Subcomplexes of Ancestral Respiratory Complex I Subunits Rapidly Turn Over in Vivo as ...

12 Pages·2013·1.66 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.288,NO.8,pp.5707–5717,February22,2013
©2013byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Subcomplexes of Ancestral Respiratory Complex I Subunits
Rapidly Turn Over in Vivo as Productive Assembly
Intermediates in Arabidopsis*□S
Receivedforpublication,October31,2012,andinrevisedform,December8,2012Published,JBCPapersinPress,December27,2012,DOI10.1074/jbc.M112.432070
LeiLi‡§1,ClarkJ.Nelson‡§,ChrisCarrie‡,RyanM.R.Gawryluk¶,CorySolheim‡§,MichaelW.Gray¶,JamesWhelan§,
andA.HarveyMillar‡§2
Fromthe‡AustralianResearchCouncilCentreofExcellenceinPlantEnergyBiologyandthe§CentreforComparativeAnalysisof
BiomolecularNetworks,M316,UniversityofWesternAustralia,35StirlingHighway,Crawley,WA6009,WesternAustralia,Australia
andthe¶CentreforComparativeGenomicsandEvolutionaryBioinformatics,DepartmentofBiochemistryandMolecularBiology,
DalhousieUniversity,Halifax,NovaScotiaB3H4R2,Canada
Background:PlantcomplexIcontains(cid:1)-CAsubunitswhoseroleisunclear.
Results:15NlabelingandimportshowCIassemblyviaarapidlyturningover(cid:1)-CAsubcomplex.
Conclusion:(cid:1)-CAsformanancientpathwayforCIassembly.
Significance:TheassemblypathwayofcomplexIinplantsisdifferentfromthatinanimalsandmorecloselyrepresentsthe
ancestralenzyme.
SubcomplexesofmitochondrialrespiratorycomplexI(CI;EC dative phosphorylation assembly and is also the major entry
1.6.5.3)areshowntoturnoverinvivo,andweproposearolein pointofelectronsfromtheoxidationofNADHintotherespi-
anancestralassemblypathway.ByprogressivelylabelingArabi- ratory chain. CI contains 14 core subunits that represent the
dopsis cell cultures with 15N and isolating mitochondria, we minimalactiveformandthatareconservedfromprokaryoticto
have identified CI subcomplexes through differences in 15N eukaryotic organisms. In the mitochondria of eukaryotes, CI
incorporation into their protein subunits. The 200-kDa sub- containsanadditional31–38accessorysubunits,anumberof
complex, containing the ancestral (cid:1)-carbonic anhydrase which differ among organisms (1–4). Single-particle electron
((cid:1)-CA),(cid:1)-carbonicanhydrase-like,and20.9-kDasubunits,had microscope imaging analysis of isolated CI from Arabidopsis
asignificantlyhigherturnoverratethanintactCIorCI(cid:2)CIII . and Polytomella identified a specific matrix-exposed domain
2
InvitroimportofprecursorsfortheseCIsubunitsdemonstrated formed by (cid:1)-carbonic anhydrase ((cid:1)-CA) subunits that are
rapidgenerationofsubcomplexesandrevealedthattheirspe- attachedtothemembranearmofmitochondrialCIbytheinte-
cificabundancevariedwhendifferentancestralsubunitswere gral membrane protein CA2 (5). In Arabidopsis, this protein
imported.Timecoursestudiesofprecursorimportshowedthe domain contains at least three different (cid:1)-CA proteins, CA1
furtherassemblyofthesesubcomplexesintoCIandCI(cid:2)CIII , (At1g19580),CA2(At1g47260),andCA3(At5g66510),having
2
indicatingthatthesubcomplexesareproductiveintermediates conserved active site regions, and two less well conserved
ofassembly.Thestrongtransientincorporationofnewsubunits (cid:1)-CA-likeproteins,CAL1(At5g66510)andCAL2(At3g48680),
into the 200-kDa subcomplex in a (cid:1)-CA mutant is consistent which contain alterations to the primary structure of typical
with this subcomplex being a key initiator of CI assembly in (cid:1)-CAs (6). Links between these subunits and photosynthetic
plants.Thisevidencealongsidethepatternofcoincidentoccur- functionhavebeenpostulated(7),aroleinphotomorphogen-
rence of genes encoding these particular proteins broadly in esisinplantshasbeenshownbygeneknock-outstudies(8),and
eukaryotes,exceptforopisthokonts,providesaframeworkfor overexpression leads to male sterility in plants (9), but a CA
theevolutionaryconservationoftheseaccessorysubunitsand activityfromtheseproteinshasnotbeenexperimentallycon-
evidenceoftheirfunctioninancestralCIassembly. firmed. Although at first these subunits were thought to be
specifictoCIfromphotosyntheticorganisms(10,11),evidence
hasemergedof(cid:1)-CAproteinsinmitochondriaandinisolated
NADH-ubiquinone oxidoreductase (complex I (CI),3 EC CI from a range of other eukaryotic lineages, including slime
1.6.5.3)isthelargest((cid:1)1000kDa)proteincomplexoftheoxi- moldsandamoebaethatlackphotosynthesis(12).Broaderphy-
logeneticstudieshavealsofoundhomologybetweenotherCI
accessorysubunitsoncethoughttobespecies-orlineage-spe-
*ThisworkwassupportedbyAustralianResearchCouncil(ARC)Centreof cific(4).
ExcellenceinPlantEnergyBiologyGrantCE0561495(toA.H.M.andJ.W.). GiventhedivergenceofCIcompositionamongeukaryotes,
□S ThisarticlecontainssupplementalDataSets1–4.
both the assembly pathway and the biological importance of
1SupportedbyScholarshipInternationalResearchFees,aUniversityInterna-
tionalStipend(UIS),andaTopUpScholarshipforUIS. accessorysubunitsinassemblyintermediatescouldbeconsid-
2An ARC Australian Future Fellow (Grant FT110100242). To whom corre-
spondence should be addressed. Tel.: [phone]; Fax: 61-8-
64884401;E-mail:[email protected]. roxymethyl)ethyl]glycine; TES, 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)eth-
3Theabbreviationsusedare:CI,complexI;CA,carbonicanhydrase;CAL,car- yl)amino]ethanesulfonic acid; ROS, reactive oxygen species; NA and H
bonicanhydrase-like;BN,bluenative;Tricine,N-[2-hydroxy-1,1-bis(hyd- peptide,naturalabundanceandheavylabeledpeptide,respectively.
FEBRUARY22,2013•VOLUME288•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5707
This is an Open Access article under the CC BY license.
AssemblyofMitochondrialComplexIinArabidopsis
erablydifferentamongmajortaxonomicgroupsofeukaryotes. washthe7-day-oldcellculturethreetimesbeforeitwastrans-
In CI mutants of Neurospora crassa, analysis of CI assembly ferred to 15N growth medium. Ammonium-15N nitrate-15N
using radiolabeling pulse-chase has revealed that the matrix (1.69g/liter)andpotassiumnitrate-15N(1.92g/liter)(98%15N;
andmembranearmsassembleindependentlyviaseparatepath- Sigma) were added to growth medium without nitrogen to
ways (13–17). The membrane subunit 20.9, which is absent make the 15N growth medium. Cells labeled for 24, 120, and
frommammalianCI,wasidentifiedsomeyearsagoasafactor 168hwith15Nwerecollectedontofilterpaperusingvacuum
essentialforassemblyofthemembranearminfungi(18).Based filtration.Threebiologicalreplicatesforeachtimepointwere
onacombinationofradiolabelingpulse-chaseexperiments,in performed.
vitromitochondrialimportandmonitoringoftaggedCIsub- Protoplast Preparation and Mitochondrial Isolation from
unitsinassembly-disturbedsystems,severalCIassemblymod- CellCulture—Freshcells(20–25g)foreachsingletimepoint
els have been proposed for human cells (14, 19, 20). In this replicateweredispersedindigestionsolution(0.4%(w/v)Ono-
process, mammalian assembly factors not conserved in other zuka cellulase RS, 0.05% (w/v) Kyowa pectolyase Y-23, 0.4 M
species have been identified (13, 14, 19). Subcomplexes con- mannitol,0.7g/literMES,pH5.7)andgentlyshakeninthedark
tainingCA2havebeenreportedusingantibodies(11)indeter- for3hatroomtemperature.Thesolutionwascentrifugedat
gentdisassemblystudiesofCIinplants(21),inArabidopsisCI 800 (cid:2) g for 10 min. The supernatant was decanted, and the
subunitknock-outmutants(22),andinwildtypeArabidopsis protoplast pellet resuspended in digestion solution without
mitochondrialproteomesinsaturationmappingoftwo-dimen- enzymes by swirling and repeated rounds of centrifugation.
sionalbluenative(BN)gels(23).KnockoutsofCA2andCA3do Washed protoplasts were then resuspended in extraction
notyieldaclearphenotypeinArabidopsis(5,11),whichmight buffer(0.4Msucrose,50mMTris,3mMEDTA,and0.1%BSA
be a consequence of functional redundancy in activities (w/v), pH 7.5) and homogenized 5–9 times with a Dounce
betweentheseproteins.However,suspensioncellculturelines 0.12-mm homogenizer. Extraction buffer containing broken
derivedfromthemutantca2showedreducedgrowthratesand protoplasts was then centrifuged at 2500 (cid:2) g for 5 min, the
lowerCIabundance(11),whichindicatesthat(cid:1)-CAsubunits supernatantwasrecovered,andthenthissupernatantwassub-
could be important for CI assembly or structural stability in jectedtocentrifugationat20,000(cid:2)gfor20min.Theresultant
plants. pelletwasresuspendedin0.5–1mlofmannitolwashbuffer(0.3
However, reports of studies to date have not been able to Mmannitol,10mMTES,and0.1%(w/v)BSA,pH7.5)usinga
distinguish between productive and non-productive interme- paintbrush,andthesuspensionwaspipettedontotwo35-ml
diates,anessentialdistinctioninbuildingarigorousCIassem- Percollstepgradientscomprising40,25,and18%(w/v)Percoll
bly model. Specifically, we needed to determine if subcom- in mannitol wash buffer. The gradients were centrifuged at
plexeswerepresentinvivooriftheywereformedinvitrodueto 40,000(cid:2)gfor60minanddeceleratedwithslowbraking.Mito-
detergentsolubilizationorelectrophoresis;iftheywereincor- chondria,whichformayellowbandatthe40and25%interface,
poratedintothesmall/earlyassemblyintermediates;andifthey were collected and diluted in sucrose wash buffer (0.3 M
wereproductiveintermediatesthatgoontoformCIorwere sucrose,10mMTES,pH7.5).Dilutedmitochondriawerecen-
simply non-productive subcomplexes that accumulated in trifuged at 24,000 (cid:2) g for 15 min and decelerated with slow
mitochondria. We have combined a progressive 15N labeling braking.Pelletwascollectedanddilutedagaininwashbuffer.
strategyinvivo(24)withBNpolyacrylamidegelelectrophoresis Washedmitochondriaweredilutedin0.5–1mlofsucrosewash
(BN-PAGE)andinvitro[35S]Metimportandassemblyassays buffer. Isolated mitochondria were quantified with the Brad-
toaddresstheseissues.Thisapproachhasallowedanalysisof fordassayandwerethenusedforimportassaysormadeinto
the incorporation rate of subunits into subcomplexes, 1-mgproteinaliquotsandrunonBNgelsorstoredat(cid:3)80°C
mature CI, and supercomplexes of plant mitochondria to forfurtherexperiments.
investigate their role in assembly and to provide key evi- Arabidopsis Plant Growth and Mitochondrial Preparation—
dence for the existence of a productive CI subcomplex in Wild typeArabidopsis (Col-0) orca2 mutant seeds ((cid:1)30 (cid:2)g)
vivo in plants. The subunits of the identified assembly sub- (11)werewashedin70%(v/v)ethanolfor2min,andthenthe
complex, lost in animal lineages, are widely conserved in seeds were placed in a sterilization solution (5% (v/v) bleach,
other eukaryotic lineages, providing evidence for their role 0.1%(v/v)Tween20)for15minwithperiodicalshaking.Seeds
asancestralaccessorysubunits. were then washed in sterile water 5 times before being dis-
pensedintoa250-mlplasticvesselcontaining80mlofgrowth
EXPERIMENTALPROCEDURES
medium(half-strengthMurashigeandSkoogmediumwithout
ArabidopsisCellCultureGrowthand15NLabeling—Arabi- vitamins, half-strength Gamborg B5 vitamin solution, 5 mM
dopsis cell suspension was cultured in growth medium (1(cid:2) MES,2.5%(w/v)sucrose,pH5.8).Arabidopsisseedlingswere
Murashige and Skoog medium without vitamin, 3% sucrose grownundera16/8-hlight/darkperiodwithlightintensityof
(w/v),0.5mg/liternaphthaleneaceticacid,0.05mg/literkine- 100–125(cid:2)molm(cid:3)2s(cid:3)1at22°Cfor2weeks.Tenvesselsofwild
tin,pH5.8)at22°Cundercontinuouslightconditionswitha typeorca2mutantseedlingswerecollectedformitochondrial
lightintensityof90(cid:2)molm(cid:3)2s(cid:3)1andwithorbitalshakingat preparations. Seedlings (50–100 g) were homogenized with
120 rpm. Cultures were maintained in 250-ml Erlenmeyer mortarandpestle,andmitochondriawereisolatedasdescribed
flasksbytheinoculationof20–25mlof7-day-oldcellsinto100 previously (25). The protein content of freshly isolated mito-
mlofnewmedium.Thesamegrowthmediumwithoutnitrogen chondriawasquantifiedwithaBradfordassayprotocolbefore
(i.e. no ammonium nitrate or potassium nitrate) was used to beingusedforimportexperiments.
5708 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER8•FEBRUARY22,2013
AssemblyofMitochondrialComplexIinArabidopsis
Two-dimensional BN/SDS-PAGE—Mitochondrial aliquots ulations, MALDI-TOF/TOF MS data were exported to
werewashedwith2mlofsucrosewashbuffer(0.3Msucrose,10 mzXML files (CompassXport version 3.0 (Bruker)) and were
mMTES,pH7.5)andthencentrifugedat14,300(cid:2)gfor10min. subsequently converted to text files using the Proteome-
Pellets were dissolved in 100 (cid:2)l of digitonin solubilization Commons.orgIOFrameworkversion6.21.Textfileswerethen
buffer(30mMHEPES,pH7.4,150mMpotassiumacetate,10% parsedusingascriptwritteninMathematicaversion7.0(Wol-
(v/v)glycerol,50mg/mldigitonin).Sampleswerecentrifugedat fram Research) for further analysis. Isotopic envelopes were
18,000 (cid:2) g for 30 min after incubation for 20 min on ice to fittedusingatwo-populationmodelconsistingofnaturalabun-
remove insoluble material and were subsequently supple- danceand15N-labeledfractionswiththeopensourceprogram
mentedwith5(cid:2)lofServaBlueG250(5%(w/v)CoomassieBlue Isodist(29)aswehavedescribedpreviously(24).
in750mMaminocaproicacid).CoomassieBlue-treatedprotein Proteindegradationrates(K )werecalculatedasdescribed
D
samplesweredirectlyloadedontoBNgels.Two-dimensional previously(24).Thetotalproteinabundanceinthecellculture
BN/SDS-PAGE was carried out as described previously (26). atdifferenttimepointswasquantifiedusingImageJofstained
Gradientgels(4.5–16%(w/v)acrylamide)wereusedforthefirst protein gels and by Amido Black analysis as described previ-
BN dimension. Second dimension Tricine-SDS-polyacryl- ously(24).AnaverageK amongsubunitswithinCI(cid:4)III ,CI,
D 2
amidegels,composedofa4%stackinggelanda12%separation and 650- and 200-kDa subcomplexes was separately deter-
gel,wereusedforseparationofproteincomplexsubunits.Elec- minedat24,120,and168h.Half-livesofCI(cid:4)III ,CI,and650-
2
trophoresis parameters and Coomassie staining of BN and and200-kDasubcomplexesat24,120,and168hweredeter-
BN/SDS-PAGEwerecarriedoutasdescribedpreviously(27). minedfromK ashalf-life(cid:7)ln2/K .ThefinalK valuesand
D D D
In-gel Digestion and MALDI-TOF/TOF Analysis—Protein half-livesforCI(cid:4)III ,CI,and650-and200-kDasubcomplexes
2
spotsinCI(cid:4)CIII ,CI,andCIsubcomplexlanesfromBNgels weredeterminedbyaveragingthevaluesobtainedat24,120,
2
were excised and subjected to in-gel digestion with trypsin and168h.
usingpreviouslydescribedmethods(28)butusingonlyaquar- Import in Cell Culture and Plant Mitochondria—The full
terofthetrypsinconcentrationinordertodecreasetheeffectof cDNAs of Arabidopsis thaliana CA1 (At1g19580), CA2
trypsin autolysis peaks on MS data interpretation. Peptides (At1g472660), CA3 (At5g66510), 20.9-(At4g16450), CAL1
were analyzed with an UltraFlex III MALDI-TOF/TOF mass (At5g63510),CAL2(At3g48680),andATPE(At1g51650)were
spectrometer(BrukerDaltonics).In-geldigestedpeptideswere clonedintopDest14(Invitrogen)usinggatewaycloningtech-
reconstitutedwith5(cid:2)lofasolutioncontaining5%(v/v)aceto- niques(Invitrogen)forinvitrotranscriptionandtranslationas
nitrile,0.1%(v/v)trifluoroaceticacid(TFA)indouble-distilled describedpreviously(30).Weusedimportsofthe(cid:4)subunitof
H O. Two (cid:2)l of each sample was spotted onto a MTP 384 ATPsynthase(At1g51650),whichdoesnotreadilygetincorpo-
2
MALDI target plate and mixed with 2 (cid:2)l of spotting matrix ratedintonativecomplexesinplantmitochondria,asacontrol
(90% acetonitrile, 10% saturated (cid:3)-cyano-4-hydroxycinnamic forradiolabeledpatternsintheisolatedmitochondria.Equiva-
acidinTA90(90%acetonitrile,0.01%TFA)).Driedspotswere lentquantitiesofmitochondriaisolatedfromcellculture,wild
overlaidwith10(cid:2)lofcoldwashingbuffer(10mMNH H PO , type,andca2knock-outArabidopsiswereusedinimportreac-
4 2 4
0.1% TFA) and allowed to stand for 10 s before removal by tionsasdescribedpreviously(30).
pipette.Spotswereanalyzedat50–85%laserintensitywithup
RESULTS
to1200shotsforMSanalysisperspot.Ionsbetween700and
4000m/zwereselectedforMS/MSexperimentsusing3%addi- Higher Turnover Rates of Specific Subunits in the 200- and
tional laser power. Masses corresponding to trypsin autolysis 650-kDa CI Subcomplexes—Arabidopsis cells were grown for
products were excluded from analysis. A maximum of 15 24,120,and168hin98%15N-enrichedMSmedium(24),mito-
molecular ions were analyzed by MS/MS from each gel spot. chondrialoxidativephosphorylationcomplexeswereprepared
TandemmassspectrometrydatawereanalyzedusingBiotools andseparatedbyBN-PAGE,andthemajorproteinspotswere
(BrukerDaltonics)andanin-houseArabidopsisdatabasecom- identified by mass spectrometry. These data were consistent
prisingATH1.pep(release9)fromtheArabidopsisInformation with the reported protein complex proteome of plant mito-
ResourceandtheArabidopsismitochondrialandplastidpro- chondria(23).ThisconfirmedseveralreportedCIsubunit-con-
tein sets (33,621 sequences; 13,487,170 residues), using the tainingproteincomplexes,includingCI(cid:4)CIII andthemature
2
Mascotsearchengineversion2.3.02andutilizingerrortoler- CI holoenzyme (10, 21) (Fig. 1). Peptide mass spectrometry
ancesof(cid:5)1.2DaforMSand(cid:5)0.6DaforMS/MS;“MaxMissed analysis revealed 22 different CI-specific subunits in the CI
Cleavages” set to 1; variable modifications of oxidation (Met) (ND1,ND7,ND9,75kDa,51kDa,39kDa,20.9kDa,18kDa,
andcarbamidomethyl(Cys).Onlyproteinmatcheswithmore ASHI,B14,B13,B14.7,B16.6,B17.2,CA2,CA3,CAL1,NDU10,
thantwopeptidesandwithionscoresgreaterthan40wereused NDU12,PDSW,PSST,andTYKY)and23differentsubunitsin
foranalysis(p(cid:6)0.05).Forallspots,the0and24htimepoints theCI(cid:4)CIII (ND1,ND7,ND9,75kDa,51kDa,39kDa,20.9
2
were used for both MS and MS/MS analyses for protein IDs, kDa,18kDa,ASHI,B14,B13,B14.7,B16.6,B17.2,CA2,CA3,
whereas only the MS data were analyzed for 120 and 168 h. CAL1,NDU10,NDU11,NDU12,PDSW,PSST,andTYKY).All
SearchresultswereexportedtoCSVformatforeachspot. ofthesesubunitsareconfirmedsubunitsintheplantCIholoen-
Quantification of Protein Turnover as NA/(NA(cid:4)H) and zyme(3,10,23,31).Interestingly,theCIsubunitCA2wasalso
Protein Degradation as K and Protein Half-life—In order to foundinspot37,whichwasavisiblestainedregioninourBN
D
calculateheavylabelincorporationandtherelativeabundance gels(Fig.1).CIsubunitshavepreviouslybeenobservedat650
ofNA(naturalabundance)andH(heavylabeled)peptidepop- kDa(22),at400–450kDa(3,22,23),andat200kDa(3,22,23)
FEBRUARY22,2013•VOLUME288•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5709
AssemblyofMitochondrialComplexIinArabidopsis
24 h 120 h
CA2 CA2
CI+CIII2 CI+CIII2 ^
CI CI
650 kDa * 650 kDa *
200 kDa * 200 kDa *
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6
NA/(NA+H) NA/(NA+H)
CAL1 CAL1
CI+CIII2 ^ CI+CIII2
CI ^
650 kDa *
650 kDa
*
200 kDa * ^
0.0 0.2 0.4 0.6 0.8 1.0 200 kDa *
NA/(NA+H) 0.0 0.1 0.2 0.3 0.4 0.5 0.6
NA/(NA+H)
20.9 kDa subunit 20.9 kDa subunit
CI+CIII2 CI+CIII2
CI CI
200 kDa * ^ 650 kDa *
0.0 0.2 0.4 0.6 0.8 1.0 200 kDa *
NA/(NA+H)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
FIGURE1.CoomassiestainingofBNandBN/SDS-PAGEseparationsof NA/(NA+H)
mitochondrialmembraneproteinsubunitsandidentificationofcom-
plexIsubunits.PositionsofsubunitsfromCIandCI(cid:4)CIII andthecorre- FIGURE2.IsotopeincorporationratiosofsubunitsinCI-containingcom-
2 plexesafter24and120hof15Nlabeling.Shownisthe15Nincorporation
sponding650-and200-kDasubcomplexesareshown.Ellipsesandnumbers, ratio(NA/(NA(cid:4)H))inpeptidesofCA2,CAL1,and20.9-kDasubunitat24and
identifiedCIsubunits(evidenceprovidedinsupplementalDataSetS1);red 120h.Errorbars,S.E.valuesofbiologicalreplicates(n(cid:7)3orelsemarkedwith
indicatesthepositionofthethreenotedCIsubunitsacrosscomplexes.
acaret).Differentcolorsshowthe200-kDasubcomplex(red),650-kDasub-
complex(darkred),CI(yellow-green),andCI(cid:4)CIII (green).*,statisticallysig-
nificantlowerNA/(NA(cid:4)H)ratiointhesubcompl2exescomparedwithintact
inArabidopsismitochondrialBNgelanalyses.Analysisofgel CI(24hCAL1iscomparedwithCI(cid:4)CIII )(p(cid:6)0.05,ttest).Forcalculations,see
2
regionsatthesenativemolecularmassesandatmassesofthe supplementalDataSetS2.
complex I subunits observed in the CI holoenzyme and
CI(cid:4)CIII supercomplex was then carried out. This revealed for the same proteins were derived from CI and CI(cid:4)CIII .
2 2
thatCIsubunitsCAL1and20.9kDacouldbefoundalongwith These results indicate a significantly higher turnover rate for
CA2at650and200kDa(Fig.1andsupplementalDataSetS1). the200-and650-kDasubcomplexes(p(cid:6)0.05)thanforCIand
A wider range of protein bands was analyzed, but only those CI(cid:4)CIII . These differences were clearly apparent after just
2
including significant identifications of CI subunits are anno- 24hof15Nlabelingandwereveryevidentafter120hoflabeling
tatedinFig.1. (Fig.2).Notably,thedifferencesbetweenCIandCI(cid:4)CIII were
2
High scoring peptides by the Mascot algorithm (p (cid:6) 0.05) notstatisticallysignificantforanyofthethreesubunitsexam-
were further analyzed to determine what proportion of each ined(Fig.2andsupplementalDataSetS2).
peptideinmassspectrawasderivedfromtheNAofnitrogenin TheturnoverratesindicatedbyNA/(NA(cid:4)H)valuesdonot
theoriginalcellcultureandwhatproportionderivedfromthe definethespecificcontributionofsynthesisof15Nproteinand
incorporationof15N(H)intonewproteinsynthesizedafterthe thedegradationofpre-existing14Nproteinstoturnover.How-
growth medium switch. These values (as outlined under ever,dataonthegrowthrateofthecellcultureallowedusto
“ExperimentalProcedures”andinRef.24)wereusedtocalcu- subsequentlycalculatethedegradationrate(K )ofCIandsub-
D
latetheNA/(NA(cid:4)H)ratioforspecificpeptidesfromeachgel complexes and convert this to protein half-life. The averaged
spot.CalculationsofNA/(NA(cid:4)H)forallofthepeptidesofa K valueforthe200-kDasubcomplex(0.42(cid:5)0.04day(cid:3)1)was
D
givenproteinwereaveragedtodetermineafinalNA/(NA(cid:4)H) about10timesfasterthanforCI(cid:4)III (0.04(cid:5)0.01day(cid:3)1)and
2
ratio(cid:5)S.E.Thisvaluewasusedasaproxyforeachprotein’s CI(0.04(cid:5)0.02day(cid:3)1)andabout1.6timesfasterthanforthe
turnoverrate(Fig.2andsupplementalDataSetS2).Subunits, 650-kDasubcomplex(0.26(cid:5)0.07day(cid:3)1).Theaveragedpro-
includingCA2,CAL1,and20.9kDa,hadasignificantlylower teinhalf-lifeforthe200-kDasubcomplex(1.7(cid:5)0.3day)was
NA/(NA(cid:4)H)ratiowhenthepeptideswerederivedfromthe thus about 8% of that of CI(cid:4)III (22.0 (cid:5) 5.7 days) and CI
2
200-and650-kDasubcomplexesthanwhenthesamepeptides (23.9(cid:5)11.9days)and60%ofthatofthe650-kDasubcomplex
5710 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER8•FEBRUARY22,2013
AssemblyofMitochondrialComplexIinArabidopsis
A. CI 120 h C. CI 168 h
20.9 kDa * 20.9 kDa In
PCDAS2W CNADL91 crea
CNADL71 ^ ND1 sin
B13 g
ND9 ND7 T
ND1 u
B13 CA2 rn
B351916 kk.DD6aa * * ^ B351916 kk.DD6aa * * * over R
75 kDa * 75 kDa * ate
0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.05 0.1 0.15 0.2 0.25 0.3
NA/(NA+H) NA/(NA+H)
B. CI+CIII2 120 h D. CI+CIII2 168 h
20.9 kDa * 20.9 kDa
CAL1 CAL1
ND1
ND7 NDU10
CA2 ^ ND7
B13 ND1
ND9
B16.6 * B13 ^
NDU12 * NDU12
39 kDa * B16.6
75 kDa * 39 kDa
18 kDa * 75 kDa *
B17.2 * *
0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.05 0.1 0.15 0.2 0.25 0.3
NA/(NA+H) NA/(NA+H)
E. CI 168 h grouped F. CI+CIII2 168 h grouped
CA domain * * CA domain * * *
* * *
Mito- Mito-
* * *
Encoded * Encoded *
Membrane Membrane
* * * *
Matrix Int * Matrix Int * *
Matrix ** ** ** Matrix ** ** **
0 0.05 0.1 0.15 0.2 0.25 0.3 0 0.05 0.1 0.15 0.2 0.25 0.3
NA/(NA+H) NA/(NA+H)
FIGURE3.IsotopeincorporationratiosforCIsubunitswithinmatureCIandCI(cid:2)CIII after120and168hof15Nincubation.A–D,theNA/(NA(cid:4)H)ratio
foreachsubunitisshownforpeptidesisolatedfromCIandCI(cid:4)CIII following120and2168hofincubationwith15N.Errorbars,S.E.valuesamongbiological
replicates(n(cid:7)3orelsemarkedwithacaret).*,statisticalsignificance2(p(cid:6)0.05)comparedwithCAL1.CIsubunitsaredividedintofourgroupsbasedoncolors.
Red,matrixgroup(18kDa,B17.2,39kDa,51kDa,and75kDa);yellow,membraneandmatrixinteractiongroup(B16.6,B13,PDSW,NDU10,andNDU12);blue,
mitochondrion-encodedgroup(ND1,ND7,andND9);green,(cid:1)-CAdomaingroup(CA1,CA2,CAL1,and20.9kDa).Errorbars,S.E.valuesamongbiological
replicates(n(cid:7)3,orelsemarkedwithacaret).*,statisticalsignificance(Student’sttest,p(cid:6)0.05)comparedwiththeCAL1subunit.EandF,statistical
comparisonofthefourgroupsinCIandCI(cid:4)CIII after168hof15Nincubation.Errorbars,S.D.amongmeasurementsofdifferentsubunitswithinagroup(n(cid:7)
6–11).One-wayanalysisofvarianceandTukey’2sposthocmultiplecomparisonsamongfourgroupswereused;**,p(cid:6)0.01;*,p(cid:6)0.05.Forcalculations,see
supplementalDataSetS3.
(3.0(cid:5)0.7days).ThelowerNA/(NA(cid:4)H)valuesofsubunitsin relatively high NA/(NA(cid:4)H) ratios and thus lower turnover
subcomplexesarethusduetotheirsignificantinvivoturnover rates. The plant 20.9-kDa subunit, which has been found in
duringthecourseofthe7daysoftheexperimentratherthan bothmatrixandmembranemodulesofCI(3,22),doesnothave
justaprocessofdilutionthroughgrowth. adirecthomologinmammalsbutisrelatedtofungalNUXM
Matrix Arm Subunits Have Higher Turnover Rates than subunits and the 13- and 21-kDa CI subunits in other
Membrane-associated and Mitochondrion-encoded CI Sub- eukaryotesandwasthemoststableproteinsubunitstudiedin
units—Toinvestigatewhethersubunitshavedifferentturnover bothCIandCI(cid:4)CIII .
2
rates compared with each other within CI and CI(cid:4)CIII , the Todetermineiftheseobservationsofvarying15Nincorpora-
2
NA/(NA(cid:4)H)ratiosforavarietyofCIsubunitsafter120and tionratesfordifferentmodulesofthecomplexwerestatistically
168hof15Nlabelingwerecompared(Fig.3,A–D).Matrixarm significant, CI subunits were divided into four groups as
subunits,includingthe75-kDa,51-kDa,39-kDa,18-kDa,and detailed in the legend to Fig. 3. One-way analysis of variance
B17.2 subunits, exhibited relatively low NA/(NA(cid:4)H) ratios and Tukey’s post hoc multiple comparisons among the four
andthusrelativelyhigherturnoverratesthanothersubunitsin groupsrevealedthatthematrixgrouphadasignificantlyhigher
bothCIandCI(cid:4)CIII .Thespecificmatrix-exposedarmcom- 15Nincorporationratethaneveryothergroup(p(cid:6)0.01).The
2
ponents,includingCA2,CAL1,and20.9kDa,andmitochon- membrane/matrix interaction group also had a significantly
drion-encoded subunits, including ND1, ND7, and ND9, had higher15Nincorporationratethanthespecific(cid:1)-CAdomain
FEBRUARY22,2013•VOLUME288•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5711
AssemblyofMitochondrialComplexIinArabidopsis
FIGURE4.ImportofCAdomainsubunitsand20.9-kDasubunitintowildtypeArabidopsismitochondria.A,[35S]Met-labeledprecursors(P)wereincu-
batedwithmitochondria(M)for60minwithandwithoutPKtreatmentandwereseparatedbySDS-PAGE.B,[35S]Met-labeledprecursorswereincubatedwith
freshmitochondria,followingwhichproteinsfromPK-treatedmitochondriawereseparatedbyBN-PAGE.RadiolabeledbandsalignedtoaCoomassie-stained
BN-polyacrylamidegelofthesamematerialandthepositionsofcomplexesandsubcomplexesareshown.Molecularmassesofcomplexesarereportedagainst
standardizedmasses(23).*,locationof200–300-kDacomplexIsubcomplexes.Thecontrollaneisanequivalentimportassaywiththe(cid:4)subunitofATPsynthase
(At1g51650),whichdoesnotreadilygetincorporatedintocomplexesandservesasareferenceforradiolabelingpatternsonSDS-polyacrylamidegelimages.
group(p(cid:7)0.04inCI,p(cid:6)0.01inI(cid:4)III )(Fig.3,EandF,and degreesintoCIandCI(cid:4)CIII (Fig.4B).Thisefficientassembly
2 2
supplemental Data Set S3). Notably, there was no difference of labeled subunits might explain why the smaller subcom-
betweentheincorporationrateof15NintotheCIholoenzyme plexes are less defined in CA1 and CA2. Comparison of the
andtheCI(cid:4)CIII supercomplex,indicatingthatthetwoappear radiolabeledimportgelswithCoomassie-stainedgelsshowed
2
tobeinequilibrium. thatthe200–300-kDacomplexes(Fig.4B,*)seenintheimport
(cid:1)-CAs,(cid:1)-CALs,and20.9-kDaSubunitPrecursorProteinsAre experiments were not exactly the same size, when compared
Imported and Assembled into a Variety of Subcomplexes in with the abundant 200-kDa subcomplex comprising CA2,
Mitochondria—Todeterminewhether(cid:1)-CAdomainsubcom- CAL1,and20.9-kDasubunitsthathadbeenobservedtoaccu-
plexesareproducedrapidlyafterimport,weperformedinvitro mulateinFig.1;however,theyweresimilarinsizetothemul-
import assays with [35S]Met-labeled CA1, CA2, CA3, CAL1, tiplesubcomplexescontaining(cid:1)-CAsfoundbyCIdisassembly
CAL2, and 20.9-kDa subunit precursors into isolated Arabi- (3).The(cid:1)-CALand20.9-kDaproteinsdifferedwithrespectto
dopsis cell culture mitochondria. Analysis of autoradiographs thesubcomplexesintowhichtheyprogressed,comparedwith
ofSDS-PAGE-separatedsamplesshowedthatallsubunitswere the(cid:1)-CAproteins.Overall,theseresultsdemonstratedthatthe
importedintomitochondriaduringa60-minincubationperiod CAdomainsubunitswerenotsimplyaddedasaccessorycom-
andthatimportedproductswereresistanttoproteinaseK(PK) ponents to fully or nearly fully assembled CI, but to different
digestion (Fig. 4A). The CAL1 subunit was clearly processed degreestheywerepresentinthefullrangeofsubcomplexesthat
upon import, leading to a (cid:1)2-kDa decrease in its apparent areknowntoincludecomponentsofthemembranearmofCI
molecular mass, and only the processed forms were PK-pro- (3,22).Importofthe(cid:4)subunitofATPsynthase,whichfailsto
tected.Theothersubunitsassayedwerenotclearlyprocessed, assemble into a protein complex, was used as a radiolabeled
leadingtochangesinmolecularweight,butmembrane-bound controlimportforcomparisonoflabelingongelimages.
proteinswerePK-protectedafter60minoftheimportreaction CA2 Is Imported and Assembled into a Transient 200-kDa
(Fig. 4A). Separation of PK-protected proteins by BN-PAGE Subcomplexinca2MutantArabidopsisMitochondria—Tofur-
revealedthatCA3,CAL1and-2,and20.9-kDasubunitcouldbe therunderstandthenatureofthe(cid:1)-CAdomainsubcomplexes
clearly assembled into 200–300-kDa subcomplexes and the inimports,timeseriesimportswerecomparedbetweenmito-
650-kDa subcomplex. CA1–CA3 were assembled to differing chondriaisolatedfromwildtypeandca2knock-outplantsthat
5712 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER8•FEBRUARY22,2013
AssemblyofMitochondrialComplexIinArabidopsis
ofopisthokonts(animals(cid:4)fungi)(12).Extendingthisanalysis,
wehavefoundthatinorganismsinwhichdeepgenomeand/or
transcriptomesequencinghasbeenperformed,multiple(cid:1)-CA
genesareoftenfound(Table1andsupplementalDataSetS4).
Wheretheinferredproteinsequencesaredeemedtobecom-
pleteattheNterminus,manyofthese(cid:1)-CAshaveapredicted
mitochondriallocalization(Table1).Abioinformaticsurveyto
assess the phylogenetic distribution of the 20.9-kDa protein
sequenceshowsasimilarlywideoccurrenceofthecorrespond-
inggeneacrossthevariouseukaryoticsupergroups(Table1).
However,incontrasttowhatisseenwithanimals,the20.9-kDa
subunitisverywidelypresentinfungi(whereithasaknown
roleinCIassembly(18).Outsideofopisthokonts,(cid:1)-CAsand
the20.9-kDasubunitexhibitastrikinglysimilarpatternofco-
occurrence(Table1).AsidefromArabidopsis,directevidence
for the presence of both (cid:1)-CAs and the 20.9-kDa subunit in
mitochondrialCIisavailableforthegreenalga,Chlamydomo-
nasreinhardtii(32),aswellasAcanthamoebacastellanii(33).
FIGURE 5. Import of CA2 into ca2 mutant mitochondria. Mitochondria
Variousofthesecomponentshavealsobeenidentifiedinpro-
werepreparedfrom2-week-oldhydroponicallygrownwildtypeandca2
knock-outArabidopsis.A,CA2precursorswereincubatedwithwildtypeand teomicscreensofpurifiedmitochondriafromadditionalnon-
ca2knock-outmitochondriafor5,10,20,30,and60min;mitochondriawere photosynthetic protists, including Tetrahymena thermophila
thentreatedwithPK;andmitochondrialproteinswereseparatedbySDS-
(34),Dictyosteliumdiscoideum(35),andTrypanosomabrucei
PAGE.B,mitochondriaweretreatedwithPK,andsolubilizedcomponents
werethenseparatedbyBN-PAGE.RadiolabeledbandsalignedtoCoomassie- (36, 37) (Table 1). Evidently, the subunits of the subcomplex
stainedBN-polyacrylamidegelsofthesamesamples,andthepositionsof
describedhereinArabidopsisarewidelyconservedandcoinci-
complexesandsubcomplexesareindicated.*,locationof200–300-kDacom-
plexIsubcomplexes. dentinCIacrosseukaryotes.
have been previously reported by others (11) and have been
DISCUSSION
propagatedinourlaboratory.Analysisinthemutanthadthe
CIMatrixArmandMitochondrion-encodedSubunitsHave
potential advantage that [35S]Met-labeled precursors did not
DifferentTurnoverRates—Theassemblyofthelargelynucleus-
havetocompetewithunlabeledones,andinitialassemblypro-
encoded matrix arms and the set of mitochondrion-encoded
cesses and assembly factors might be more abundant in ca2
proteinsthataremainlyinthemembranearmofCIoccurvia
knockouts, where the abundance of fully assembled CI is
independentprocessesinotherorganisms(13–17,19,20,38);
reduced(11).Atimeseriesof5,10,20,30,and60minofCA2
thus,itisconceivablethatthesesubcomplexesmightturnover
precursorimportwasconducted,andthesamplesweresepa-
atdifferentratesinplants.Ourdatashowthatthematrixarm
ratedbySDS-PAGEandBN-PAGE.MoreCA2precursorwas
(includingmembersofboththeN-module(51kDaand75kDa)
imported and PK-protected in mitochondria isolated from
Arabidopsis ca2 knockouts than in the equivalent wild type and the Q-module (39 kDa), as defined for the mammalian
mitochondria(Fig.5A).OnBNgels,itwasclearthatCA2pre- complex(39))haveahigherturnoverratethaneitherthemito-
cursors were rapidly assembled into a discrete 200-kDa sub- chondrion-encodedsubunitsorthe(cid:1)-CAdomaincomponents
complex in mitochondria from the ca2 knockout (Fig. 5B). ofthemembranearm.Elevatedproteinturnovercanarisefrom
Incomparison,mitochondriafromwildtypeshowedassembly high synthesis or high degradation rates or a combination of
of CA2 into the same 200–300-kDa range of complexes both processes (24). As matrix arm subunits form the major
observedduringimportsintocellculturemitochondria(Fig.4B ROS production sites in the respiratory chain, these compo-
versus Fig. 5B). Whereas the [35S]Met incorporation into CI nentsmaybedamagedmorereadilyandrequirereplacement
steadilyincreasedintheca2knockoutduringtheimporttime morefrequently.ROS-mediatedproteinoxidationcansensitize
course,theabundanceofthe200kDabandpeakedat20min proteinstoproteolyticattack(40,41).ItispossiblethattheROS
and declined thereafter, indicating that it was further assem- releasedfromCIcouldleadtoincreasedoxidationofsubunits
bledintointactCIandwasthusaproductiveintermediateof in the vicinity or that ROS damage in the matrix is generally
assembly (Fig. 5B). CI(cid:4)CIII could not be visualized until 20 greaterinmagnitudethaninthemembrane(41).Thematrix
2
min,andthenitexhibitedthesamesteadilyincreasingpattern arm subunits are also likely to be more accessible to matrix
of [35S]Met incorporation seen for CI. A similar but weaker proteasesthanthemainlymembranesubunits.Itisalsopossi-
patternwasobservedinwildtypemitochondriaforprogressive blethatthenaturalbreakdownofintactCIsimplybeginswith
labelingfromsubcomplexestoCIandCI(cid:4)CIII (Fig.5B). thedegradationofmatrixarmsubunits,irrespectiveofdamage
2
ConservationandLossesof20.9,(cid:1)-CA,and(cid:1)-CALProteins oraccessibility.Whicheverthereason,thehigherturnoverrate
among Eukaryotic Lineages—Although (cid:1)-CAs were initially of matrix arm subunits than of both the mitochondrion-en-
consideredplant-specificsubunitsofCI(6,10,11),morerecent coded and the specific (cid:1)-CA domain subunits demonstrates
work has demonstrated the widespread occurrence of (cid:1)-CA that different functional modules of CI have distinct protein
homologs throughout eukaryotes, with the notable exception half-livesinplantcells.
FEBRUARY22,2013•VOLUME288•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5713
AssemblyofMitochondrialComplexIinArabidopsis
TABLE1
Phylogeneticdistributionof(cid:1)-CAand20.9-kDasubunitsofCI
MajortaxonomicgroupsarebasedonFig.2ofBrownetal.(51).Shadedandboldfaceentriesindicatethatthereisdirectevidenceforoneormoreofthecorresponding
componentsinmitochondriaand/orisolatedCI,asdescribedunder“Results”(furtherdetailsarealsoprovidedinsupplementalDataSetS4).(cid:4)/(cid:3),presence/absenceofthe
20.9-kDasubunitgeneinagivenorganism.Datainthetablearetakenlargelyfrompubliclyavailablegenome/transcriptomesequencingprojects,wheregenecoverageis
likelytobehigh.Additionalsequencesforotherorganismsaswellasotherinformation(accessionnumbers,mitochondrialtargetingprobabilities)maybefoundin
supplementalDataSetS4.
a(cid:1)-CAhomologscanbeclassifiedintotwotypes,basedonahighdegreeofconservation(Type1)ornot(Type2)intheN-terminalregion(12).Plant(cid:1)-CAsfallintothe
Type1category,whereasplant(cid:1)-CALsareincludedamongType2(cid:1)-CAs.Anasteriskdenotesthatthe(cid:1)-CATypeassignmentistentative.
b(cid:1)-CAmultiplicityattributabletogenomeduplication(s).
cUnpublisheddatafromtheA.godoyigenomeproject.
5714 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER8•FEBRUARY22,2013
AssemblyofMitochondrialComplexIinArabidopsis
Supercomplex Formation Does Not Significantly Stabilize mustcompetewiththecorrespondingsubunitspresentinvivo
ArabidopsisComplexI—Ithaspreviouslybeenproposedthat and could lessen the subcomplex signal or even change the
formationofsupercomplexI(cid:4)III mightstabilizecomplexIin originalassemblyprocess,especiallywhenredundantsubunits
2
Arabidopsis(42).Loweringproteinturnoverbysupercomplex competewithsubunitshavinghighsequencesimilarity.Mito-
formation could provide a functional significance to these chondriafromCIsubunitmutantshaveprovedtobeauseful
superassembliesintheelectrontransportchain,beyondtheir system for detection of assembly intermediates in mammals
roleinelectrontransportcatalysis.Thisproposalissupported (14,19).WefoundthatmoreCA2precursorcouldbeimported
by evidence from mice that knockout of complex III (43) or into ca2 mutant Arabidopsis mitochondria and that a strong
complexIV(44)subunitsdecreasesthecontentofcomplexI, 200-kDasubcomplexbandcouldbedetected,whichweakened
suggestingthatsupercomplexformationstabilizescomplexI.It after 20 min as the radiolabel was transferred into CI and
hasalsobeenproposedinhumanmitochondriathatsupercom- CI(cid:4)CIII .Thus,(cid:1)-CAsand(cid:1)-CALsappeartoassembleintoa
2
plexformationisrequiredforassemblyofcomplexIaswellas productive membrane 200-kDa subcomplex, which serves as
foritsstabilization(45).Inaddition,structuralstabilizationof theinitiatorofCIassembly.Ourdataprovidekeyexperimental
labile membrane protein complexes is proposed as a major evidencetojustifythemodelrecentlyproposedinplants(22)
function of supercomplex formation in Paracoccus denitrifi- thatsubunits(includingatleastND1andND7andmatrixsub-
cans(46).Aswepointedoutunder“Results,”however,wedid unitsof75and39kDa)areaddedtoaplant-specific200-kDa
not show any impact of supercomplex formation on protein subcomplex to form a 650-kDa subcomplex that is further
turnoverrateinArabidopsiscells(Fig.3).Hence,thesedirect assembledintoCI.
measurementsarenotabletoconfirmaroleforsupercomplex OurdatadonotprovethatCA,CAL,andthe20.9-kDasub-
formation in enhanced stabilization of complex I. Rather, it unitaretheonlysubunitsofthis200-kDasubcomplex.Previous
appearsthatplantcomplexIisinequilibriumbetweenthecom- surveysofBN-PAGEhavefoundevidenceforAt2g28430and
plexIandthesupercomplexI(cid:4)III states.Higherordercom- ND9inthisregiononBNgels(23).Althoughwewerenotable
2
plexesandrespirasomescouldnotbedetectedinouranalysisof toconfirmthisinourdata,thepossibilityofotherCIsubunits
Arabidopsis membranes, so we are not able to determine if inthis200-kDacomplexcannotbeexcludedandislikelygiven
thesemighthaveenhancedstability,butthistechniquecould itsmass.However,thelowabundanceofthecomplexhasnot
be used in systems where larger supercomplexes are evident allowedustodatetomakeanyprogressinfindingotherrapid
andprovideinvivoevidenceofrelativestability. turnoversubunitsinthismassrange.
ASpecificSubcomplexofUnknownFunctionProteinsHasa ConservationoftheSubunitsof200-kDaSubcomplexSuggests
ProductiveRoleinCIAssembly—Arangeofknock-outmutants an Ancestral Feature of CI Important for CI Assembly—The
forCIsubunitsleadingtotheaccumulationofcertaininterme- patternsofconservationofgenesfor(cid:1)-CAsandthe20.9-kDa
diates has previously been exploited to propose that CA2 is subunit and the evidence for their conserved presence in CI
incorporatedattheearlystageofCIassembly(22),and(cid:1)-CA- (Table 1) argue that both protein types are ancestral compo-
containingsubcomplexeshavebeendetectedinBN-polyacryl- nentsofCI,presentattheearlieststagesofeukaryoticcellevo-
amidegelsofwildtypemitochondria(23).Usinga15Nlabeling lution;notably,however,thisco-occurrencepatternisnotpre-
approach,wehaveshownthehigher15Nincorporationrateof served in opisthokonts (fungi (cid:4) animals) (Table 1). Of
subunitscontainedinsubcomplexesthaninCIandCI(cid:4)CIII , particular note is the finding that several protists included
2
revealingthatthesestructureswerenotcreatedinvitrobysol- withintheassemblageHolozoa(animalsandtheirspecificuni-
ubilization or electrophoresis artifacts but rather are in vivo cellularrelatives(47))areseentoencodeeither(cid:1)-CAsorthe
intermediatesofdisassemblyorassemblyofCI. 20.9-kDa subunit, although (like fungi) not both (Table 1).
The CI subcomplexes could conceivably be disassembly Intriguingly, genes for both (cid:1)-CAs and the 20.9-kDa subunit
intermediatesthathaveahighdegradationrate.However,the can also be retrieved from the recently determined genome
higher15NincorporationinsubcomplexesascomparedwithCI sequence of the apusomonad Thecamonas trehans, arguably
andCI(cid:4)CIII meansthisexplanationonlyholdsifdissociation theclosestneighbortoopisthokonts(48).Theseobservations
2
of the complex is biased toward newly assembled CI or indicate that progressive loss of components of this putative
CI(cid:4)CIII .Itisnotclearwhatcouldcausesuchabias.Also,our assemblysubcomplexbeganinopisthokontsatthebaseofthis
2
calculationsbasedonCoomassiestainingabundancetogether lineage, after its divergence from its closest related lineages,
withtheproportionofpeptide15Nrevealthattheamountof ApusozoaandAmoebozoa.InHolozoa,lossofallofthesub-
15N-labeled subunits within subcomplexes exceeds the total units known to be in the subcomplex appears to have been
amountof15NlabelinbothCIandCI(cid:4)CIII combined.Hence, completebeforetheemergenceofmulticellularanimals(Meta-
2
thebalanceofthedata,from15Nincorporationalone,isinfavor zoa). Outside of eukaryotes, (cid:1)-CAs are prominent among
ofthe200-kDasubcomplexesfoundinwildtypebeingassem- (cid:3)-proteobacteria, and the (cid:1)-CAs of these organisms are the
blyratherthandisassemblyintermediates. mostsimilarinsequencetotheeukaryotic(cid:1)-CAs,althoughin
Theimportexperimentsusing(cid:1)-CAs,(cid:1)-CALs,and20.9-kDa thiscasetheydonotappeartobeassociatedwithCI(49).Con-
subunitsshowedthatallofthesesubunitscanbeimportedinto sideringthe(cid:3)-proteobacterialancestryofmitochondria(50),it
cell culture mitochondria and that they form a series of sub- ispossible(perhapsprobable)thatthisbacterialgroupwasthe
complexesranginginsizefrom100to300kDa,althoughthe evolutionarysourceofmitochondrial(cid:1)-CAs,whichwerethen
200-kDacomplexfoundinwildtypedidnotpredominate(Fig. recruited to serve in CI assembly early in eukaryotic cell
1 versus Figs. 4 and 5). Imported [35S]Met-labeled precursors evolution.
FEBRUARY22,2013•VOLUME288•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5715
AssemblyofMitochondrialComplexIinArabidopsis
18. Schulte,U.,Fecke,W.,Kru¨ll,C.,Nehls,U.,Schmiede,A.,Schneider,R.,
Acknowledgments—WethankDr.IanCastleden(CentreforCompu-
Ohnishi,T.,andWeiss,H.(1994)Invivodissectionofthemitochondrial
tationalSystemsBiology,UniversityofWesternAustralia)forexper-
respiratoryNADH:ubiquinoneoxidoreductase(complexI).Biochim.Bio-
tiseandassistanceinfileformatconversionsandconcatenation.We phys.Acta1187,121–124
alsothankDr.MarekElias(CharlesUniversity,Prague)forpermis- 19. Lazarou,M.,McKenzie,M.,Ohtake,A.,Thorburn,D.R.,andRyan,M.T.
siontoincludeunpublisheddatafromtheon-goingAndaluciagodoyi (2007)Analysisoftheassemblyprofilesformitochondrial-andnuclear-
genomesequencingproject(Table1andsupplementalDataSetS4). DNA-encodedsubunitsintocomplexI.Mol.Cell.Biol.27,4228–4237
20. Vogel,R.O.,vandenBrand,M.A.,Rodenburg,R.J.,vandenHeuvel,L.P.,
Tsuneoka,M.,Smeitink,J.A.,andNijtmans,L.G.(2007)Investigationof
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