The Arabidopsis Metacaspase9 DegradomeC W

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LARGE-SCALE BIOLOGY ARTICLE
Arabidopsis
The Metacaspase9 DegradomeCW
LianaTsiatsiani,a,b,c,d,1EvyTimmerman,c,dPieter-JanDeBock,c,dDominiqueVercammen,a,b,2SimonStael,a,b,c,d
BrigittevandeCotte,a,bAnStaes,c,dMarcGoethals,c,dTineBeunens,a,bPetraVanDamme,c,dKrisGevaert,c,d
andFrankVanBreusegema,b,3
aDepartmentofPlantSystemsBiology,VIB,9052Ghent,Belgium
bDepartmentofPlantBiotechnologyandBioinformatics,GhentUniversity,9052Ghent,Belgium
cDepartmentofMedicalProteinResearch,VIB,9000Ghent,Belgium
dDepartmentofBiochemistry,GhentUniversity,9000Ghent,Belgium
Metacaspasesaredistantrelativesofthemetazoancaspases,foundinplants,fungi,andprotists.However,incontrastwith
caspases,informationaboutthephysiologicalsubstratesofmetacaspasesisstillscarce.BymeansofN-terminalcombined
fractional diagonal chromatography, the physiological substrates of metacaspase9 (MC9; AT5G04200) were identified in
youngseedlingsofArabidopsisthalianaontheproteome-widelevel,providingadditionalinsightintoMC9cleavagespecificity
andrevealingapreviouslyunknownpreferenceforacidicresiduesatthesubstrateprimesitepositionP19.Thefunctionalitiesof
theidentifiedMC9substrateshintedatmetacaspasefunctionsotherthanthoserelatedtocelldeath.Theseresultsallowedus
toresolvethesubstratespecificityofMC9inmoredetailandindicatedthattheactivityofphosphoenolpyruvatecarboxykinase1
(AT4G37870),akeyenzymeingluconeogenesis,isenhanceduponMC9-dependentproteolysis.
INTRODUCTION anddifferentiationbyactingonalargenumberofdifferentcellular
substrates (Crawford et al., 2012). Based on their structural
Proteolysis is an irreversible, protease-catalyzed protein modifi-
similarity to the caspase catalytic domain, metacaspases have
cation with diverse effects on protein function and cellular or been identified in fungi, protozoa, and plants, whereas para-
organismalfate.Proteasesmediateproteinstability,turnover,and
caspaseshavebeenfoundinmetazoansandintheslimemold
activityandassistinthecorrecttargetingandimportofproteins
Dictyosteliumdiscoideum(Aravindetal.,1999;Urenetal.,2000;
todifferentorganelles.Earlydevelopment,xylemformation,seed
Vercammen et al., 2007). Like caspases, metacaspases and
maturation,nutrientsupplymobilization,responsestobioticand
paracaspasescarrythehallmarksoftheCysproteasesoftheCD
abiotic stresses, and programmed cell death are facilitated by
clan,includingthecatalyticdyadHis/Cysandthehemoglobinase
proteases(Avcietal.,2008;vanderHoorn,2008;Colletal.,2011;
fold(RawlingsandBarrett,1993;AravindandKoonin,2002).
Hara-Nishimura and Hatsugai, 2011; Vartapetian et al., 2011;
Inviewofthecrucialroleofcaspasesinapoptosis,initialefforts
Pesquet, 2012). Plant genomes code for numerous proteases
assessedmainlywhethermetacaspaseswereresponsibleforthe
(over700annotatedinArabidopsisthaliana),buttheirsubstrates
caspase-likeactivitiesdetectedinplantsduringcelldeathevents
remainpoorlyidentified(Tsiatsiani etal.,2012). Nevertheless, in
(Bonneau et al., 2008). However, biochemical studies using re-
the near future, this knowledge gap is expected to be filled by
combinantmetacaspasesorproteinextractsfromloss-of-function
theuseofemergingtechnologiesthatallowtheproteome-wide
orgain-of-functionmutantsclearlydemonstratedthatmetacaspases,
identificationofproteasesubstrates(HuesgenandOverall,2012). incontrastwiththeAsp-specificcaspases,cleavesyntheticpep-
Mammalian caspases are cysteinyl proteases that, besides
tidesubstratesafterArgandLysresidues(Vercammenetal.,2004,
functioninginapoptosis,mediateinflammation,cellproliferation,
2006;WatanabeandLam,2005;Bozhkovetal.,2005;González
etal.,2007;Mossetal.,2007;Ojhaetal.,2010).Therefore,met-
acaspasesareprobablynotdirectlyresponsibleforthecaspase-
1Current address: Biomolecular Mass Spectrometry and Proteomics,
Utrecht Institute for Pharmaceutical Sciences and Bijvoet Centre for likeactivitiesinplants,butratheractupstreamoftheAsp-specific
Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH proteases(Meslinetal.,2011).
Utrecht,TheNetherlands. Besides the multifunctionality of metacaspases, which is ap-
2Currentaddress:deVGen,Technologiepark30,9052Ghent,Belgium.
parentmostlyinorganismsencodingsinglemetacaspasegenes,
[email protected].
Theauthorresponsiblefordistributionofmaterialsintegraltothefindings suchasSaccharomycescerevisiae(yeast)andLeishmaniamajor,
presentedinthisarticleinaccordancewiththepolicydescribedinthe redundant and antagonistic functions have been demonstrated
Instructions for Authors (www.plantcell.org) is: Frank Van Breusegem inorganismsencodingmultiplemetacaspases.Theyeastmeta-
([email protected]). caspase,termedYeastCaspase1(YCA1),isrequiredforvarious
CSomefiguresinthisarticlearedisplayedincoloronlinebutinblackand
stress-induced cell death events, including oxidative, osmotic,
whiteintheprintedition.
WOnlineversioncontainsWeb-onlydata. toxic,andvirus-inducedstressesandaging(Madeoetal.,2002;
www.plantcell.org/cgi/doi/10.1105/tpc.113.115287 Herkeretal.,2004;IvanovskaandHardwick,2005;Chahomchuen
ThePlantCellPreview,www.aspb.orgã2013AmericanSocietyofPlantBiologists.Allrightsreserved. 1of17
2of17 ThePlantCell
etal.,2009).YCA1isalsoinvolvedincellcycleregulation(Lee
et al., 2008) and clearance of insoluble protein aggregates
(Leeetal.,2010).TheL.majormetacaspaseMCAisinvolved
incellcycleregulationandinoxidativestress–inducedcelldeath
(Ambit et al., 2008; Zalila et al., 2011). Three of the five Trypa-
nosoma brucei metacaspases are essential for viability of the
bloodstreamformoftheparasite(Helmsetal.,2006).Redundant
functionsintheregulationofsenescence-associateddeathwere
alsoshownfortwoPodosporaanserinametacaspases(Hamann
etal.,2007)andintheresistancetoendoplasmicreticulumstress
fortwoAspergillusfumigatusmetacaspases(Richieetal.,2007).
Interestingly,antagonisticfunctionsforhomologousmetacaspases
werefoundinT.bruceiandArabidopsisintheregulationofcell
death events or cell proliferation and differentiation (Coll et al.,
2010;Laverrièreetal.,2012).
Metacaspases are posttranslationally regulated by multiple
mechanisms,andtheiractivitydependsonspecificcellularcon-
ditions.TheproteolyticactivityofArabidopsisMC9isregulatedby
S-nitrosylation,byaSerproteaseinhibitor(Serpin1),andbyau-
tocatalytic processing (Vercammen et al., 2004, 2006; Belenghi
et al., 2007). Furthermore, the activities of the Norway spruce
(Piceaabies)metacaspase(mcII-Pa)andtheArabidopsisMC1,
MC4, and MC5 are calcium dependent (Bozhkov et al., 2005;
Watanabe and Lam, 2005, 2011a). Recently, a T. brucei meta-
caspase (MCA4) without detectable proteolytic activity, but
necessaryforparasiticproliferationandvirulence,wasfoundto
beprocessedbyanothermetacaspase,MCA3(Protoetal.,2011).
Despite these results, only a handful of substrates have been
identified so far (Tsiatsiani et al., 2011). As knowledge on the
correspondingsubstratesofproteaseswithunknownfunctioncan
provide insights into their potential function or role in a specific
Figure 1. Expression Characteristics of the MC9 Gene and Molecular
process, new tools that facilitate substrate identification should CharacterizationoftheMC9TransgenicLinesUsedinThisStudy.
assistinexpandingourunderstandingofplantproteasefunctions.
(A)to(D)GUSstainingofrootcapof2-d-oldseedlings(A),rootcapof
Here,weassessedthedegradome(Overalletal.,2004)ofthe
1-week-oldseedling(B),trachearyelementsinexpandingcotyledon(C),
Arabidopsis metacaspase 9 (MC9; AT5G04200) by N-terminal
andpetalspriortoabscission(D).
combinedfractionaldiagonalchromatography(COFRADIC).The
(E) Schematic representation of the T-DNA insertion of the GK-540H0
physiologicalsubstratesofthisplantproteasewereidentifiedon allele.ThecatalyticHisandCys,thep20andp10MC9proteasesub-
the proteome-wide level, and the concrete substrate cleavage units, and the linker region between the two catalytic subunits are
sites were characterized by means of positional proteomics. depicted.
Thankstothesedata,thesubstratespecificityofMC9couldbe (F)ImmunoblotanalysisofArabidopsisseedlings’proteinswithaMC9
resolvedinmoredetail,andtheactivityofphosphoenolpyruvate polyclonalantibody.Full-length35.5-kDMC9isdetectedinthewildtype
carboxykinase 1 (PEPCK1; AT4G37870), a key enzyme in glu- (Col-0)and35S:MC9overexpressor(Vercammenetal.,2006),butnotin
the mc9 knockout line (GK-540H0 allele). An actin antibody directed
coneogenesis,wasfoundtobeenhanceduponMC9-dependent
againstacommonepitopeofallactinisoforms(Immuno,cloneC4)was
proteolysis.
usedasgelloadingcontrol.
(G)TranscriptlevelsofallArabidopsismetacaspasegenes(MC1toMC9)
in 2-d-old seedlings. Values represent the mean of three measure-
RESULTS ments 6 SD relative to the MC9 gene transcript level in the wild type
(Col-0).
ExpressionCharacteristicsoftheArabidopsisMC9Gene
ThespatiotemporalexpressionofArabidopsisMC9wasstudied stage0.5to0.7,includinga3-dstratificationperiod)(Boyesetal.,
in ProMC9:GUS reporter lines by means of b-glucuronidase 2001)(Figure1A),remainedthesamein1-week-oldseedlings,
(GUS) histochemical analysis (for a detailed description, see and was observed also in epidermal cells (Figure 1B). In hy-
Supplemental Methods 1 online). Several independent homo- pocotyls and cotyledons, but not in rosette leaves, discrete
zygous alleles were analyzed with similar results (see represen- differentiatingprimaryandsecondarytrachearyelementswere
tative GUS staining patterns in Figures 1A to 1D). Interestingly, alsostained(Figure1C).Inpetals,astrongsignalappearedjust
the MC9 promoter activity was already obvious in root caps of prior to abscission (Figure 1D). Anther connectives also dis-
young 2-d-old seedlings (corresponding to the developmental playedstrongMC9promoteractivity(Figure1D).
Metacaspase9Degradome 3of17
By quantitative RT-PCR, we assessed the expression of all Furthermore, such N-terminal peptides would imply that the
nine Arabidopsis metacaspases in 2-d-old seedlings of wild- particular protein (form) was not up- or downregulated in the
type Columbia-0 (Col-0) and in the T-DNA insertion knockout tested proteomes. (2) Some neo-N-terminal peptides would
(mc9) and MC9 overexpression (35S:MC9) (Vercammen et al., occurinbothsamples,butwithasignificantlyhigherintensity
2006)transgeniclines(foradetaileddescription,seeSupplemental inthesamplewitheithertheactiveMC9orthesupplemented
Methods2online)(Figures1Eto1G).BothMC9gain-of-function
or loss-of-function plants did not display any obvious aberrant
phenotypes under normal vegetative or reproductive growth
conditions. MC4 expression was the highest (Figure 1G), con-
sistentwithapreviousreportthatitisthemostexpressedmet-
acaspaseatvariousgrowthstages(WatanabeandLam,2011a).
In young seedlings, corresponding to a developmentally active
stage with a dynamic proteome, the MC9 expression was the
secondhighest;therefore,thisstagewaschosenforanalysisof
theMC9degradome(Figure1G).
IdentificationofMC9ProteinProcessingEventsby
N-TerminalCOFRADIC
ToexplorethefunctionofMC9throughidentificationofitsphys-
iological protein substrates, we used the N-terminal COFRADIC
technique(Gevaertetal.,2003;Staesetal.,2011).Incontrastwith
mostproteomicapproaches,N-terminalCOFRADICisapositional
proteomics technology that uses sequential peptide chromatog-
raphytoenrichforthosepeptidesthatcontaintheproteinNtermini
out of thewhole peptide mixture obtained by, for instance, full
trypsindigestionofproteomes.Here,thisso-calledN-terminome
consisted of all peptides that possessed the mature protein
Nterminiandtheneo-N-terminithathadbeengeneratedupon
proteolysis by MC9. Neo-N-termini that emerged at MC9 pro-
cessingsitesdirectlyconveyedtheMC9proteolyticeventsand
theexactsitewheretheproteincleavageoccurred(VanDamme
et al., 2005). We analyzed the in vivo N-terminome of 2-d-old
seedlings of wild-type and MC9 gain-of-function and loss-
of-function transgenic plants (Figures 1E and 1F). First, the
N-terminome of the T-DNA insertion knockout (mc9) seedlings
wascomparedwiththatofwild-typeseedlingsand,second,to
that of seedlings overexpressing MC9 (35S:MC9). Additionally,
a mc9 seedling proteome to which Arabidopsis recombinant
MC9 (rMC9) (Vercammen et al., 2004) had been added in vitro
Figure 2. Illustration of the Different Categories of Isolated (Neo)-N-
was studied versus an untreated mc9 control. To compare the TerminalPeptides.
N-terminomes,N-hydroxysuccinimideestersoflightandheavy
MasstaggingallowedthedifferentialcomparisonofN-terminomesde-
isotopicvariantsofbutyricacidwereusedtomasstagthe(neo)-
rived from samples in which MC9 was active (Col-0 or 35S:MC9 or
N-terminal peptides (see Supplemental Figure 1 online). This dif-
addedrMC9)orabsent(controlsamplemc9).MSspectraofN-terminal
ferential labeling strategy was possible because MC9 introduced peptideswere,inmostcases,identifiedasdoubletionsoflight(inblue)
novel, primary a-amino groups that were uniquely tagged with andheavy(inred)isotopes.m/z,mass-to-chargeratio.
either a light or heavy variant of butyric acid. After this mass (A)Proteincleavedinbothsamplestothesameextent(i.e.,bytrypsin;
tagging,equalamountsoftheproteomepreparationsweremixed yellowarrow)andfragmentsbearingthematureproteinNterminus.As
and digested with trypsin, and (neo)-N-terminal peptides were theamountsofthesepeptideswereapproximatelyequal,noMC9-driven
isolatedwithN-terminalCOFRADIC(seeSupplementalFigure1 proteolysiscouldbededuced.
online). Mass tagging ofneo-N-termini enabled ustodetermine (B)Proteincleavedinbothsamples,butatahigherfrequencyintheMC9
sample(largeredarrow).Theneo-N-terminusfragmentwasgeneratedin
theproteomeoriginofeachlabeledpeptide.Therespectiveion
both samples, but in significantly higher amounts in the MC9 sample.
signal intensities were further used to identify the MC9 pro-
Proteolysisofthetargetinthecontrolsamplemighthaveoccurredby
cessedsites.
aredundantproteaseactivity(bluearrow).Thefragmentisidentifiedas
The following MS readouts were expected: (1) Some (neo)-
adoubletwiththehighestintensityionderivedfromtheMC9sample.
N-terminal peptides would be found at equal intensity in both (C)ProteinsolelycleavedbyMC9(redarrow)inthesamplesofactiveMC9
samples.SuchpeptidesmightcarrythematureproteinNterminus proteases.Thereby,afragmentwithaneo-N-terminuswasgenerated,later
and would not be indicative of MC9 proteolysis (Figure 2A). identifiedasuniqueintheMC9N-terminomeand,thus,asasingletonion.
4of17 ThePlantCell
rMC9. This outcome would mean that the corresponding pro- of the Arg and Lys P1-specific cleavage sites (Schechter and
teinswerealsocleavedinthecontrolsample(lackingMC9ac- Berger,1967)andanalyzedthefrequenciesofspecificresidues
tivity),althoughtoalesserextent,byanotherprotease,hintingat afterstatisticalcorrectionbymeansofthenaturaloccurrenceof
aredundantproteaseactivity(Figure2B).Sucheventscouldbe aminoacidsinArabidopsisproteins.Thespecificityinformation
explainedbyascenarioinwhichtheMC9perturbationactivated derived from the three data sets was very similar. The large in
otherproteases(i.e.,metacaspases).(3)Someneo-N-terminal vitro COFRADIC data set showed that the acidic residues Asp
peptides(so-calledsingletons)wouldbeuniquelyfoundinthe andGluwereoftenfoundatneighboringpositionstothebasic
proteome either with the active MC9 (Col-0 or 35S:MC9 pro- (Arg or Lys) cleavage site. For instance, in 46 and 47% of the
teome) or supplemented with rMC9, implying that the corre- Lys-cleaved and Arg-cleaved sequences, respectively, the P19
spondingproteinswerecleavedbyMC9(Figure2C). position(C-terminallytothecleavageP1position)wasoccupied
Peptideswereanalyzed withanLTQOrbitrap XLmass spec- byAsporGluand,likewise,theP2positionin23and22%ofthe
trometer. The obtained tandem mass spectrometry (MS/MS) Lys-cleavedandArg-cleavedsequences,respectively.However,
spectra were searched against The Arabidopsis Information onlyin5and7%oftheLys-cleavedandArg-cleavedsequences,
Resource database (release 8) with the Mascot algorithm and respectively, both P2 and P19 were acidic (Figure 3A; see
subsequently mapped to the TAIR10 database with DBToolkit SupplementalFigures3and4Aonline).Attheprimesite,Asp
(Martensetal.,2005)(foradetaileddescription,seeSupplemental orGluwasalsoobservedin27%ofthecasesatP39.AtP29,the
Methods3online).Intotal,75,310MS/MSspectraweregenerated smallaminoacidsThr,Ala,andValoccurredin38%ofallcases,
inthemc9/Col-0analysis,69,388inthemc9/35S:MC9analysis, whereasAlawastoleratedateveryposition.Analogoustothein
and 75,042 in the in vitro analysis, identifying 3781, 2879, and vivoCOFRADIC-derivedspecificity(Figure3B),ValandGluwere
3050peptidesandconvergingto1705,1407,and1138proteins, preferredatP4andLysandArgwerefrequentlyfoundatP3.Ala
respectively.Theratiosoflight/heavypeptidesignalsforalliden- andThrwerethesolepreferredresiduesatP2intheinvivo–cleaved
tified peptides were calculated with the Mascot Distiller software substrates,butinvitro–cleavedproteinsadditionally(23%ofthe
and were individually checked for possible neo-N-terminal pep- cleavedsubstrates)toleratedAspandGluatthisposition(Fig-
tidespointing to MC9 processing. We focused on two classes ures 3A and 3B; see Supplemental Figures 3 and 4 online).
of peptides: the significantly enriched neo-N-terminal peptides RegardingtheprimesitesubstratespecificityofMC9,theinvivo
(Z-score#22.33orZ-score$2.33,dependingonthelabeling andinvitrodatawerelargelysimilar,exceptfortheaminoacid
usedtocalculatethelight/heavyintensityratios)andtheunique toleranceatP29:Inthelatter,smallresidueswerepresentatthis
peptides (singletons) in the Col-0, 35S:MC9, or rMC9-treated position, whereas aromatic residues, such as Phe and Tyr and
seedlingproteomesincomparisontomc9(Figures2Band2C). thealiphaticIleandLeu,werefoundalsointheformer.
Astheformermightalsoappearbecauseofdifferentialprotein Inconclusion,theMC9specificityforbasicresiduesattheP1
levelsin theinput proteomes, wescanned thedataforthecorre- sitewas accompanied bya strong preference for acidicamino
spondingprecursorproteinNterminiorotherneo-N-terminalpep- acids at the P19 site (see Supplemental Figure 4 online). This
tidesfromtheseprecursorproteins,whichwerenotcleavedby particular trend of acidic-basic amino acid combinations was
MC9.Comparisonofthesepeptidelevelsledustoconcludethatno also seen at other positions at the nonprime site (P4-P3),
largeproteomechangesoccurredbetweenthedifferentproteomes whereasAlawasgenerallytoleratedateveryposition.Arelative
assayed.Foranoverviewofallcleavagesites,theircorresponding preferenceoccurredforLysoverArgattheP1siteandinvivo,
substrates,andtheirprecursorproteinNterminiidentifiedinthe often (27% of the cases) a second Arg or Lys at P3. For in-
threeanalyses,seeSupplementalDataSets1Ato1Conline. stance, when Lys was present at P1, 24% of these substrates
AfterfilteringthedatawiththeknownMC9specificityforArg containedasecondLysatP3.
andLys,72%ofallprocessedsitesmatchedtheexpectedMC9 Previously, in a positional scanning of a synthetic combina-
specificity (28 and 44% cleavage C-terminally to Arg and Lys, toriallibrary(PS-SCL)of7-amino-4-methylcoumarin(AMC)–labeled
respectively), without significant enrichment for a particular synthetic tetrapeptides spanning the positions P4-P1, VRPR
aminoacidintheotherprocessingevents.Intotal,551cleavage was found as the most preferred rMC9 tetrapeptide substrate,
eventsafterArgorLys(116inmc9/Col-0,103inmc9/35S:MC9, whereas IISK, the best cleaved peptide with Lys at P1, was
and 332 in vitro) (see Supplemental Figure 2A online) were fivefold lessefficiently cleavedthanVRPR (forVal-Arg-Pro-Val)
identifiedin392proteins(97inmc9/Col-0,84inmc9/35S:MC9, (Vercammenetal.,2006).Ourcurrentresultssomewhatcontrast
and211invitro)(seeSupplementalFigure2Bonline),fromwhich withthestrongerpreferenceforArgoverLysatP1,buttheyare
approximately one-third (160 sites) were reported as singleton in agreement with the preferred presence of a dibasic amino
peptides in either Col-0, 35S:MC9, or rMC9-treated proteomes acid motif (such as RxR) as in VRPR. When the two in vitro
(16inmc9/Col-0,11inmc9/35S:MC9,and133invitro)and391 approaches (in vitro COFRADIC and PS-SCL) were compared
assignificantlyenrichedpeptidesinthesameproteomes(100in (Figure 3C), Val was the most prevalent residue at P4 in both
mc9/Col-0,92inmc9/35S:MC9,and199invitro)(seeSupplemental data sets of Arg-cleaved substrates. However, a major differ-
DataSets1Ato1Conline) ence was observed at the P2 site: The COFRADIC data in-
dicated that Thr and Asp were preferred and the PS-SCL data
PositionalAminoAcidCleavageSpecificityofMC9 showedapreferenceforhydrophobicresiduesorPro.Basedon
the in vivo and in vitro amino acid frequencies at the P4-P39
Using iceLogo (Colaert et al., 2009), we inspected the amino substrate positions derivedfrom theCOFRADIC analyses (Fig-
acidsequencesattheprime andnonprimesubstrate positions ure 3; see Supplemental Figures 3 and 4 online), two revised
Metacaspase9Degradome 5of17
Figure3. MC9SpecificityMonitoredviaItsProteome-WideSubstrateProfiling.
AnalysiswasperformedwithiceLogo(Colaertetal.,2009)aftermultiplesequencealignmentandstatisticalcorrectionwiththenaturaloccurrenceof
aminoacidsinArabidopsisproteins.HeatmapsillustratingtheaminoacidfrequenciesattheP4-P39positionsintheMC9substratesasobservedinthe
invivomc9/Col-0andmc9/35S:MC9COFRADICstudies(A),theinvitroCOFRADIC(B),andthePS-SCLapproach(C).Rsoftwarewasusedtobuild
thePS-SCLheatmapswiththepublishedvalues(Vercammenetal.,2006).Arrowsmarkthecleavedscissilebonds.
6of17 ThePlantCell
consensus sequences of the MC9 substrate specificity are identical to the COFRADIC-deduced siteR214↓E. As for GRP7,
proposedforeithercleavageafterArgandLys,namely,[V]-[R]- thepeptide precursor wasoriginally expected tobecleaved at
[T/Q]-[R]-↓-[G/D/E]-[V/F/L/I]-[D/E] and [E/Q]-[K/A]-[T/E]-[K]-↓-[D/E]- theMK64↓DsitebutwasadditionallyprocessedatEK61↓A,asimilar
[Y/T]-[D/E]todetecttheMC9activityinbiochemicalassays.By site,becauseinbothcases,Kwasadjacenttoanacidicresidue
meansofGenomeNet(http://www.genome.jp)andMotifSearch, (AsporGlu).Furthermore,theconsensussequenceforLys-cleaved
wescreenedtheArabidopsisproteomeforthepresenceofthese peptidessupportedthepreferenceforacidicresidueseitheratthe
sequences.Thenumberofproteinhitsvarieddependingonthe N-terminalorC-terminalsideofthecleavedLys.
lengthofthemotif.Infact,theP4-P1’sequences[V]-[R]-[T/Q]-[R]- To validate experimentally the substrate cleavages at the
↓-[G/D/E] and [E/Q]-[K/A]-[T/E]-[K]-↓-[D/E] were found in more proteinlevel,weemployedinvitrotranscriptionandtranslation
than20and380proteinhits,respectively(seeSupplementalData coupledassays.Intotal,wesuccessfullytranslated16proteins,
Set1Donline).Nevertheless,theseobservationsarebasedonly all of which were cleaved by rMC9. Although the precursor
on the sequences of the queried proteins and do not take into proteinlevelsclearlydecreasedorevenwerecompletelynegli-
accountwhetheranyofthepredicted motifsaresolventacces- gible after the rMC9 treatment, for some substrates, cleavage
sibleorresideinasecondarystructureelementnot readilyrec- fragments were not always observed. It is possible that the
ognizedbytheprotease. substrate fragments were not detectable by autoradiography
because they did not contain a radiolabeled Met or that pro-
teolyticfragments<20kDmighthavebeentoosmallforproper
ExperimentalValidationofMC9ProteinSubstrates
separation and visualization by the SDS-PAGE system used.
FortheexperimentalvalidationoftheMC9substrates,weprior- Thesubstratesthatwereprocessedintofragmentsofcorrectly
itized all in vivo singleton peptides together with those proteins predicted molecular weight based on the cleavage site(s) pre-
bearingcleavagesitesidentifiedinmorethanoneproteomicanal- viouslyidentifiedbyCOFRADICandMS,wereGRF5,PEPCK1,
ysis.Inthismanner,99sitesin74differentproteinswereselected nicotinamideadeninedinucleotide(NAD)synthetase(AT1G55090),
forcleavagevalidation(Table1),fromwhich18siteswereiden- GDP-mannose 4,6-dehydratase 2 (GMD2; AT3G51160), penta-
ticalinthethreedifferentsetups,35sitesinbothinvivoanalyses, tricopeptide repeat (PPR)–containing protein (AT1G02150), and
19sitesbetweenthemc9/Col-0invivoandtheinvitroanalysis, citrate synthase3 (CSY3; AT2G42790) (Figure 4). Furthermore,
and finally 15 sites between the mc9/35S:MC9 in vivo and the these proteins were not cleaved by the inactive rMC9 mutant,
invitroanalysis.Theremaining12siteswerereportedasinvivo rMC9C147AC29A(Belenghietal.,2007),indicatingthattheob-
singletonpeptides(Table1;seeSupplementalFigure2Aonline). served fragments were indeed due to rMC9 and not to other
Two independent validation approaches were chosen. First, (contaminating)proteasespresentintheassaymixture.
18–amino acid peptides harboring the identified cleavage sites
were synthesized for six randomly selected candidate protein
MC9CleavesPEPCK1inVivo,LeadingtoIncreased
substratesandusedforinvitrorMC9cleavageassays.Second,
PEPCK1Activity
in vitro–transcribed and translated, radiolabeled (35S-Met) pro-
teins, generated from available cDNA clones (ABRC), were in- PEPCK, which catalyzes the reversible ATP-dependent de-
cubatedwithrMC9andtheirdigestionproductswereanalyzed carboxylationofoxaloacetatetophosphoenolpyruvate,isinvolved
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ingluconeogenesis,photosyntheticCO -concentratingmecha-
2
(SDS-PAGE)andautoradiography. nisms during C4 photosynthesis (Edwards et al., 1971), Cras-
AfterincubationwithincreasingconcentrationsofrMC9(0,31, sulaceanacidmetabolism(Dittrichetal.,1973),andnitrogenous
125,and500nM),syntheticpeptidesandtheirdigestionproducts compoundmetabolism(LeegoodandWalker,2003).AsPEPCK
wereseparatedbyreverse-phaseHPLCandanalyzedbymatrix- is posttranslationaly regulated by reversible phosphorylation
assisted laser desorption/ionization-time-of-flight mass spec- (WalkerandLeegood,1995)andpronetorapidproteolyticcleav-
trometry (for a detailed method description, see Supplemental ageinplanta(Walkeretal.,1995,1997;Maloneetal.,2007),which
Methods 4 online). Peptides spanning the MC9 cleavage sites gives rise to fragments with sizes that correlate to the cleavage
identified in trypsin inhibitor 2 (AT1G47540), general regulatory sitesobservedbyN-terminalCOFRADIC,wefocusedontheMC9-
factor5(GRF5;AT5G16050),Gly-richRNAbindingprotein(GRP7; dependentPEPCK1cleavage.Tothisend,weassessedwhether
AT2G21660),andprotochlorophyllideoxidoreductaseB(PORB; MC9wasresponsibleforinplantacleavageofPEPCK1,whichis
AT4G27440) were cleaved by rMC9 at the sites identified by highlyactiveduringgerminationatthe2-d-oldseedlingstageof
COFRADIC.Thedegreeofpeptideprocessingincreasedwithin- thestudiedproteome(Rylottetal.,2003).
creasingrMC9concentrations(seeSupplementalFigure5online). PEPCK1 polypeptides (74 and 73 kD) were previously immu-
Forproteinscontainingmorethanoneidentifiedcleavagesite, nodetectedinArabidopsiscotyledonswithapolyclonalantiserum
suchasthelate-embryogenesisabundant(LEA)domain–containing raised against PEPCK from Guinea grass (Panicum maximum)
protein(AT2G42560),PEPCK1(AT4G37870),andGRP7,twosyn- leaves (Malone et al., 2007). Similarly, in pea (Pisum sativum)
theticpeptidesweretested(seeSupplementalFigure6online).All cotyledons, besides the mature 72-kD form, a smaller 68-kD
thesepeptideswerecleavedbyrMC9atthesitesidentifiedby PEPCK polypeptide was detected that was attributed either to
COFRADIC.However,additionalalternativecleavagesiteswere a genuine isoform or to a regulatory protein processing event
observedaswell,probablyduetosequencesimilaritieswiththe (Delgado-Alvaradoetal.,2007).Recently,thepineapple(Ananas
COFRADIC-deducedsites.Forinstance,theadditionalcleavage comosus) PEPCK had been found to have two active poly-
siteR212↓EintheLEAdomain–containingproteinprecursorwas peptide variants of 74 and 65 kD in vivo (Martín et al., 2011).
Metacaspase9Degradome 7of17
Table1.SelectionofNeo-N-TerminalPeptidesIdentifiedinatLeastTwoIndependentAnalyses.
No.ofCleavage
Sites Accession P4-P39 Sequence Description
Identifiedinallthreeanalyses
1 AT1G12000 AVTR(10)DLT DLTAVGSPENAPAKGR Phosphofructokinasefamilyprotein
2 AT1G49240 MNQK(53)DAY DAYVGDEAQSKR Actin8
3 AT2G21060 HMAR(150)ECS ECSQGGGGYSGGGGGGR Gly-richprotein2B
4 AT2G42560 VGSK(454)AVD AVDLTKEKAAVAADTVVGYTAR LEAdomain–containingprotein
5 AT3G02480 MKEK(51)AQG AQGAADVVKDKTGMNKSH LEAfamilyprotein
6 AT3G09840 KSKK(15)DFS DFSTAILER Celldivisioncycle48
7 AT3G17520 EKAK(184)EAK EAKEAAKR LEAfamilyprotein
8 AKDK(262)ASQ ASQSYDSAAR
9 AT3G53040 QKTK(111)ETA ETADYTADKAR Putative/LEAprotein
10 DKTK(155)ETA ETAEYTAEKAR
11 AT4G02930 GKAK(99)AIA AIAFDEIDKAPEEKKR GTPbindingElongationfactorTufamilyprotein
12 AT4G21020 EKAK(156)DYA DYAEDTMDNAKEKAR LEAfamilyprotein
13 AT4G37870 SFPK(20)GPV GPVMPKITTGAAKR PEPCK1
14 AT5G16050 EAPK(254)EVQ EVQKVDEQAQPPPSQ Generalregulatoryfactor5
15 AT5G44310 ERTK(70)DYA DYAEQTKNKVNEGASR LEAfamilyprotein
16 EKTK(110)DYA DYAEEAKDKVNEGASR
17 EKTK(179)NYA NYAEQTKDKVNEGASR
18 AT5G47210 RYAK(341)EAA EAAAPAIGDTAQFPSLG Hyaluronan/mRNAbindingfamily
Identifiedinbothinvivoanalyses
1 AT1G04750 VLDR(156)GEK GEKIELLVDKTENLR Vesicle-associatedmembraneprotein721
2 AT1G07780 SVAK(109)DIS DISKVAR Phosphoribosylanthranilateisomerase1
3 AT1G09310 TEAK(165)EAV EAVAIKEAVAVKEAA Proteinofunknownfunction,DUF538
4 AT1G11480 ARPR(404)ELV ELVLKER Eukaryotictranslationinitiationfactor-related
5 AT1G29350 QNAR(525)ELD ELDFQYSPFSAQQSMQSR Kinase-relatedproteinofunknownfunction
(DUF1296)
6 AT1G47540 AYDR(31)KCL KCLKEYGGDVGFSYCAPR Trypsininhibitor2
7 AT1G49240 SIEK(241)NYE NYELPDGQVITIGAER Actin8
8 AT1G49970 FEKR(363)DYD DYDGTLAQR CLPproteaseproteolyticsubunit1
9 AT1G51710 VATR(157)ELF ELFGELDR Ubiquitin-specificprotease6
10 AT1G55490 TVAR(114)EVE EVELEDPVENIGAKLVR Chaperonin60b
11 AT1G56070 ICLK(544)DLQ DLQDDFMGGAEIIKSDPVVSFR RibosomalproteinS5/ElongationfactorG/III/V
familyprotein
12 AT2G01250 VESK(6)VVV VVVPESVLKKR RibosomalproteinL30/L7familyprotein
13 AT2G18960 GSYR(902)ELS ELSEIAEQAKR H(+)-ATPase1
14 AT2G19730 SVNK(88)SIL SILKKEFPR RibosomalL28eproteinfamily
15 AT2G26250 IVNR(18)GIE GIEPSGPNAGSPTFSVR 3-Ketoacyl-CoAsynthase10
16 AT2G28000 PRGR(82)NVV NVVLDEFGSPKVVNDGVTIAR Chaperonin-60a
17 TIAR(103)AIE AIELPNAMENAGAALIR
18 AT2G32240 VKSR(709)DID DIDLSFSSPTKR Unknownprotein
19 AT2G36620 NIPK(149)SAA SAAPKAAKMGGGGGR RibosomalproteinL24
20 AT2G42560 ARAK(324)DYT DYTLQKAVEAKDVAAEKAQR LEAdomain–containingprotein
21 AT3G13300 VGER(708)NLD NLDVSSVEEISR Transducin/WD40repeat-likesuperfamilyprotein
22 AT3G53040 TVLK(449)EAD EADQMTGQTFNDVGEIDDEEKVR Putative/LEAprotein
23 AT3G61260 SLDR(92)DVK DVKLADLSKEKR Remorinfamilyprotein
24 AT4G15410 LRSR(109)GGA GGAGENKETENPSGIR Ser/Thrproteinphosphatase2A
25 AT4G20360 ERAR(127)GIT GITINTATVEYETENR RABGTPasehomologE1B
26 AT4G27440 YITK(337)GYV GYVSETESGKR ProtochlorophyllideoxidoreductaseB
27 AT4G31700 IVKK(119)GEN GENDLPGLTDTEKPR RibosomalproteinS6
28 AT5G04200 LFGR(216)DAG DAGLKFR Metacaspase9
29 AT5G06140 EQPR(9)NIS NISGSMQSPR Sortingnexin1
30 AT5G12140 GGVR(15)DID DIDANANDLQVESLAR Cystatin-1
31 AT5G44310 EKTK(150)DFA DFAEETKEKVNEGASR LEAfamilyprotein
32 AT5G52440 TLER(145)EIG EIGLDDISTPNVYNQNR Bacterialsec-independenttranslocationprotein
mtta/Hcf106
33 AT5G54190 YITK(341)GYV GYVSESEAGKR ProtochlorophyllideoxidoreductaseA
34 AT5G56000 SSKK(604)TME TMEINPENSIMDELR Heatshockprotein81.4
(Continued)
8of17 ThePlantCell
Table1. (continued).
No.ofCleavage
Sites Accession P4-P39 Sequence Description
35 AT5G62575 TREK(42)ALL ALLAEDSALKR Unknownprotein
Identifiedinthemc9/35S:MC9andtheinvitroanalysis
1 AT1G13270 RKMR(78)ELE ELETKSKVR Metaminopeptidase1B
2 AT1G54870 EKIK(288)NFG NFGSEVPMKR Aldehydereductase
3 AT1G72370 LGTK(33)NCN NCNYQMER 40sribosomalproteinSA
4 AT2G21660 KAMK(64)DAI DAIEGMNGQDLDGR Gly-rich,RNAbindingproteinatgrp7
5 AT2G42560 MRER(214)EGK EGKESAGGVGGR LEAdomain–containingprotein
6 ATEK(268)GKE GKEAGNMTAEQAAR
7 EKGK(270)EAG EAGNMTAEQAAR
8 VEAK(335)DVA DVAAEKAQR
9 AT3G17520 ESAK(260)DKA DKASQSYDSAAR LEAfamilyprotein
10 AT3G22640 TRSK(231)EIG EIGQGIIR Cupinfamilyprotein
11 AT3G51160 TAPK(20)ADS ADSTVVEPR Gdp-D-mannose-4,6-dehydratase2,gmd2
12 AT3G53040 DKTK(133)ETA ETADYAAEKAR Putative/LEAprotein
13 AT4G37870 SLTR(102)ESG ESGPKVVR PEPCK1
14 AT4G39260 DLQR(25)TFS TFSQFGDVIDSKIINDR Cold,circadianrhythm,andRNAbinding1
15 AT5G10360 IVKK(119)GVS GVSDLPGLTDTEKPR Ribosomalproteins6e
Identifiedinthemc9/Col-0andtheinvitroanalysis
1 AT1G47540 CAPR(49)IFP IFPTFCDQNCR Trypsininhibitor2
2 AT1G55490 VLTK(382)ETS ETSTIVGDGSTQDAVKKR Chaperonin60b
3 AT1G65090 TSER(162)TLQ TLQDDKKSGNAKSEEVQEQPEKR Unknownprotein
4 AT2G21660 TDDR(23)ALE ALETAFAQYGDVIDSKIINDR Gly-rich,RNAbindingproteinatgrp7
5 AT2G36640 QKTR(338)EST ESTESGAQKAEETKDSAAVR Embryoniccellprotein63
6 AT2G40490 VTER(47)KVS KVSATSEPLLLR Uroporphyrinogendecarboxylase
7 AT2G42560 QTTK(607)NIV NIVIGDAPVR LEAdomain–containingprotein
8 AT3G02480 QQMK(49)EKA EKAQGAADVVKDKTGMNKSH LEAfamilyprotein
9 AT3G11710 QTTK(11)ALS ALSELAMDSSTTLNAAESSAGDGAGPR Lysyl-tRNAsynthetase1
10 AT3G12960 EKGK(58)EQS EQSAASGDQTQIQR Unknownprotein
11 AT3G13470 VLTK(378)EMT EMTTIVGDGTTQEAVNKR TCP-1/cpn60chaperoninfamilyprotein
12 AT3G15280 TEYR(135)GVE GVEDLHQQTGGEVKSP Unknownprotein
13 AT3G15670 IKNK(134)AQD AQDAAQYTKETAQGAAQYTKETAEAGR LEAfamilyprotein
14 QYTK(143)ETA ETAQGAAQYTKETAEAGR
15 AT3G48870 ISDR(501)FLP FLPDKAIDLIDEAGSR ClpATPase
16 AT4G02510 VSSR(607)EFS EFSFGGKEVDQEPSGEGVTR Transloconattheouterenvelopemembraneof
chloroplasts159
17 AT4G21020 EKAK(116)DTA DTAYNAKEKAKDYAER LEAfamilyprotein
18 ESTK(218)NAA NAAQTVTEAVVGPEEDAEKAR
19 AT4G38630 QKDK(258)DGD DGDTASASQETVAR Regulatoryparticlenon-ATPase10
Fractionofsingletonneo-N-terminalpeptidesthatweresolelyfoundinoneoftheinvivoanalyses
1 AT1G02150 VTSK(474)EAV EAVLELLR Pentatricopeptiderepeat–containingprotein
2 AT1G55090 NSNK(710)EIG EIGVVAANSGDPSAGL NADsynthetase
3 AT3G23990 YAAK(35)EIK EIKFGVEAR Heatshockprotein60
4 AT3G45190 VHDR(558)DYD DYDLAGLANNLNQFR SIT4phosphatase-associatedfamilyprotein
5 AT2G42790 KEDK(86)GLK GLKLYDPGYLNTAPVR Citratesynthase3,CSY3
6 AT2G47470 YIEK(320)GSD GSDYASKETER Thioredoxinfamilyprotein
7 AT3G23990 LMLK(49)GVE GVEDLADAVKVTMGPKGR Heatshockprotein60
8 AT4G05180 DQAR(110)DFS DFSLALKDR PhotosystemIIsubunitQ-2
9 AT4G34450 GIEK(27)GAV GAVLQEAR Coatomerg-2subunit,putative
10 AT5G16130 LTGK(181)DVV DVVFEYPVEA RibosomalproteinS7efamilyprotein
11 AT5G35910 ESTR(578)DLI DLIMGAANTNEGR Polynucleotidyltransferase
12 AT5G54310 QSGK(469)DFD DFDFSSLMDGMFTKH ARF-GAPdomain5
Theequivalentexperiments,peptidesequence,accessionnumber,andproteindescriptionaregiven.AminoacidpositionsoftheP1’sitesareindicated
inparenthesesintheP4-P39column.
Metacaspase9Degradome 9of17
proteaseactivityordegradation.Takentogether,ourdataindicate
thatoverexpressionofMC9increasedthePEPCK1processing
invivo.
To assess the effect of the MC9-dependent PEPCK1 cleav-
age, we measured the carboxylation activity of PEPCK1 in
youngseedlingextractsofCol-0,pepck1(Penfieldetal.,2004),
andMC9gain-of-functionandloss-of-functionmutants(Figure
6A).PEPCK1activitywasthehighestin35S:MC9plantextracts
(46 and 71% higher than that of Col-0 and mc9, respectively),
revealing that MC9 cleavage enhanced PEPCK1 activity and
that proteolysis might remove the putative PEPCK1 inhibitory
phosphorylation site (Ser-62). As PEPCK1 is involved in Suc
synthesis from lipids during oilseed plant germination, the
pepck1 mutants are impaired in hypocotyl elongation when
grown in the dark in Suc-deprived growth media (Rylott et al.,
2003;Penfieldetal.,2004).Hence,undertheseconditions,we
monitoredthehypocotylelongationofpepck1,mc9,Col-0,and
35S:MC9 for a period of 10 d after germination. As expected,
pepck1 hypocotyls were the smallest and the mc9 hypocotyl
growth was significantly compromised (P value < 0.05) in com-
parisontothatofCol-0.Reciprocally,35S:MC9hypocotylswere
thelongest(17.4,36.9,and56.3%longerthanCol-0,mc9,and
pepck1hypocotyls,respectively)(Figures6Band6C).Thesere-
sultsareindicativeofapositiveregulatoryeffectofMC9onthe
PEPCK1activityduringgluconeogenesis.
MC9SubcellularLocalization
Because of the apparent discrepancy between the cytosolic
Figure 4. In Vitro–Transcribed and Translated Proteins Incubated with
localizationofPEPCK1andthedetectionofMC9intheapoplast
Wild-Type Recombinant MC9 Protease and the Catalytically Inactive
of35S:MC9lines(Vercammenetal.,2006),wereassessedthe
MutantrMC9CACA.
subcellular localizations of MC9 and PEPCK1 by transiently
Arrowheads indicate the proteolytic fragments with molecular masses expressing N- and C-terminal green fluorescent protein (GFP)–
matchingthoseidentifiedbyCOFRADIC. tagged MC9 and PEPCK1 in tobacco (Nicotiana benthamiana)
leaves(foradetaileddescription,seeSupplementalMethods5
online).PEPCK1fusionproteinswereexclusivelylocalizedinthe
In cucumber (Cucumis sativus), an in vivo phosphorylation site cytosol(Figure7A)andN-andC-terminalMC9fusionproteins
is located in the proteolytically removed N-terminal fragment in the nucleocytoplasm (Figures 7B and 7C), but not in the
(WalkerandLeegood,1995).InGuineagrass,theactivityofthe apoplast, possibly dueto theGFPinstability in theacidicapo-
full-length phosphorylated PEPCK was lower than that of the plast environment (Ward, 1981; Patterson et al., 1997). To ex-
full-length or truncated dephosphorylated PEPCK, indicating clude that the nuclear GFP signal was due to diffusion of free
thatremovalofthephosphorylationsite(s)–containingN-terminal GFP after proteolytic processing of the fusion protein, we eval-
regionmightrestorethePEPCKactivity(Walkeretal.,2002). uatedtheintegrityoftheMC9fusionproteinsbyproteingelblot
Cleavage of PEPCK1 by MC9 in vivo and in vitro at Lys-19 analysis(Figure7D).An;45-kDGFP:MC9proteinwasfoundthat
and Arg-101 (Figures 4 and 5A; see Supplemental Figure 6 corresponded with the 25-kD GFP protein fused with the large
online)ledtoC-terminalfragmentsof72and63kD,respectively p20subunitofMC9(20kD).Inthiscase,thesmallp10subunit
(Figure5B).Withrabbitpolyclonalantibodiesraisedagainstthe (15kD)wasremovedafterautocatalyticactivationoftheprotease
recombinant Arabidopsis PEPCK1, we monitored the PEPCK1 (Vercammenetal.,2004),indicatingthatMC9wasactiveinthe
processing in MC9 gain-of-function and loss-of-function mu- observed subcellular compartments. For the C-terminal MC9:
tants.Proteinextractsofmc9,Col-0,and35S:MC9from2-d-old GFPfusion,an;60-kDproteincorrelatedwiththefulllengthof
seedlings were prepared under postlytic proteolysis-inhibiting thefusionprotein,revealingthattheGFPfusionattheCterminus
conditions, directly mixed with the SDS-PAGE loading buffer, of MC9 might have interfered with the proteolytic separation of
and heated before protein gel blot analysis with anti-PEPCK1. the two subunits. However, MC9 localized in the same cellular
The 74-kD precursor protein was processed in the 35S:MC9 compartmentsirrespectiveoftheGFPfusionconformations.
extracts, whereas in Col-0 or mc9 extracts, it remained pre- To confirm the nucleocytoplasmic localization of MC9, we
dominantlyintact(Figure5C).Smallercross-reactivefragments additionally analyzed Arabidopsis roots of the ProMC9:MC9:
of 57to 68 kDwereequally abundant in mc9,Col-0,and 35S: GFP reporter lines. Interestingly, the localization of MC9 in the
MC9 extracts, implying that they could result from another nucleusandcytoplasmofepidermalcellswasconfirmed(Figure
10of17 ThePlantCell
Figure5. CleavageofthePEPCK1ProteinasDeducedfromtheCOFRADICDataandinVivoValidatedbythePEPCK1ImmunodetectionintheWild
TypeandtheMC9Gain-andLoss-of-FunctionMutantExtracts.
(A)MassspectraofthePEPCK1proteolysisreporterpeptidesasidentifiedinmc9/Col-0,mc9/35S:MC9,andtheinvitrostudies.Mass/chargevalues
foreachisotopearegivenontopofeachpeptideion.Butyrylatedresiduesareunderlined.
(B)SchematicrepresentationofPEPCK1proteolysisbyMC9attheidentifiedcleavagesitesandgenerationoffragmentswithvariablesize.
(C)Immunodetectionof(i)PEPCK1proteininextractsof2-d-oldseedlingsfrompepck1,35S:MC9,Col-0,andmc9,(ii)full-lengthMC9zymogen,and
(iii)CAT2(AT4G35090)loadinggelcontrol.ThePEPCK1proteolyticfragmentsof57to68kDaremarkedwitharrowheads.
7E). Altogether, our localization study demonstrated that MC9, (Moss et al., 2007). As EF-Tu was cleaved upon the bacterial
besides its apoplastic location, is also present in nucleus and production of the recombinant Tb MCA2, the EF-Tu cleavage
cytoplasm. had not been demonstrated to be a physiologically relevant
proteolytic event (Moss et al., 2007). Interestingly, we also
identifiedanelongationfactorTufamilyprotein(AT4G02930)to
DISCUSSION
be cleaved in vitro and in vivo by MC9 (Table 1). Furthermore,
Todate,ourknowledgeonmetacaspasesubstratesislimitedto althoughthecleavagesiteswerenotidenticalasreported pre-
theAtSerpin1inhibitorofAtMC9(Vercammenetal.,2004),the viously,theArabidopsisTSN-1(AT5G07350),TSN-2(AT5G61780),
udor staphylococcal nuclease (TSN) target of Norway spruce andGAPDHBsubunit(AT1G42920),aproteinsharing37%amino
mcII-pa(Sundströmetal.,2009),theglyceraldehyde-3-phosphate acidsequencesimilaritytotheYCA1substrateGAPDH,wereall
dehydrogenase (GAPDH) target of YCA1 (Silva et al., 2011), the cleavedbyrMC9invitroatconservedArgorLysresidues(see
metacaspaseTbMCA4targetofTbMCA3(Protoetal.,2011),and Supplemental Data Set 1C online). Therefore, it is tempting to
theEscherichiacolielongationfactor(EF-Tu)targetofTbMCA2 hypothesizethatmetacaspasesubstratesareconservedacross