Human CLPP reverts the longevity phenotype of a fungal ClpP deletion strain.

0.36 MB·English

by  Fischer, Fabian

ARTICLE
Received27Jul 2012 |Accepted 18 Dec 2012 |Published 29 Jan 2013 DOI:10.1038/ncomms2397 OPEN
Human CLPP reverts the longevity phenotype
of a fungal ClpP deletion strain
Fabian Fischer1, Andrea Weil1, Andrea Hamann1 & Heinz D. Osiewacz1
Mitochondrial maintenance crucially depends on the quality control of proteins by various
chaperones,proteasesandrepairenzymes.Whilemostoftheinvolvedcomponentshavebeen
studied in some detail, little is known on the biological role of the CLPXP protease complex
locatedinthemitochondrialmatrix.HereweshowthatdeletionofPaClpP,encodingtheCLP
protease proteolytic subunit CLPP, leads to an unexpected healthy phenotype and increased
lifespan of the fungal ageing model organism Podospora anserina. This phenotype can be
reverted by expression of human ClpP in the fungal deletion background, demonstrating
functionalconservationofhumanandfungalCLPP.Ourresultsshowthatthebiologicalroleof
eukaryotic CLP proteases can be studiedin an experimentally accessible model organism.
1JohannWolfgangGoetheUniversity,FacultyforBiosciences&ClusterofExcellence,‘MacromolecularComplexes’Frankfurt,InstituteofMolecular
Biosciences,Max-von-Laue-Stra(cid:2)e9,60438Frankfurt,Germany.CorrespondenceandrequestsformaterialsshouldbeaddressedtoH.D.O.
(email:[email protected]).
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M
itochondriaareorganellesofeukaryoticorganismsthat demonstrating that overexpression of the gene encoding the
are involved in a number of vital processes, including mitochondrial LON proteasehomologuePaLON1in P. anserina
the assembly of iron/sulphur clusters, biosynthesis of results in robust lifespan extension10. In this context, the
lipids and amino acids and the generation of ATP via oxidative DPaClpP strain served as a control to assess the contribution of
phosphorylation. Therefore, it is not surprising that mitochon- PaCLPP to the mitochondrial matrix serine protease activity but
drial dysfunction is associated with numerous adverse phenom- was not further characterized.
ena such as ageing1–3 or degenerative diseases like Parkinson’s Here we report this characterisation. After verification of
andAlzheimer’sdisease4.Tomaintainafunctionalpopulationof DPaClpP by Southern blot analysis (Fig. 1b), absence of the
mitochondria, a complex network of quality control pathways is protein in the deletion strain was confirmed by western blot
active1–3.Inthisnetwork,proteinqualitycontrolisfundamental. analysis(Fig.1c).Understandardgrowthconditionsat271Cand
In the mitochondrial matrix of most eukaryotic organisms, two constant light, the PaClpP deletion strain displayed no
soluble AAAþ (ATPase associated with diverse cellular impairments of its vital functions, that is, growth rate and
activities) serine proteases termed LON and CLPXP are located. female fertility (Fig. 1d,e) and was morphologically
While the former is a homo-oligomeric complex with each indistinguishable from the wild-type. Furthermore, susceptibility
subunitcontainingaproteaseandchaperonedomain5,CLPXPis of DPaClpP to various oxidative stressors thought to impair
a hetero-oligomeric complex consisting of individual CLPP mitochondrial and/or cellular protein homoeostasis, including
proteolytic and CLPX chaperone subunits6. The cellular paraquat, copper sulphate and H O , as well as against
2 2
function of LON proteases, including their relevance for ageing dithiothreitol-induced endoplasmic reticulum (ER) stress was
processes, has been studied in some detail in a number of unchanged(SupplementaryFig.S1).Remarkably,comparedwith
eukaryotic models7–12.In contrast, inpart because ofthe lackof thewild-type(n¼59),lifespanofDPaClpP(n¼57)wasfoundto
suitable model organisms, data on the biological role of the be significantly increased (mean lifespan:þ71%, maximum
mitochondrial CLPXP protease complex, aside from its implied lifespan:þ167%; P¼5.8E-18 by two-tailed Wilcoxon rank-sum
function in the so-called ‘mitochondrial unfolded protein test;Fig.1f).Thisresult,thoughcounter-intuitiveatfirstglance,is
response’13–15, is scarce and to date no physiological substrates reminiscentofthehealthylongevityphenotyperesultingfromthe
of any eukaryotic CLP protease have been identified. deletionofPaIap,encodingthemembrane-boundmitochondrial
The filamentous ascomycete P. anserina which, in contrast to i-AAA protease in P. anserina17.
other filamentous fungi, is characterized by a limited lifespan,
displays a clear mitochondrial aetiology of ageing and has been
extensively investigated to unravel the molecular basis of ageing DPaClpPlongevityisindependentofi-AAAandLONprotease.
processes16. Recent work demonstrated a clear impact of TheobservationthatbothDPaClpPandthePaIapdeletionstrain
mitochondrial protein quality control on the regulation of (DPaIap) are long-lived at 271C prompted us to investigate
lifespan and stress adaptation in this model organism10,12,17. whethertheseproteasesmightbeinvolvedinthesamemolecular
Inthepresentstudy,weinvestigatedtheeffectsofdeletingthe pathwaysregulatinglifespan.Tothisend, wegeneratedadouble
gene encoding the CLP protease proteolytic subunit CLPP in mutant (DPaClpP/DPaIap) in which both genes, that is, PaClpP
P. anserina. Unexpectedly, the PaClpP deletion strain is viable and PaIap, are deleted (Supplementary Fig. S2) and analysed its
and characterized by an extended lifespan without any distinct lifespan in comparison with the wild-type and the respective
impairment. We further demonstrate that the longevity pheno- single deletion strains (Fig. 1f). A lifespan extension similar to
type can be reverted by expression of human ClpP, thus that of DPaClpP was verified for DPaIap (mean lifespan:þ74%,
identifying P. anserina as a promising in vivo model to study maximum lifespan:þ89%; n¼15), corresponding to the results
the biological role of the poorly characterized mitochondrial obtainedinanearlierstudy17.Interestingly,lifespanofthedouble
CLPXP protease complex. deletion strain (n¼89) was even further increased (mean
lifespan:þ225%, maximum lifespanþ530%) and significantly
longer than that of either DPaClpP (P¼3.1E-17 by two-tailed
Results
Wilcoxon rank-sum test) or DPaIap (P¼4.2E-08 by two-tailed
In silico identification of CLP protease components. While
Wilcoxon rank-sum test). This additive lifespan extending effect
CLPP is absent in several lower eukaryotes, including the yeasts
Saccharomyces cerevisiae and Schizosaccharomyces pombe6, the followingtheconcomitantdeletionofPaClpPandPaIapsuggests
at least two possible scenarios: absence of the encoded
genomeofP.anserinacodesforaCLPPhomologue,aswellasfor
components influences pathways acting in parallel to promote
homologues of the CLP chaperones CLPA, CLPB and CLPX
longevity or lack of either PaIAP or PaCLPP causes mild
(http://podospora.igmors.u-psud.fr). With the exception of the
mitochondrialstress,aneffectwhichisexacerbatedinthedouble
chaperone PaCLPA, all proteins have a predicted probability for
deletionstrain,therebyactivatingcommondownstreampathways
mitochondrial localization of 490%. PaCLPP displays the
that are responsible for the observed extension of lifespan.
expected distribution of the canonical catalytic residues
In addition, to examine the epistatic relationship between
(Ser, His and Asp) and is highly conserved compared to CLPP
PaClpP and PaIap in more detail, we analysed the lifespans and
from other species, sharing about 60% similarity with the
growth rates of the single and double deletion strains at 371C
homologues from human, Caenorhabditis elegans and
(Supplementary Fig. S3). While deletion of PaIap had already
Escherichia coli (Fig. 1a). The deduced PaCLPP pre-protein is
been demonstrated to result in a pronounced heat-sensitive
composed of 254 amino acids and contains a putative
phenotype17, the PaClpP deletion strain displayed only a minor
mitochondrial targeting sequence. After import into mitochon-
impairmentoffitnessatelevatedtemperatures.Interestingly,heat
dria,cleavageofthemitochondrialtargetingsequenceisexpected
to lead to a mature protein of B25kDa. stress sensitivity of the DPaClpP/DPaIap double deletion strain
wasslightlyincreasedcomparedwitheithersingledeletionstrain.
This observation argues for potentially overlapping functions
Deletion of PaClpP extends lifespan. To investigate the biolo- of PaCLPP and PaIAP in a cellular heat stress response in
gical role of the mitochondrial CLPXP protease complex in P. anserina.
P. anserina, we analysed a PaClpP deletion strain (DPaClpP), As it was previously reported that overexpression of the gene
which was generated in the course of an earlier study codingfortheP.anserinaLONproteasehomologuePaLON1led
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PaCLPP Wild-tΔypPeaClpP 0.7
254 aa 0.6
PaClpP
CeHCsLCPLPP-P1 277 aa 5670 %% ble wth rateper day) 000...345
EcCLPP 20272 a1a a a 58 % Wild-typeΔ PaClpMPr(K) Gro(cm 00..12 Wild-type ΔPaClpP
MTS Catalytic residues (Ser, His, Asp) Anti-PaCLPP - 26
Anti-PaPOR - 34
1.0
Wild-type
e ΔPaClpP
ale fertility (%) 111146802460000000 Relative survival rat 0.5 ΔΔPPaaICalppP/ΔPaIap
em 20
F
Wild-type ΔPaClpP 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Age (days)
Figure1|DeletionofClpPinP.anserinaextendsthehealthylifespanat271C.(a)TheCLPPhomologuesfromP.anserina,Homosapiens,C.elegansand
E.colidisplayaconserveddistributionofthecanonicalcatalyticresiduesSer,HisandAsp.Additionally,alleukaryoticCLPPscontainamitochondrial
targetingsequence(MTS).ThelengthofthedifferentCLPPhomologuesvariesfrom207aminoacidsinE.colito277aminoacidsinH.sapiens.Theamino-
acidsequenceofPaCLPPshares57%,60%and58%similaritywiththatofHsCLPP,CeCLPP-1,andEcCLPP,respectively.(b)Southernblotanalysisof
HindIIIdigestedgenomicDNAfromwild-typeandDPaClpP.ThePaClpP-specifichybridizationprobedetectsthe4995-bpfragmentcontainingPaClpPonlyin
wild-typegenomicDNA.A2454-bpfragmentcontainingthephleomycinresistancegene(ble)isdetectedbyable-specifichybridizationprobeonlyin
genomicDNAofDPaClpP.(c)Westernblotanalysisofmitochondrialproteinextractsfromwild-typeandDPaClpP.ThePaCLPP-specificantibodydetects
theB25-kDaPaCLPPmonomeronlyinthewild-typesample.PaPORIN(PaPOR)wasdetectedasaloadingcontrol.(d)Growthrateofwild-type
(0.60±0.04;n¼59)andDPaClpP(0.58±0.04;n¼55;P¼0.07bytwo-tailedWilcoxonrank-sumtest)isolatesat271C.Datagiveninparenthesesare
meangrowthrate±s.e.incentimetresperday.(e)Femalefertilityofwild-type(100±14.7;n¼11)andDPaClpP(126.1±20.3;n¼11;P¼0.26bytwo-tailed
Wilcoxonrank-sumtest)isolatesat271C.Datagiveninparenthesesaremeanfemalefertility±s.e.inpercentage.(f)Lifespanofwild-type(21.7±0.5;
n¼59),DPaClpP(37.1±1.5;n¼57;P¼5.8E-18),DPaIap(37.9±1.8;n¼15;P¼2.7E-09)andDPaClpP/DPaIap(70.6±3.4;n¼90;P¼8.5E-25)isolatesat
271C.Datagiveninparenthesesaremeanlifespan±s.e.indays.P-valuesweredeterminedincomparisonwiththewild-typesamplebytwo-tailed
Wilcoxonrank-sumtest.
toanincreaseinhealthspansimilartothatofDPaClpP10,wealso experiments (Supplementary Fig. S5). Western blot analysis of
investigatedwhetherthedeletionstrain’slongevityphenotypecan mitochondrial protein extracts from DPaClpP/HsClpP_OEx1
beaccountedforbyacompensatoryupregulationofthisprotease. with an HsCLPP-specific antibody confirmed the presence of
Both CLPXP and LON are soluble proteases located in the matureHsCLPPwithanapparentmolecularmassofB26kDain
mitochondrial matrix and it is thus well conceivable that they mitochondria of the transgenic mutant strain (Fig. 2a).
possess overlapping substrate specificities and may at least Next, we examined whether HsCLPP forms functional oligo-
partially be able to compensate for dysfunction or even loss of mers in the mitochondria of P. anserina. The proteolytic core of
eachother.Yet,weobservednosignificantchangeintherelative the mitochondrial CLPXP protease complex is composed of two
PaLON1 protein abundance in mitochondrial protein extracts heptameric CLPP rings, which congregate to form a self-com-
from the PaClpP deletion strain (n¼6) when compared with partmentalised, cylindrical structure reminiscent of the 20S pro-
those of the wild-type strain (n¼6; P¼0.63 by two-tailed teasome core particle. Only if HsCLPP were able to form this
Student’st-test;SupplementaryFig.S4),suggestingthatPaLON1 structureinsidethefungalmitochondria,complementationofthe
does not have a major role in the extended lifespan of DPaClpP. deletion strain could be expected. To detect possible oligomers,
mitochondrial protein extracts from DPaClpP/HsClpP_OEx1
were solubilised with the mild detergent digitonin and separated
Human CLPP reverts the DPaClpP longevity phenotype. by blue native-polyacrylamide gel electrophoresis (BN-PAGE)18.
Having demonstrated that deletion of PaClpP is non-lethal and After western blotting and incubation with an HsCLPP-specific
leadstoaspecificphenotype,weaskedthequestionofwhetheror antibody, two distinct oligomers could be detected in the
notthisphenotypecanberevertedbyheterologousexpressionof DPaClpP/HsClpP_OEx1 sample (Fig. 2b). The apparent
aconstructcodingforhumanCLPP.Toensurestableexpression molecular mass ratio of these two oligomers was 1:2, as
ofthetransgene,avectorcontainingthefull-lengthopenreading expected for the single heptameric CLPP ring and the CLPP
frame (ORF) of the human ClpP complementary DNA (cDNA) proteolytic core cylinder formed by two such heptamers. After
undercontrolofaconstitutive,P.anserina-specificpromoterwas heat denaturation of the DPaClpP/HsClpP_OEx1 sample the
constructedandsubsequentlytransformedintoprotoplastsofthe signals disappeared, confirming the presence of bona fide
PaClpPdeletionstrain.Theresultingtransformantswereverified HsCLPP oligomers inside mitochondria of the fungal
by Southern blot analysis and two independent transformants transformants. However, the oligomers appeared roughly 20%
(DPaClpP/HsClpP_OEx1and2),eachwithasingleintegrationof larger than one would expect from calculating their molecular
the vector into their genome, were selected for subsequent massbasedonthatofasingleHsCLPPmonomer.Thisapparent
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a b HsClpPH_sOClEpxP1_OEx1(den.) tloHoefssnCthg)ul.epmvTPitha_ynOispEChaxLlem2nP:ooPtsPytto¼pceop2mbr.4oypEvHli-ed0tse9eCtLrhbePeyvPeinrtcswilvoeoianv-rtolaoyiflfueandDrgcPutWiaeoCsnillsfcpooPorx’fsotPhnpearCorcaLnanpPokauP-bnsiaculniemtddy
ClpP_OEx1 Mr(K) Wild-tΔypPeaClpΔPPaClΔpPP/aC2lp ×P/ Heptamer tfuhnugsidtoemhounmstarnast.es the functional conservation of CLPP from
Hs 480 -
Wild-typΔePaClpΔPPaCMlprP(/K) 242 - Discussion
Anti-HsCLPP - 26 The biological role of eukaryotic CLP proteases is as yet only
Anti-PaPOR - 34 146 - Heptamer poorly understood. Unfortunately, the unicellular ascomycete
Anti- Anti- S. cerevisiae, the most thoroughly studied and accessible
PaCLPP HsCLPP eukaryotic model organism20, lacks a CLPP homologue6 and
c
1.0 thus is not suitable to investigate eukaryotic CLP proteases.
Strikingly,thegenomeofthefilamentousascomyceteP.anserina
e Wild-type
urvival rat 0.5 ΔΔΔPPPaaaCCClllpppPPP//HHssCCllppPP__OOEExx12 emthniacttoodcdheesolneatdiorCinaLlPoCPfLPPhaocCmhlpaoPploegraounned,eatCshLuwPsXela.lbOlaasutiroanrehsooumfltsomloditgeoumcehoononsfdtrrtaihtaeel
elative s CexLtePnXsPionprootefasteheacthiveiatylthleyadlsifetospaanduuanldpehrensottaynpdea,rtdhatgrios,watnh
R
conditions but slightly impaired fitness at elevated
temperatures. Very similar effects have previously been
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 observed in a PaIap deletion strain17. As the concomitant
Age (days) deletion of both genes enhances either effect, it can be assumed
Figure2|HeterologousexpressionofhumanClpPcomplementsthe that, firstly, the P. anserina CLPP and i-AAA protease are
longevityphenotypeofDPaClpP.(a)Representativewesternblotanalysis involvedinparallelpathwaysregulatinglifespanand/orthattheir
ofmitochondrialproteinextractsfromwild-type,DPaClpPandDPaClpP/ absence under standard growth conditions causes mild but
HsClpP_OEx1.TheHsCLPP-specificantibodydetectstheB26kDaHsCLPP tolerable stress leading to mitochondrial hormesis (that is, the
monomeronlyintheDPaClpP/HsClpP_OEx1sample.PaPORIN(PaPOR)was beneficial activation of maintenance and repair pathways that
detectedasaloadingcontrol.(b)Westernblotanalysisofthesame ultimately results in the observed lifespan extension21) and,
mitochondrialproteinextractsasin(a)followingBN-PAGE.Incubationof secondly,thatPaCLPPandPaIAParecooperativelyinvolvedina
thewesternblotmembranewithaPaCLPP-specificantibodyvisualizedtwo mitochondria-specific heat stress response. The underlying
oligomerswithapparentmolecularmassesofB170and340kDainthe mechanisms, however, remain to be understood and have to be
wild-typesample.IntheDPaClpP/HsClpP_OEx1sample,twodistinct addressed in future studies. Similarly, the role of PaCLPX needs
oligomerswithapparentmolecularmassesofB220and440kDawere to be investigated in detail. As S. cerevisiae possesses a CLPX
detectedafterincubationwithanHsCLPP-specificantibody.Additionally, homologue despite the absence of CLPP22, it is probable that
theDPaClpP/HsClpP_OEx1(den.)samplewasheatdenaturedfor10minat eukaryotic CLPX chaperones perform functions independent of
951C,afterwhichtheHsCLPPoligomerscouldnotbedetectedanymore. theirroleintheCLPXPproteasecomplex.Indeed,humanCLPX
(c)Lifespanofwild-type(23.3±0.4;n¼39),DPaClpP(39.8±1.9;n¼37; has recently been shown to be involved in mitochondrial DNA
P¼7.2E-13),DPaClpP/HsClpP_OEx1(26.2±0.7;n¼40;P¼6.8E-04)and maintenance through interaction with the mitochondrial
DPaClpP/HsClpP_OEx2(26.2±0.7;n¼39;P¼2.5E-03)isolatesat271C. transcription factor TFAM23. Comparative analysis of DPaClpP
Datagiveninparenthesesaremeanlifespan±s.e.indays.P-valueswere and a PaClpX deletion strain is likely to generate important
determinedincomparisonwiththewild-typesamplebytwo-tailed insightsintopossibleprotease-independentfunctionsofCLPXin
Wilcoxonrank-sumtest. P. anserina.
Overall, the finding that deletion of PaClpP leads to an
unexpected effect on lifespan, which can be reverted by human
discrepancy can most likely be attributed to the migration of CLPP, identifies the potential of P. anserina to be used as an
proteinsduringBN-PAGEbeingsignificantlyinfluencedbytheir experimentally accessible system to study the yet unknown
native shape, their intrinsic charge and the amount of bound functionsofeukaryoticCLPproteases,possiblyincludingthoseof
Coomassie dye19. As an additional control, the western blot thehumanhomologues.Itthusmaybepossibletodevelopinvivo
membrane was incubated with a PaCLPP-specific antibody, approaches for the characterization of substrate recognition
thereby visualizing endogenous PaCLPP oligomers in the mechanisms, to identify CLPXP-specific substrates, to unravel
mitochondrial protein extract from the wild-type strain. The interactions of CLPXP with other components of the mitochon-
apparent molecular mass of these oligomers was more in accord drial protein quality control system and to integrate the
with their theoretically calculated size. elaborated mechanisms into the complex molecular network of
Finally, lifespans of the transgenic DPaClpP/HsClpP_OEx mitochondrial pathways governing development and biological
mutant strains were analysed in comparison with their parental ageing1–3.
strain DPaClpP and the wild-type(Fig. 2c). The PaClpPdeletion
strain (n¼37) consistently showed an increase in its lifespan
(mean lifespan:þ71%, maximum lifespan:þ167%) compared Methods
with the wild-type (n¼39; P¼7.2E-13 by two-tailed Wilcoxon P.anserinastrainsandcultivation.Inthepresentstudy,theP.anserinawild-type
rank-sumtest).Strikingly,lifespanofthetwoindependentstrains strain‘s’24,thePaClpPdeletionstrainDPaClpP(ref.10),thePaIapdeletionstrain
DPaClpP/HsClpP_OEx1(n¼40)and2(n¼39)wasonlyslightly DPaIap(ref.17),anewlygeneratedDPaClpP/DPaIapdoubledeletionstrainanda
longer than that of the wild-type (mean lifespan:þ12%, newlygeneratedPaClpPdeletionstrainoverexpressinghumanClpP(DPaClpP/
HsClpP_OEx)wereused.Allmutantstrainsareinthegeneticbackgroundofthe
maximum lifespan:þ44%) and significantly reduced compared
wild-typestrain‘s’.StrainsweregrownonstandardcornmealagarBiomalz-Mais-
with DPaClpP (DPaClpP/HsClpP_OEx1: P¼9.5E-10, DPaClpP/ Medium(BMM)at271Cunderconstantlight25.
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Lifespandetermination.Todeterminethelifespanofthestrainsusedinthis Southernblotanalysis.TotalDNAofP.anserinawasisolatedfollowingthe
study,monokaryoticascosporeswereisolatedfromindependentcrossesofthe protocoldevelopedbyLecellierandSilarforrapidextractionofnucleicacidsfrom
respectivestrainsandgerminatedfor2daysat271CinthedarkonBMMsup- filamentousfungi27.DNAdigestion,gelelectrophoresisandSouthernblottingwere
plementedwith60mMammoniumacetate.Aftergermination,piecesofthe performedaccordingtostandardprotocols.ForSouthernblothybridizationand
resulting2daysoldmyceliawereplacedonM2agarracetubes(M2medium: detection,Digoxigenin-labelledhybridizationprobes(DIGDNALabellingand
0.25gl(cid:2)1KHPO,0.3gl(cid:2)1KHPO,0.25gl(cid:2)1MgSO (cid:3)7HO,0.5gl(cid:2)1urea, DetectionKit,RocheAppliedScience)wereusedaccordingtothemanufacturer’s
2 4 2 4 4 2
and10gl(cid:2)1yellowdextrin.Additionof2.5mgl(cid:2)1biotin,50mgl(cid:2)1thiamine, protocol.
5mgl(cid:2)1citricacid(cid:3)1HO,5mgl(cid:2)1ZnSO (cid:3)7HO,1mgl(cid:2)1Fe(NH) TheHsClpP-specifichybridizationprobewasamplifiedbyPCRusingthe
2 4 2 42
(SO) (cid:3)6HO,2.5mgl(cid:2)1CuSO (cid:3)5HO,25mgl(cid:2)1MnSO (cid:3)1HO,50mgl(cid:2)1 oligonucleotidesHsClpP_1for(50-ATGTGGCCCGGAATATTG-30)and
42 2 4 2 4 2
NaMoO (cid:3)2HO,and50mgl(cid:2)1HBO aftersterilizationofthebasalmedium) HsClpP_2rev(50-GCGAGTAGATGTCATAGG-30)andcorrespondedto
2 4 2 3 4
andincubatedat271Cunderconstantlight.Forlifespandeterminationunderheat nucleotides124-355oftheHsClpPcDNA(GenBank:BC002956.1).ThePaClpP-
stressconditions,theisolateswereallowedtogrowfor2daysat271Candthen specifichybridizationprobecorrespondedtoa634-bpPstI-fragmentcontaining
transferredto371C(HeraeusBK6160climatechamber,ThermoScientific)forthe partofthePaClpPgeneandits30-untranslatedregion.ThePaIap-specific
remainderoftheirlife.Theperiodoflineargrowthwasrecordedaslifespanin hybridizationprobecorrespondedtoa1287-bpNcoI-fragmentcontainingpartof
days.Growthratewasmeasuredasgrowthofthemyceliaincentimetresperday. thePaIapgene.Asahybridizationprobespecificforthephleomycinresistance
gene(ble),the1293bpBamHI-fragmentoftheplasmidpKO3(ref.28)wasused.
Fertilityanalysis.Assessmentoffemalefertilitywasessentiallyperformedas
describedpreviously17.Isolatesofwild-typeorDPaClpP,originatingfrom Westernblotanddensitometryanalysis.Mitochondrialproteinextractsfrom
monokaryoticascospores,weregrownonM2agarplatesat271Cunderconstant P.anserinastrainswereisolatedaccordingtoapreviouslydevelopedprocedure29.
lightfor13days.Todeterminetheabilityofwild-typeorDPaClpPtoformfruiting SeparationofproteinsbySDS–PAGEandsubsequenttransferofproteinsto
bodiesafterexposuretoheatstress,thesameisolateswereallowedtogrowforeight polyvinylidenedifluoride(PVDF)membranes(Immobilon-FL,Millipore)were
daysat271Candthentransferredto371C(KBW400climatechamber,BINDER) performedfollowingstandardprotocols.Blockingandantibodyincubationof
for5days.Followingspermatisationatday13,allplateswereincubatedforan blottedPVDFmembraneswereperformedaccordingtotheOdyssey‘WesternBlot
additionalthreedaysat271C,afterwhichthetotalnumberofperithecia Analysis’handbook(LI-COR).
developingoneachplatewascounted.Themeannumberofperitheciadeveloping PrimaryantibodieswereraisedagainstaPaLON(UniProt:B2AZ54)synthetic
perplateovergrownwiththewild-typestrain‘s’at271Cor371Cwassetas100% peptide([H]-CDKIGRGYQGDPS-[OH];Sigma)correspondingtoaminoacids
femalefertility. 677–688(antibodydilution:1:1,500),aPaCLPP(UniProt:B2B591)synthetic
peptide([Ac]-CGTMLSADAKEGKH-[OH];NEP)correspondingtoaminoacids
242–254(antibodydilution:1:400)andthePaPORIN(UniProt:B2B736)full-
Measurementofgrowthrateunderstressconditions.Toassessthesuscept- lengthprotein(NEP;antibodydilution:1:5,000).ThehumanCLPPantibody
ibilityofDPaClpPtovariousoxidativestressorsanddithiothreitol,monokaryotic (abcam,productcode:ab56455)wasraisedagainstanHsCLPP(UniProt:Q16740)
ascosporeswereisolatedfromindependentcrossesofthewild-typeandDPaClpP recombinantfragmentcorrespondingtoaminoacids178–278(antibody
andgerminatedfor2daysat271CinthedarkonBMMsupplementedwith60mM concentration:1mgml(cid:2)1).
ammoniumacetate.Aftergermination,piecesoftheresulting2daysoldmycelia Inallanalyses,secondaryantibodiesconjugatedwiththeinfrareddyesIRDye
wereplacedonM2agarplatessupplementedwithdifferentconcentrationsof 800CWorIRDye680CW(LI-COR)wereused(antibodydilution:1:15,000).The
paraquat(0,20,80or150mM),CuSO4(0,100,200or400mM),H2O2(0,0.04,0.06 OdysseyInfraredImagingSystem(LI-COR)wasusedfordetectionand
or0.1%),ordithiothreitol(0,0.5,5or10mM)andincubatedat271Cunder quantificationwasperformedusingthemanufacturer’ssoftwareasrecommended.
constantlight.PlatescontainingHO werekeptinthedarktoprotecthydrogen
2 2
peroxidefromdegradation.Growthwasrecordedfor4daysandgrowthratewas
measuredasgrowthofthemyceliaincentimetresperday. BluenativePAGE.BN-PAGEwasperformedaccordingtotheprotocoldeveloped
byScha¨ggerandcolleagues18.Forsamplepreparation,300mgofmitochondrial
proteinextractwassolubilisedusingadigitonin/proteinratioof4:1(w/w).Linear
CloningproceduresandgenerationofP.anserinamutants.ThehumanClpP gradientgels(4–13%)overlaidwith3.5%stackinggelswereusedforseparationof
cDNAopenreadingframewasamplifiedbyPCRusingtheoligonucleotides thesolubilisedsamples.ForwesternblottingofBN-PAGEgels,0.05%SDSwas
BamHI-HsClpP_3for(50-TAGGATCCATGTGGCCCGGAATATTG-30)and addedtothetransferbufferandthetransferwasperformedfor16hat25Vand
XbaI-HsClpP_4rev(50-ATTCTAGATCAGGTGCTAGCTGGGAC-30),introdu- 41C.Blocking,antibodyincubationandantibodydetectionofblottedPVDF
cingBamHIandXbaIrestrictionsites(underlined).Theampliconwasclonedinto membraneswereperformedasdescribedintheprecedingsection.
thepExMtterhph10backbone(BamHI/XbaIdigested)undercontrolofthestrong
constitutivemetallothioneinpromoterandthemetallothioneinterminator.The
resultingplasmidpHsClpPEx1wassubsequentlytransformedintoP.anserina Statisticalanalysis.Forstatisticalanalysisofwesternblotdata,two-tailedStu-
DPaClpPspheroplasts.TransformantswereselectedforhygromycinBresistance dent’st-testwasused.Forstatisticalanalysesoflifespan,growthrateandfemale
andverifiedbySouthernblotanalysis.Strainscontainingasingleintegrationof fertility,two-tailedWilcoxonrank-sumtestwasused.Ifnotexplicitlystated
pHsClpPEx1weretermedDPaClpP/HsClpP_OEx. otherwise,therespectivesampleswerecomparedwiththeappropriatewild-type
ToconstructaDPaClpP/DPaIapdoubledeletionstrain,amonokaryotic sample.P-valueso0.05wereconsideredstatisticallysignificant.
DPaClpPisolatewascrossedwithamonokaryoticDPaIapisolate.Meioticoffspring
containingtheconcomitantdeletionofbothgeneswereidentifiedbytheir
phleomycinresistanceandverifiedbySouthernblotanalysis. References
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