trypanosoma bruceiwith partially overlapping substrate specificities (perry et al., 2008, mani et...
TRANSCRIPT
1
TbLOK1/ATOM19isanovelsubunitofthenon-canonical
mitochondrialoutermembraneproteintranslocaseof
Trypanosomabrucei
SilviaDesy1),JanMani1),AnkeHarsman1),SandroKäser1)andAndréSchneider1)*
1)DepartmentofChemistryandBiochemistry,UniversityofBern,Freiestrasse3,
CH-3012Bern,Switzerland
*Towhomcorrespondenceshouldbeaddressed:
AndréSchneider;Tel:+41316314253;E-mail:[email protected]
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
References
Adl,S.M.,A.G.Simpson,M.A.Farmer,R.A.Andersen,O.R.Anderson,J.R.Barta,S.S.Bowser,G.Brugerolle,R.A.Fensome,S.Fredericq,T.Y.James,S.Karpov,P.Kugrens,J.Krug,C.E.Lane,L.A.Lewis,J.Lodge,D.H.Lynn,D.G.Mann,R.N.McCourt,L.Mendoza,O.Moestrup,S.E.Mozley-Standridge,T.A.Nerad,C.A.Shearer,A.V.Smirnov,F.W.Spiegel&M.F.Taylor,(2005)Thenewhigherlevelclassificationofeukaryoteswithemphasisonthetaxonomyofprotists.J.Eukaryot.Microbiol52:399-451.
Bochud-Allemann,N.&A.Schneider,(2002)MitochondrialsubstratelevelphosphorylationisessentialforgrowthofprocyclicTrypanosomabrucei.J.Biol.Chem.277:32849-32854.
Burki,F.,(2014)Theeukaryotictreeoflifefromaglobalphylogenomicperspective.ColdSpringHarbPerspectBiol6:a016147.
Cavalier-Smith,T.,(2010)KingdomsProtozoaandChromistaandtheeozoanrootoftheeukaryotictree.Biol.Lett.6:342-345.
Chacinska,A.,C.M.Koehler,D.Milenkovic,T.Lithgow&N.Pfanner,(2009)Importingmitochondrialproteins:machineriesandmechanisms.Cell138:628-644.
Cristodero,M.,T.Seebeck&A.Schneider,(2010)MitochondrialtranslationisessentialinbloodstreamformsofTrypanosomabrucei.Mol.Microbiol.78:757-769.
Dean,S.,M.K.Gould,C.E.Dewar&A.C.Schnaufer,(2013)SinglepointmutationsinATPsynthasecompensateformitochondrialgenomelossintrypanosomes.Proc.Natl.Acad.Sci.USA110:14741-14746.
Friedman,J.R.&J.Nunnari,(2014)Mitochondrialformandfunction.Nature505:335-343.
Gray,M.W.,(2012)Mitochondrialevolution.ColdSpringHarb.Perspect.Biol.4:a011403.Hamilton,V.,U.K.Singha,J.T.Smith,E.Weems&M.Chaudhuri,(2014)Trypanosome
alternativeoxidasepossessesbothanN-terminalandinternalmitochondrialtargetingsignal.EukaryotCell13:539-547.
Harsman,A.,M.Niemann,M.Pusnik,O.Schmidt,B.M.Burmann,S.Hiller,C.Meisinger,A.Schneider&R.Wagner,(2012)Bacterialoriginofamitochondrialoutermembraneproteintranslocase:Newperspectivesfromcomparativesinglechannelelectrophysiology.J.Biol.Chem.287:31437-31445.
Hauser,R.,M.Pypaert,T.Häusler,E.K.Horn&A.Schneider,(1996)InvitroimportofproteinsintomitochondriaofTrypanosomabruceiandLeishmaniatarentolae.J.CellSci.109:517-523.
He,D.,O.Fiz-Palacios,C.J.Fu,J.Fehling,C.C.Tsai&S.L.Baldauf,(2014)Analternativerootfortheeukaryotetreeoflife.CurrBiol24:465-470.
Hewitt,V.,F.Alcock&T.Lithgow,(2011)Minormodificationsandmajoradaptations:theevolutionofmolecularmachinesdrivingmitochondrialproteinimport.Biochim.Biophys.Acta.1808:947-954.
Lithgow,T.&A.Schneider,(2010)Evolutionofmacromolecularimportpathwaysinmitochondria,hydrogenosomesandmitosomes.Philos.Trans.R.Soc.Lond.BBiol.Sci.365:799-817.
Maćasev,D.,J.Whelan,E.Newbigin,M.C.Silva-Filho,T.D.Mulhern&T.Lithgow,(2004)Tom22',an8-kDatrans-sitereceptorinplantsandprotozoans,isaconservedfeatureoftheTOMcomplexthatappearedearlyintheevolutionofeukaryotes.Mol.Biol.Evol.21:1557-1564.
Mani,J.,C.Meisinger&A.Schneider,(2015)PeepingatTOMs-DiverseEntryGatestoMitochondriaProvideInsightsintotheEvolutionofEukaryotes.MolBiolEvol.33:337–351.
22
Mani,J.,S.Desy,M.Niemann,A.Chanfon,S.Oeljeklaus,M.Pusnik,O.Schmidt,C.Gerbeth,C.Meisinger,B.Warscheid&A.Schneider,(2016)NovelmitochondrialproteinimportreceptorsinKinetoplastidsrevealconvergentevolutionoverlargephylogeneticdistances.NatureCommun.6:6646.
McCulloch,R.,E.Vassella,P.Burton,M.Boshart&J.D.Barry,(2004)TransformationofmonomorphicandpleomorphicTrypanosomabrucei.MethodsMol.Biol.262:53-86
Perry,A.J.,J.M.Hulett,V.A.Likić,T.Lithgow&P.R.Gooley,(2006)Convergentevolutionofreceptorsforproteinimportintomitochondria.Curr.Biol.16:221-229.
Perry,A.J.,K.A.Rimmer,H.D.Mertens,R.F.Waller,T.D.Mulhern,T.Lithgow&P.R.Gooley,(2008)Structure,topologyandfunctionofthetranslocaseoftheoutermembraneofmitochondria.PlantPhysiolBiochem46:265-274.
Povelones,M.L.,C.Tiengwe,E.Gluenz,K.Gull,P.T.Englund&R.E.Jensen,(2013)Mitochondrialshapeandfunctionintrypanosomesrequirestheoutermembraneprotein,TbLok1.Mol.Microbiol.87:713-729.
Pusnik,M.,F.Charrière,P.Mäser,R.F.Waller,M.J.Dagley,T.Lithgow&A.Schneider,(2009)ThesinglemitochondrialporinofTrypanosomabruceiisthemainmetabolitetransporterintheoutermitochondrialmembrane.Mol.Biol.Evol.26:671-680.
Pusnik,M.,J.Mani,O.Schmid,M.Niemann,S.Oeljeklaus,F.Schnarwiler,B.Warscheid,T.Lithgow,C.Meisinger&A.Schneider,(2012)Anessentialnovelcomponentofthenon-canonicalmitochondrialoutermembraneproteinimportsystemoftrypanosomatids.Mol.Biol.Cell23:3420-3428.
Pusnik,M.,O.Schmidt,A.J.Perry,S.Oeljeklaus,M.Niemann,B.Warscheid,T.Lithgow,C.Meisinger&A.Schneider,(2011)Mitochondrialpreproteintranslocaseoftrypanosomatidshasabacterialorigin.Curr.Biol.21:1738-1743.
Schmidt,O.,N.Pfanner&C.Meisinger,(2010)Mitochondrialproteinimport:fromproteomicstofunctionalmechanisms.Nat.Rev.Mol.Cell.Biol.11:655-667.
Schnarwiler,F.,M.Niemann,N.Doiron,A.Harsman,S.Käser,J.Mani,A.Chanfon,C.E.Dewar,S.Oeljeklaus,C.B.Jackson,M.Pusnik,O.Schmidt,C.Meisinger,S.Hiller,B.Warscheid,A.C.Schnaufer,T.Ochsenreiter&A.Schneider,(2014)TrypanosomalTAC40constitutesanovelsubclassofmitochondrialβ-barrelproteinsspecializedinmitochondrialgenomeinheritance.ProcNatlAcadSciUSA111:7624-7629.
Schneider,A.,N.Bouzaidi-Tiali,A.-L.Chanez&L.Bulliard,(2007a)ATPproductioninisolatedmitochondriaofprocyclicTrypanosomabrucei.MethodsMol.Biol.372:379-387.
Schneider,A.,F.Charrière,M.Pusnik&E.K.Horn,(2007b)IsolationofmitochondriafromprocyclicTrypanosomabrucei.MethodsMol.Biol.372:67-80.
Schulz,C.,A.Schendzielorz&P.Rehling,(2015)Unlockingthepresequenceimportpathway.TrendsCellBiol25:265-275.
Sharma,S.,U.K.Singha&M.Chaudhuri,(2010)RoleofTob55onmitochondrialproteinbiogenesisinTrypanosomabrucei.Mol.Biochem.Parasitol.174:89-100.
Waizenegger,T.,S.Schmitt,J.Zivkovic,W.Neupert&D.Rapaport,(2005)Mim1,aproteinrequiredfortheassemblyoftheTOMcomplexofmitochondria.EMBORep.6:57-62.
Williams,T.A.,P.G.Foster,C.J.Cox&T.M.Embley,(2013)Anarchaealoriginofeukaryotessupportsonlytwoprimarydomainsoflife.Nature504:231-236.
Wirtz,E.,M.Hoek&G.A.M.Cross,(1998)RegulatedprocessivetranscriptionofchromatinbyT7RNApolymeraseinTrypanosomabrucei.NucleicAcidsRes.26:4626-4634.
Wirtz,E.,S.Leal,C.Ochatt&G.A.Cross,(1999)Atightlyregulatedinducibleexpressionsystemforconditionalgeneknock-outsanddominant-negativegeneticsinTrypanosomabrucei.Mol.Biochem.Parasitol.99:89-101.
23
Zarsky,V.,J.Tachezy&P.Dolezal,(2012)Tom40islikelycommontoallmitochondria.CurrBiol.22:R479-R481.
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