ADimericPINK1-containingComplexonDepolarized ...ADimericPINK1-containingComplexonDepolarized...

A Dimeric PINK1-containing Complex on DepolarizedMitochondria Stimulates Parkin Recruitment*

Received for publication, August 9, 2013, and in revised form, October 28, 2013 Published, JBC Papers in Press, November 4, 2013, DOI 10.1074/jbc.M113.509653

Kei Okatsu‡§, Midori Uno¶, Fumika Koyano‡§, Etsu Go‡, Mayumi Kimura‡, Toshihiko Oka¶, Keiji Tanaka‡1,and Noriyuki Matsuda‡2

From the ‡Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku 156-8506, Tokyo, the§Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8561, and the ¶Department of Life Science, Collegeof Science, Rikkyo University, Nishi-Ikebukuro, Tokyo 171-8501, Japan

Background: PINK1 functions on depolarized mitochondria. However, details regarding its mechanism remain limited.Results:We reveal the formation of a high molecular weight complex composed of two phosphorylated PINK1 molecules thatstimulates Parkin recruitment.Conclusion: The dimeric PINK1-containing complex is important for mitochondrial quality control.Significance: The PINK1 molecular process is reminiscent of receptor kinase dimerization in signal transduction.

Parkinsonism typifiedby sporadicParkinsondisease is a prev-alent neurodegenerative disease. Mutations in PINK1 (PTEN-induced putative kinase 1), a mitochondrial Ser/Thr proteinkinase, or PARKIN, a ubiquitin-protein ligase, cause familialparkinsonism. The accumulation and autophosphorylation ofPINK1 on damaged mitochondria results in the recruitment ofParkin, which ultimately triggers quarantine and/or degrada-tion of the damaged mitochondria by the proteasome andautophagy.However, themolecularmechanismofPINK1 indis-sipation of the mitochondrial membrane potential (��m) hasnot been fully elucidated. Here we show by fluorescence-basedtechniques that the PINK1 complex formed following adecrease in ��m is composed of two PINK1 molecules and iscorrelated with intermolecular phosphorylation of PINK1. Dis-ruption of complex formation by the PINK1 S402A mutationweakened Parkin recruitment onto depolarized mitochondria.Themost disease-relevantmutations of PINK1 inhibit the com-plex formation. Taken together, these results suggest that for-mation of the complex containing dyadic PINK1 is an importantstep for Parkin recruitment onto damaged mitochondria.

Parkinsonism is a pervasive neurodegenerative diseasecaused by a loss of dopaminergic neurons in the substantianigra. The predominant form of parkinsonism, clinicallydefined as Parkinsondisease (PD),3 is sporadic. Less prevalent is

the heritable familial early onset parkinsonism. Although thesymptoms of hereditary parkinsonism are not the same as spo-radic PD, they share a number of clinical features (1). Thus, thefunctional study of genes/proteins involved in familial parkin-sonism can also expand our understanding of the pathogenicmechanisms underlying PD.The mitochondrial Ser/Thr kinase PINK1 (PTEN-induced

putative kinase 1) was identified as a causal gene for autosomalrecessive early onset parkinsonism (2) and has been studiedgenetically using PINK1-deficient model organisms. For exam-ple, Drosophila melanogaster lacking pink1 is characterized byflight muscle degeneration, short life span, and male sterility(3–5). Furthermore, mitochondria in the pink1 mutants havemorphological and functional defects. Factors that regulatemitochondrial morphology have been shown to interact genet-ically with pink1 (5–7), and the pink1-deficient phenotype inDrosophila can be rescued by a component of the mitochon-drial electron transport chain complex or amitochondrial elec-tron carrier (8, 9). Respiratory chain defects have been reportedin Pink1�/� mouse embryonic fibroblasts (10), and some mor-phological and functional defects of mitochondria have beenobserved in the striatum of Pink1 null mice; Pink1 knock-outmice, however, do not exhibit any obvious morphologicalchanges or loss of dopaminergic neurons in the substantia nigra(11, 12). These findings indicate that PINK1 contributes tomitochondrial integrity.Reductions in the activity of the electron transport chain

complex I and mitochondrial DNA mutations have beenobserved in sporadic PD (13, 14). Further, toxic parkinsonism iscaused by various inhibitors of the mitochondrial respiratorychain, such as rotenone andMPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Thus, a correlation between parkinsonismand mitochondrial impairment has been well established.Recent studies revealed that when the mitochondrial mem-

brane potential (��m) decreases, PINK1 accumulates and is

* This work was supported by Japan Society for the Promotion of Science(JSPS) KAKENHI Grant 23687018 and MEXT KAKENHI Grants 24111557 and25112522, the Takeda Science Foundation, and the Tomizawa Jun-ichi andKeiko Fund for Young Scientists (to N. M.); by JSPS KAKENHI Grant23570232 and the grant for CREST from JST (to T. O.); by JSPS KAKENHIGrant 23�6061 (to K. O.); and by KAKENHI Grant 21000012 and the TakedaScience Foundation (to K. T.).

1 To whom correspondence may be addressed. Tel.: 81-3-5316-3123 (ext.2720); Fax: 81-3-5316-3152; E-mail: [email protected].

2 To whom correspondence may be addressed. Tel.: 81-3-5316-3123 (ext.2720); Fax: 81-3-5316-3152; E-mail: [email protected].

3 The abbreviations used are: PD, Parkinson disease; ��m, mitochondrialmembrane potential; OMM, outer mitochondrial membrane; TOM, trans-locase of the outer membrane; IB, immunoblotting; CCCP, carbonylcyanide p-chlorophenylhydrazone; NAMOS, native antibody-based

mobility shift; BN-PAGE, blue native PAGE; CN-PAGE, clear native PAGE; KD,kinase-dead; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 51, pp. 36372–36384, December 20, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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phosphorylated on the outermitochondrialmembrane (OMM)by escaping ��m-dependent degradation (15–17). PINK1simultaneously forms a high molecular weight complex withthe translocase of the outer membrane (TOM)machinery (18).The ubiquitin ligase (E3) Parkin, another causal gene in auto-somal recessive early onset parkinsonism (19, 20), is subse-quently recruited to the depolarized mitochondria and func-tions in the sequestration and/or elimination of damagedmitochondria in cultured cells (21), iPS-derived neurons (1, 22,23), and mouse primary neurons (24–26). PINK1 is essentialfor recruiting Parkin to the depolarized mitochondria; thus, itacts as an upstream factor of Parkin (15, 16, 27–30).When bothhealthy and damaged mitochondria coexist in the same cell,Parkin selectively localizes on the damaged mitochondria (16,21, 31).The transport of inner mitochondrial membrane or mito-

chondrial matrix proteins generally utilizes��mas the drivingforce (32). In this transport process, the proteins are cleaved byvarious proteases. Numerous studies have shown that understeady-state conditions, PINK1 is cleaved by MPP (mitochon-drial processing peptidase), PARL (presenilin-associated rhom-boid-like protein), ClpXP, and AFG3L2 (33–37) and then rec-ognized by the cytoplasmic N-end rule degradation pathway,which defines the rapid PINK1 turnover (38). The suppressionof PINK1 transport into the inner mitochondrial membrane bya decrease in ��m triggers an accumulation of PINK1 onOMM (18). Phosphorylation of Ser-228 and Ser-402 activatesthe accumulated PINK1, triggering the recruitment of Parkin tothe depolarized mitochondria (17). Thus, PINK1 is not onlyquantitatively but also qualitatively regulated bymitochondrialconditions. However, the dynamics of PINK1 on depolarizedmitochondria have not been fully elucidated.In this study, we established a multicolor detection method

for resolving with high sensitivity the dynamic interactions ofPINK1. Using thismethod, we demonstrated that PINK1 formsa high molecular weight complex containing a PINK1 dimer.Complex formation is correlated with intermolecular phosphor-ylation. Defects in PINK1 complex formation significantlyreduced Parkin translocation onto depolarized mitochondria,suggesting that PINK1 complex formation might be a patho-physiologically significant process for Parkin-mediated mito-chondrial quality control.

EXPERIMENTAL PROCEDURES

Plasmids and Antibodies—Plasmids used in this study aresummarized in Table 1. For the native antibody-based mobilityshift (NAMOS) assay, the following antibodies were used: anti-PINK1 (product number BC100-494; Novus; 1:10 dilution),anti-Tom20 (product number FL-145; Santa Cruz Biotechnol-ogy, Inc.; 1:10 dilution), anti-Tom22 (clone 1C9-2; Sigma; 1:10dilution), anti-Tom40 (gift from Dr. Mihara’s laboratory;1:10 dilution), anti-Tom70 (gift form Dr. Mihara’s laboratory;1:10 dilution), anti-GFP (product number A6455 and clone 3E6(Invitrogen; 1:10 dilution) and product number ab6556(Abcam; 1:10 dilution)), anti-mCherry (clone 3G5; MBL; 1:10dilution), and anti-V5 (product number 46-0705; Invitrogen;1:10 dilution). For immunoblotting (IB), anti-FLAG/DDDDK(clone FLA-1; MBL; 1:1,000 dilution), anti-PINK1 (product

number BC100-494; Novus; 1:1,000 dilution), anti-actin (cloneAC-40; Sigma; 1:500 dilution), anti-Tom20 (product numberFL-145; SantaCruzBiotechnology; 1:200 dilution), anti-Tom40(gift from Dr. Mihara’s laboratory; 1:1,000 dilution), anti-Tim23 (product number 611222; BD Biosciences; 1:500 dilu-tion), and anti-HSP60 (clone N-20; Santa Cruz Biotechnology;1:1,000 dilution) antibodies were used.Cells and Transfections—HeLa cells were cultured at 37 °C

with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM;Sigma) containing penicillin, streptomycin and L-glutamine(Invitrogen), 1� nonessential amino acids (Invitrogen), 1�sodium pyruvate (Invitrogen), and 10% fetal bovine serum(Invitrogen). HeLa cells stably expressing GFP-Parkin wereestablished by infecting HeLa cells transiently expressingmCAT1 with recombinant retroviruses. Recombinant retrovi-rus was made using PLAT-E cells as reported previously (16,17). Various plasmids were transfected with Fugene 6 (Pro-mega), and PINK1 siRNAwas introduced intoHeLa cells stablyexpressing GFP-Parkin by using Lipofectamine 2000 (Invitro-gen). To decrease��m, the cells were treated for varying timeswith 10–15 �M CCCP (Sigma).Cell Fractionation and Proteinase K-resistant Assay—HeLa

cells transfected with expression plasmids were treated withCCCP for 1 h at 37 °C, suspended in fractionation buffer (0.25 M

sucrose, 20mMHEPES-NaOH (pH8.1), and protease and phos-phatase inhibitor mixture (Roche Applied Science)) and dis-rupted by passaging (30 times) through a 25-gauge needle witha 1-ml syringe. Debris was removed by centrifugation at1,000 � g for 7 min. The resulting supernatant was centrifugedat 10,000 � g for 10 min at 4 °C to separate the mitochondria-rich fractions from the cytosolic fraction. The mitochondria-rich fractions were incubated with 50 �g/�l proteinase K(Wako) on ice for 30 min, and the reaction was terminated bythe addition of 1 mM PMSF prior to electrophoresis.Phos-tag Assay—To detect phosphorylated proteins via

PAGE, 7.5% polyacrylamide gels containing 50 �M Phos-tagacrylamide (Wako) and 100 �M MnCl2 were used as reportedpreviously (17). After electrophoresis, the gels were initiallyincubated with gentle shaking in transfer buffer containing0.01% SDS and 1 mM EDTA for 10 min and then incubated intransfer buffer containing 0.01% SDSwithout EDTA for 10minaccording to the manufacturer’s protocol. Proteins were blot-ted onto PVDF membranes and analyzed by IB.Blue Native PAGE (BN-PAGE), Clear Native PAGE (CN-

PAGE), andNAMOSAssay—BN-PAGEwas performed follow-ing the manufacturer’s protocol, whereas CN-PAGE based onWittig and Schagger’s method (39) was optimized to facilitateobservation of the PINK1 complex. The samples for BN- andCN-PAGE analysis were harvested using a NativeTM PAGESample Prep Kit (Invitrogen). Cells were extracted with 1�NativeTMPAGEbuffer for 15min at 4 °C, pipetted up and down10 times, and centrifuged at 20,000 � g for 30 min at 4 °C.BN-PAGE and CN-PAGE were performed using NativeTMPAGE Running Buffer (Invitrogen) containing either 0.002%G-250 (Invitrogen) or 0.05% sodium cholate (Wako) and 0.01%n-heptyl-�-D-thioglucoside (DOJINDO), respectively. The flu-orescence signal in clear native polyacrylamide gels wasdetected with a Typhoon 9400 fluorescence scanner (GE

Dimeric PINK1-containing Complex on Mitochondria

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Healthcare). For BN-PAGE, gels were shaken in denaturationbuffer (10mMTris-HCl pH 6.8, 1% SDS, and 0.006% 2-mercap-toethanol) for 15 min at 60 °C after electrophoresis and thentransferred to PVDF membranes for IB analysis. For theNAMOS assay, cell lysates were prepared as before, and anti-bodies were added to the lysate. The samples were incubatedat room temperature for 1 h. Samples were subjected to CN-PAGE and fluorescence-detected as before.Two-dimensional BN/SDS-PAGE—Two-dimensional gel

electrophoresis was performed using BN-PAGE in the firstdimension. The resulting gels were initially incubated withshaking in a reducing solution (SDS sample buffer with 50 mM

DTT) before being transferred to an alkylating solution (SDSsample buffer with 50 mM N,N-dimethylacrylamide) at 60 °Cfor 15 min. Alkylation was terminated by incubating the gels ina quenching solution (SDS sample buffer with 5 mM DTT and20% methanol) according to the manufacturer’s protocol. Theequilibrated gel strip was then immediately subjected to seconddimension electrophoresis using Phos-tag SDS-PAGE.

siRNAAssay—siRNAwas designed to a portion of the PINK1mRNA 3�-UTR comprising 3�-GGGUCAGCACGUUCAGU-UAdTdT-5� and 5�-UAACUGAACGUGCUGACCCAdT-3�.HeLa cells stably expressing GFP- or HA-Parkin were pre-treated with PINK1 siRNA and then transfected with CMV d1promoter-driven PINK1WT, S228A, or S402A mutants. After42 h, the cells were treatedwithCCCP (10�M) for the indicatedtimes (1.5 or 16 h), and the number of cells with Parkin-positivemitochondria orTom20was determined in�100 cells. Becausethe siRNA targeted the 3�-UTR of PINK1, it had no effect onexpression of the exogenous PINK1 mutants.

RESULTS

Development of a Multicolor Detection Method to Resolve aHigh Molecular Weight PINK1 Complex in Response to aDecrease in ��m—Under ordinary cellular conditions, PINK1on healthy mitochondria is immediately cleaved and degradedin the presence of a normal ��m. On depolarized mitochon-dria, however, PINK1 eludes ��m-dependent degradation,

TABLE 1Plasmids used

Vector Description Source

Plasmids used in conjunction with CN-PAGE and two-dimensionalBN/SDS-PAGE (Figs. 1–3)

pcDNA3.1-non tagged PINK1WT For transient expression of non-tagged WT PINK1 Ref. 58pEGFP-N1-PINK1WT-GFP For expression of WT PINK1-GFP Ref. 17

Plasmids used to obtain stoichiometric evidenceof PINK1 complex (Figs. 1 and 2)

pCMV14-PINK1WT-GFP-3FLAG For expression of WT PINK1-GFP-3FLAG This studypCMV14-PINK1WT-mCherry-3FLAG For expression of WT PINK1-mCherry-3FLAG This studypcDNA3.1-PINK1WT-V5/His For expression of WT PINK1-V5/His This study

Plasmids used to transiently express pathogenic mutantsof PINK1-GFP (Fig. 4)

pEGFP-N1-PINK1 KD-GFP For expression of KD PINK1-GFP Ref. 17pEGFP-N1-PINK1 G92F-GFP For expression of G92F PINK1-GFP This studypEGFP-N1-PINK1 A168P-GFP For expression of A168P PINK1-GFP This studypEGFP-N1-PINK1 E240K-GFP For expression of E240K PINK1-GFP This studypEGFP-N1-PINK1 H271Q-GFP For expression of H271Q PINK1-GFP This studypEGFP-N1-PINK1 G309D-GFP For expression of G309D PINK1-GFP This studypEGFP-N1-PINK1 L347P-GFP For expression of L347P PINK1-GFP This studypEGFP-N1-PINK1 G386A-GFP For expression of G386A PINK1-GFP This studypEGFP-N1-PINK1 G409V-GFP For expression of G409V PINK1-GFP This studypEGFP-N1-PINK1 E417G-GFP For expression of E417G PINK1-GFP This studypEGFP-N1-PINK1 534insQ-GFP For expression of 534insQ PINK1-GFP This study

Plasmids used to transiently express pathogenic mutantsof PINK1–3FLAG (Figs. 5 and 6)

pCMV14-PINK1WT-3FLAG For expression of WT PINK1-3FLAG Ref. 51pCMV14-PINK1 KD-3FLAG For expression of KD PINK1-3FLAG Ref. 59pCMV14-PINK1 C92F-3FLAG For expression of A168P PINK1-3FLAG Ref. 17pCMV14-PINK1 A168P-3FLAG For expression of A168P PINK1-3FLAG Ref. 17pCMV14-PINK1 E240K-3FLAG For expression of E240K PINK1-3FLAG Ref. 17pCMV14-PINK1 H271Q-3FLAG For expression of H271Q PINK1-3FLAG Ref. 51pCMV14-PINK1 G309D-3FLAG For expression of G309D PINK1-3FLAG Ref. 51pCMV14-PINK1 L347P-3FLAG For expression of L347P PINK1-3FLAG Ref. 59pCMV14-PINK1 G386A -3FLAG For expression of G386A PINK1-3FLAG Ref. 17pCMV14-PINK1 G409V-3FLAG For expression of G409V PINK1-3FLAG Ref. 59pCMV14-PINK1 E417G-3FLAG For expression of E417G PINK1-3FLAG Ref. 51pCMV14-PINK1 534insQ-3FLAG For expression of 534insQ PINK1-3FLAG Ref. 17

Plasmids used in retrovirus-mediated expression (Fig. 6)pMXs-puro-GFP-Parkin For retrovirus-mediated expression of GFP-Parkin Ref. 16

Plasmids used to analyze the relationship betweenautophosphorylation and PINK1 complex formation (Fig. 6)

pcDNA3.1-non tagged PINK1WT For expression of WT PINK1 Ref. 58pcDNA3.1-non tagged PINK1 S228A For expression of S228A PINK1 Ref. 17pcDNA3.1-non tagged PINK1 S402A For expression of S402A PINK1 Ref. 17pEGFP-N1-PINK1 S228A-GFP For expression of S228A PINK1-GFP This studypEGFP-N1-PINK1 S402A-GFP For expression of S402A PINK1-GFP This studypCMV(d1)TNT-PINK1WT-3HA For weak expression of WT PINK1-3HA Ref. 17pCMV(d1)TNT-PINK1 S228A-3HA For weak expression of S228A PINK1-3HA This studypCMV(d1)TNT-PINK1 S402A-3HA For weak expression of S402A PINK1-3HA This study




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and the accumulated PINK1 recruits cytosolic Parkin to thedamaged mitochondria (15, 16). To observe the dynamics ofPINK1 on depolarizedmitochondria, we performedBN-PAGE,a method for detecting supermolecular complexes of mem-brane proteins (40). In BN-PAGE, Coomassie G-250 functionsas the negatively charged molecule that binds to the non-dena-tured proteins and drives their migration toward the anodeduring electrophoresis.WhenHeLa cells transiently expressingnon-tagged PINK1 (�60 kDa) were treated with CCCP todecrease ��m, exogenous PINK1 formed a supermolecularcomplex (predicted size of �850 kDa; referred to as 850-kDacomplex hereafter) (Fig. 1A) (18, 41). We next used differentialcentrifugation to examine whether this complex is formed onmitochondria. HeLa cells transiently expressing non-taggedPINK1were treatedwithCCCP, and the resulting extracts werecentrifuged to obtain a mitochondria-rich fraction. BN-PAGE

analysis and concomitant immunoblotting indicated that thesupermolecular PINK1 complex was indeed recovered in themitochondrial fraction (Fig. 1B). Moreover, we confirmed thatendogenous PINK1 formed a similar 850-kDa complex inCCCP-treated cells as well as exogenous PINK1 (Fig. 1C).We next performed CN-PAGE (39, 42), in which sodium

cholate is used as the negatively charged molecule. Similar toBN-PAGE, proteins bound to sodium cholate migrate towardthe anodewithout denaturation. Because sodium cholate is col-orless and the native folding structure of proteins is maintainedduring electrophoresis, it is possible to use fluorescently taggedproteins (e.g. GFP and mCherry) for in gel detection in CN-PAGE (39). When HeLa cells expressing PINK1-GFP weretreated with CCCP and then subjected to BN- and CN-PAGE,the fluorescently tagged protein co-localizedwith the supermo-lecular complex (Fig. 1D, middle lane). This band disappeared

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FIGURE 1. A, exogenous PINK1 forms a supermolecular complex following CCCP treatment. HeLa cells expressing non-tagged PINK1 were treated with CCCP(10 �M, 1 h), and the resulting cell lysates were subjected to BN-PAGE and immunoblotted with an anti-PINK1 antibody. The blue arrowhead indicates thesupermolecular PINK1 complex (note that this designation will be used hereafter unless otherwise specified). B, mitochondria-rich fractions isolated from cellstreated as before were analyzed by BN-PAGE and immunoblotted. C, endogenous PINK1 also forms a supermolecular complex following CCCP treatment. HeLacells were treated with CCCP (10 �M, 12 h) and subjected to BN-PAGE and immunoblotted. D, the presence of the PINK1 complex following a decrease in ��mwas detected using an anti-PINK1 antibody or GFP fluorescence. HeLa cells expressing PINK1-GFP were treated with CCCP (10 �M, 3 h) and subjected toBN-PAGE or CN-PAGE. PINK1 was detected using an anti-PINK1 antibody following blotting to a PVDF membrane (left and middle) or was detected directly ingel by GFP fluorescence using a fluorescence imaging scanner (right). E, dual color imaging of PINK1 using distinct florescent proteins. HeLa cells expressingPINK1-GFP and/or PINK1-mCherry were treated with CCCP (10 �M, 3 h) and subjected to CN-PAGE, and fluorescence signals derived from GFP and mCherrywere detected.




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when ��mwas recovered with CCCP washout (Fig. 1D, right).CN-PAGE in gel GFP fluorescence offered higher resolutionand sensitivity for detecting the PINK1 complex than immuno-blotting using an anti-PINK1 antibody (Fig. 1D, compare theright panel with the middle panel). Similar to PINK1-GFP,PINK1-mCherry likewise formed the 850-kDa complex follow-ing a decrease in ��m and was detected by the multicolordetection method (Fig. 1E). These results confirmed the utilityof themulticolor fluorescence detectionmethod and suggestedthat this approach would be advantageous for monitoringPINK1 dynamics.The 850-kDaHighMolecularWeight Complex Contains Two

Molecules of PINK1—We next performed a NAMOS assay (43)to determine the component constituency and stoichiometry ofthe supermolecular complex. In this assay, an antibody againstone component causes the complex to shift to a higher molec-ular weight position in native PAGE. HeLa cell extracts con-taining PINK1-GFPwere subjected toCN-PAGEwith either ananti-PINK1 or anti-GFP antibody, followed by detection ofPINK1-GFP fluorescence (Fig. 2A). In this experiment, we usedmultiple antibodies, including a polyclonal anti-PINK1 anti-body, BC100-494 (immunoreactive to amino acids 175–250 ofPINK1); polyclonal anti-GFP antibodies A6455 and ab6556(which recognize full-length GFP); and the monoclonal anti-GFP antibody 3E6. When the anti-PINK1 antibody was incu-bated with the 850-kDa complex, a shift in the PINK1 complexwas clearly observed (Fig. 2, A and B, lane 2). When the mono-clonal antibody 3E6 was utilized, PINK1-GFP resolved as a sin-gle band (probably because the antibody recognizes a uniqueGFP site), whereas PINK1-GFP resolved asmultiple bands withthe polyclonal antibodies (e.g. BC100-494, A6455, and ab6556)(Fig. 2A). We next determined if components of the TOMmachinery were also included in the PINK1 complex (18). Inthis assay, antibodies against Tom20, Tom22, and Tom40caused a shift in the PINK1 complex in CN-PAGE (Fig. 2B,lanes 3–5), suggesting that all three TOM components areincluded in the PINK1 complex. In contrast, an anti-Tom70antibody had no effect on the position of PINK1 fluorescence(lane 6). Although we cannot exclude the possibility thatTom70 interacts with the PINK1 complex in a digitonin-sensi-tive manner or a Tom70 antibody recognition site is masked,this result suggests that the complex is devoid of Tom70 (see“Discussion”).We next used PINK1 proteins fused to a distinct tag (V5,

GFP, ormCherry) to obtainmore detailed stoichiometric infor-mation regarding the number of PINK1molecules contained inthe complex. PINK1-GFP and PINK1-mCherry were co-ex-pressed in HeLa cells, treated with CCCP, and subjected to theNAMOS assay using anti-GFP and anti-mCherry monoclonalantibodies. If the 850-kDa complex is composed of only PINK1-GFP, then the addition of an anti-mCherry antibody shouldhave no effect on the position of the PINK1-GFP fluorescentsignal in CN-PAGE. A shift in the PINK1-GFP signal by theanti-mCherry antibody, however, would indicate that the 850-kDa complex contains both PINK1-mCherry and PINK1-GFP.When PINK1-GFP alone was expressed in cells, the GFP fluo-rescence signal did not change following the addition of theanti-mCherry antibody (Fig. 2C, lane 7). In contrast, when

co-expressed with PINK1-mCherry, the PINK1-GFP-de-rived fluorescence signal was noticeably shifted upward fol-lowing the addition of the anti-mCherry antibody (Fig. 2C,lane 9).The above findings revealed that the 850-kDa complex con-

tains at least two PINK1 molecules. We next sought to deter-mine the stoichiometry. PINK1-GFP was co-expressed withboth PINK1-V5 and PINK1-mCherry, subjected to a NAMOSassay using anti-V5 and anti-mCherry monoclonal antibodies,and detected by GFP-derived fluorescence in CN-PAGE. If the850-kDa complex contains two PINK1 molecules, only a one-step shift in GFP-fluorescence (equivalent to one antibody)would be observed under the aforementioned conditionsregardless of the type and combination of antibodies used,whereas, if the 850-kDa complex contains more than twoPINK1 molecules, a two-step shift in GFP fluorescence wouldbe observed with the anti-V5 and anti-mCherry antibodies.HeLa cells transfectedwith PINK1-GFP alone, PINK1-GFP andPINK1-V5, PINK1-GFP and PINK1-mCherry, or all three con-structs were treated with CCCP and then subjected to theNAMOS assay using anti-V5 and anti-mCherry antibodies. Notwo-step shift in GFP-fluorescence was observed (Fig. 2D),strongly suggesting that the 850-kDa complex contains onlytwo PINK1 molecules. A two-step shift was only observed fol-lowing the inclusion of an anti-GFP antibody along with theanti-V5 and anti-mCherry antibodies, thus confirming that thesupermolecular complex is limited to two PINK1 molecules(Fig. 2D, far right panel).The High Molecular Weight PINK1 Complex Is Composed of

Phosphorylated PINK1—PINK1 rapidly accumulates on depo-larized mitochondria and undergoes autophosphorylation fol-lowing a decrease in ��m (17, 44). Hence, we next examinedthe phosphorylation state of PINK1 with polyacrylamide gelsconjugated to a 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-olato diMn(II) complex (referred to here as Phos-tag (45)).Phos-tag specifically retards the migration of phosphorylatedproteins; thus, phosphorylated PINK1 resolved as a slowermigrating band compared with non-phosphorylated PINK1(Figs. 3, A and B). To determine if PINK1 in the high molec-ular weight complex is phosphorylated, we performed two-dimensional electrophoresis in which the proteins were firstresolved using BN-PAGE and then subsequently Phos-tagSDS-PAGE. Following CCCP treatment, almost all of thePINK1 in the complex underwent a molecular shift in thePhos-tag SDS-PAGE, revealing that PINK1 in the highmolecular weight complex is the phosphorylated form (Fig.3, C and D). These results suggest that PINK1 complex for-mation is related to PINK1 phosphorylation.Disease-relevant Mutations of PINK1 Inhibit Complex

Formation—We next sought to determine if various patho-genic mutations or a kinase-dead (KD) mutation consisting ofthe triple missense mutation K219A/D362A/D384A (46)inhibit formation of the PINK1 complex in cells. Constructsharboring the PINK1 KD mutation or one of 10 pathogenicmutations, including C92F, A168P, E240K, H271Q, G309D,L347P, G386A, G409V, E417G, and a pathogenicmutation thatresults in the insertion of glutamine at amino acid position 534(referred to hereafter as 534insQ), were generated. These




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PINK1 mutants, with the exception of C92F, partially or com-pletely impaired phosphorylation and inhibited Parkin recruit-ment onto depolarized mitochondria without altering mito-chondrial localization (16, 17, 47). HeLa cells expressingPINK1-GFP harboring the aforementioned mutations weresubjected to CN-PAGE following CCCP treatment. Formationof the 850-kDa PINK1 complex was completely or severelyinhibited by the KD mutation as well as the A168P, E240K,H271Q, L347P, G386A, E417G, and 534insQ mutations (Fig.4A). In contrast, the C92F, G309D, and G409V PINK1mutants

formed the 850-kDa complex equivalent to wild type (WT)PINK1 (Fig. 4A). Immunoblotting by conventional SDS-PAGEwith an anti-PINK1 antibody confirmed that themutant PINK1proteins were almost equally expressed (Fig. 4B). The NAMOSassay revealed that the C92F, G309D, and G409V PINK1mutants interact with components of theTOMcomplex aswellas PINK1 WT (Fig. 4C). Taken together, the mutation datademonstrated that various PINK1mutants were unable to formthe 850-kDa complex in cells, suggesting an etiological impor-tance of PINK1 complex formation.

480

1236

1048

720

1236

1048

720

1236

1048

720(kDa)

MergeGFP

mCherry

12361048

720

(kDa)

1236

1048

720(kDa)

PINK1-V5

PINK1-GFP

no antibody

1 2 3 4 5 6

1 2 3 4 5

7 8 9 10 11 12

+ antibody + anti-V5 + anti-mCherry+ anti-V5,

anti-mCherry

PINK1-mCherry

+ anti-V5,anti-mCherry,

anti-GFP

GFP ( Fluorescence )

mCherry(Fluorescence)

GFP(Fluorescence)

non-shift1st-shift2nd-shift




PINK1-mCherry

PINK1-GFP

no antibody+ antibody + anti-GFP + anti-mCherry+ anti-GFP,anti-mCherry

+ +

++

-

- ++ - ++ - ++ -

++ + ++ + ++ + ++ + ++ ++ + ++ +

+- - -+ - -+ + +- + -- -+ + +- +-- + ++ - -- + ++ - +- -- + ++ -

+ +- + +- +-

GFP (Fluorescence)

shiftedband

A

D

* *

* *

no a

ntibo

dy

BC100-

494

(pAb)

A6455

(pAb)

3E6

(mAb)

ab65

56 (p

Ab)+ anti-PINK1 or

anti-GFP antibody

480

12361048

720

(kDa) 1 2 3 4 5 6

+ an

ti-PIN

K1

+ an

ti-Tom

20

+ an

ti-Tom

22

+ an

ti-Tom

40

+ an

ti-Tom

70

GFP (Fluorescence)

shiftedband

+ antibody

no a

ntibo

dy

PINK1-GFP, CCCP

B PINK1-GFP, CCCP

+

C

FIGURE 2. Two PINK1 molecules comprise the PINK1 complex on depolarized mitochondria. A, cell extracts from HeLa cells expressing PINK1-GFP, whichhad been treated with CCCP (10 �M, 3 h), were incubated with an anti-PINK1 (BC100 – 494) or anti-GFP (A6455, 3E6, or ab6556) antibody and then subjected toCN-PAGE. The red arrowheads and lines indicate a shift in the PINK1 complex following interaction with the antibody. B, a NAMOS assay was used to identifycomponents of the PINK1 complex. HeLa cells expressing PINK1-GFP were treated with CCCP (10 �M, 3 h), and the cellular extracts were incubated withanti-Tom20, anti-Tom22, anti-Tom40, or anti-Tom70 antibodies and then subjected to CN-PAGE with PINK1-GFP fluorescence detected as before. C, a NAMOSassay was used to determine the stoichiometry of PINK1 in the complex. HeLa cells expressing PINK1-GFP and/or PINK1-mCherry treated with CCCP (10 �M, 3 h)were incubated with anti-GFP and/or anti-mCherry antibodies and subjected to CN-PAGE, and GFP or mCherry fluorescence was detected as before. The greenasterisks indicate a PINK1-GFP shift caused by interaction between the anti-mCherry antibody and PINK1-mCherry. The red asterisks indicate a PINK1-mCherryshift caused by the anti-GFP antibody. D, HeLa cells expressing PINK1-GFP, PINK1-mCherry, and/or PINK1-V5 treated with CCCP (10 �M, 3 h). These cell extractswere incubated with anti-mCherry, anti-V5, and/or anti-GFP antibodies, and GFP fluorescence was detected as before.




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PINK1Complex Formation Is Correlatedwith IntermolecularPhosphorylation of PINK1 following the Reduction of ��m—We next used CCCP exposure and subsequent washout todetermine which regulation process is correlated with PINK1complex formation. To date, several aspects of PINK1 regula-tion following a change in ��mhave been reported e.g. PINK1accumulates on OMM and undergoes ��m dissipation-de-pendent phosphorylation but is reimported and degradedwhen��m is recovered followingCCCPwashout (Fig. 5A) (reviewedin Ref. 48). Hence, we examined the relationship between theseprocesses and formation of the 850-kDa complex. First, HeLacells expressing PINK1 WT and the pathogenic mutants weretreated with CCCP, homogenized, and subjected to an in vitroproteinase K-resistant assay. In this assay, OMM proteins areeasily degraded, whereas matrix proteins are more resistant.We used Tom20 (a single spanning OMM protein), Tom40 (a�-barrel protein in OMM), Tim23 (an inner mitochondrialmembrane protein facing the intermembrane space), andHSP60 (a matrix protein) as control proteins (Fig. 5B, depictedon the right). We observed that the accumulation of somePINK1 mutants on mitochondria was attenuated; however,there was no correlation between protease resistance and com-plex formation (Fig. 5B). Complete degradation of PINK1 byproteinase K was similar to that of Tom20, suggesting thatPINK1 WT and the pathogenic mutants localize to OMM(41, 49).We also examined the reimport and degradation ability of

various PINK1mutants in response to��mrecovery followingCCCP washout. Similar toWT PINK1, all of the PINK1 patho-

genic mutants accumulated following CCCP treatment. Fur-thermore, whenCCCPwas washed out, all of the PINK1-FLAGpathogenicmutants examined underwent degradationwith thecleaved form of PINK1 (�1) increasing (Fig. 5C) despitemost ofthemutants being unable to form the PINK1 complex (Fig. 4A).This result suggests that the correlation between PINK1 reim-port activity and PINK1 association with the TOM complex istenuous under our experimental conditions.Last, we examined the correlation between the supermolec-

ular complex formation and intermolecular phosphorylation(17) of PINK1. PINK1-FLAG WT or the pathogenic mutantswere co-expressed with WT PINK1-GFP, the cells wereexposed to CCCP, and the phosphorylation state of PINK1-FLAG was determined. Although both phosphorylated andunphosphorylated forms of PINK1 resolve as a single band on

1D: BN-PAGE

66148

242

480

720

1048

1236

(kD

a)

top

botto

m

Input(2D only)CCCP +-

A

2D: S

DS

-PA

GE

(w

ithou

t Pho

s-ta

g)

1D: BN-PAGE

64

148

98

(kDa)

IB: PINK1

IB: PINK1

SDS-PAGE(with Phos-tag)

CCCP +-

SDS-PAGE(without Phos-tag)

Δ1FL

pPINK1

Δ1FL

pPINK1

66148

242

480

720

1048

1236

(kD

a)

2D: S

DS

-PA

GE

2D

: SD

S-P

AG

E

(with

Pho

s-ta

g)

top

botto

m

Input(2D only)

pPINK1

pPINK1

Δ1FL

Δ1FL

B

C

D

FIGURE 3. The PINK1 supermolecular complex is composed of phospho-rylated PINK1. A–D, HeLa cells expressing non-tagged PINK1 were treatedwith CCCP (10 �M, 1 h), and lysates were subjected to Phos-tag SDS-PAGE (Aand B) or two-dimensional electrophoresis consisting of BN-PAGE and Phos-tag SDS-PAGE (C and D). The same sample as that in Fig. 1A was used. Total celllysates were electrophoresed (shown in the leftmost panel) as controls in Cand D. Red arrowheads, phosphorylated PINK1 (pPINK1); black arrowheads,non-phosphorylated full-length PINK1 (FL) or the cleaved form (�1).

97

64

+-A

66

148

242

480

720

1048

1236

WT

G40

9V

G38

6A

L347

P

G30

9D

H27

1Q

E24

0K

A16

8P

C92

F

KD

534i

nsQ

E41

7GPINK1-GFP

CN-PAGE, GFP (Fluorescence)

SDS-PAGE, IB: PINK1

WT

CCCP

Δ1FL

(kDa)

(kDa)

+-

WT

G40

9V

G38

6A

L347

P

G30

9D

H27

1Q

E24

0K

A16

8P

C92

F

KD

534i

nsQ

E41

7GPINK1-GFP W

T

CCCPB

12361048

720

(kDa)

+ a

nti-T

om20

+ a

nti-T

om22

+ a

nti-T

om40

+ antibody

no a

ntib

ody

GFP (Fluorescence)

shiftedband

+ a

nti-T

om20

+ a

nti-T

om22

+ a

nti-T

om40

no a

ntib

ody

+ a

nti-T

om20

+ a

nti-T

om22

+ a

nti-T

om40

no a

ntib

ody

+ a

nti-T

om20

+ a

nti-T

om22

+ a

nti-T

om40

no a

ntib

ody

PINK1-GFP WT C92F G309D G409V

C

FIGURE 4. Most of the pathogenic PINK1 mutants prevent formation ofthe 850-kDa complex. A, HeLa cells expressing PINK1-GFP with KD or variouspathogenic mutations were treated with CCCP (10 �M, 3 h) and subjected toCN-PAGE with GFP fluorescence detected as before. The blue arrowheadsindicate a PINK1 complex similar to the complex containing WT PINK1-GFP. B,the same samples were subjected to conventional PAGE and immunoblottedusing an anti-PINK1 antibody. The black arrowheads indicate the position ofthe full-length (FL) or cleaved form (�1) of PINK1. C, components of the TOMmachinery are included in mutant PINK1 (C92F, G309D, and G409V)-contain-ing complexes.




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commercially available precast gels, such as NuPAGE 4–12%BisTris gels (Figs. 4B and 5 (B andC)), PINK1 can be resolved asa doublet using hand-made 7.5% Tris-glycine gels with phos-phorylated PINK1migrating as a highermolecular weight bandas shown in Fig. 6 (top) andRef. 17.We also performedPhos-tagPAGE and immunoblotted using an anti-FLAGantibody to dis-tinguish between phosphorylated PINK1-FLAG and degradedPINK1-GFP (Fig. 6, fourth panel). PINK1WTand theC92F andG309D mutants underwent phosphorylation irrespective ofPINK1-GFP co-expression (lanes 5, 9, and 17). The KD, A168P,E240K, H271Q, L347P, E417G, and 534insQ PINK1 mutantsshowed no detectable phosphorylation in the absence ofPINK1-GFP (lanes 7, 11, 13, 15, 19, 25, and 27). TheG386A andG409V PINK1 mutants exhibited an aberrant phosphorylationpattern in the absence of PINK1-GFP (lanes 21 and 23; compare

the position of the WT phosphorylated band with that in lane5). Interestingly, the G409V mutant, which can interact withthe 850-kDa complex (Fig. 4), exhibited an aberrant phosphor-ylation pattern in the absence ofWT PINK1 (lane 23) but dem-onstrated the expected phosphorylation-derived shift whenPINK1(WT)-GFP was co-expressed (Fig. 6, fourth panel, lane24, blue arrowhead). These results suggest that the two PINK1molecules in the 850-kDa complex contribute to intermolecu-lar phosphorylation.PINK1 Complex Formation Is Important for Efficient Parkin

Recruitment on Mitochondria with Decreased ��m—We pre-viously identified Ser-228 and Ser-402 in PINK1 as the sitesphosphorylated following a decrease in ��m; a S228A/S402APINK1 double mutant was not phosphorylated despite localiz-ing on depolarized mitochondria (17). We consequently exam-

P＋＋＋＋＋＋

ψ ↓A

B

C

OMM

IMM OMM OMM

PINK1 PINK1 PINK1

Location onto OMM Intermolecular phosphorylation

P P

Re-import and degradation

PINK1

mitochondrial Δ

Recovery

OMM

IMM

Tom40 Tom20

Tim23

HSP60

IB: Tom40

IB: Tom20

IB: Tim23

IB: HSP60

IB: PINK1

IB: PINK1

IB: actin

FL

Δ1

PINK1-Flag

CCCPWash-out

WT KD C92F A168P E240K H271Q G309D L347P G386A G409V E417G 534insQ

WT KD C92F A168P E240K H271QG309D L347P G386AG409VE417G534insQ

- + +- +-

- + +- +-

- + +- +-

- + +- +-

- + +- +-

- + +- +-

- + +- +-

- + +- +-

- + +- +-

- + +- +-

- + +- +-

- + +- +-

(kDa)

(kDa)

51

64

51

39

64

14

51

3928

19

64

51

PINK1-FlagPK - + - + - + - + - + - + - + - + - + - + - + - +

m

＋＋＋＋＋＋

＋＋＋＋＋＋

FIGURE 5. A, the proposed mechanisms described are illustrated. A reduction in ��m results in the accumulation of PINK1 and cross-phosphorylation of thetwo PINK1 molecules. Reestablishment of ��m following CCCP washout results in the reimport and degradation of PINK1. B, HeLa cells expressing PINK1-FLAGWT, KD, or various pathogenic mutants were treated with CCCP (10 �M, 1 h) and fractionated. Mitochondria-rich fractions were incubated with 50 �g/�lproteinase K (PK) and subjected to SDS-PAGE using precast 4 –12% BisTris gels and immunoblotted using an anti-PINK1, anti-Tom20, anti-Tom40, anti-Tim23,or anti-HSP60 antibody. C, HeLa cells expressing PINK1-FLAG WT, KD, or pathogenic mutants were treated with CCCP (10 �M, 3 h). The CCCP was subsequentlywashed out for 1 h, and SDS-PAGE was performed using precast 4 –12% BisTris gels and then immunoblotted using anti-PINK1 and anti-actin antibodies.




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ined whether mutations in Ser-228 and Ser-402 affect forma-tion of the PINK1 complex. The PINK1 S228Amutation had noeffect on formation of the PINK1 complex in CN-PAGE,whereas the S402Amutation strongly hindered formation (Fig.7A); however, both sites affect the phosphorylation pattern ofPINK1 (Fig. 7B) (17). Single S228A or S402A mutations areinsufficient to block PINK1 phosphorylation, which is com-pletely abolished by the S228A/S402A double mutation (17).Although the PINK1 S402A mutation inhibited formation ofthe 850-kDa complex (Fig. 7A), this result does not necessarilymean that phosphorylation at Ser-402 is essential for complexformation. Indeed, in cells expressing G409V alone, Ser-402-dependent phosphorylation was not observed (Fig. 6, top, lane23; a highermolecularweight band equivalent to Ser-402-phos-phorylated PINK1 was not observed in non-Phos-tag PAGE),but formation of the complex was equivalent to WT PINK1(Fig. 4A). These mutants should thus enable us to examinewhether or not formation of the PINK1 complex is required forParkin function on damaged mitochondria.We also examined the Parkin recruitment activity of the

PINK1 S228A or S402A mutants following CCCP treatment.HeLa cells stably expressing GFP-Parkin were treated withPINK1 siRNA to deplete endogenous PINK1. Those cells werethen transfected with plasmids containing PINK1WT and theS228A or S402Amutants under the control of a modified cyto-megalovirus (CMV) promoter (referred to as CMV d1), whichcontains a deletion that reduces the strength of the promoterand thus lowers overall expression. Because the siRNA wasdesigned to a portion of the PINK1 3�-UTR, there should be noeffect on expression of the exogenous PINK1, which lacks thecorresponding sequence. Although excessive overproductionof PINK1 under the normal CMV promoter targets Parkin tomitochondria irrespective of a reduction in ��m, and thusregardless of PINK1 complex formation (15, 50), co-expressionof CMV d1 promoter-driven PINK1 and Parkin results in

CCCP-dependent mitochondrial localization of Parkin (17).PINK1 reduction using siRNA inhibited Parkin recruitmentonto depolarized mitochondria even following CCCP treat-ment (Fig. 7C, yellow arrowheads), whereas reintroduction ofWT PINK1 rescued the Parkin recruitment defect (Fig. 7C,white asterisks). Expression of the CMV d1 promoter-drivenPINK1 was too weak to be detected by immunocytochemistryusing the anti-PINK1 antibody, and thus the transfected cellswere marked by co-expression with DsRed. Reintroduction ofthe control vector did not complement the Parkin recruitmentdefect (Fig. 7C, red asterisks). Following CCCP treatment (10�M, 1.5 h), the number of cells with Parkin-positive mitochon-dria was determined in �100 transfected DsRed-positive cells.Interestingly, the PINK1S228Amutant, which undergoes com-plex formation, complementedmislocalization of Parkin equiv-alent toWTPINK1,whereas the S402Amutant, which does notform the complex, exhibited significantly reduced Parkinrecruitment to depolarized mitochondria (Fig. 7D).We next examined the subsequent effect of thesemutants on

mitochondrial degradation. Endogenous PINK1 in HeLa cellsstably expressing HA-Parkin was depleted by siRNA, and theS228A or S402Amutants PINK1were introduced as before. Tomonitor OMM protein degradation, Tom20 was immuno-stained following prolonged exposure to CCCP (10�M, 16 h) asreported previously (16, 21), and the number of Tom20 immu-noreactive cells was determined in�100 transfected (GFP-pos-itive) cells. The complex formation-competent PINK1 S228Amutant accelerated OMM protein degradation equivalent toWT PINK1, whereas the complex formation-deficient S402Amutant exhibited significantly reducedOMMprotein degrada-tion (Fig. 7, E and F). Taken together, these results suggest thatformation of the PINK1 complex plays an important, albeitnot essential, role in Parkin recruitment and mitochondrialdegradation.

IB: PINK1

1 262524232221201918171615141312111098765432 27 28

FL

FL

Δ1PINK1-GFPPINK1-Flag

PINK1-Flag

PINK1(WT)-GFPCCCP

WT KD C92F A168P E240K H271Q G309D L347P G386A G409V E417G 534insQWT --- +

+- +

-++++ -

+++ -

+++ -

+++ -

+++ -

+++ -

+++ -

+++ -

+++ -

+++ -

+++ -

+++

(kDa)

64

98

-

FL

pFL PINK1-Flag

PINK1-Flag

FLPINK1-GFP

pFL

Phos-tag(-)

Phos-tag(+)

IB: PINK1

IB: Flag

FL

(kDa)

64

98

FL

pFL

Phos-tag(-)

Phos-tag(+)

IB: Flag

FIGURE 6. Formation of the PINK1 complex is correlated with the intermolecular phosphorylation of PINK1. HeLa cells expressing the indicated PINK1mutants were treated with CCCP (10 �M, 1 h), subjected to �Phos-tag SDS-PAGE using hand-made 7.5% Tris-glycine gels, and immunoblotted using either ananti-PINK1 or anti-FLAG antibody. The red and blue arrowheads indicate phosphorylated PINK1-FLAG (see “Results”). FL, pFL, and �1, the full-length, phosphor-ylated full-length, and cleaved forms of PINK1, respectively.




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DISCUSSION

In this study, we revealed the stoichiometry and functionalsignificance of a dyadic PINK1-containing complex that wasformed following a decrease in ��m. Although Lazarou et al.(18) previously reported formation of the PINK1 complex ondepolarized mitochondria and suggested a role in the reimportprocess following recovery of ��m, we show that the PINK1complex also has an important function on damaged mito-chondria. First, we confirmed that exogenous and endogenousPINK1 form a complex following CCCP treatment (Fig. 1, A–C)(18, 41).Thereafter,we identified components of this PINK1com-plex. Conventionally, immunoprecipitation was performed todetermine the components of the protein complex. However, our

initial immunoprecipitation attempts to identify components ofthe CCCP-treated PINK1 complex suffered from poor reproduc-ibility. Exogenous PINK1 exists in two forms, a high molecularweight complex depending on reduction of ��m and a lowmolecular species irrespective of mitochondrial state that isobserved as a broad smear (Figs. 1A and 3 (C andD)). Separationand characterization of only the highmolecularweight complex isdifficult by immunoprecipitation and thus might account for thepoor reproducibility. To address this problem, we developed anexperimental system to detect the PINK1 complex using fluores-cence in combination with CN-PAGE and NAMOS assays (Fig.2B). This allowed us to determine the component constituencyand stoichiometry of this multimeric complex.

CCCP + + +-

A

66

148

242

480

720

10481236

S22

8A

WT S40

2A

Non-taggedPINK1

IB: PINK1

GFP-Parkin stable HeLa cellswith siRNA for PINK1 3’UTR

HA-Parkin stable HeLa cells with siRNA for PINK1 3’UTR

DsRed + Vector

GFP-Parkin DsRed

Tom20

Vector WT S228A S402A

GFP(transfection

marker)

+ CCCP

+ CCCP

+ CCCP

GFP-Parkin DsRed

DsRed + PINK1 (no 3’UTR)

+ PINK1(no 3’UTR)

+ Phos-tag

NonPhos-tag

FL

pPINK1

FLpPINK164

98

(kDa)

CCCP + + +-

B

D

S22

8A

WT

100

* **

p = 0.2191 (NS) p = 0.0011

80

60

40

20

**

0W

T

Vec

tor

S40

2AS

228A

S40

2A

Non-tagged PINK1

GFP-Parkin on mitochondria/ transfected cells (%)

CN

-PA

GE

GF

P(F

luor

esce

nce)

*

64

98

(kDa) SD

S-P

AG

EIB

: PIN

K1

*

* ** *

***

E

F

* p = 0.884 (NS), ** p = 0.0004

Cells with no Tom20 signal/ transfected cells (%)

100

80

60

40

20

**

0

WT

Vec

tor

S22

8A

S40

2A

*

C

FIGURE 7. PINK1 Ser-402 is essential for PINK1 complex formation and its dysfunction specifically affects Parkin recruitment onto depolarizedmitochondria. A, HeLa cells expressing PINK1-GFP WT, S228A, or S402A mutants were treated with CCCP (10 �M, 3 h) and subjected to CN-PAGE, and GFPfluorescence was detected as before. B, HeLa cells expressing non-tagged PINK1 WT, S228A, or S402A mutants were treated with CCCP (10 �M, 3 h), subjectedto SDS-PAGE using 7.5% Tris-glycine gels with or without Phos-tag, and then immunoblotted using an anti-PINK1 antibody. C, HeLa cells stably expressingGFP-Parkin were pretreated with PINK1 siRNA and then transfected with CMV d1 promoter-driven PINK1 WT or a control vector. DsRed was used as transfectionmarker. After 42 h, these cells were treated with CCCP (10 �M, 1.5 h) and subjected to microscopic observation. Bar, 20 �m. D, HeLa cells stably expressingGFP-Parkin were pretreated with PINK1 siRNA and transfected with CMV d1 promoter-driven PINK1 WT, S228A, S402A, or a control vector, and then the numberof cells with Parkin-positive mitochondria was determined in �100 transfected (DsRed-positive) cells. Bars, mean � S.D. (error bars) of three experiments.Statistical significance was calculated using Student’s t test. NS, not significant. E, PINK1 knockdown HeLa cells stably expressing HA-Parkin were transfectedwith CMV d1 promoter-driven PINK1 WT, S228A, and S402A. Cells were treated with CCCP for 16 h, and then the number of Tom20-immunoreactive cells wasdetermined using �100 transfected (GFP-positive) cells. Bars, mean � S.D. values of three experiments. Statistical significance was calculated using a Student’st test. NS, not significant. F, representative images from E demonstrating the Tom20 signal loss. Bar, 10 �m.




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Several studies have proposed a direct interaction betweenPINK1 and Parkin (50–52). We have also observed that over-expressed PINK1 results in Parkin recruitment to mitochon-dria even without CCCP treatment (15, 17, 50), which is easilyexplainable if PINK1 physically interacts with Parkin. We andothers have reported that PINK1phosphorylates Parkin (44, 53,54). In addition, Lazarou et al. observed that ectopically over-expressed PINK1 translocates Parkin to the same organelle(18). These results seemingly suggest that PINK1 and Parkininteract directly. If so, does the 850-kDa PINK1 complex con-tain Parkin? In our initial experiments, we failed to detect Par-kin in the complex following CCCP treatment,4 which is con-sistent with Lazarou et al. (18). In addition, PINK1 reimportand degradation following ��m recovery precedes the disen-gagement of Parkin from polarized mitochondria (18). Thesedata are inconsistent with the hypothesis that Parkin localizeson mitochondria via stable interactions with PINK1. BecauseParkin is not incorporated into the complex, we surmise thatPINK1 does not stably interact with Parkin but rather acceler-ates the interaction betweenParkin and themembrane. Furtherstudies are needed to comprehensively understand this issue.We also examined the complex-forming ability of patho-

genic PINK1 mutants that cause hereditary early onset parkin-sonism. Although it has been reported, based on an in vitromitochondrial import assay, that several pathogenic PINK1mutants formed the complex (18), we found that most PINK1mutants, with the exception of C92F, G309D, andG409V, com-pletely or partially lost the complex-forming ability (Fig. 4A).The PINK1 G409V mutant exhibits typical complex formation(Fig. 4) but failed to undergo complete phosphorylation (Fig. 6)and inhibited Parkin recruitment and activation on depolarizedmitochondria (17, 47).We thus surmise that the principle func-tional defect of this mutant is in the process of PINK1 auto-phosphorylation or substrate recognition. Various PINK1mutantswere unable to form the complex in cells, suggesting anetiological importance of PINK1 complex formation.We have previously reported that PINK1 undergoes inter-

molecular phosphorylation (17). In this study, we noticed thatthe complex-forming ability of PINK1 correlated well with theintermolecular phosphorylation of PINK1 (Figs. 4 and 6). Inaddition, using a novel procedure, we determined that the 850-kDa complex contains two PINK1 molecules (Fig. 2). Thisresult seems reasonable because two PINK1 molecules mustlocalize in physical proximity to perform trans-phosphoryla-tion. Interestingly, when we performed the NAMOS assayusing an anti-GFP monoclonal antibody, 3E6, the secondmolecular weight shift was not observed (Fig. 2A). Nonetheless,the data obtained from the NAMOS assay using cells co-ex-pressing PINK1-GFP and PINK1-mCherry clearly showed thattwo molecules of PINK1 are present in the 850-kDa complex(Fig. 2C). We surmise that PINK1 forms an asymmetric dimersuch that one of the antibody recognition sites is masked byeither the second PINK1 molecule or the TOM complex.Indeed, some cell surface receptor kinases dimerize asymmet-rically following specific ligand binding prior to the subsequent

autophosphorylation event (55, 56). To our knowledge, this isthe first report to demonstrate PINK1 dimerization in responseto a decrease in ��m. The formation of a complex containingtwo PINK1molecules might play a pivotal role in intermolecu-lar phosphorylation.Although two groups have reported that PINK1 forms a

supermolecular complex after dissipation of ��m (18, 41), theincorporation of TOM components in the PINK1 complex wascontroversial. In general, the TOMcomplex consists of distinctimport receptors (Tom20 or Tom70) and a core complex (com-mon import receptor Tom22, pore Tom40, and organizerTom5, -6, and -7). The complex imports variousmitochondrialproteins by distinct mechanisms. For example, proteins con-taining a presequence are imported in a Tom20-dependentmanner, whereas carrier proteins and proteins containingmul-tiple �-helical transmembrane segments are imported in aTom70-dependent manner. Moreover, �-barrel proteins areintegrated into the mitochondrial outer membrane by thecooperation of Tom20- and Tom70-dependent processes (32,57). Using immunoprecipitation in the presence of a cross-linker and with Tom20 and Tom22 under native conditions,Lazarou et al. reported that PINK1 interacts with Tom20,Tom22, Tom40, and Tom70 (18). Meanwhile, Becker et al.showed that an immunodetected Tom40 signal did not co-mi-grate with the PINK1 complex and thus concluded that thecomplex is unlikely to contain TOM components (41). Ourdata, which consist of two independent assays (the NAMOSassay andCN-PAGE), are consistentwith the report by Lazarouet al. (18), although we did not observe the incorporation ofTom70 (Fig. 2).We surmise that the difference in experimentalconditions influenced the interaction between Tom70 andPINK1. Although the physiological significance of TOM com-ponents in the PINK1 complex remains obscure, TOMcompo-nents might assist in orientation of the PINK1 pair to facilitatethe intermolecular phosphorylation.Finally, to confirm the close relationship between PINK1

complex formation and Parkin function on depolarized mito-chondria, we analyzed the complex-forming ability of PINK1Ser-228 or Ser-402 mutants. We observed that the PINK1S402A mutant, which is unable to form the 850-kDa complex,partly but significantly reduced mitochondrial localization ofParkin following CCCP treatment. In contrast, the S228Amutant, which is a component of the 850-kDa complex,recruited Parkin onto depolarized mitochondria equivalent towild type PINK1 (Fig. 7). Complex formation and concomitantintermolecular phosphorylation of PINK1 is not imperative forParkin translocation because the complex-deficient S402Amutant still partially recruited Parkin to the mitochondria(�40%; Fig. 7D). Similarly, peroxisome-targeted PINK1 letsParkin localize on the peroxisome, which is devoid of TOMcomponents (18). Nevertheless, our finding indicates that theprocess is important for the efficient retrieval of Parkin to themitochondria.In conclusion, we propose that formation of a highmolecular

mass (850 kDa) supermolecular complex containing dimericPINK1 contributes to intermolecular phosphorylation and theeffective recruitment of Parkin onto depolarizedmitochondria.4 K. Okatsu, and N. Matsuda, unpublished data.




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Acknowledgments—We thankDr. T. Kitamura for providing PLAT-Ecells, Dr. N. Fujita for the mCAT1 plasmid, and Dr. N. Hattori for thePINK1-V5/His plasmids.

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Keiji Tanaka and Noriyuki MatsudaKei Okatsu, Midori Uno, Fumika Koyano, Etsu Go, Mayumi Kimura, Toshihiko Oka,

Parkin RecruitmentA Dimeric PINK1-containing Complex on Depolarized Mitochondria Stimulates

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