Trypanosoma bruceiwith partially overlapping substrate specificities (Perry et al., 2008, Mani et al. 2015). Most of our knowledge on mitochondrial protein import systems stems from

1

TbLOK1/ATOM19isanovelsubunitofthenon-canonical

mitochondrialoutermembraneproteintranslocaseof

Trypanosomabrucei

SilviaDesy1),JanMani1),AnkeHarsman1),SandroKäser1)andAndréSchneider1)*

1)DepartmentofChemistryandBiochemistry,UniversityofBern,Freiestrasse3,

CH-3012Bern,Switzerland

*Towhomcorrespondenceshouldbeaddressed:

AndréSchneider;Tel:+41316314253;E-mail:andre.schneider@dcb.unibe.ch

Runningtitle:TbLOK1isanATOMsubunit

Keywords:mitochondria,mitochondrialproteinimport,TOMcomplex,Trypanosoma

source: https://doi.org/10.7892/boris.92436 | downloaded: 17.2.2021

2

Summary

TbLOK1haspreviouslybeencharacterizedasatrypanosomatid-specific

mitochondrialoutermembraneproteinwhoseablationcausedacollapseofthe

mitochondrialnetwork,disruptionofthemembranepotentialandlossof

mitochondrialDNA.HereweshowthatablationofTbLOK1primarilyabolishes

mitochondrialproteinimport,bothinvivoandinvitro.Co-immunprecipitations

togetherwithbluenativegelanalysisdemonstratethatTbLOK1isastableand

stoichiometriccomponentofthearchaicproteintranslocaseoftheouter

membrane(ATOM),thehighlydivergedfunctionalanalogueoftheTOMcomplex

inotherorganisms.Furthermore,weshowthatTbLOK1togetherwiththeother

ATOMsubunitsformsacomplexfunctionalnetworkwhereablationofindividual

subunitseithercausesdegradationofaspecificsetofothersubunitsortheir

exclusionfromtheATOMcomplex.Insummarytheseresultsestablishthat

TbLOK1isanessentialnovelsubunitoftheATOMcomplexandthusthatits

primarymolecularfunctionislinkedtomitochondrialproteinimportacrossthe

outermembrane.Thepreviouslydescribedphenotypescanallbeexplainedas

consequencesofthelackofmitochondrialproteinimport.Wethereforesuggest

that,inlinewiththenomenclatureoftheATOMcomplexsubunits,TbLOK1should

berenamedtoATOM19.

3

Introduction

Mitochondriaperformanumberofimportantfunctionsandareessentialforall

eukaryoticlife(Friedman&Nunnari,2014).Theoriginofmitochondriacanbetraced

backtotheengulfmentofα-proteobacteriumbyanarchealhostcell(Williamsetal.,

2013).Duringevolutiontheendosymbioticancestorofmitochondriawasconverted

intoanorganellethatlargelyisunderthecontrolofthenucleus.Crucialforthis

conversionwastheacquirementofaproteinimportsystemthatallowedthe

endosymbionttomakeuseofgenesthatpreviouslyhadbeentransferredtothenucleus

(Lithgow&Schneider,2010,Hewittetal.,2011,Gray,2012).Todaymorethan95%of

themorethan1000mitochondrialproteinsaresynthesizedinthecytosoland

subsequentlyimportedacrossoneorbothmitochondrialmembranes.

Mitochondrialproteinimportandthemembranecomplexesthatmediateithavebeen

studiedingreatdetail(Chacinskaetal.,2009,Schmidtetal.,2010,Schulzetal.,2015).

Theproteintranslocaseofthemitochondrialoutermembrane(TOM),isofspecial

interestasitisthefirsttranslocasewithwhichimportedproteinshavetoengage.In

yeastandhumanstheTOMcomplexconsistsof7subunits.Theβ-barrelmembrane

proteinTom40,amemberoftheVDAC-likeproteinfamily(Pusniketal.,2009),forms

thetranslocationporeandistightlyassociatedwiththesinglemembranespanning

proteinTom22thatfunctionsasTOMcomplexorganizerandasasecondaryreceptor.

Tom7,Tom6andTom5areimportantforassemblyoftheTOMcomplexandregulateits

dynamics.Tom20andTom70,finally,areinpartredundantproteinimportreceptors

withpartiallyoverlappingsubstratespecificities(Perryetal.,2008,Manietal.2015).

Mostofourknowledgeonmitochondrialproteinimportsystemsstemsfromstudies

doneinyeast,Neurosporaandhumans,whichonaglobalscalearecloselyrelatedand

onlyrepresentaverysmallsegmentoftheeukaryoticdiversity(Burki,2014,Adletal.,

2005).Bioinformaticanalysisandexperimentalstudiesinplantsandmorerecentlyin

4

theparasiticprotozoaTrypanosomabruceihaveshownthatsurprisinglyofallTOM

subunitsonlyTom40andTom22areconservedinalleukaryotes(Manietal.2015).

ThemosthighlydivergedTOMcomplexfoundtodateisthearchaictranslocaseofthe

outermembrane(ATOM)oftrypanosomatids(Pusniketal.,2011),whichareoneofthe

mostearlydivergingeukaryotesthathavemitochondriacapableofoxidative

phosphorylation(Cavalier-Smith,2010,Heetal.,2014).Arecentstudyhasshownthat

theATOMcomplexconsistsofsixsubunits(Pusniketal.,2011,Manietal.2015).

ATOM40formsthehighlydivergedproteinimportchannelandshowsweaksequence

similaritytoboththeVDAC-likeandtheOmp85-likeproteinfamily(Pusniketal.,2011,

Zarskyetal.,2012).BesidesTom40ofyeast,Neurosporaandhumans,allofwhich

belongtothesameeukaryoticsupergroupoftheOpisthokonts,trypanosomalATOM40

istheonlyTOMcompleximportporethathasbeenanalyzedbyelectrophysiological

methods.TheseexperimentsshowedthatrecombinantATOM40behavedmoresimilar

toOmp85-likeproteinsratherthantoyeastTom40inregardofitsoligomericstateand

itsfastflickeringconductancestates(Harsmanetal.,2012).Thesecondconserved

subunitisATOM14.ItisaremoteorthologueofTom22which,similartotheplant

Tom22orthologueTom9,hasaveryshortcytosolicdomainthatlacksaclusterofacidic

residues(Manietal.2016).TheremainingfoursubunitsarespecificforKinetoplastids.

Theyinclude:ATOM11andATOM12whichregulateATOMcomplexassembly;ATOM69

whichcontainsaCS/Hsp20-likedomainandtetratricopeptiderepeat(TPR)motifs;and

ATOM46whichhasanarmadillorepeatdomain.ATOM69andATOM46areprotein

importreceptorsthatrecognizemitochondrialtargetingsignals.Interestingly,despite

thefunctionalconservationoftheimportsignalsbetweenyeastandKinetoplastidsthe

receptorsthatrecognizethem,Tom20/Tom70inyeastandATOM69/ATOM46in

trypanosomes,evolvedindependentlyofeachother(Manietal.2015).Thesame

appearstobethecasefortheproteinimportreceptorsthathavebeencharacterizedin

plantmitochondria(Perryetal.,2006).

5

InthepresentstudyweshowthatTbLOK1,arecentlycharacterizedmitochondrial

morphologyfactor(Povelonesetal.,2013),infactisanadditionalcoresubunitofthe

ATOMcomplex.Interestingly,unlikeanyotherTOMcomplexsubunitsinyeast,plantsor

trypanosomes,itlikelyhastwotransmembranedomains.

6

Results

TbLOK1isessentialunderallconditions

ArecentstudyinT.bruceicharacterizedakinetoplastid-specificproteinof19kDathat

wastermedTbLOK1forlossofkinetoplastDNA(kDNA),themitochondrialgenomeof

trypanosomes(Povelonesetal.,2013).AblationofTbLOK1interferedwithnormal

mitochondrialmorphologyandwiththemaintenanceofthesingleunitkDNA.Moreover,

theproteinwasshowntobeessentialforanintactmitochondrialmembranepotential

andtranscriptionofthekDNA.BiochemicalexperimentsindicatedthatTbLOK1isan

integralproteinofthemitochondrialoutermembrane.

InordertostudythefunctionofTbLOK1inmoredetailweproducedaninducibleRNAi

celllineforthebloodstreamformofT.brucei.Fig.1AshowsthatTbLOK1isalso

essentialinthislifecyclestage.Moreover,asobservedinprocyclicforms,ablationofthe

proteinaffectsthemorphologyofthemitochondrion(Fig.S1).Mitochondrialtranslation

andthusthekDNAareessentialforthesurvivalofT.bruceithroughoutitslifecycle

(Cristoderoetal.,2010).Tofindoutwhetherthegrowtharrestobservedininduced

TbLOK1-RNAicellsislinkedtokDNAmaintenanceoranotherasyetunknownfunction,

weusedanengineeredcelllinethatasabloodstreamformcangrowintheabsenceof

mitochondrialDNA(Deanetal.,2013).Thisispossiblebecauseitcarriesasinglepoint

mutationinthenuclear-encodedγ-subunitofthemitochondrialATPasethatallowsto

compensateforthelackofthemitochondriallyencodedsubunit6oftheATPase.Inthis

celllineablationofgenesinvolvedinmitochondrialgeneexpressionwillthereforenot

causeagrowtharrest.However,TbLOK1remainsessentialalsointhiscellline(Fig.1B),

whichstronglysuggeststhattheprimaryfunctionofTbLOK1isnotlinkedto

mitochondrialgeneexpression.

7

AblationofTbLOK1interfereswithmitochondrialproteinimport

TheonlyknownessentialmitochondrialoutermembraneproteinsinT.bruceithatare

notdirectlylinkedtomitochondrialgeneexpressionaresubunitsoftheATOMcomplex

(Manietal.,2015a),Sam50(Sharmaetal.,2010,Schnarwileretal.,2014)andpATOM36

(Pusniketal.,2012),allofwhichareinvolvedinmitochondrialproteinimport.We

thereforewonderedwhetherTbLOK1mightalsobeinvolvedinmitochondrialprotein

import.Totestthiswepreparedproteinextractsfromaprocyclictetracycline-inducible

TbLOK1-RNAicelllineatdifferenttimesafterinductionandanalyzedthemon

immunoblotsusingapanelofantibodiesrecognizingimportedmitochondrialproteins

(Fig.2A).Ithaspreviouslybeenshownthatformanysubstratesinvivoablationof

proteinimportfactorscausescytosolicaccumulationofunprocessedprecursorproteins

and/oradecreaseofthemitochondriallylocalizedmatureforms(Pusniketal.,2011).It

shouldbenotedthoughthataccumulationofprecursorsisnotseenforallsubstrates

becausemislocalizedcytosolicprecursorproteinsareoftenrapidlydegraded.

TheresultsinFig.2AshowthatablationofTbLOK1,simultaneouslywiththegrowth

arrest,resultsinadecreaseoftheimportedmitochondrialproteinsRNAediting

associatedprotein1(REAP1),initiationfactor2(IF2),cytochromeoxidasesubunitIV

(CoxIV)andvoltagedependentanionchannel(VDAC).Moreover,forREAP1andCoxIV

accumulationofunprocessedprecursorformsisobserved.Cytosolicelongationfactor

1a(Ef1a)howeverisnotaffected.

Inordertoexcludethattheobservedgrowtharrestandtheconcomitantaccumulation

oftheunprocessedCoxIVprecursorareduetoofftargeteffects,wecomplementedthe

TbLOK1-RNAicelllinewitharecodedversionoftheTbLOK1geneexpectedtobe

resistanttotheRNAiFig.S2showsthatinthiscelllineadditionoftetracyclinestill

causesablationoftheendogenousTbLOK1mRNA.However,duetoexpressionofthe

recodedTbLOK1thecellsgrownormallyandaccumulationofCoxIVprecursorisnot

observedanymore.

8

Mitochondrialproteinimportwasalsoassayedinvitrousingmitochondriaisolatedfrom

theuninducedandinducedTbLOK1-RNAicellline.Topreventpleiotropiceffects,

organellesfrominducedcellswereisolatedonly1.5daysafterinductionofRNAiprior

totheonsetofthegrowtharrest(Fig.S3).Fig.2Bshowsthatimportoftheinvitro

translatedchimericproteinconsistingofthepresequence-containing150N-terminal

aminoacidsoflipoamidedehydrogenase(LDH)fusedtomousedihydrofolatereductase

(DHFR)(Hauseretal.,1996)aswellasofthetrypanosomalinnermembraneprotein

alternativeoxidase(TAO)(Hamiltonetal.,2014)wasessentiallyabolishedin

mitochondriaisolatedfromtheinducedcellline.

Insummary,theseresultsshowthatTbLOK1playsaprominentroleinmitochondrial

proteinimport.

TbLOK1isasubunitoftheATOMcomplex

ThemitochondrialOMofT.bruceicontainstwoproteintranslocases:theATOMcomplex

(Manietal.,2015),whichisthegeneralentrygateforessentiallyallmitochondrial

proteins,andthemorespecializedSAMcomplex,whichmediatesbiogenesisofβ-barrel

proteins(Sharmaetal.,2010).Moreover,aproteintermedpATOM36thatisinvolvedin

importofasubsetofproteinshasalsobeenidentified(Pusniketal.,2012).Theresults

inFig.2showthatablationofTbLOK1affectsimportofmatrix,innermembraneaswell

asβ-barrelproteinsveryearlyafterinductionofRNAi.Thiscouldbeexplainedif

TbLOK1isimplicatedinthefunctionoftheATOMcomplex.

Apreviousbluenativepolyacrylamideelectrophoresis(BN-PAGE)analysishasshown

thatATOM40,theporeformingsubunitoftheATOMcomplex,migratesinahigh

molecularweightcomplexofapproximately700kDa(Pusniketal.,2011).Inorderto

testwhetherTbLOK1isaphysicalcomponentoftheATOMcomplexweperformedBN-

PAGEandsubsequentimmunoblotingusingantiseraagainstTbLOK1andthedifferent

ATOMsubunits.TheresultinFig.3AshowsthatTbLOK1co-migrateswithallATOM

9

complexsubunitsexcepttheperipheralreceptorATOM69.Aspreviouslyshown

ATOM69ispresentinhighermolecularweightcomplexthatonlycontainslittleofthe

ATOM40protein(Manietal.,2015).Similarexperimentswereperformedinthe

bloodstreamformofT.brucei.TheyshowedthatalsointhislifecyclestagetheTbLOK1-

containingcomplexesco-migratewiththeATOM40complexwhenanalyzedbyBN-

PAGE(Fig.S4).Furthermore,reciprocalco-immunoprecipitations(co-IPs)usinga

procycliccelllineexpressingMyc-taggedTbLOK1incombinationwithC-terminally

hemagglutinine(HA)-taggedATOM40demonstratethatpullingoneithertheMyc-or

theHA-tagresultsintheco-precipitationoftheothertaggedprotein,irrespectivelyof

whetherTbLOK1isN-orC-terminallytagged(Fig.3B).Aco-IPwasalsoperformed

usingacelllinewhichexpressesN-terminallyMyc-taggedTbLOK1inawild-type

backgroundandtheresultingfractionswereanalyzedusingantibodiesagainstthe

differentATOMsubunits.AsshowninFig.3CallATOMsubunitsco-immunoprecipitate

withthetaggedTbLOK1,whereasVDAC,whichservesacontrol,remainedinthe

supernatant.TheresultsinFig.3Ccannotbeexplainedbyanartefactualassociationof

taggedTbLOK1withtheATOMcomplexduetoitsoverexpression,sinceapulldown

usingtaggedATOM12recoversnativeuntaggedTbLOK1andATOM40,respectively(Fig.

3D).

Insummary,theseresultsshowthatTbLOK1isapreviouslyunidentifiedcomponentof

theATOMcomplex.

AblationofTbLOK1affectsATOMsubunitsandtheATOMcomplex

TorevealthefunctionalconnectionsbetweenTbLOK1andtheotherATOMsubunits,we

measuredhowitsablationaffectstheabundanceoftheothersubunitsasdetectedby

sodiumdodecylsulfate(SDS)–PAGEandsubsequentimmunoblotting(Fig.4,top

panels).InadditionweanalyzedthesamesamplesbyBN–PAGEtoinvestigatehow

TbLOK1affectsthesteadystatelevelsoftheATOMcomplexes(Fig.4,bottompanels).

10

Tominimizeindirecteffectsintheseexperiments,theTbLOK1-RNAicelllinewas

analyzedveryearly,1.5daysafterinduction,whichis1.5dayspriortotheonsetofthe

growtharrest(Fig.S3).TheresultsshowthatablationofTbLOK1leadstoablationof

ATOM12,whereasallotherATOMsubunitsremainstable.Thus,TbLOK1isrequiredfor

thestabilityorforimportofATOM12(Fig.4,toppanels).However,whereasATOM14,

ATOM11andATOM46arestableintheabsenceofTbLOK1theirassemblyintohigh

molecularweightcomplexesismuchreduced(Fig.4,bottompanels).Theassemblyof

theβ-barrelporeATOM40andthereceptorsubunitATOM69isalsoaffectedbuttoa

muchlesserextent.

Interestingly,theATOM40-containingcomplexesthatarestillpresentininducedcells

aremoreheterogenousduetotheaccumulationofvariouslowermolecularweight

forms(Fig.4secondpanel,Fig.S5,control).Thiscouldeitherbeduetolessefficient

assemblyortoadecreasedstabilityofTbLOK1-lackingcomplexes.IndeedtheBN-PAGE

inFigS5showsthatwhileATOM40-containingcomplexesthatlackTbLOK1remain

stableinthepresenceofincreasingconcentrationsoflauryl-β-D-maltoside(DDM)they

weredestabilizedatelevatedtemperature.Theseresultssuggestthatunderthese

conditionsTbLOK1exertsastabilizingfunctionontheATOMcomplex.

AsexpectedifTbLOK1isindeedanessentialsubunitoftheATOMcomplexweshould

seesimilareffectsinthebloodstreamformsincemitochondrialproteinimportis

essentialthroughoutthelifecycleoftheparasite.Fig.S6showsthatthisisthecaseas

ablationofTbLOK1inthebloodstreamformcausesadecreaseinthelevelofATOM

complexsubunitsaswellasofothermitochondrialproteins.

AblationofATOMsubunitsaffectsTbLOK1

Wealsodidthecomplementaryexperiments.Thus,thelevelsofTbLOK1asdetermined

bySDS-PAGEandimmunoblotsaswellasitsassemblystateasrevealedbyBN-PAGE

wereanalyzedincelllinesinwhicheachoftheATOMsubunitswasindividuallyablated

11

(Fig.5).TheresultsshowthatablationofATOM40andATOM14resultsinthe

degradationofTbLOK1(Fig.5,toppanels).Alternativelythereductioninthesteady

statelevelsofTbLOK1mightbeduetofactthatimportofTbLOK1directlydependson

ATOM40andATOM14.However,asshownabove,thedependenceofTbLOK1on

ATOM40andATOM14isnotreciprocalsincebothproteinsremainstableeveninthe

absenceofTbLOK1(Fig.4,toppanels).Inallothercelllinesablatedforthedifferent

ATOMsubunitsTbLOK1remainsstable.Wehavepreviouslyperformedanextensive

analysisoftheeffectsthatablationofthedifferentATOMsubunitshasontheATOM

complexesasanalyzedbyBN-PAGE(seeFig.S4in(Manietal.,2015)).Lookingatthe

presenceofTbLOK1inthesametypeofexperimentsconfirmsthepicture.Inthe

ATOM11andATOM46RNAicelllinesTbLOK1accumulatesinalowermolecularweight

complexof440kDa(Fig.5,bottompanels).Itisworthmentioningthatforunknown

reasonsthislowmolecularweightcomplexissometimes(Fig.5,bottompanels)butnot

always(Fig.3AandFig.4,bottompanels)alsovisibleintheuninducedRNAicelllines.

Asshownpreviously(Manietal.,2015)ablationofATOM12resultsinashiftofthe

ATOMcomplextoahighermolecularweightthatcontainsthemajorfractionofATOM69

(Fig.3A).TbLOK1getsshiftedinthesameway.AblationofATOM69ontheotherhand

doesnotappeartoaffectTbLOK1-containingcomplexes.IntheATOM46/ATOM69

doubleknockdowncellline,finally,theresultlookslikeintheATOM46RNAicellline

alone.

Insummary,theseresults(Fig.5)togetherwiththeanalysisoftheTbLOK1-RNAicell

line(Fig.4)suggestthatTbLOK1isacoresubunitoftheATOMcomplex.

12

Discussion

TbLOK1wasdiscoveredbyaglobalRNAiscreenforproteinswhoseablationcausesloss

ofkDNA.FurtherworkshowedthatTbLOK1isanintegralmitochondrialouter

membraneproteinspecificforKinetoplastids(Povelonesetal.,2013).Itsablation

causedagrowtharrestintheprocyclicformofT.brucei.Thefirstdetectedphenotypes

intheinducedRNAicellswereacollapseofthemitochondrialnetworkthatwas

followedbyalossofthemitochondrialmembranepotentialandadeclineinthe

respirationrate(Povelonesetal.,2013).Latereffectsincluded:asymmetricdivisionof

thekDNAnetworkresultinginsmallerkDNAsindaughtercells;areductionof

transcriptionofthemaxicircleDNAandasubsequentlossofthekDNA.Basedonthese

resultsitwasarguedthatTbLOK1mightbeamorphologyfactorthatregulates

mitochondrialfissionand/orfusionandbydoingsowouldinfluencemitochondrial

function.Alternativelyitwassuggestedthatthemorphologychangesmightbecaused

byanindirecteffectofTbLOK1suchasmodulatingthelipidcompositionofthe

mitochondrialmembranes(Povelonesetal.,2013).

HereweshowthatTbLOK1likelyisanovelandessentialcomponentofthe

trypanosomalATOMcomplexthatmediatesimportofproteinsacrossthemitochondrial

outermembrane(Manietal.,2015).Thisconclusionisbasedonthefollowing

evidences:i)AblationofTbLOK1interfereswithmitochondrialproteinimportbothin

vivoandinvitro;ii)Co-IPanalysisshowsthatTbLOK1isfoundinthesamecomplexas

allotherATOMsubunits,aresultthatissupportedbytheobservationthatTbLOK1co-

migrateswithATOM40,ATOM46,ATOM14,ATOM12andATOM11onBN-gels;iii)

TbLOK1isfunctionallyconnectedtotheotherATOMsubunitsinacomplexnetworkof

interdependenciesandiv)TbLOK1isessentialinabloodstreamformcelllinethatcan

growintheabsenceofthekDNA.Alloutermembraneproteinsthatbehavelikethatin

othersystemsareinvolvedinmitochondrialproteinimport.

13

Asinothereukaryotesmorethan1000proteinsareimportedintothemitochondrionof

T.brucei(Manietal.,2016).DepletionofTbLOK1reducestheabundanceoftheATOM

complex,whichabolishesproteinimportandleadstoadepletionofnuclear-encoded

mitochondrialproteins.Theselikelyincludefactorsinvolvedinthemaintenanceof

mitochondrialmorphology,inoxidativephosphorylationaswellasinthereplication

andsegregationofthekDNAwhichfullyexplainsthepreviouslyreportedphenotypes.

ThesubunitsoftheATOMcomplexwereoriginallydefinedbytheoverlapbetweenthe

T.bruceioutermembraneproteomeandtheproteinsidentifiedinco-IPsfromcells

expressingHA-taggedATOM40(Manietal.,2015).Fortheco-IPstheelutionwaseither

doneunderdenaturingconditionsorundernativeconditionswithsubsequentsize

selectionbyBN–PAGE.TbLOK1waspresentintwoofthesedatasets,butforunknown

reasonsitwasabsentintheco-IPsamplethatwaselutedundernativeconditions,which

explainswhyitwasmissedinthepreviousstudy(Manietal.,2015).

ThepresentstudynowrevealsthatTbLOK1isanessentialsubunitoftheATOM

complex,thegeneralproteintranslocaseoftheoutermembrane.Wethereforesuggest,

inlinewiththenomenclatureoftheotherATOMsubunits(Manietal.,2015),torename

TbLOK1toATOM19,thenumberreflectingitspredictedmolecularweight.

ATOMsubunitscanbedividedintothreegroups:i)ThecoresubunitsATOM40and

ATOM14,whichareremoteorthologuesoftheyeastTom40andTom22;ii)Thesmall

subunitsATOM12andATOM11whichinhibitorpromotetheassemblyofthecomplex

andiii)TheperipheralimportreceptorsATOM46andATOM69,whoseablation

doesnotaffectthestabilityofanyoftheothersubunits(Manietal.,2015).

ATOM19/TbLOK1bestfitsintothesecondgroupasitdoesnotshowhomologytoany

knownproteinimportfactorandappearstoinfluencetheassemblyand/orthestability

oftheATOMcomplex.Itappearstohavetwotransmembranedomainsaccordingto

variouspredictionalgorithms.Thisishighlyunusualforasubunitofthemitochondrial

14

outermembraneproteintranslocase,sinceexceptfortheβ-barrelporeTom40,allTOM

subunitsinalleukaryotesthathavebeenanalyzedtodatehaveonlyasinglemembrane-

spanningdomaineach.

Moderneukaryoticphylogenyresolvesfivetosixsupergroupsthatdivergedveryearly

inevolution(Burki,2014,Adletal.,2005).However,biochemicalandfunctionaldatais

onlyavailableontheTOMcomplexesoftheyeast,N.crassa,higherplantsandmost

recentlyfromT.brucei.Theseorganismsrepresentthethreesupergroups:

Opisthokonts,ArcheaplastidaeandExcavates.

Acomparativeanalysisshowsthattheonlysubunits,thataresharedbetweenallthree

TOMcomplexes,areaβ-barrelmembraneproteinoftheVDAC-likeproteinfamily

(Tom40inyeastandplants,ATOM40intrypanosomatids)thatformstheimport

channelandacloselyassociatedconservedprotein(Tom22inyeast,Tom9inplant,

ATOM14intrypanosomatids)thatactsasasecondaryimportreceptorandasan

organizerofthewholecomplex(Manietal.,2015).OrthologuesofTom5,Tom6and

Tom7arefoundinboththeTOMcomplexesofyeastandplantsbutareabsentfromthe

trypanosomalATOMcomplex(Maćasevetal.,2004,Manietal.,2016).Finally,TOM

complexesofallthreesystemshavetworeceptorsubunitseach:Tom20/Tom70in

yeast;Tom20/OM64inplantsandATOM46/ATOM69intrypanosomatids.Theydonot

sharesequencesimilarityandinsomecasesnoteventhesametopologyindicatingthat

thethreeproteinreceptorpairsaroseindependentlyofeachotherbyconvergent

evolution.

ThediscoveryofTbLOK1/ATOM19asanovelsubunitoftheATOMcomplexthatis

specificforKinetoplastidsandthathastwotransmembranedomainthereforesetsthe

trypansomaloutermembraneproteintranslocaseevenfurtherapartfromits

counterpartsinyeastandplants.Thefactthat5outof7ATOMsubunitsarespecificfor

15

Kinetoplastidssuggeststhattheyareadeep-branchingcladewithinthesupergroupof

theExcavatesorthatthelatterhavepolyphyleticevolutionaryorigin.

Thefunctionsofthemitochondrialproteinimportsystem,toimportmorethan1000

differentproteinsandtosortthemtothecorrectintramitochondriallocalizationare

conservedbetweenyeast,plantandtrypanosomatids.Revealingthedetailedfunctionof

TbLOK1/ATOM19andtheotherATOMsubunitsinthecontextofproteinimportwill

helpustodefinetheirreduciblefeaturesoftheimportmachinerythatareconserved

amongalleukaryotesandthuswillprovideuswithadeeperinsightintothephysical

andbiochemicalconstraintsthatshapemitochondrialproteinimport.

16

Experimentalprocedures

Transgeniccelllines

ProcyclicRNAicelllinesofTrypanosomabruceiwerebasedonthe29-13strain(Wirtzet

al.,1998,Wirtzetal.,1999)andgrownat27°CinSDM-79supplementedwitheither5

or10%offetalcalfserum(FCS).BloodstreamformRNAicelllineswerebasedontheT.

bruceiNewYorksinglemarkerstrainoraderivativethereofthatcangrowinthe

absenceofkDNA,termedF1γL262P(Deanetal.,2013).Allbloodstreamformcellswere

grownat37°CinHMI-9supplementedwith10%FCS.

RNAicelllinesdirectedagainstATOMsubunitshavebeendescribedbefore(Manietal.,

2015).RNAiofTbLOK1(Tb927.9.10560)wasdonebyusingapLEW100-derivedstem-

loopbasedplasmid(Wirtzetal.,1999,Bochud-Allemann&Schneider,2002).Asinsert,

weuseda396bplongfragmentcoveringnucleotides48-443oftheORF.The

complementationoftheTbLOK1-RNAicelllinewasdoneusingasynthetically

synthesizedTbLOK1gene(GeneScript)predictedtoberesistanttoRNAi(Fig.S2)that

wasclonedintopLEW100.TbLOK1wasepitopetaggedusingpLEW100derived

plasmidsdesignedtoallowforadditionofatripleMyc-tagateithertheN-orC-terminus

oftheprotein(Manietal.,2015).ThecomplementationoftheTbLOK1RNAicelllines

wasdoneusingasyntheticallysynthesizedgene(GeneScript)predictedtoberesistant

toRNAithatwasclonedintopLEW100.Thesameplasmidswereusedtotransfectacell

lineexpressinganinsituC-terminallyHA-taggedATOM40(Manietal.,2015)which

yieldedcelllinesexpressingbothHA-taggedATOM40andthecorrespondingMyc-

taggedTbLOK1.Transformationandselectionofclonesweredoneasdescribedin

(McCullochetal.,2004).Toobtaingrowthcurvesthecellswerecounteddailyusinga

Neubauerhematocytometerandsubsequentlydilutedto4x106fortheprocyclicform

and105cells/mlforthebloodstreamform.

17

IsolationofMitochondria

MitochondriaofthecelllinesablatedfortheATOMcomplexsubunitswereisolatedat

thetimeoftheonsetofthegrowthphenotype.MitochondriafromtheinducedTbLOK1-

RNAicelllinewereisolatedafter1.5daysoftetracyclineinduction(theonsetofthe

growtharrestwasobservedat3.5daysofinduction),inordertoavoidsecondary

effects.

MitochondriausedforCo-IPexperimentscontainedMyc-taggedTbLOK1.Expressionof

thetaggedproteinwasinducedfor3days.Mitochondriafromallcelllinesmentioned

abovewereisolatedusingtheisotonicprocedureaspreviouslydescribed(Schneideret

al.,2007b,Hauseretal.,1996)andstoredin20mMTrispH7.5,0.6Msorbitol,2mM

EDTAcontaining10mg/mlfattyacidfreebovineserumalbumin.

Invitroproteinimport

35S-MetlabeledsubstrateproteinsweresynthesizedusingtheTNTT7QuickCoupled

Transcription/TranslationSystem(Promega,ProductNoL1170).FortheLDH-DHFR

precursor,thesameplasmidasdescribedbefore(Pusniketal.,2011)encodingfora

chimericproteinconsistingofthefirst150aminoacidsoftheT.bruceiLDHfusedto

mouseDHFRwasused.ToproducetheTAOprecursoraPCRproductconsistingofthe

completeORFoftheprotein(Tb927.10.7090)thatwasflankedbyaT7-RNApolymerase

promoterand3'DNAstretchencodingapolyA-tailwasusedasatemplate.

Invitroproteinimportwasbasicallydoneasdescribed(Hauseretal.,1996).25µgof

isotonicallyisolatedmitochondriawereincubatedin20mMHEPES-KOHpH7.4,0.6M

sorbitol,25mMKCl,10mMMgCl2,1mMEDTA,2mMKH2PO4,5mg/mlfattyacidfree

BSAcontaining4mMATPpH7.0.5μgcreatinekinaseand20mMphosphocreatine.The

radiolabeledprecursorproteinswereaddedfortheindicatedtimes.Thetotalreaction

volumewas25µl.Thereactionswerestoppedbyadding0.5µlof0.2mMvalinomycine

and5mMofcarbonylcyanidem-chlorophenylhydrazone.Allsamplesweredigested

18

with80µg/mlofproteinaseKfor10minonice.ProteinaseKwasinactivatedbythe

additionof3.6mMofphenylmethylsulfonylfluoride.Mitochondriawerereisolatedby

centrifugationandtheresultingpelletswereprocessedforSDS-PAGEanalysis.

AutoradiographsoftheSDS-PAGEwereanalysedusingaPhosphoImager(FujiFilm

FLA3000).

BluenativePAGE

Isotonicallyisolatedmitochondria(Schneideretal.,2007b,Hauseretal.,1996)or

pelletsfromdigitoninfractionationscorrespondingtocrudemitochondria(Fig.S4)

weresolubilizedin20mMTris-HCl,pH7.4,50mMNaCl,10%glyceroland0.1mMEDTA

containing1to1.5%(w/v)digitonin.Thesampleswerecentrifugedwith20'800gat4°C

andtheresultingsupernatantswereseparatedon4-13%gradientBNgels.Before

blottingthegelsonpolyvinylidenefluoridemembranes(MerckMillipore,ProductNo

IPFL00010)thegelsweresoakedinSDSrunningbuffer(25mMTris,190mMglycine,

1mMEDTA,0.05%(w/v)SDS).Proteinsweredetectedusinghorseradishperoxidase-

coupledsecondaryantibodywiththeSuperSignalWestPicoChemiluminescence

SubstrateorFemtoMaximumSensitivitySubstrateKits(ThermoScientific,ProductNo

34080or35095).

Co-Immunoprecipitations

Thereciprocalco-IPsshowninFig.3Bweredoneaspreviouslydescribedfortheother

ATOMcomplexsubunits(Manietal.,2015).Inshort,digitonin-purifiedcrude

mitochondrialfractionsweresolubilizedinlysisbuffer(20mMTris-HClpH7.4,0.1mM

EDTA,100mMNaCl,25mMKCl)containing1%(w/v)digitoninand1xprotease

inhibitormix(Roche,ProductNo.11873580001).After15minincubationonice,the

lysatewasclearedbya10mincentrifugationstepat20'800gandincubatedwitheither

anti-Mycbeads(ClontechLaboratories,Inc.,ProductNo.631208)oranti-HAbeads

19

(Roche,ProductNo.11815016001)thathadpreviouslybeenequilibratedwithlysis

buffercontaining0.1%(w/v)digitonin.Theresultingmixtureswereincubatedfor2hat

4°Cunderconstantmixing,washedthreetimeswithlysisbuffer,containing0.1%(w/v)

digitoninand1xproteaseinhibitormix,andelutedbyboilinginSDS-PAGEsample

buffer.

TheprocedurefortheCo-IPsshowninFig.3Cwasessentiallyidenticalasdescribed

above,exceptthatinsteadofdigitonin-purifiedcrudemitochondrialfractions520µgof

isotonicallyisolatedmitochondriapurifiedfromtheMyc-TbLOK1-expressingcellswere

used.Themitochondrialpelletwassolubilizedin600µllysisbufferasdescribedabove.

Incubationwaswith100µlof1:1slurryofanti-Mycbeads(Sigma,ProductNoE6654).

Washesandelutionweredoneasabove.

Miscellaneous

Toprepareacrudemitochondrialfractioncellswereextractedwithlowconcentrations

ofdigitonin(Schneideretal.,2007a).Inshort,108cellswereharvested,washedin

phosphatebufferedsalineandresuspendedin0.5ml1xSoTE,prewarmedtoambient

temperature.Next0.5mlofice-cold1xSoTEcontaining0.03%(w/v)ofdigitoninwas

added.Themixturewasincubatedonicefor5minandcentrifugedat4°Cfor5minwith

6'800g.Theresultingpelletscorrespondtothecrudemitochondrialfractionsusedin

someoftheexperimentsmentionedabove.

TheimmunofluorescenceanalysisshowninFig.S1wasdoneasdescribed(Manietal.,

2015).Tostainthemitochondrionpolyclonalrabbitanti-ATOM40(dilution1:1'000)

andpolyclonalmouseanti-mtHsp60(dilution1:200)antiserawereused.Thesecondary

antibodiesusedweregoatanti-rabbitFITC(dilution1:100,SigmaProductNo.F0382)

andgoatanti-mouseAlexaFluor633(dilution1:1'000,ThermoScientificProductNo.A-

21052).

20

ImageswereacquiredwithaDVC360FXmonochromecamera(LeicaMicrosystems)

mountedonaDMI6000Bmicroscope(LeicaMicrosystems).Downstreamanalysisand

deconvolutionwasdoneusingtheLeicaApplicationSuiteXsoftware(Leica

Microsystems).

ThepolyclonalTbLOK1rabbitantiserausedforimmunoblotswasproduced

commerciallybyEurogentec.Theantigen,ahis-taggedversionofTbLOK1,was

expressedinE.coli.SubsequentlyTbLOK1-containinginclusionbodieswereisolated

andtheproteinwasfurtherpurifiedbySDS-PAGE.ThebandcorrespondingtoTbLOK1

wascutoutandusedforimmunization.ForimmunoblotsderivedfromBNgelsblotting,

apreviouslydescribedTbLOK1antibodywasused(Povelonesetal.,2013).

Acknowledgments

WethankProf.AdrianHehl,UniversityofZurichandProf.RobJensen,JohnHopkins

UniversitySchoolofMedicineforprovidinguswithantibodies.ResearchinthelabofA.

Schneiderwassupportedbygrant138355andinpartbytheNCCR"RNA&Disease"

bothfundedbytheSwissNationalScienceFoundation.

Authorcontributions

S.D.andJ.M.carriedoutmostoftheexperimentsandanalyzedthedata.S.K.discovered

thatTbLOK1migratesinahighmolecularweightcomplexonBN-gels.A.H.performed

thecomplementationexperiments.A.S.analyzedthedataandwrotethepaper.

21

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24

Figurelegends

Fig.1.TbLOK1isessentialinacelllinethatdoesnotdependonmitochondrial

DNA.Growthcurvesofuninduced(-Tet)andinduced(+Tet)bloodstreamform

TbLOK1-RNAicelllines.Toppanel:bloodstreamformTbLOK1-RNAicellline.Solidand

brokenlinesdepicttwoindependentexperiments.Bottompanel:TbLOK1-RNAicellline

basedontheF1γL262PbloodstreamstrainthatcangrowintheabsenceofthekDNA.

Meanandstandarddeviationoftriplicateexperimentsareindicated.InsetWestern

blotsshowthesuccessfuldownregulationofTbLOK1aftertwodaysofinduction.Ef1ais

usedasloadingcontrol.Thecrossindicatesthatafterthreedaysofinductionnoliving

cellscouldbedetected.

Fig.2.AblationofTbLOK1interfereswithmitochondrialproteinimport.A.Upper

panel:InvivoanalysisofmitochondrialproteinimportduringablationofTbLOK1.

Immunoblotsofwholecellextractsshowingthesteadystatelevelsofthemitochondrial

proteinsREAP1,IF2,CoxIV,VDACandTbLOK1intheprocyclicTbLOK1-RNAicellline.

CytosolicEf1aservesasaloadingcontrol.Timeofinductionindays(days+Tet)is

indicatedatthetop.Blacktriangleindicatestheonsetofthegrowtharrest.OM,outer

membrane;IM,innermembrane;Cyt,cytosol.Precursor(p)andimportedmature

proteins(m)areindicated.Lowerpanel:quantificationofimmunoblotsoffour

independentexperimentsdemonstratingtheTbLOK1-dependentreductionofthe

steadystatelevelsoftheindicatedmitochondrialproteins,relativetoEf1a.Standard

errorsareindicated.B.Invitroimportofthe35S-methionine-labeledprecursorproteins

LDH-DHFRandTAOintomitochondriaisolatedfromtheprocyclicTbLOK1-RNAicell

linegrownintheabsenceorthepresence(1.5days)oftetracycline.IN,input.Precursor

25

(p)andimportedmatureproteins(m)areindicated.Thesampleswherethemembrane

potential(ψ)wasinactivatedservedasnegativecontrols.SegmentsoftheCoomassie-

stainedgelsthatdonotcoincidewiththegelscontainingthelabeledproteinsareshown

asloadingcontrols.

Fig.3.TbLOK1isasubunitoftheATOMcomplex.A. BN-PAGE immunoblots of

mitochondrial membrane extracts from procyclic wildtype cells were probed with antisera

against all six ATOM subunits and TbLOK1. Molecular weight markers (kDa) are indicated.

B. Reciprocal Co-IPs of C-terminally tagged ATOM40 and N- as well as C-terminally tagged

TbLOK1. Mitochondrial membrane extracts of procyclic cell lines co-expressing C-

terminally HA-tagged ATOM40 and N- or C-terminally Myc-tagged TbLOK1, respectively,

were subjected to Co-IPs using anti-HA or anti-Myc antisera, respectively. Immunoblots

containing 5% input (IN), 100% eluate (IP) and 5% flow through fractions (FT) were probed

for the presence of HA-tagged ATOM40 and the Myc-tagged TbLOK1, respectively. As a

control the panel stained for Myc-tagged TbLOK1 was reprobed for the abundant

mitochondrial OM protein VDAC. C. Mitochondrial membrane extracts of a procyclic cell

line expressing N-terminally Myc-tagged TbLOK1 was subjected to IP using an anti-Myc

antiserum. The resulting fractions were probed for tagged TbLOK1, for all ATOM subunits

and for VDAC which serves as a control. D. Mitochondrial membrane extracts of a procyclic

cell line expressing C-terminally Myc-tagged ATOM12 was subjected to IP using an anti-

Myc antiserum. The resulting fractions were probed for native untagged TbLOK1 and

ATOM40. VDAC served as a control.

Fig.4.AblationofTbLOK1affectsATOMsubunitsandtheATOMcomplex.Top

panels:mitochondrialmembraneextractsoftheuninduced(-Tet)andinduced(+Tet)

procyclicTbLOK1-RNAicelllinewereseparatedbySDS-PAGEandtheresulting

immunoblotswereprobedusingantiseraagainstTbLOK1andtheindicatedATOM

26

subunits.Bottompanels:thesameextractswereseparatedbyBN-PAGEandthe

resultingimmunoblotswereanalyzedasinthetoppanel.Theprecentageofthe

remainingcomplexafterRNAiisindicatedforeachcellline.

Fig.5.AblationofATOMsubunitsaffectsTbLOK1.Toppanels:mitochondrial

membraneextractsoftheindicateduninduced(-Tet)andinduced(+Tet)procyclicRNAi

celllineswereseparatedbySDS-PAGEandtheresultingimmunoblotswereprobed

usingtheTbLOK1antiserum.Bottompanels:thesameextractswereseparatedbyBN-

PAGEandtheresultingimmunoblotswereanalyzedasinthecorrespondingpanelon

thetop.Adetailedcharacterizationofthecelllinesallowinginducibleablationofeach

individualATOMsubunitincludinggrowthcurvesandimmunoblotshasbeenpublished

before(Manietal.,2015).

27

0 1 2 3104

105

106

107

108

109

days

cum

ulat

ive

cell

num

ber

(cel

ls/m

l)

combined cSD1.2 curves

A BSF

-Tet

+Tet

0 1 2 3 4 5 6104105106107108109

101010111012

days

cum

ulat

ive

cell

num

ber

(cel

ls/m

l)

BSF F1aL262P

+Tet

-Tet

B

Figure 1

-

EF1a

+ TbLOK1Tet

EF1a

- + TbLOK1Tet

28

A

OM

Matrix

Ef1a

VDAC

REAP1

IF2

TbLOK1

IM CoxIVpm

pm

0 1 2 3 4 5days +Tet

Figure 2

Cyt

REAP1 IF2Cox

IVVDAC

0.0

0.5

1.0

1.5

rela

tive

prot

ein

leve

l [%

]

VDACCox

IVIF2

REAP1

rela

tive

prot

ein

leve

l

IN 3 9 27 3 9 27 2727 min

-+ + + -+ + + s

LDH-DHFR

TAO

pm

-Tet +Tet

35S-Met

Coom.

pm

35S-Met

Coom.

B

29

ATOM

40

ATOM

69AT

OM46

ATOM

14AT

OM11

ATOM

12

TbLO

K1

669

440

232

140

kDa

A

B bait ATOM40-HA

Myc-TbLOK1 TbLOK1-Myc

HAVDACMyc

bait Myc-tagged TbLOK1

HA

VDACMyc

TbLOK1-MycMyc-TbLOK1

FTIPIN FTIPIN

FTIPINFTIPIN

ATOM14

ATOM40

ATOM46

ATOM69

VDAC

ATOM11

ATOM12

Myc

FTIPINbait Myc-TbLOK1C

bait ATOM12-MycD

TbLOK1

Myc

ATOM40

VDAC

IN IP FT

Figure 3

30

669

440

232

140

TbLOK1 ATOM40 ATOM14 ATOM11 ATOM12 ATOM46 ATOM69

- + - + - + - + - + - + - +TbLOK1 RNAi

Tet (1.5d)

kDa

Figure 4

BN-PAGE

SDS-PAGE

100 4 100 62 100 2910018 100 34 100 28 100 48 %

ATOM40 ATOM69ATOM14 ATOM12ATOM11 ATOM46

TbLOK1

669

440

232

ATOM46/69 TbLOK1

RNAi cell line

kDa

TbLOK1BN-PAGE

SDS-PAGE

Figure 5

- + - +- + + - - + - + - + - +Tet