentES24and/orES41withinthe25SrRNA(Gumieroetal.
2016),consistentwithasalt-sensitivebindingofSsbtovacantribo-somes(Pfundetal.
1998).
Incontrast,Ssbbind-ingtoribosomesexposinganascentchain(RNCs)isresistanttohighsaltconcentrations,presumablybecauseofhydrophobicSsb–na-scentchaininteractions(PowersandWalter1996;Pfundetal.
1998;Raueetal.
2007).
Asec-ondpositivelychargedregionwithintheSsbSBDβinadditioncontributestoribosomebind-ing(Hanebuthetal.
2016).
Cross-linkingexper-imentsrevealedthatSsbcontactsribosomalpro-teinsuL29,eL39,andeL19incloseproximityofthetunnelexit(Gumieroetal.
2016).
Togetherwiththecrystalstructureoffull-lengthC.
ther-mophilumSsb(intheATP-boundstate),apic-tureemergesinwhichanexactpositioningofSsbontheribosomeclosetotheexittunneliscriticalforitsfunction(Gumieroetal.
2016;Hanebuthetal.
2016).
ThepositioningofSsbontheribo-someismodulatedbyRAC(aswellasthenucle-otide-statusofSsbitself)indicatingdynamicchangesofSsbontheribosomeonATPhydro-lysis(Gumieroetal.
2016).
Intriguingly,inthepresenceofRAC,autonomousribosomebind-ingofSsbisnotessentialforproteinfolding,suggestingaRAC-mediatedinteractionofSsbwithRNCs(Gumieroetal.
2016;Hanebuthetal.
2016).
Thisismostlyconsistentwiththesituationinorganismsotherthanfungi,inwhichnodedicatedautonomouslyribosome-anchoredHsp70existsandRACcollaborateswithacyto-solicHsp70instead(Fig.
1;Nelsonetal.
1992;Gautschietal.
2002).
Tworecentstudiesinvestigatedthecotrans-lationalsubstratesofSsbonaglobalscale(Will-mundetal.
2013;Dringetal.
2017).
IntherststudybyWillmundandcolleagues,ribosome–Ssbcomplexeswereisolatedandtheribosome-associatedmRNAanalyzedusinggenome-widemicroarrays.
Thisstudyrevealedthatthena-scentchainsubstratepoolofSsbisverybroad,encompassing80%ofcytosolicandnuclearproteins.
GeneralfeaturesoftheSsb-boundpro-teinswerethepresenceoflargerdomainsandagenerallylargesize.
Ssbsubstrateswerealsoen-richedforsubunitsofoligomericcomplexes(e.
g.
,TRiC/CCT,proteasome,andribosomalparticle),whichinmostcasesdonotrepresentlargeproteins,butusuallyinteractwithseveralothersubunitswithinthecomplex.
Furtherfea-E.
Deuerlingetal.
12AdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a033977onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/DownloadedfromturescommontomostSsbsubstrateswereagenerallowtranslationrate,andanenrichmentofβ-sheets,andshortlinearhydrophobicele-ments.
Together,thesedatasuggestthatSsbas-sistscotranslationalfoldingoflargerproteinsharboringcomplicatedstructuresorthatrequirebindingpartnersfortheirstability(Willmundetal.
2013).
Inthesecondanalysis,SeRP(Beckeretal.
2013)wasusedtoanalyzeSsbinteractionwithnascentchainsonaproteome-widescaleatnear-codonresolution(Dringetal.
2017).
ConsistentwithWillmundetal.
(2013),Ssbboundto80%ofcytosolicandnuclearproteinsintheSeRPstudy.
Furthermore,andindissentwithWillmundetal.
(2013),80%ofnascentmitochondrialproteinsandmorethan40%ofER-targetedproteinswerefoundassociatedwithSsb,suggestingafunctionofSsbintargeting/translocationoftheseproteins(Dringetal.
2017).
BecauseSsbwasfoundtobindnascentmitochondrialproteinspreferentiallyatlengthsof100residues,ithasbeenspeculatedthatSsbmightincreasethetargetingefciencyoftheseproteinsbypreventingprematurefoldingandmisfolding(Dringetal.
2017).
Suchascenarioissupportedbythefactthatasubsetofnuclear-encodedmitochondrialproteinsaggregatedintheabsenceofSsb(Willmundetal.
2013).
TheSeRPstudyfurthershowedthatSsbbindsposi-tivelycharged,degeneratesequencesclosetotheribosomalsurface,whenthesubstraterecogni-tionmotifextends5aafromthetunnelexit,wherebydisorderedregionsseemtobedisfa-vored.
Ssbnascentchainbindingclosetothetunnelexitisingoodagreementwithawealthofpreviousbiochemicalandstructuraldata(Pfundetal.
1998;Gautschietal.
2002;Hundleyetal.
2002;Gumieroetal.
2016).
Notably,Ssbengagesmostsubstratesbymultiplebinding-re-leasecycles(Dringetal.
2017).
AsSsbbindspreferentiallysegmentsthatwillbesurfaceex-posedorformthehydrophobiccoreofthefold-edprotein,Ssblikelyassistsdomain-wisefoldingbyretardingpremature/unproductivefolding(Dringetal.
2017).
ChallengingthecommonviewthatSRPistherstandonlycytosolicinteractorofnascentchainsofthecotranslationaltranslocationpath-waybeforedockingtothetranslocon,atleastforasubsetofSRP-targetednascentchains,Ssben-gagementbeforeSRPbindinghasbeenobserved(Dringetal.
2017).
WhetherthisimpliesahandoverofnascentchainsfromSsb–RACtoSRPand/ortheexistenceofanalternativetar-getingroutetotheERmembraneiscurrentlyunknown(Dringetal.
2017).
AninuenceofRAConthesubstratespeci-cityofSsbwasobservedinbothstudies(Will-mundetal.
2013;Dringetal.
2017).
Theab-senceofRACseverelyimpairedSsbbindingtoemergingrecognitionmotifsanddelayedRNCengagementofSsb(Dringetal.
2017).
Inaddition,deletionofRACwasfoundtorelaxthespecicityofSsb(Willmundetal.
2013).
Intriguingly,SsbbindingtoRNCscoincideswithanincreasedtranslationspeed,whichcanbeattributedtobothmRNAfeaturesaswellasnascentchainfeatures(Dringetal.
2017).
Ithasbeenspeculatedthatfastertrans-lationcouldreducethenumberofribosomesrequiredtomaintainproteinsynthesisandmaythereforerepresentastrategytoeconomizeproteinsynthesis(Dringetal.
2017).
Eitherway,thisndinghighlightstheclosecoordina-tionofproteinsynthesisandcotranslationalproteinfolding.
WhereasSsbisuniquetofungi,homologsofRACarepresentinmammalsaswell(Hundleyetal.
2005;Ottoetal.
2005).
ThisindicatesthatthepresenceofHsp70/40chaperonesonribo-somesiscommonintheeukaryoticworld.
Zuo1homologsinmammals(MPP11)containtwoadditionalSANTdomainsattheircarboxylter-minus(Hundleyetal.
2005;Ottoetal.
2005;Chenetal.
2014).
SANTdomainsarenormallyinvolvedinDNAbindingandchromatinre-modeling(Boyeretal.
2004),theirexactroleinMPP11howeveriscurrentlyunknown.
Knock-downofhumanMPP11inHeLacellsresultsingrowthdefectsandsensitivitytowarddrugssim-ilartowhatwasobservedforyeastcellslackingRAC(Jaiswaletal.
2011),suggestingthatRACfulllssimilarfunctionsinyeastaswellasinmetazoans.
However,becausehighereukaryoteslackadedicatedribosome-anchoredHsp70-likeSsb,itwassuggestedthatcytosolicHsp70sactasfunctionalpartnersforRACinhighereukary-ChaperoneInteractionsattheRibosomeAdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a03397713onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/Downloadedfromotes(Jaiswaletal.
2011).
Interestingly,althoughZuohomologsarefoundinalleukaryoticspe-ciesinwhichthewholegenomeissequenced,notallofthemalsorevealanSsz-likeHsp70(e.
g.
,thereisnoSszhomologfoundinplants),suggestingthattheRACsystemmayhaveamoreversatilesettingthantheyeastandmammalianversionscharacterizedsofar.
INTERPLAYWITHOTHERRIBOSOME-ASSOCIATEDFACTORS:SRP,PDF,MAP,ANDNATTheribosome-associatedchaperonesystemspresentedinthisreviewareintegralandessentialpartsofagreatercotranslationalproteostasismachinerythatcontrolsthequalityandlocali-zationofnewlysynthesizedproteins.
Numerousothercytosolicproteinbiogenesisfactors,in-cludingpeptidedeformylase(PDF),methionineaminopeptidases(MAPs),NATs,andtheSRPalsobindincloseproximitytotheribosomeexitsite,andmanyofthesefactorsusepartiallyoverlappingbindingsurfacesonribosomes.
Anemergingquestionishowthesefactorsgainreg-ulatedaccesstonascentpolypeptidesandwheth-ertheycompeteand/orcollaborateattheribo-someexitsite(GamerdingerandDeuerling2014;Glogeetal.
2014).
Theonlytunnelexit-bindingfactorsthatareatleastasabundantasribosomesareTFinbac-teriaandNACineukaryotes(Merzetal.
2006;Raueetal.
2007).
Thesemajorcotranslationalsystemsaresupposedtoplayanimportantroleinorchestratingnascentchain-processingeventsattheribosomeexitsite.
AmolecularinterplaybetweenTFandSRPiswelldocumentedinvitro.
TFgenerallypreventsSRPbindingtoribosomes,excepttothoseRNCspresentingsignalse-quences(Bornemannetal.
2014).
ThisRNCprelterfunctionbyTFcouldexplainwhyasmallamountofSRP(ratioofSRPtoribosomeabout1:100)issufcientforeffectivetargetingofmembraneproteins(JensenandPedersen1994).
Moreover,TFalsoenhancesthespecicityofSRP-dependentproteintargeting.
Bothfactorscanbindsimultaneouslytosignalsequence-containingRNCs,andTFregulatesSRPfunctionatmultipletargetingsteps,includinginitialbinding,targetingofRNCtothemembraneviaSRP–SRPreceptor(FtsY)assembly,andremovalofSRPfromRNCsexposingnascentchainsexceedingacriticallengththatcompromisescotranslationaltranslocation(Buskiewiczetal.
2004;Bornemannetal.
2014;Ariosaetal.
2015).
Together,theseactivitiesenhancethe-delityofsubstrateselectionbySRPandpromotetheefciencyofmembraneproteintransportinthecell.
IncontrasttoSRP,TFseemsnottomodulatedirectlyPDFandMAPbindingtotranslatingribosomes,indicatingthattheseen-zymescanprocesstheirnascentsubstratesbe-foreorinparallelwithTFbinding(Bornemannetal.
2014).
TheregulatoryfunctionofTFincotransla-tionalproteintransportpartiallyresemblesthatofNACineukaryotes.
Asoutlinedabove,NACiscriticalforaccuratesubstrateselectionbytheSec61translocon,butalsomodulatesthebind-ingspecicityofSRP(delAlamoetal.
2011;Gamerdingeretal.
2015).
Thepresenceofanot-yet-exposedsignalanchorsequenceinsidetheribosomaltunnelincreasestheafnityofSRPforribosomes,andtheearlybindingofSRPtothoseRNCsdependsonNAC(Berndtetal.
2009;Zhangetal.
2012).
ThisearlySRPrecruitmentislikelymediatedbysubtlestruc-turalalterationsoftheribosomeandNACattheexitsitesuchthatSRPbindingisfavored.
More-over,intheabsenceofNAC,SRPpartiallybindstononsecretoryRNCstherebyperturbingtheaccuratesubstrateselectionofMAP1byblock-ingitsribosomalaccess(delAlamoetal.
2011;NyathiandPool2015).
Thus,althoughNACandMAP1canbindsimultaneouslytoribo-somesanddonotdirectlymodulateeachother'sbindingproperties(NyathiandPool2015),uncontrolledbindingofathirdfactorcancauseunforeseenproblemsinproteinbiogenesis.
Thisexampleshowsthedelicatenessofthemolecularinterplayatthetunnelexitandhighlightsthedynamicbindingandreleaseofribosome-asso-ciatedproteinbiogenesisfactorsthroughacon-certedaction.
However,weareonlybeginningtounderstandthecomplexinterdependencyofthesefactorsandhowthisaffectsthespecicandtimelytargetingofnascentchainsintothecorrectproteinbiogenesispathway.
E.
Deuerlingetal.
14AdvancedOnlineArticle.
CitethisarticleasColdSpringHarbPerspectBioldoi:10.
1101/cshperspect.
a033977onMarch8,2019-PublishedbyColdSpringHarborLaboratoryPresshttp://cshperspectives.
cshlp.
org/DownloadedfromCONCLUDINGREMARKSThemyriadofrecentlyaccumulateddataontheinvivonascentsubstrates,structures,andmech-anismsofribosome-associatedchaperonesfromprokaryoticandeukaryotickingdomssuggeststhattheyareversatileandvitalelementsofthechaperonenetworkcrucialtocontrolthequalityandtransportofnewlysynthesizedproteins.
Inaddition,notdiscussedhereatall,thereiscul-minatingevidencethatthesechaperonesmaydisplayadditionalfunctionsofftheribosome,forexample,TFandRAC–Ssbhavebeensug-gestedtopromotetheassemblyofribosomalparticlesandNACwasfoundtoenhancetherefoldingofaggregatedluciferaseinC.
elegans(Martinez-HackertandHendrickson2009;Koplinetal.
2010;Kirstein-Milesetal.
2013).
Thefuturechallengewillbetodissecttheirindi-vidualrolesduringproteinbiogenesisandgainadeeperstructuralandmechanisticunderstandingoftheirribosomalandnonribosomalactivities.
ACKNOWLEDGMENTSWeapologizethatwecouldnotdiscussallas-pectsofdenovofoldingandchaperonefunc-tionsindepth.
Moreover,weapologizetoallourcolleagueswhoseresearchwasnotoronlyverybrieydiscussedornotcited.
WethankDr.
ChristinaSchlatterforassistanceingureprep-arationandSandraFriesforproofreadingthemanuscript.
M.
G.
,S.
G.
K.
,andE.
D.
aresupport-edbytheDeutscheForschungsgemeinschaft(DFG)-fundedcollaborativeresearchcenterSFB969onthe"ChemicalandBiologicalPrin-ciplesofCellularProteostasis.
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