Journal of Cell Science Accepted manuscript

47 Pages·2014·8.35 MB·English

by  松田 憲之

(cid:2)2015.PublishedbyTheCompanyofBiologistsLtd|JournalofCellScience(2015)128,964–978doi:10.1242/jcs.161000
RESEARCH ARTICLE
Unconventional PINK1 localization to the outer membrane of
depolarized mitochondria drives Parkin recruitment
Kei Okatsu1, Mayumi Kimura1,2, Toshihiko Oka3, Keiji Tanaka2,* and Noriyuki Matsuda1,*
ABSTRACT disease. Genetic studies using Drosophila melanogaster lacking
PINK1 showed a contribution of PINK1 to mitochondrial
Dysfunction of PTEN-induced putative kinase 1 (PINK1), a Ser/Thr
integrityinvivo(Clarket al.,2006;Parket al.,2006;Yangetal.,
kinase with an N-terminal mitochondrial-targeting sequence (MTS),
2006). Drosophila lacking pink1 have abnormal mitochondrial
causes familial recessive parkinsonism. Reduction of the
morphology in flight muscles, short life span and male sterility
mitochondrial membrane potential limits MTS-mediated matrix
(Clark et al., 2006; Park et al., 2006; Yang et al., 2006). These
import and promotes PINK1 accumulation on the outer
phenotypes are rescued by a component of the mitochondrial
mitochondrial membrane (OMM) of depolarized mitochondria. electrontransportchaincomplex,amitochondrialelectroncarrieror
PINK1 then undergoes autophosphorylation and phosphorylates apositiveregulatorformitochondrialprotectivegenes(Kohetal.,
ubiquitin and Parkin, a cytosolic ubiquitin ligase, for clearance of 2012; Vilain et al., 2012; Vos et al., 2012). Genes regulating
damagedmitochondria.ThemolecularbasisforPINK1localizationon mitochondrial morphology such as Marf, opa1, fzo1 and drp1
the OMM of depolarized mitochondria rather than release to the interactgeneticallywithPINK1(Dengetal.,2008;Parketal.,2009;
cytosol is poorly understood. Here, we disentangle the PINK1 Pooleetal.,2008;Yangetal.,2008).Inaddition,Pink1-knockout
localization mechanism using deletion mutants and a newly miceexhibitmorphologicalandfunctionalmitochondrialdefectsin
established constitutively active PINK1 mutant. Disruption of the striatum (Gautier et al., 2008; Kitada et al., 2007; Kitada et al.,
MTSthroughN-terminalinsertionofasparticacidresiduesresultsin 2009). These reports suggest that PINK1 plays important roles in
OMMlocalizationofPINK1inenergizedmitochondria.Unexpectedly, maintainingmitochondrialrobustness.
theMTSandputativetransmembranedomain(TMD)aredispensable Recentcell-basedandinvivostudieshaverevealedthatPINK1
forOMMlocalization,whereasmitochondrialtranslocaseTom40(also acts upstream of another gene product that is relevant to
Parkinson’s disease, Parkin (Clark et al., 2006; Geisler et al.,
known as TOMM40) and an alternative mitochondrial localization
2010; Kitada et al., 1998; Matsuda et al., 2010; Narendra et al.,
signalthatresidesbetweentheMTSandTMDarerequired.PINK1
2010;Parketal.,2006;Rakovicetal.,2010;Vives-Bauzaetal.,
utilizes a mitochondrial localization mechanism that is distinct from
2010;Yang etal., 2006; Ziviani et al., 2010).PINK1 selectively
thatofconventionalMTS proteinsandthatpresumablyfunctionsin
recruits Parkin on depolarized mitochondria and phosphorylates
conjunction with the Tom complex in OMM localization when the
both Parkin and ubiquitin, which leads to Parkin activation and
conventionalN-terminalMTSisinhibited.
the subsequent ubiquitylation of outer mitochondrial membrane
KEYWORDS:Mitochondria,Parkin,Parkinson’sdisease,PINK1 (OMM)proteinsonthedamagedmitochondria(Chanetal.,2011;
Iguchi et al., 2013; Kane et al., 2014; Kazlauskaite et al., 2014;
Kondapallietal.,2012;Koyanoetal.,2014;Okatsuetal.,2012a;
INTRODUCTION Sarraf et al., 2013; Shiba-Fukushima et al., 2012; Tanaka et al.,
Parkinson’s disease and its relative parkinsonism are pervasive 2010). Degradation of the ubiquitylated mitochondria is thought
neurodegenerative diseases. PTEN-induced putative kinase 1 to proceed through the proteasome (Yoshii et al., 2011) and
(PINK1), a mitochondrial Ser/Thr kinase, was identified as a autophagy, a process referred to as mitophagy (Narendra et al.,
causalgeneforfamilialrecessiveearly-onsetparkinsonism(Valente 2008; Okatsu et al., 2010).
et al., 2004). Because patients with familial parkinsonism present During the aforementioned process, PINK1 recognizes a
symptoms similar to sporadic Parkinson’s disease, functional collapse of the membrane potential (DYm) in mitochondria and
analysis of familial Parkinson’s disease-related proteins such as signals this reduction to Parkin. In mitochondria with a normal
PINK1 provides insights into the pathogenic mechanism of the DYm, the positively charged mitochondrial-targeting sequence
(MTS) of PINK1 is imported into the mitochondrial matrix and
PINK1 undergoes stepwise cleavage; first by the mitochondrial
1ProteinMetabolismProject,TokyoMetropolitanInstituteofMedicalScience, e
Setagaya-ku,Tokyo156-8506,Japan.2LaboratoryofProteinMetabolism,Tokyo processing peptidase (MPP), possibly with cooperation from c
n
MetropolitanInstituteofMedicalScience,Setagaya-ku,Tokyo156-8506,Japan. ClpXP, and then intramembrane cleavage by presenilin- e
3DepartmentofLifeScience,CollegeofScience,RikkyoUniversity, associated rhomboid-like protein (PARL) and possibly AFG3L2 ci
Nishi-Ikebukuro,Tokyo171-8501,Japan. S
(Deasetal.,2011;Greeneetal.,2012;Jinetal.,2010;Meissner
*Authorsforcorrespondence([email protected]; et al., 2011). Exposure of the phenylalanine (Phe) residue at ell
[email protected]) C
position 104 of the N-terminus of processed PINK1 following
f
o
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution PARL-mediated cleavage acts as a signal for ‘N-end rule
License(http://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse,distribution pathway’-mediated degradation (Yamano and Youle, 2013). al
andreproductioninanymediumprovidedthattheoriginalworkisproperlyattributed. n
PINK1 is subsequently subjected to proteasomal degradation ur
Received31July2014;Accepted12January2015 (LinandKang,2008;LinandKang,2010;Narendraetal.,2008) Jo
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RESEARCHARTICLE JournalofCellScience(2015)128,964–978doi:10.1242/jcs.161000
andthePINK1signalisturnedoffundersteady-stateconditions.By m-chlorophenylhydrazine (CCCP). Consistent with previous
contrast, dissipation of DYm hinders movement of the positively reportsusingotherN-terminalepitopetagssuchasMyc(Takatori
charged MTS through the inner mitochondrial membrane (IMM), et al., 2008), N-FLAG–PINK1 localized on mitochondria even
preventing exposure of the crucial Phe104 N-terminal processing without CCCP treatment (Fig. 1A), whereas C-terminally tagged
site. PINK1 thus bypasses DYm-dependent degradation, which and non-tagged PINK1 localized on mitochondria only following
triggerstheaccumulationofPINK1ontheOMM,interactionwith CCCP treatment (Fig. 1A–C) as reported previously (Matsuda
the translocase of the outer membrane (TOM) complex, PINK1 etal.,2010).WenextverifiedwhetherN-FLAG–PINK1underwent
dimerization and autophosphorylation (Lazarou et al., 2012; phosphorylationusingphosphorylated-protein-affinitySDS-PAGE
Matsudaetal.,2010;Narendraetal.,2010;Okatsuetal.,2012b). (Phos-tag SDS-PAGE). The small molecule Phos-tag binds to
As a consequence, the PINK1 signal is turned on when DYm acrylamideand two Mn2+ ions, such thatphosphorylated proteins
decreases.Apoorlyunderstoodaspectofthisprocessisthatwhen are capturedby the Mn2+-Phos-tagand migrate more slowly than
theDYm-drivenmatrixtargetingofMTSisinhibited,PINK1isnot non-phosphorylated proteins (Kinoshita et al., 2006). Phos-tag
releasedinto the cytosol but isratherretainedon the OMM.This SDS-PAGE showed that N-FLAG–PINK1 is constitutively
contrasts with many matrix proteins that relocate to the cytosol phosphorylated,whereasC-FLAG–PINK1andnon-taggedPINK1
following a decrease in DYm. Consequently, the mechanism arephosphorylatedonlyfollowingCCCP-treatment(Fig. 1D).
underlying PINK1 targeting to the OMM is crucial for PINK1
function.ThemolecularbasisforPINK1retentionintheOMMof ThePINK1N-terminuscontainsaMTS
depolarizedmitochondriaandthedomain(s)thatarecrucialtothis ToaddressthemolecularbasisforN-FLAG–PINK1accumulation
processhavenotbeenconclusivelyresolved. on mitochondria, we first focused on the N-terminal region of
To date, various data on the mitochondrial localization signal PINK1, because the import of mitochondrial proteins frequently
and submitochondrial localization of PINK1 have been reported. requiresanintactMTSattheN-terminus.BothTargetP1.1(http://
For example, the submitochondrial localization of PINK1 varies www.cbs.dtu.dk/services/TargetP/)andPSORTII(http://psort.hgc.
from the OMM (Gandhi et al., 2006; Narendra et al., 2010; jp/form2.html) predict mitochondrial localization for PINK1. The
Weihofen et al., 2009; Zhou et al., 2008) to the intermembrane expectedvaluesformitochondriallocalizationare0.791and73.9%
space(IMS)andIMM(Marongiuetal.,2009;Muqitetal.,2006; (the maximum values are 1 or 100%) in TargetP and PSORT II,
Silvestrietal.,2005).Moreover,thereareconflictingconclusions respectively(supplementarymaterialFig.S1A).MTSfunctionality
regarding the extreme N-terminus of PINK1 (,34 amino acids). requires a positively charged amphiphilic a-helix within the N-
Thisregionhasbeenreportedtobesufficientforthemitochondrial terminus.SecondarystructurepredictionofthePINK1N-terminus
localizationofareporterprotein(Muqitetal.,2006;Silvestrietal., region utilizing Jpred3 (http://www.compbio.dundee.ac.uk/www-
2005; Takatori et al., 2008), but deemed dispensable for PINK1 jpred/) and PHD (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.
mitochondrial localization (Zhou et al., 2008). To more fully pl?page5/NPSA/npsa_phd.html) indicated the presence of a 20-
understandtheunderlyingprocesses,wemustdistinguishbetween amino-acid residue helical segment (supplementary material Fig.
the PINK1 mitochondrial localization mechanisms utilized for S1B). Visualization of this region as a helical wheel using the
energizedmitochondriaanddepolarizedmitochondria. Phyre2proteinhomology/analogyrecognitionengine(Phyre2:http://
Here,wedissectthemultiplemitochondriallocalizationsignals www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id5index) suggested
ofPINK1anddisentanglethecomplicated mechanismsbyusing amphiphilicity(supplementarymaterialFig.S1C).Inaddition,the
various deletion mutants and a newly established constitutively PINK1N-terminalregionissimilartothea-helixinthebacterial
active mutant. We show that inactivation of the PINK1 N- microcompartment shell protein PduB (4FAY) (supplementary
terminus MTS alone is sufficient to promote PINK1 localization material Fig. S1D,E), suggesting that the PINK1 N-terminus
on the OMM through a second ‘latent’ outer mitochondrial containsagenuineMTS.
membranelocalizationsignal(OMS)thatsubsequentlyenhances The34N-terminalaminoacidresidues(N34)ofPINK1,which
the recruitment and activation of Parkin. Our results provide a include the predicted amphiphilic a-helix, were fused with GFP
molecular basis for how inhibition of PINK1 import through the andthesubcellularlocalizationoftheresultingchimera(referred
IMM autonomously triggers PINK1 OMM localization, Parkin toasPINK1-N34–GFP)wasexamined.Consistentwithprevious
recruitment and mitochondrial degradation. reports (Muqit et al., 2006; Takatori et al., 2008), PINK1-N34–
GFPlocalizedtothemitochondriainHeLacells,similartoSu9–
RESULTS GFP (Ishihara et al., 2003), an accepted mitochondrial matrix
AnN-terminaltaginducesmitochondriallocalizationand marker (Fig. 2A, upper panels). Incubation with CCCP inhibited
autophosphorylationofPINK1 the mitochondrial import of Su9–GFP, with fluorescence being
Although PINK1 has been shown to localize on the OMM of limited to the cytosol and nucleus. The mitochondrial import of
depolarized mitochondria and undergo autophosphorylation- PINK1-N34–GFP was likewise inhibited by CCCP treatment;
dependent activation in response to a decrease in DYm, however,thereticularfluorescencepatterndifferedslightlyfrom
e
(Matsuda et al., 2010; Narendra et al., 2010; Okatsu et al., the cytosolic and nuclear pattern of Su9–GFP (Fig. 2A, lower c
n
2012b),themolecularbasisofthisprocesshasyettobeelucidated. panels). Because the processed form of PINK1-N34–GFP was e
To begin to address this, we utilized a PINK1 construct with an almost undetectable following CCCP treatment (Fig. 2C), we ci
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epitopetagfusedattheN-terminusthatlocalizesonmitochondria believe that the unprocessed hydrophobic PINK1-N34–GFP
intheabsenceofmitochondrialdepolarization(Beilinaetal.,2005; localized in the membrane. Indeed, following CCCP treatment, ell
C
Takatorietal.,2008).HeLacellsweretransfectedwithnon-tagged the PINK1-N34–GFP fluorescence signal partially colocalized
f
o
PINK1orPINK1withanN-terminalFLAGtag(referredtoasN- with the plasma-membrane-targeted mKO1 signal (Fig. 2B). To
FLAG–PINK1)oraC-terminaltag(referredasC-FLAG–PINK1), further confirm import into energized mitochondria, HeLa cells al
n
and the localization of PINK1 was observed in the presence and transfected with GFP, Su9–GFP and PINK1-N34–GFP were ur
absence of the mitochondrial uncoupler carbonyl cyanide treated with CCCP and subjected to immunoblotting. Su9–GFP o
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RESEARCHARTICLE JournalofCellScience(2015)128,964–978doi:10.1242/jcs.161000
Fig.1. AdditionofanN-terminalFLAGtagcausesPINK1accumulationandactivationonenergizedmitochondria.(A,B)HeLacellsexpressingN-orC-
terminallyFLAG-taggedPINK1(N-FLAG–PINK1orC-FLAG–PINK1)wereimmunostainedusinganti-PINK1(greeninmerge)andanti-Tom20(redinmerge)
antibodiesintheabsence(A)orpresence(B)ofCCCPtreatment.N-FLAG–PINK1localizedtomitochondriaevenundersteady-stateconditions.Scale
bars:10mm.(C)ThenumberofcellswithPINK1localizedtothemitochondriawascountedin100cells.Datarepresentthemean6s.d.ofatleast
threeexperiments.(D)TheinsertionoftheN-FLAGtagconvertsPINK1toitsauto-phosphorylatedform.Cellsexpressingnon-tagged,N-FLAG-taggedorC-
FLAG-taggedPINK1weresubjectedtoSDS-PAGEwith(+)orwithoutphos-tagandwereimmunoblotted(IB)forPINK1.Theredasterisksindicate
phosphorylatedPINK1(pPINK1).
was cleaved in mitochondria under steady-state conditions, but expressing PINK1-N34–GFP and incubated this with proteinase
remained intact when Su9–GFP import was inhibited by CCCP K (Pro K) before immunoblotting. PINK1–N34–GFP and the
(Fig. 2C, lanes 5 and 6). PINK1-N34–GFP cleavage was matrix protein Hsp60 are resistant to Pro K, whereas the OMM
similarly inhibited by blocking its import with CCCP (Fig. 2C, proteinTom20andIMSproteinAIF(alsoknownasAIFM1)are
lanes 3 and 4). Five OPA1-immunoreactive bands were detected susceptibletoProKdigestion(Fig. 2E).Usinganinvitroimport
under steady-state conditions (Fig. 2C, odd lanes), whereas only assay, Becker et al. also reported that the PINK1 N-terminus
three bands were detected following CCCP treatment (Fig. 2C, functions as a pre-sequence for mitochondrial import and that it
even lanes), confirming the dissipation of DYm in this passes through the IMM in a DYm-dependent manner (Becker
experiment (Ishihara et al., 2006). et al., 2012). Our data indicate that the N-terminal region of
ToexaminewhetherPINK1-N34–GFPlocalizestothematrix, PINK1 functions as a MTS for mitochondrial matrix import.
we immunostained HeLa cells with distinct permeabilization
methods andperformed aproteinase K (ProK)-protection assay. TheimportpotentialofPINK1MTSisinhibitedbythe
AlthoughTritonX-100(1%)permeabilizesbothOMMandIMM, insertionofnegativelychargedaminoacids
digitonin (50 mg/ml)permeabilizes onlythe OMM.Tom20(also Because N-FLAG–PINK1 localized on mitochondria and
known as TOMM20; an OMM protein) and cytochrome C (IMS underwent autophosphorylation (Fig. 1), we hypothesized that
protein) were clearly recognized by their respective antibodies additionofextraaminoacidresiduesattheN-terminusofPINK1
afterpermeabilizationwithdigitonin,andtheirsignalsoverlapped would impair the mitochondrial import potential of PINK1 N34.
well (supplementary material Fig. S2, left panel). Meanwhile, We tried to inactivate the PINK1 MTS sequence without
permeabilization with digitonin was insufficient to allow disrupting the amphiphilic a-helix structure by inserting
detection of the FoF1 ATP synthase b subunit (also known as multiple Ala (non-polar) or Asp (negatively charged) amino
e
ATP5B; exposed to matrix protein; Wang and Oster, 1998); acid residues into the PINK1 N-terminus (Fig. 3A). Mutants c
n
instead, permeabilization with Triton X-100 was required containing 3Ala, 5Ala, 3Asp and 5Asp yielded TargetP and e
(supplementary material Fig. S2, right panel). We then prepared PSORT II mitochondrial localization prediction scores of 0.905 ci
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HeLacellsexpressingSu9–GFPandPINK1-N34–GFP,inwhich and 69.6%, 0.913 and 34.8%, 0.385 and 4.3%, and 0.202 and
the GFP fluorescence was observed irrespective of the 4.3%, respectively (Fig. 3B). We verified the predictions by ell
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permeabilization conditions. Anti-GFP immunoreactivity was observingthelocalizationofthesePINK1mutantsinHeLacells.
f
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absent from both Su9–GFP- and PINK1-N34-GFP-expressing The Ala insertions had minimal effects on PINK1-N34–GFP
cellsafterpermeabilizationwithdigitonin,butbecamedetectable localization, whereas the Asp insertions proportionally inhibited al
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following Triton X-100 permeabilization (Fig. 2D). We next mitochondrial localization in relation to the number of residues ur
prepared a mitochondria-enriched fraction from HeLa cells inserted (Fig. 3C–E). To confirm these results, we examined the o
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Fig.2. TheN-terminal34aminoacidsinPINK1functionasamitochondrial-matrix-targetingsignalthatisdependentonDYm.(A)HeLacellsexpressing
PINK1-N34–GFPorSu9(mitochondrialmatrixmarker)MTS–GFPwith(+)orwithout(2)CCCPtreatment.CellswerepretreatedwithCCCPfor2hpriorto
transfectionwithSu9–GFPorPINK1-N34–GFP,andwerefurtherincubatedinthepresenceofCCCP.(B)PINK1-N34–GFPpartiallycolocalizedwiththeplasma-
membranemarkermKO1(PM-mKO1)followingCCCPtreatment.(C)HeLacellsexpressingGFP,PINK1-N34–GFPorSu9–GFPweretreatedwithorwithout
CCCPandthensubjectedtoimmunoblotting(IB).TheredarrowheadsindicateDYm-dependentprocessedforms.OPA1isdetectedasthreebandsfollowinga
decreaseinDYm.a–eindicateOPA1mRNAsplicedisoforms;aandbareprocessedindepolarizedmitochondria.Tom22representsthetotalamountof
mitochondria.ThesizeofGFPcleavedfromN34–GFPappearstobegreaterthanthatofnativeGFPbecauseamulti-cloning-site-derivedpeptideisaddedattheN-
terminusofGFP.(D)HeLacellsexpressingSu9–GFPorPINK1-N34–GFPwereimmunostainedwithananti-GFPantibodyafterpermeabilizationwithdigitoninor
TritonX-100,andthentheimmunofluorescenceimageswerecomparedwiththeGFPautofluorescenceimages.Scalebars:10mm.(E)Mitochondria-rich
fractionscollectedfromHeLacellsexpressingPINK1-N34–GFPwereincubatedwithProteinaseK(ProK:250mg/ml)andsubjectedtoimmunoblottingusinganti-
GFP,anti-Tom20(OMMprotein),anti-AIF(IMSprotein)andanti-Hsp60(matrixprotein)antibodies.TheredarrowheadsindicatethePro-K-resistantbands.
processing activity of the MTS by immunoblotting. The PINK1–GFP or 5Asp-inserted PINK1–GFP was observed in
processed band was present in the 5Ala insertion mutant but relation to the DYm-dependent dye TMRE. Under steady-state
absent from the 5Asp mutant (Fig. 3F), further indicating that conditions, wild-type PINK1–GFP did not localize on energized
e
Asp insertion almost completely inhibited mitochondrial import. mitochondria stained by TMRE, but did localize on TMRE- c
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negative mitochondria following CCCP treatment (Okatsu et al., e
DisruptionoftheN-terminalMTSinducesanalternative 2012b) (Fig. 4B, left two panels). Interestingly, insertions of ci
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signal-dependentmitochondriallocalizationmechanismand 3Asp or 5Asp induced PINK1 accumulation on TMRE-positive
autophosphorylationofPINK1 polarizedmitochondria(Fig. 4B,righttwopanels). ell
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Because the Asp insertions inhibited PINK1-N34–GFP Becausethefull-lengthPINK1thataccumulatesondepolarized
f
o
mitochondrial import, we examined the subcellular localization mitochondriaisphosphorylated(Okatsuetal.,2012b),weexamined
and phosphorylation of full-length PINK1–GFP when three or the phosphorylation of PINK1–GFP with Asp insertions using al
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fiveAspresidueswereinsertedatthePINK1N-terminus(Fig.4A). Phos-tagSDS-PAGE.Whereaswild-typePINK1–GFPundergoes ur
The subcellular localization of PINK1–GFP, 3Asp-inserted phosphorylationonlyfollowingCCCPtreatment(Fig. 4C,lanes1 o
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Fig.3. Theadditionofnegatively
chargedaminoacidstotheN-
terminusofPINK1MTSinhibitsits
mitochondrialimportactivity.
(A)Schematicdiagramofthe
constructsused.(B)Mitochondrial
localizationpredictionscoresfrom
TargetP1.1andPSORTII.
(C)AutofluorescenceimagesofHeLa
cellsexpressingPINK1-N34–GFP
withN-terminalinsertionofthe
indicatedaminoacids.(D)HeLacells
expressingPINK1-N34–GFP
mutantswith3or5asparticacid
(‘D’)insertionswerestainedwiththe
DYm-dependentdyeTMRE.Scale
bars:10mm.(E)Graphsindicatethe
fluorescenceintensityofthewhite
linesinthemergesinD.Greenand
redlinesindicatethefluorescence
intensityofGFPandTMRE
(mitochondria),respectively.(F)The
processedformofPINK1-N34–GFP
thatwasdetectedbyimmunoblotting
(IB)withananti-GFPantibodywas
notdetectedinthe5Dinsertion.The
OPA1patternindicatesthepresence
ofDYm,andTom22representstotal
mitochondriaasinFig.2C.a–e
indicateOPA1mRNAspliced
isoforms;aandbareprocessedin
depolarizedmitochondria.Theblack
arrowheadindicatestheDYm-and
matrix-import-dependent
processedform.
and 2), PINK1–GFP with either of the Asp-repeat insertions was (supplementary material Fig. S3A). Jpred3, PHD and Phyre2
phosphorylatedevenundersteady-stateconditions(Fig. 4C,lanes search engines also suggest a helical structure for this region
3and4).TheseresultssuggestthatdisruptionofthePINK1MTS (supplementary material Fig. S3B). Moreover, TargetP 1.1 and
by insertion of the negatively charged amino acids induced PSORTIIestimatethatresidues67–94ofPINK1,aregionproximal
mitochondrial localizationand autophosphorylationof full-length to the TMD, has the potential to function as a mitochondrial
PINK1.BecausetheFLAGepitopetagcontainsfiveAspresidues localization domain (supplementary material Fig. S3C). We next
(DYKDDDDK), it is reasonable to assume that placement of the constructedseveralfluorescentchimerasofGFPfusedwithvarious
FLAGtagattheN-terminusdisruptsthePINK1MTSandinduces deletionsofthe110aminoacidscomprisingthePINK1N-terminus
mitochondriallocalizationandautophosphorylation(Fig. 1). (Fig. 4D),andexaminedtheirsubcellularlocalization.N35–110of
Results shown in Fig.4B,C contrast with the largely cytosolic PINK1, which lacks a MTS, clearly targeted GFP to the
localization data of PINK1-N34–GFP with the Asp insertions mitochondria (Fig.4E), as did N35–90 albeit with a weaker
e
(Fig.3C). However, given that PINK1 possesses an alternative signal(Fig.4F).Theseresultsrevealthat,inadditiontothetypical c
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OMMlocalizationsignalthatfunctionswhentheN-terminalMTS N-terminal MTS, PINK1 contains a second mitochondrial e
isinhibited,thisseeminglycontradictoryresultisexplainable.We localization signal spanning residues 35–110. This is consistent ci
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sought to define the second PINK1 mitochondrial localization with a previous study reporting that PINK1 lacking the first 34
signal. As in the case of N-terminal MTS identification residuesstilllocalizesonthemitochondria(Zhouetal.,2008). ell
C
(supplementary material Fig. S1), we first tried in silico
f
identification. A Kyte–Doolittle plot (Kyte and Doolittle, 1982) LossoftheMTSintheN-terminusinducesmitochondrial o
suggestedthatinadditiontotheMTSandputativetransmembrane localizationandautophosphorylationofPINK1 al
n
domain (TMD; residues 94–110), there is a weak hydrophobic Because functional disruption of the PINK1 MTS (N34) causes ur
region around residues 70–95 of the PINK1 N-terminus mitochondrial localization and activation of PINK1 (Fig. 4), we o
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RESEARCHARTICLE JournalofCellScience(2015)128,964–978doi:10.1242/jcs.161000
Fig.4. DisruptionoftheN-terminalMTSinducesalternativesignal-dependentmitochondriallocalizationandautophosphorylationofPINK1.
(A)SchematicdiagramoftheconstructsusedinBandC.WT,wildtype.(B)HeLacellsexpressingfull-lengthPINK1–GFPwiththe3Dor5Dinsertionswere
imagedusingGFPautofluorescence(green)andtheDYm-dependentdyeTMRE(red).(C)ThelysatesweresubjectedtoPAGEwith(+)orwithout(2)PhosTag
andimmunoblotted(IB)withanti-PINK1,anti-OPA1andanti-Tom22antibodies.a–eindicateOPA1mRNAsplicedisoforms;aandbareprocessedin
depolarizedmitochondria.TheredarrowheadsindicatephosphorylatedPINK1(pPINK1).(D)SchematicdiagramoftheconstructsusedinEandF.(E)N35–110
ofPINK1,whichlackstheMTS,clearlytargetedGFPtothemitochondria.(F)N35–90ofPINK1,whichlacksboththeMTSandTMD,stillpartiallyrecruitedGFP
tothemitochondria.EandFrevealthatthePINK1N-terminuscontainsanalternativemitochondriallocalizationsignalotherthantheMTS.Scalebars:10mm.
next constructed PINK1 lacking the MTS (N34) and examined MitochondrialtranslocaseTom40isimportantin
its localization and phosphorylation. We performed mitochondriallocalizationandphosphorylationof
immunocytochemistry using an anti-PINK1 antibody (the constitutivelyactivePINK1mutants
antigenic region corresponds to amino acids 175–250), an anti- In general, mitochondrial proteins containing MTS are imported
Tom20 antibody (mitochondrial marker), and MitoTracker through Tom40, a channel component of the translocase of the
Orange CM-H TMROS (a fixable DYm-sensitive dye). We outermembrane(TOM)complex.Thus,weexpectedthatTom40
2
confirmed that there was no crosstalk between the respective knockdownwouldinhibit MTS-dependentmitochondrialimport,
fluorescent signals (supplementary material Fig. S4A). Full- resulting in PINK1 constitutive mitochondrial localization,
lengthPINK1specificallylocalizedondepolarizedmitochondria similar to MTS disruption (Fig. 4) or MTS deletion
e
as inFig. 4B, whereas PINK1 lacking N34 (referred to as DN34 (supplementary material Fig. S4). To test this, HeLa cells were c
n
PINK1)localizedonmitochondriawithanintactDYmasdidN- transfectedwithbothTom40smallinterfering(si)RNAandnon- e
FLAG–PINK1 (supplementary material Fig. S4B,C). We next tagged PINK1, and the localization and phosphorylation state of ci
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analyzed the phosphorylation state of DN34 PINK1 using Phos- PINK1 was examined. In contrast to PINK1 MTS disruption
tagPAGE.Althoughthephosphorylationsignalwasweakerthan (Fig. 4B) and MTS deletion (supplementary material Fig. S4B), ell
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that of N-FLAG–PINK1 (Fig. 1D), DN34 PINK1 was PINK1neitherlocalizedonenergizedmitochondria(Fig. 5A,left
f
also phosphorylated in the absence of CCCP treatment twopanels)norwasphosphorylated(Fig. 5B,lane6).Rather,the o

(supplementary material Fig. S4D, lane 3). These results downregulation of Tom40 hampered PINK1 localization al
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indicate that removal of the MTS promotes mitochondrial (Fig. 5A, right two panels) and phosphorylation (Fig. 5B, lane ur
localization and autophosphorylation of full-length PINK1. 8) on depolarized mitochondria. We also observed the effect of o
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Fig.5.Seenextpageforlegend. ell
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Tom40knockdownonOMMlocalizationandautophosphorylation autophosphorylation (Fig. 5F–H). Import and MTS processing of
ofMTS-deficientPINK1mutants.Themitochondriallocalization Su9–GFP and PINK1-N34–GFP were also inhibited (Fig. 5I,J), al
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ofDN34PINK1,N-FLAG–PINK1and5AspPINK1wereblocked confirming that knockdown of Tom40 indeed hampers matrix ur
in cells treated with Tom40 siRNA (Fig. 5C–E), as was targetingoftheMTS.UnlikePINK1MTSdysfunctionfollowing o
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Fig.5. AutophosphorylationandOMMlocalizationofPINK1dependon knockdown hampered mitochondrial localization of 5D PINK1-
Tom40.(A,B)HeLacellspretreatedwithTom40siRNAweretransfectedwith N90–GFP,confirmingthatTom40isrequiredforthefunctionof
non-taggedPINK1,andthentreatedwithorwithoutCCCP(10mM,1h). the alternative OMM localization signal (Fig. 6D).
Thesecellsweresubjectedtoimmunostainingwithanti-PINK1andanti-
Tom20antibodies(A),andlysateswereimmunoblotted(IB)withanti-PINK1
TheputativeTMDisdispensableforOMMlocalizationof
andanti-Tom40antibodiesafterSDS-PAGEwith(+)orwithout(2)Phos-tag
(B).(C–H)HeLacellstreatedwithTom40siRNAweresubsequently PINK1ondepolarizedmitochondria
transfectedwithN-terminallydeletedDN34PINK1(C,F),N-terminallyFLAG- Next,weexaminedwhethertheputativeTMD(residues94–110)
taggedPINK1(D,G)orN-terminally5D-insertedPINK1–GFP(E,H).These is essential for OMM localization following CCCP treatment,
cellswerefixedandimmunostainedwithanti-PINK1andanti-Tom20 becausethishydrophobicregionwasdefinedaprioriasaPINK1
antibodies(C,D)orwereimagedusingGFPautofluorescenceandTMREin TMD(Silvestrietal.,2005).Weexpressednon-taggedwild-type
livingcells(E).Thecelllysateswereimmunoblottedwithanti-PINK1and
PINK1 or PINK1 without the putative TMD (referred to as
anti-Tom40antibodiesafterSDS-PAGEwithorwithoutPhos-tag(F–H).The
DTMDPINK1)incells(Fig. 7A)andexaminedtheirsubcellular
redandblackarrowheadsindicatephosphorylatedfull-lengthPINK1
(pPINK1)ornon-phosphorylatedfull-lengthPINK1,respectively. localization. Under steady-state conditions in the absence of
(I,J)Mitochondriallocalization(I)andMTSprocessing(J)ofboththe CCCPtreatment,anti-PINK1immunoreactivityofDTMDPINK1
mitochondrialmatrixmarkersSu9–GFPandPINK1-N34–GFPwereinhibited was barely detectable after permeabilization with digitonin, but
byTom40knockdown,confirmingthatTom40knockdownindeedhampers was readily apparent following Triton X-100 permeabilization
matrixtargetingofMTS.Scalebars:10mm.
(Fig. 7B, panels 4 and 5). These results revealed that the
hydrophobic TMD domain (residues 94–111) of PINK1
insertionof5AsporMTSdeletion(e.g.DN34),MTSdysfunction functions as stop-transfer signal to inhibit passage through the
in response to Tom40 knockdown did not enhance PINK1 IMMinenergizedmitochondria,assuggestedpreviously(Becker
localization and activation in OMM. Tom40 is thus involved in etal.,2012),andthatitsupportsIMM-basedcleavageatPhe104
OMM localization and autophosphorylation of PINK1, as and subsequent PINK1 degradation (Yamano and Youle, 2013).
suggested by previous reports (Hasson et al., 2013; Lazarou We then examined whether this putativeTMD isresponsible for
etal.,2012;Okatsuetal.,2013)(seeDiscussion). PINK1 localization in the OMM of depolarized mitochondria.
Surprisingly, DTMD PINK1 still localized on mitochondria
ThesecondmitochondriallocalizationsignalallowsPINK1to following CCCP treatment (Fig. 7B, panel 3). PINK1 DTMD
localizeonOMMwhenthetypicalMTSisinhibited received autophosphorylation following CCCP treatment
We next examined whether the second mitochondrial localization (Fig. 7C), suggesting that this mutant PINK1 localizes correctly
signal is essential for OMM localization of PINK1. As shown in as does wild-type PINK1. Moreover, when DTMD PINK1 was
Fig.2D, in the case of matrix-localized PINK1-N34–GFP, anti- coexpressed with GFP–Parkin in PINK1-knockout cells, DTMD
GFPimmunoreactivitywasabsentafterdigitoninpermeabilization, PINK1 assisted the recruitment of GFP–Parkin to depolarized
butbecamedetectablefollowingTritonX-100permeabilization.By mitochondria and its activation, equivalent to wild-type PINK1
contrast, inthe case of PINK1-N90–GFP and PINK1-N110–GFP, (Fig. 7D,E). These results further confirm that DTMD PINK1
their anti-GFP antibody immunostaining pattern was not affected correctlylocalizesontheOMMofdepolarizedmitochondria,and
bythepermeabilizationconditions(Fig. 6A).Theseresultsindicate that TMD is dispensable for the OMM retention of PINK1 in
that the domain downstream of the N-terminal MTS promoted depolarized mitochondria.
PINK1 retention in the OMM (Fig. 6A), whereas PINK1 lacking Data shown in Figs 2–7 suggest that PINK1 possesses an
thisdomain(suchasPINK1-N34–GFP)passedthroughtheIMMto alternativemitochondriallocalizationsignalbetweenresidues34
the matrix in energized mitochondria (Fig.2D). Because N110– and 90, and that this signal presumably interacts with the Tom
GFPcontainstheTMDdomainforPARL-mediatedprocessing,itis complexinOMMlocalizationwhentheprimaryN-terminalMTS
possible that Phe104 is exposed by processing and N110–GFP is is inhibited, i.e. following a decrease in DYm (see Discussion).
subjected to N-end-rule-catalyzed degradation. However, N110–
GFP seemed not be degraded, probably because an unidentified InhibitionordeletionofthePINK1MTSdrivesParkin
domainjustbehindtheTMDisessentialfordegradationorbecause mitochondriallocalizationandactivation
theproximalGFPtaginhibitsPARL-mediatedprocessingowingto Parkin recruitment and E3 activity on depolarized mitochondria
structuralhindrance. completelydependonPINK1.Thus,wefinallyexaminedwhether
We then examined the subcellular localization of the chimera PINK1 that is localized on the OMM and autophosphorylated
proteins under DYm dissipation conditions. Although pre- followingMTSdysfunction(i.e.N-terminaltagorMTSdeletion)
treatment with CCCP caused PINK1-N34–GFP to localize to recruits and activates Parkin on energized mitochondria. To
the cytosol and plasma membrane (Fig. 6B, panel 2, also shown confirm whether N-FLAG–PINK1 or DN34 PINK1 activates
inFig. 2),mitochondriallocalizationwasretainedinthePINK1- Parkin under steady-state conditions, we tried to examine the
N90–GFP and N110–GFP chimeras following CCCP treatment localizationandE3activityofParkinwhenthesePINK1mutants
e
(Fig. 6B, panels 3 and 4). We also examined localization were expressed. However, overexpression of exogenous PINK1 c
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following the insertion of 5Asp at the N-terminus to inhibit itself(eventhewild-typeform)triggersmitochondriallocalization e
MTS function (Fig. 3). As before, the insertion changed the and autoubiquitylation of Parkin even in the absence of CCCP ci
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mitochondrial localization of PINK1-N34–GFP to a dispersed (Matsudaetal.,2010;Narendraetal.,2010),andthuscomparisons
cytosolicpattern(Fig. 6C,panel2,alsoshowninFig. 3),whereas of the effect that the PINK1 mutants have on Parkin are ell
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the mitochondrial localization of PINK1-N90–GFP and N110– problematic. To overcome this limitation, we have previously
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GFPremainedunchanged(Fig. 6C,panels3and4).Theseresults established more appropriate experimental conditions for the
suggestthatthesecondmitochondriallocalizationsignalresiding coexpression of PINK1 and Parkin using a weakened CMV al
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between residues 35–90 maintains PINK1 localization in the promoter – CMV(d1) (Okatsu et al., 2012b). Overexpression of ur
OMM when the MTS is inhibited. By contrast, Tom40 wild-type PINK1 using an intact CMV promoter causes Parkin o
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Fig.6. TheN-terminal90aminoacidsofPINK1aresufficient
formitochondriallocalizationwhentypicalMTSfunctionis
inhibited.(A)GFPfusedtotheN-terminal90or110aminoacidsof
PINK1doesnotpassthroughtheIMM.Permeabilizationconditions
didnotaffecttheimmunostainingpatternofeitherPINK1-N110–
GFPorN90–GFPasdetectedbyananti-GFPantibody.
(B)MitochondriallocalizationofPINK1-N90–GFPandN110–GFP
wasconservedevenfollowingCCCPtreatment.(C)Mitochondrial
localizationofPINK1-N90–GFPandN110–GFPwasunchanged
whenfiveAspresidueswereinsertedattheN-terminustoinhibit
MTSfunction.(D)Tom40knockdownhamperedthemitochondrial
localizationof5DPINK1-N90–GFP,suggestingthatTom40is
requiredforfunctionofthealternativemitochondriallocalization
signal.Scalebars:10mm.
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mitochondrial localization and activation irrespective of DYm, N-terminally or C-terminally tagged PINK1 under control of the
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whereas expression of wild-type PINK1 using the CMV(d1) CMV(d1)promoter,andthensubjectedtoTMREstaining.Incells
promoter results in Parkin recruitment and activation that are expressingC-terminally3HA-taggedPINK1,Parkinwascytosolic al
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dependent on a decrease in DYm (Okatsu et al., 2012b). HeLa under steady-state conditions and mitochondria were stained ur
cells stably expressing GFP–Parkin were transfected with with TMRE. By contrast, CCCP treatment promoted Parkin o
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Fig.7. TheTMDofPINK1isrequiredforstop-transferintheIMMinenergizedmitochondriabutisdispensableforOMMlocalizationindepolarized
mitochondria.(A)Schematicdiagramoftheconstructsusedinthisfigure.WT,wild-type.(B)Subcellularlocalizationofnon-taggedwild-typePINK1orDTMD
PINK1(lackingtheputativeTMD,residues94–110)undernormalandDYmdissipationconditions.TheTMDfunctionsasastop-transfersignaltoinhibit
passagethroughtheIMMinenergizedmitochondria,whereastheTMDisdispensableforlocalizationindepolarizedmitochondria.(C)DTMDPINK1undergoes
autophosphorylationfollowingCCCPtreatment.RedarrowheadsindicatephosphorylatedPINK1(pPINK1).(D)PINK1-knockoutcellscoexpressingGFP–Parkin
andtheindicatedPINK1mutantswereimmunostainedwithanti-GFPandanti-Tom20antibodies.Parkinwasrecruitedtodepolarizedmitochondriaby
DTMDPINK1,confirmingthatDTMDPINK1correctlylocalizedontheOMMofdepolarizedmitochondria.Scalebars:10mm.(E)PINK1-knockoutcelllysates
coexpressingGFP–Parkinandwild-typeorDTMDPINK1wereimmunoblotted(IB)withanti-Parkin,anti-PINK1andanti-actin(loadingcontrol)antibodies.
TheredbarindicatesubiquitylatedGFP–Parkin.Althoughthepositionofthemolecular-massmarker(64kDa)changesalittlebetweenahand-madenon-
phostaggel(C)andthecommercialprecastgel(E),PINK1bandsshowninEcorrespondtothefull-lengthPINK1.
localization on unstained mitochondria (Fig. 8A, upper and the canbeusedasanindexofParkinactivation(Matsudaetal.,2010).
middle panels). Interestingly, transfection of N-terminal FLAG- N-FLAG–PINK1andDN34PINK1undertheCMV(d1)promoter
tagged PINK1 triggered Parkin recruitment on energized accelerated autoubiquitylation of GFP–Parkin, whereas C-
mitochondria stained with TMRE (Fig. 8A, lower panels). terminal 36HA-tagged PINK1 under the same CMV(d1)
e
Similarly, DN34 PINK1 recruited GFP–Parkin to energized promoter did not (Fig. 8D, lanes 3–5). Indeed, Parkin E3 c
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mitochondria even without CCCP treatment, whereas wild-type activity was more pronounced with the N-terminal-tagged and e
PINK1 did not recruit Parkin to mitochondria under the same N-terminal-deleted PINK1 than with over-produced wild-type ci
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experimentalconditions(Fig. 8B).StatisticalanalysisusingHeLa PINK1(Fig. 8D,comparelanes2withlanes4and5),eventhough
cells co-transfected with GFP–Parkin and N-FLAG–PINK1 or thePINK1levelofthetwomutantswaslowerthanthatofwild- ell
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DN34 PINK1 confirmed their Parkin recruitment activity typePINK1underthefullCMVpromoter(Fig. 8D,lowerpanel).
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(Fig. 8C). We next examined whether the expression of N- We further examined whether N-FLAG–PINK1 triggers
FLAG–PINK1andDN34PINK1acceleratetheautoubiquitylation downstream events such as the recruitment of ubiquitin and LC- al
n
ofGFP–Parkin,becausethetranslocatedParkinexertsE3activity 3,anautophagicmarker(Kabeyaetal.,2000)onmitochondria.As ur
andubiquitylatesin-frame-fusedGFP,andthusautoubiquitylation has already been reported, ubiquitin and LC-3 accumulated only o
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