Recent Advances in the Composition and Heterogeneity of the Arabidopsis Mitochondrial Proteome.

0.74 MB·English

by  Lee, Chun Pong

MINIREVIEWARTICLE
published:25January2013
doi:10.3389/fpls.2013.00004
Recent advances in the composition and heterogeneity of
the Arabidopsis mitochondrial proteome
ChunPongLee1*,NicolasL.Taylor2,3 andA.HarveyMillar2,3
1DepartmentofPlantSciences,UniversityofOxford,Oxford,UK
2ARCCentreofExcellenceinPlantEnergyBiology,TheUniversityofWesternAustralia,Crawley,WA,Australia
3CentreforComparativeAnalysisofBiomolecularNetworks,TheUniversityofWesternAustralia,Crawley,WA,Australia
Editedby: MitochondriaareimportantorganellesforprovidingtheATPandcarbonskeletonsrequired
JoshuaL.Heazlewood,Lawrence to sustain cell growth. While these organelles also participate in other key metabolic
BerkeleyNationalLaboratory,USA
functions across species, they have a specialized role in plants of optimizing photosyn-
Reviewedby:
thesis through participating in photorespiration. It is therefore critical to map the protein
SebastienCarpentier,KULeuven,
Belgium composition of mitochondria in plants to gain a better understanding of their regulation
ChristianLindermayr,Helmholtz and define the uniqueness of their metabolic networks.To date, <30% of the predicted
ZentrumMünchen–German number of mitochondrial proteins has been verified experimentally by proteomics and/or
ResearchCenterforEnvironmental
GFPlocalizationstudies.Inthismini-review,wewillprovideanoverviewoftheadvancesin
Health,Germany
mitochondrialproteomicsinthemodelplantArabidopsisthalianaoverthepast5years.The
*Correspondence:
ChunPongLee,DepartmentofPlant ultimate goal of mapping the mitochondrial proteome inArabidopsis is to discover novel
Sciences,UniversityofOxford,South mitochondrialcomponentsthatarecriticalduringdevelopmentinplantsaswellasgenes
ParksRoad,OxfordOX13RB,UK. involvedindevelopmentalabnormalities,suchasthoseimplicatedinmitochondrial-linked
e-mail:[email protected]
cytoplasmicmalesterility.
Keywords:Arabidopsis thaliana, mitochondria, proteomics, heterogeneity, protein complex, post-translational
modifications,functionalproteomics
INTRODUCTION functional roles (Heazlewood et al., 2007; Cui et al., 2011). In
Mitochondria are semi-autonomous, double membrane bound comparison,themitochondrialgenomeencodesforonly57gene
organelleswithuniquemorphologiesandhighlyspecializedfunc- products (Unseld et al., 1997). The first extensive experimental
tions.Whiletheseorganellesarewell-recognizedforenergymetab- studies of the mitochondrial proteome in Arabidopsis identified
olismviacouplingtheoxidationof organicacidswithoxidative ∼100–150proteins(Kruftetal.,2001;Millaretal.,2001;Werhahn
phosphorylation (OXPHOS), they also have diverse functional and Braun, 2002; Millar and Heazlewood, 2003). Improvement
rolessuchasmetabolismofaminoacidsandbiosynthesisofcofac- of organellepurificationprocedure,availabilityof differentpro-
torsandvitamins.Mitochondriainplantsaresetapartfromtheir teinmappingstrategies,enhancedsensitivityofpeptidedetection
mammalian counterparts by their mediation of photosynthesis bymassspectrometry(MS),andimprovedgenomicresourcesand
through providing alternative electron sinks for photosynthetic peptideidentificationsoftwarehavedrivenasignificantincreasein
products and their participating in photorespiration (Padmas- thenumberofmitochondrialproteinsidentifiedacrossdifferent
ree et al., 2002). In order to fully understand the functional model species – from 843 in Arabidopsis (Table S1A in Supple-
roles of mitochondria in photosynthetic cells, it is essential to mentaryMaterial)and851inyeast(Reindersetal.,2006),to1404
establishtheirtotalproteinmake-up(proteome)andtheirpost- inmouse(Forneretal.,2009).
translationalmodifications(PTMs),aswellastogenerateapro- Given the number of proteins identified so far in Arabidop-
tein atlas that collects information about mitochondrial protein sis mitochondrion, it is clear that our understanding of its
expressionpatternsduringstressandindifferentcells,tissues,and compositionandfunctionsinplantsisfarfromcomplete.Inthis
organs. mini-review,wewillprovideanupdateonthestatusofArabidop-
Arabidopsisthaliana becamethefirstmodelsystemforplants sismitochondrialproteomicsresearchbasedonpublisheddatain
afteritsgenomewasfullysequencedandmadepubliclyavailable thepast5years(2007–2012).Wewouldreferreaderstoprevious
in 2000 (The Arabidopsis Genome Initiative, 2000). In the last reviewarticlesformorecomprehensiveoverviewsontheprogress
decade,tremendousprogresshasbeenmade,bybothexperimental ofplantmitochondrialproteomicsintheprecedingyears(Millar
andbioinformaticsapproaches,todefinethemitochondrialpro- etal.,2005,2011;Itoetal.,2007;Dudkinaetal.,2010).
teomeinthismodelplantspecies.Likeitsyeastandmammalian
counterparts, most of the mitochondrial proteins in Arabidop- HOWFARAREWEFROMCOMPILINGTHECOMPLETESETOF
sis areencodedbythenucleargenome.Basedontheanalysesof ARABIDOPSIS MITOCHONDRIALPROTEINS?
theN-terminaltargetingpeptidesequencesinArabidopsis,there A recent in-depth analysis of the proteome in Percoll-purified
are about 2500 predicted nuclear-encoded mitochondrial pro- mitochondriahasidentifiedanon-redundantsetof572proteins
teins (representing 7–10% of all encoded proteins) with broad inArabidopsis cellculture(Tayloretal.,2011).Withacombined
www.frontiersin.org January2013|Volume4|Article4|1
Leeetal. TheArabidopsismitochondrialproteome
proteomics,localization experiment and literature confirmation FUNCTIONSOFTHEMITOCHONDRIALPROTEOMEIN
approach,asetof38mitochondrialproteinshavebeenfoundin ARABIDOPSIS
or associatedwith themitochondrial outermembrane (Duncan MITOCHONDRIALPROTEINFUNCTIONSANDABUNDANCE
etal.,2011).Morerecently,atotalof66novelintegralmembrane Oftheconfirmedsetofmitochondrialproteins(TableS1BinSup-
proteinshavebeenidentifiedinmitochondriausingaMS-based plementaryMaterial),∼22%arecomponentsofpyruvatemetabo-
quantitativeenrichmentapproach(Tanetal.,2012).Anewsetof lism/TCAcycleandOXPHOS,whileasimilarnumber(∼20%)are
componentswithunknownfunctionshavealsobeenidentifiedin identifiedassubunitsofmachineryformitochondrialgeneexpres-
anumberofrecentstudies,includingtheanalysisofthemitochon- sion and maintenance (Figure 1A). In the yeast mitochondrial
drial fraction from:(i) separated protein complexes (Klodmann proteome,asimilarproportion(∼15%)ofidentifiedproteinsare
etal.,2010,2011;KlodmannandBraun,2011;Schertletal.,2012); involvedinenergymetabolism(Schmidtetal.,2010).Whencom-
(ii) enriched phospho-proteome (Ito et al.,2009); (iii) different paring the abundance of proteins in these functional categories
tissuetypes(Leeetal.,2012);(iv)varioustimepointsof adiur- usingtherecentlypublishedLC-MS/MSdata(Tayloretal.,2011),
nalcycle(Leeetal.,2010);and(v)cellssubjectedtobioticstress energymetabolismcomprisesover50%ofthetotalproteinabun-
(Livajaetal.,2008). danceinmitochondria,whereas<2%isassociatedwithprocessing
While various large-scale proteomics studies over the last mitochondrialDNA/RNA(Figure1B).Theobservedabundance
5years have led to the identification of a non-redundant set of ofproteinsinenergymetabolismisconsistentwiththemainrole
843putativemitochondrialproteins(TableS1AinSupplementary ofmitochondriainthecellandbulkofthechemicalreactionsper-
Material),itremainsdifficulttodiscriminatetruemitochondrial formedintheorganelle;incontrast,thelowabundanceofproteins
proteins from contaminants,particularly for low abundant pro- formitochondrialDNA/RNAprocessingcanprobablybeattrib-
teins,inasample.Ithasbeenestimatedthatabout11%ofthetotal utedtotheirrelativelylessstablenaturesothattheycanrespond
spotintensityona2-DmapofmitochondriafromArabidopsiscell rapidlytoexternalstimuliortochangesinenergycost(Schwan-
cultureareproteinsoriginatedfromothercompartments(Taylor hausseretal.,2011),thetransientneedfortheirfunctionsduring
etal.,2011).Byqueryingpreviousevidencefromliteratureand/or thelifeof cellsandpresumablythehighspecificactivityof their
consensussubcellularlocalizationpredictionscorefrompublicly functions. At the whole cellular level,components in this func-
availabledatabases[SUBA3(Heazlewoodetal.,2007);ARAMEM- tionalcategoryhaverecentlybeenshowntohaveahighturnover
NON7.0(Schwackeetal.,2003)],wedefineasetof504proteins rateinArabidopsis(Lietal.,2012).Mitochondrialproteinsinvolv-
whichcanbeassignedtobemitochondrial-localizedinArabidop- ingnucleicacidprocessingappeartoperformhighlyspecialized
sis withhighconfidence(TableS1BinSupplementaryMaterial). functionsanddonotseemtohaveoverlappingspecificity.Only
This approach is biased toward proteins that are highly abun- ∼12% of the proteins in the yeast mitochondrial proteome are
dantanddoesnotexplicitlyimplythattheremainingproteinsare dedicatedtogenomemaintenanceandprocessing(Schmidtetal.,
in fact contaminants from other compartments. Some of these 2010).TheproportionishigherinArabidopsisduetothepresence
proteins lack a predictable targeting presequence, may be dual- ofmultipleplant-specificpentatricopeptiderepeat(PPR)proteins
targetedtomultiplecompartmentsand/orarepresentinrelatively and/oritslargergenomesizewhichmayrequiremoreproteinsto
low amount,thus their localization should be confirmed in the maintainandprocess.EachPPRproteinrecognizesandactsona
futurethroughmultipleindependentproteomicanalysesand/or singlesiteinaspecifictranscriptsequence(Delannoyetal.,2007).
byfluorescentproteinlocalization. Severaloftheunknownproteinsidentifiedbyourearlierstudy
According to the SUBA database, a number of GFP tagging (Heazlewood et al., 2004) have since been re-assigned as plant-
studieshaverevealedthemitochondriallocalizationof 222pro- specific components of OXPHOS (Klodmann et al.,2011). The
teins that cannot be identified through proteomic approaches most nebulous subset of the known proteome is the more than
(Table S1C in Supplementary Material), most of which are low 18% of the identified proteins that remain without any func-
abundanceproteinsinvolvedintheprocessingandmaintenance tionalclass.However,whilethissubsetaregreatinnumberthey
of the mitochondrial genome. Together with the proteomics contribute to <2% of mitochondrial protein abundance. Inter-
set, 726 proteins can be confidently assigned as mitochondr- estingly, these include a number of plant-specific proteins. It is
ial, <30% of the presumed number of predicted proteins. To thereforeclearthat manymorestudiesare requiredtoelucidate
further expand the current Arabidopsis mitochondrial protein thefunctionsofthissubsetofproteinswhichcanpotentiallylead
compendium, it is essential to overcome the challenge of iden- tothediscoveryof novelplant-specificmitochondrialmetabolic
tifying low abundance proteins. To achieve this a number of pathways/functions.
approaches could be employed including protein enrichment
tools,suchasproteominer(Fröhlichetal.,2012)orproteinfrac- PROTEINCOMPLEXESANDINTERACTOME
tionation approaches including strong cation exchange (SCX) Multipleproteins/isoformsoftenassembledintolargecomplexes
or off-gel electrophoresis (OGE) prior to RP-LC-MS (Chenau which serve vital metabolic and regulatory roles. While earlier
et al., 2008; Ito et al., 2011). Together with biological fraction- reports have extensively analyzed the structure and function of
ation approaches such as investigation of pre-fractionated sub- individual enzyme complexes of interest, such as glycine decar-
mitochondrialcompartmentsorenrichmentbymetalorco-factor boxylase complex (Douce et al., 2001), it is uncertain whether
bindingapproachesandadvancesinLC-MStechniquesandequip- othermitochondrialproteinscouldalsoorganizeintomacromol-
ment, it is likely that an increasing number of low abundance ecular structures. Using 2-D blue-native/SDS-PAGE, Klodmann
proteinswillberevealed. etal.(2011)found35differentproteincomplexesinmitochondria
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Leeetal. TheArabidopsismitochondrialproteome
FIGURE1|Overviewofthenumberandabundanceofmitochondrial beenfound(Schmidtetal.,2010).Inaddition,moreproteinsareinvolvedin
proteinsacrossfunctionalcategories.(A)Piechartshowingoffunctional mitochondrialgenomemaintenance(white)inplants(∼20%)thaninyeast
categoriesoftheconfirmedsetof726mitochondrialproteins(seeTable (∼12%),duetothepresenceofnumerousplant-specificpentatricopeptide
S1B,CinSupplementaryMaterial).Acomparisonwiththemorecomplete repeat(PPR)proteinsandalargergenomesize.(B)Distributionofthe
yeastmitochondrialproteomeshowsthatsimilarproportionofproteins abundanceofproteinsthatcanbeidentifiedbygel-freeMS(Tayloretal.,2011)
involvingenergymetabolismaswellasproteinswithunknownfunctionshas acrosssevenfunctionalcategories.
fromArabidopsiscellculture.OXPHOScomplexesareamongstthe and lysine acetylation (Finkemeier et al., 2011). However, there
largestandthemostabundantproteincomplexesinmitochondria. appears to be no evidence for specific preference of PTMs to
Mitochondrialcomplexassembliesarealsodominatedbycompo- particular functional categories of identified proteins (Table 1),
nents in the TCA cycle, amino acid metabolism, PPR proteins, suggestingthatPTMshaveawidevarietyoffunctionaltargetsin
andpre-proteinimportapparatus.Whilethepreliminarycompo- themitochondrion.
sitionsoftheseproteinscomplexeshavebeenproposedbasedon ThetotalnumberofidentifiedproteinswithPTMsisverylikely
thenumberofsubunitsidentifiedandtheirmigrationonthefirst agrossunderestimationduetoanumberoftechnicalchallenges,
andseconddimension,theymustbeverifiedthroughindependent suchasthelossofPTMsduringmitochondrialpurificationpro-
biochemicalanalysis. ceduresandtherelativelylowabundanceofthemodifiedpeptides
Anumberofmitochondrialproteinsofdiversefunctionhave compared to their unmodified counterparts.Also,it is not clear
beenidentifiedtointeractwithmetalions(Tanetal.,2010)and/or howmanyproteins,includingthoselistedinTable1,arefunction-
havebindingaffinitywithATP(Itoetal.,2006)inArabidopsis.In allymodifiedthroughenzyme-catalyzedmitochondrialprocesses
contrast,studiesonthemoretransientdirectinteractions(func- in vivo. For example, degradation products observed on a 2-D
tionalandphysical)betweenmultiplemitochondrialproteinsin geloftenperceiveasartificialpost-purificationevents.Thesecon-
plants are lacking. Such detailed studies in the future will lead cernscanbeatleastpartiallyovercomebyenrichmentofmodified
to the construction of plant mitochondrial interactome, to sit peptides/proteinsand/orrepeatanalysisof multiplereplicatesto
alongsidesidethecomplexome,andhelptodefineuniquemeta- ensurethatsimilarchangescanbeobservedinallsamples.Alterna-
bolicregulationsinplantsthatdifferentiatethemfromyeastand tively,theincorporationofradioactivetracersintoproteinsinvivo
mammals. (cells)orinvitro(isolatedmitochondria)canbeusedtoidentify
proteinswithreversiblePTMs.Forinstance,18phosphoproteins
POST-TRANSLATIONALMODIFICATIONS haverecentlybeenidentifiedby[γ32P]-ATPlabelingandaffinity
ThecomplexityofArabidopsismitochondrialproteomeisfurther enrichmentofisolatedmitochondria(Itoetal.,2009).
implicatedbythedynamicregulationofPTMswhichcancontrol
activity,stability,and structural characteristics of proteins. Pro- CHANGESINTHEMITOCHONDRIALPROTEOMEIN
teinswithPTMsoftenappearasmultiplespotswithdifferentpI DIFFERENTTISSUESANDINRESPONSETOOXIDATIVE
and/ormolecularmassona2-Dgel,andtheregionofapeptide STRESS
withmodifiedresiduescanbedetectedasanalteredm/zionspecies The mitochondrial proteome is not static, but has many com-
byMS.Recentlarge-scaleproteomicstudieshavereportedanum- ponents that are dynamically regulated in order to meet energy
ber of PTMs in Arabidopsis mitochondrial proteome (Table 1), andmetabolicneedsrequiredbythecellinresponsetodevelop-
includingoxidation(Tanetal.,2010;Solheimetal.,2012),phos- mentaland/orenvironmentalchanges.Therearemanydifferent
phorylation (Ito et al.,2009;Taylor et al.,2011),S-nitrosylation cell/tissue/organ types which have functions that are unique to
(Palmierietal.,2010),N-terminalacetylation(Huangetal.,2009), plants.Thus,mitochondrialcomposition,metabolism,andstress
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Leeetal. TheArabidopsismitochondrialproteome
Table1|ThesetofmitochondrialproteinsinArabidopsiswithknownpost-translationalmodifications.
AGIaccession ProteinID PTM Reference
OXIDATIVEPHOSPHORYLATION
AT1G15120 ComplexIIIQCR6-1,Hingeprotein N-acetylation Huangetal.(2009)
AT1G22840 Cytochrome Lysacetylation Finkemeieretal.(2011)
N-acetylation Huangetal.(2009)
AT1G47420 SDH5succinatedehydrogenasesubunit5 Phosphorylation Itoetal.(2009)
AT1G51980 MPPalpha-1mitochondrialprocessingpeptidasealphasubunit Oxidation Solheimetal.(2012)
AT3G12260 NADHdehydrogenaseB14subunit N-acetylation Huangetal.(2009)
AT3G62810 Complex1familyprotein/LVRfamilyprotein N-acetylation Huangetal.(2009)
AT4G21105 COXX4 Oxidation Solheimetal.(2012)
AT5G08670 ATPsynthasebetasubunit Oxidation Solheimetal.(2012)
AT5G52840 NADHdehydrogenaseB13subunit Phosphorylation Tayloretal.(2011)
AT5G66760 SDH1-1succinatedehydrogenaseflavoproteinsubunit Phosphorylation Itoetal.(2009)
ATMG01190 ATPsynthasealpha-1subunit S-Nitrosylation Palmierietal.(2010)
Oxidation Solheimetal.(2012)
PYRUVATEMETABOLISMANDTCACYCLE
AT1G24180 E1alpha-2(pyruvatedehydrogenase) Phosphorylation Itoetal.(2009)
AT1G48030 mtLPD-1(mtlipoamidedehydrogenase-1) S-Nitrosylation Palmierietal.(2010)
Oxidation Solheimetal.(2012)
AT1G53240 Malatedehydrogenase-1 Oxidation Solheimetal.(2012)
At1G59900 E1alpha-1(pyruvatedehydrogenase) Phosphorylation Itoetal.(2009)
AT2G05710 Aconitatehydratase-2 Phosphorylation Tayloretal.,2011;reftherein
Oxidation Tanetal.(2010)
AT2G20420 Succinyl-CoAligase(GDP-forming)beta-chain Oxidation Solheimetal.(2012)
AT3G13930 E3-1(dihydrolipoamidedehydrogenase) Phosphorylation Tayloretal.,2011;reftherein
AT3G15020 Malatedehydrogenase-2 Oxidation Solheimetal.(2012)
AT3G17240 mtLPD-2(mtlipoamidedehydrogenase-2) S-Nitrosylation Palmierietal.(2010)
AT4G26970 Aconitatehydratase-1 Oxidation Tanetal.(2010)
AT5G03290 Isocitratedehydrogenase-5 Oxidation Solheimetal.(2012)
TRANSPORT
AT2G29530 Translocaseinnermembranesubunit10,TIM10 N-acetylation Huangetal.(2009)
AT3G08580 AAC1(ADP/ATPcarrier1) Oxidation Solheimetal.(2012)
AT3G46560 Translocaseinnermembranesubunit9,TIM9 N-acetylation Huangetal.(2009)
AT5G13490 AAC2(ADP/ATPcarrier2) Oxidation Solheimetal.(2012)
AT5G14040 mtphosphatetransporter Oxidation Solheimetal.(2012)
AT5G50810 Translocaseinnermembranesubunit8,TIM8 N-acetylation Huangetal.(2009)
NUCLEICACIDPROCESSINGANDPROTEINFOLDINGANDSTABILITY
AT1G74230 GR-RBP5(glycine-richRNA-bindingprotein5) Phosphorylation Itoetal.(2009)
AT3G13160 PPR8-2 Oxidation Solheimetal.(2012)
AT3G23990 HSP60-3B Oxidation Solheimetal.(2012)
AT4G02930 ElongationfactorTu Oxidation Solheimetal.(2012)
AT4G26780 Co-chaperonegrpE Phosphorylation Itoetal.(2009)
AT4G37910 HeatshockproteinHSP70-1 Phosphorylation Tayloretal.,2011;reftherein
AT5G26860 LON1(LONprotease1) Phosphorylation Itoetal.(2009)
AT5G40770 Prohibitin-3 N-acetylation Huangetal.(2009)
AT5G61030 GR-RBP3(glycine-richRNA-bindingprotein3) Phosphorylation Itoetal.(2009)
PHOTORESPIRATION
AT1G11860 GDT1aminomethyltransferase S-Nitrosylation Palmierietal.(2010)
Phosphorylation Tayloretal.(2011)
AT1G32470 GDHGlycinedecarboxylaseHsubunit S-Nitrosylation Palmierietal.(2010)
Oxidation Solheimetal.(2012)
AT2G35370 GDHGlycinedecarboxylaseHsubunit S-Nitrosylation Palmierietal.(2010)
Oxidation Solheimetal.(2012)
(Continued)
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Leeetal. TheArabidopsismitochondrialproteome
Table1|Continued
AGIaccession ProteinID PTM Reference
AT4G33010 GlycinedecarboxylaseP-protein1 S-Nitrosylation Palmierietal.(2010)
Oxidation Solheimetal.(2012)
AT4G37930 SHM1(serinetranshydroxymethyltransferase1) S-Nitrosylation Palmierietal.(2010)
Oxidation Solheimetal.(2012)
AT5G26780 SHM2(serinehydroxymethyltransferase2) Oxidation Solheimetal.(2012)
OTHERMETABOLISM
AT3G61440 Cyanoalaninesynthase Phosphorylation Tayloretal.,2011;reftherein
AT3G22200 4-Aminobutyrateaminotransferase Oxidation Solheimetal.(2012)
AT4G15940 Fumarylacetoacetatehydrolasefamilyprotein N-acetylation Huangetal.(2009)
AT5G07440 GDH2(glutamatedehydrogenase-2) Phosphorylation Itoetal.(2009)
N-acetylation Huangetal.(2009)
Oxidation Solheimetal.(2012)
AT5G14780 FDHFormatedehydrogenase Phosphorylation Itoetal.(2009)
Oxidation Solheimetal.(2012)
AT5G18170 GDH1(glutamatedehydrogenase-1) Phosphorylation Itoetal.(2009)
N-acetylation Huangetal.(2009)
AT5G50370 Adenylatekinasefamily Phosphorylation Tayloretal.(2011)
N-acetylation Huangetal.(2009)
AT5G63400 ADK1Adenylatekinase1 N-acetylation Huangetal.(2009)
PROTEINSWITHUNKNOWNFUNCTIONS
AT2G39795 mtglycoproteinfamilyprotein Phosphorylation Itoetal.(2009)
AT3G18240 Unknownprotein Phosphorylation Itoetal.(2009)
AT3G55605 Mitochondrialglycoproteinfamilyprotein Phosphorylation Itoetal.(2009)
AT4G21460 Unknownprotein Phosphorylation Itoetal.(2009)
AT4G27585 Stomatin-likeprotein Phosphorylation Itoetal.(2009)
AT4G23885 Unknownprotein N-acetylation Huangetal.(2009)
PTM,post-translationalmodification(s).
response in these cells/tissues/organs from Arabidopsis will be in flowers. In mouse,it has been reported that just over half of
differentfromwhathasbeenobservedinyeastandanimals. all proteins identified by gel-free MS approach can be found in
Analysis of the mitochondria proteome from photosynthetic alltheinvestigatedorgans(Pagliarinietal.,2008).However,the
shoots,non-photosyntheticcellculture,androotsidentifiedmajor numberof mitochondrialproteinsthatarehighlytissue-specific
differences in the abundance of enzymes of the TCA cycle and (i.e.,totallyabsentinatleastonetissue)inArabidopsisremainsto
photorespiration (Lee et al.,2008,2011). Quantitative compari- bedefined.Suchanalysiswillassistinidentifyingmitochondrial
sonofthemitochondrialproteomeacross10differenttimepoints componentsthatcausesplant-specificdevelopmentalphenotypes,
covering24-hof thelifeofArabidopsis shootsalsouncoversday e.g.,cytoplasmmalesterility.
(photosynthetic)-andnight(non-photosynthetic)-enhancedpro- Using a gel-free quantitative MS approach, Tan et al. (2012)
teins in central carbon metabolism (Lee et al., 2010). In these recentlyidentifyanumberofintegralmembraneproteinsinmito-
studies,theabundancesofOXPHOScomplexesinpurifiedmito- chondriathatarealteredinabundanceinresponsetocoldand/or
chondriagenerallyremainunalteredbuttheirrespiratorycapacity variouschemicalstresses.Theseproteinsincludethecomponents
differs depending on the choice and/or availability of substrates ofthealternativeNADHdehydrogenases,alternativeoxidase,and
(Leeetal.,2008,2011).However,onawholetissuebasisdifferences uncoupling proteins, but also several stress-sensitive subunits
inmitochondrialelectrontransportchaincomplexratiosbetween within the OXPHOS complexes. Together with a similar study
tissueshasbeenreported(Petersetal.,2012).Leeetal.(2012)have by Sweetlove et al. (2002),it is concluded that the reduction in
reportedchanges intheisolatedArabidopsis mitochondrialpro- respiration in response to chemical-induced oxidative stress is a
teomebeyonddifferencesinthecellularphotosyntheticcapacity. consequence of coordinated changes in the mitochondrial pro-
Changesintheabundanceofawidevarietyofmitochondrialpro- teome,particularlyOXPHOScomplexsubunitsandstress-related
teins can be observed from cells/tissues from various vegetative components.
and reproductive phases of development. Differences in protein
accumulationandmetabolicspecializationsofthesemitochondria APPLICATIONOFPROTEOMICSTOANALYZE
generallycoincidewiththemainphysiologicalroleofeachcorre- MITOCHONDRIALPROTEINFUNCTIONS
spondingtissuetype,suchasglycinecleavageviaphotorespiration Overthelastdecade,advancesintheunderstandingofmitochon-
in shoot and maintenance of mitochondrial redox environment drialcompositionandproteincomplexassemblyhaveledtothe
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Leeetal. TheArabidopsismitochondrialproteome
identification of many genes associated with genetic diseases in regulationofthemitochondrialproteomeinacellular/organismal
humans(CalvoandMootha,2010).Incontrast,plantproteomics context. However, further work is needed to characterize mito-
still needs to discovernovel mitochondrial components that are chondrialproteinsaccordingtotheirsub-organellarlocalization.
associated with known developmental defects in plants. Never- In-depth identification of components in the intermembrane
theless,bycombiningproteomicsandreverse-geneticsstrategies, spacehasnotbeenreportedsincetheimprovementsinMSanaly-
a number of recent studies have highlighted the unique role of sis in recent years. Recent discoveries of a pyruvate transporter
a mitochondrial component of interest in Arabidopsis that had (Herzig et al.,2012) and a calcium uniporter (Baughman et al.,
not been unraveled by other biochemical and molecular tech- 2011) in mouse mitochondria have been conducted through an
niques.Metaboliteanalysesofmalatedehydrogenase(MDH)anti- integratedproteomics,bioinformatics,andgeneticsstrategy.Thus,
senseandknockoutlinesintomatoandArabidopsis respectively identificationoflowabundanceproteinsshouldallowustocom-
show an elevated foliar ascorbate level (Nunes-Nesi et al.,2005; pletethecatalogofmitochondrialproteinsinArabidopsis,which
Tomazetal.,2010).Suchaccumulationcoincideswiththereduc- will provide us several candidates for identifying plant-specific
tionofComplexI-associatedgalacton-1,4-lactonedehydrogenase transportersormetabolicpathwaysbyasimilarapproach.
(GLDH) abundance in the mitochondrial proteome of a MDH
double mutant (mmdh1mmdh2; Tomaz et al., 2010), indicating ACKNOWLEDGMENTS
that there might be a complex metabolic regulation/interaction ThisworkwasfundedthroughagranttotheARCCentreofExcel-
betweenOXPHOS,TCAcycle,andcellularascorbatebiosynthe- lence in Plant Energy Biology (CE0561495; A. Harvey Millar).
sis.AmutationinmitochondrialLonproteaseleadstoaretarded A. Harvey Millar is funded as anARCAustralian Future Fellow
growthphenotype(Rigasetal.,2009),whichcanbeexplainedby (FT110100242) and Chun Pong Lee is a receipt of an EMBO
analteredabundanceofenzymesintheTCAcycleandOXPHOS, Fellowship(ALTF1140-2011).
adecreaseintheabundanceof breakdownproductsandasmall
increase in the number of proteins with oxidized peptides, but SUPPLEMENTARYMATERIAL
notbyheightenedoxidativestress(Solheimetal.,2012).Incon- The Supplementary Material for this article can be found
trast, knockout of the protease AtFtsH4 does not significantly online at http://www.frontiersin.org/Plant_Proteomics/10.3389/
affectArabidopsisgrowthunderlongdayconditions,butchanges fpls.2013.00004/abstract
rosette development under short-day conditions (Gibala et al.,
2009). The phenotypes correlate with elevated levels of oxida- TableS1|Mitochondrialproteinsidentifiedbymassspectrometryor
fluorescentproteinlocalizationstudies.(A)Allproteinsidentifiedfrom
tive stress, increased abundance of Hsp70 and prohibitins, and
isolatedmitochondriafromArabidopsisusingproteomicsinthelast5years.(B)
decreasedabundanceofATPsynthasesubunits.
Asetofproteinswhichhasahighprobablybeinglocatedinthemitochondrion.
Fortheinclusionofaproteininthelist,itshouldmeetthefollowingcriteria:(i)A
CONCLUSIONANDPERSPECTIVES proteinisautomaticallyconsideredmitochondrialifatleasttwostudieshave
identifieditinisolatedmitochondrialfraction.However,aproteinisconsidered
The availability of the full genome sequence of Arabidopsis for
tobenon-mitochondrialifitisidentifiedinequalnumberoformore
morethanadecade,advancesinvariousproteomictechnologies, non-mitochondrialproteomicsstudiesthanthemitochondrialones.(ii)Ifthe
as well as their wider adoption, have provided an opportunity locationofaproteinisverifiedindependentlybyfluorescentproteinlocalization
to understand the protein make-up of mitochondria and their analysis,then(ii)isignoredanditisincludedinthelist.(iii)Ifaproteinis
identifiedbyonestudy,thelocalizationbasedonSUBAconscoreand/or
underlying metabolism in this plant more than in any other.
ARAMEMNONlocalizationconsensusscoreisalsoconsidered.(C)
SignificantprogressinextractinginformationonPTMsandpro-
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teinabundanceshasalsoimprovedourinsightintothedynamic (accordingtoSUBA)onlyandnotbyproteomics.
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Leeetal. TheArabidopsismitochondrialproteome
Werhahn,W.,andBraun,H.-P.(2002). conducted in the absence of any Citation: Lee CP, Taylor NL and Copyright©2013Lee,TaylorandMillar.
Biochemicaldissectionofthemito- commercial or financial relationships Millar AH (2013) Recent advances Thisisanopen-accessarticledistributed
chondrialproteomefromArabidop- thatcouldbeconstruedasapotential in the composition and heterogene- underthetermsoftheCreativeCommons
sisthalianabythree-dimensionalgel conflictofinterest. ity of the Arabidopsis mitochondrial Attribution License,which permits use,
electrophoresis. Electrophoresis 23, proteome. Front. Plant Sci. 4:4. doi: distribution and reproduction in other
640–646. 10.3389/fpls.2013.00004 forums,providedtheoriginalauthorsand
Received: 18 November 2012; accepted: ThisarticlewassubmittedtoFrontiersin source are credited and subject to any
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