www.sciencemag.org/cgi/content/full/science.1249161/DC1

Supplementary Materials for

PINK1 Loss of Function Mutations Affect Mitochondrial Complex I

Activity via NdufA10 Ubiquinone Uncoupling

Vanessa A. Morais,* Dominik Haddad, Katleen Craessaerts, Pieter-Jan De Bock,

Jef Swerts, Sven Vilain, Liesbeth Aerts, Lut Overbergh, Anne Grünewald,

Philip Seibler, Christine Klein, Kris Gevaert, Patrik Verstreken, Bart De Strooper*

*Corresponding author. E-mail: Bart.DeStrooper@cme.vib-kuleuven.be (B.D.S.);

Vanessa.Morais@cme.vib-kuleuven.be (V.A.M.)

Published 20 March 2014 on Science Express

DOI: 10.1126/science.1249161

This PDF file includes:

Materials and Methods

Figs. S1 to S8

Tables S1 and S2

References

2

Materials and Methods

Site-directed Mutagenesis and Generation of Stable Cell Lines

Pink1-/- MEFs stably transduced with PINK1 PD-causing mutations (G309D; W437X), and the

artificial kinase inactive mutant (K219S) were previously described (6). PINK1 truncated

mutants were generated using the reverse primer 5’-cagggctgccctccatgagca-3’ and the following

forward primers: for ∆N77-hPINK1 5’-caccatgctggcggcgcggttg-3’; for ∆N113-hPINK1 5’-

ccaccatggaaaaacaggcgg-3’; for ∆N153-hPINK1 5’-caccatgctggaggagtatctg-3’. Mouse NdufA10

cDNA was obtained from Origene (USA), and for detection purposes a 3xFLAG-tag or a GFP-

tag was inserted. NdufA10 mutants were constructed using the QuickChange multisite-directed

mutagenesis kit as described by supplier’s protocol (Stratagene), where Serine250 was mutated to

either an Alanine (phosphor-deficient mutant) or to a Glutamate (phosphomimetic mutant). To

generate stable cell lines, MEFs were transduced using a replication-defective recombinant

retroviral expression system (Clontech) and where selected based on their acquired hygromycin

resistance. HeLa NdufA10 downregulated cell line were generated using shRNA against human

NdufA10 (targeted sequence 5’-cagaagaaaggagatccacatgaaatgaa-3’) according to supplier’s

protocol (Origene) and where selected based on their acquired puromycin resistance.

Human fibroblasts and iPS cells derived from PINK1 PD-causing mutations

Human fibroblasts harbouring the homozygous p.Q456X nonsense mutation (L2122) and the

homozygous p. V170G missense mutation (L1703) and age-matched controls (L2134, L2132)

were previously described (20). Where indicated human fibroblasts were electroporated with

GFP-tagged NdufA10 mutants using the NEON system (Invitrogen) according to the supplier’s

protocol.

The PINK1 derived induced pluripotent stem (iPS) cells were obtained according to the previous

described protocol (21), where previously published patient (L2124; L2122) and control (L2135)

lines and newly established control (L2134) line were analysed.

Morphometric analysis

Human derived fibroblast cells were electroporated with mitochondrial targeted red fluorescent

protein (mtRFP) corresponding to pDsRed2-Mito obtained from Clontech (Mountain View, CA)

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using the NEON system (Invitrogen) according to the supplier’s protocol. For epifluorescent

imaging of the mitochondrial network, 48h post-transfection medium was replaced with Hanks

balanced salt solution (HBBS) and 10 mM HEPES and cells were placed on the stage of an

Olympus IX81 inverted microscope equipped with a CellR imaging system (Olympus). Cells

were excited using a 525± 20nm excitation filter and emitted light was collected using a 40 x 1.4

NA Plan Apo objective (Olympus). Morphometric analysis was performed using ImageJ software

as previously described(31).

Mitochondrial isolation and Complex I immunocapture

Mitochondria were isolated from Pink1+/+ and Pink1-/- mice by standard differential

centrifugation and resuspended in Isolation buffer (IB: 0.2M sucrose, 10 mM Tris-MOPS pH 7.4,

0.1 mM EGTA-Tris pH 7.4) as previously described (32). Complex I was immunocaptured from

mitochondrial enriched fraction treated with 1% DDM according to manufacturer’s protocol

(Mitoscience).

LC-MS/MS analysis

After elution in 1% SDS, immunocaptured Complex I was analysed on an SDS-PAGE followed

by coomassie staining. The gel lanes were cut into 15 slices, and these gel slices were then

washed with water, followed by acetonitrile/water (1/1, v/v) and acetonitrile, and then vacuum-

dried. The dried gel slices were then re-swollen in 10% acetonitrile and 50 mM ammonium

bicarbonate (pH 8) containing 0.1 µg sequencing-grade modified trypsin (Promega, Madison,

WI, USA). Digestion was allowed to proceed overnight at 37°C. After digestion, the generated

peptide mixtures were vacuum-dried and re-dissolved in 20 µl of 2% acetonitrile and 0.1% TFA.

These peptide mixtures were then analyzed on an Ultimate 3000 HPLC system (Dionex,

Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap Velos mass spectrometer

(Thermo Electron, Bremen, Germany). Here, a 30 min gradient of 2% acetonitrile to 50%

acetonitrile, followed by a washing and re-equilibration step, on an in-house packed 15 cm long

and 75 µm inner diameter columns (Reprosil-Pur Basic C18-HD 3 µm, Dr. Maisch, Germany)

was used. Per LC-MS/MS analysis, 2.5 µl of the peptide mixture was consumed. Instrument

settings for LC-MS/MS analysis and the generation of MS/MS peak lists were as previously

described (33). These MS/MS peak lists were then searched using the Mascot Daemon interface

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(version 2.3.0, Matrix Science, London, UK). The Mascot search parameters were as follows.

The spectra were searched in the mouse subsection of the Swiss-Prot database. Acetylation of the

protein N-terminus, pyroglutamate formation of N‐terminal glutamine, methionine oxidation to

methionine‐sulfoxide, propionamide formation of cysteine and phosphorylation of serine,

threonine and tyrosine were set as variable modifications. The protease was set to trypsin with

one missed cleavage allowed. The mass tolerance on the precursor ion was set to ± 10 ppm and

on fragment ions to ± 0.5 Da. In addition, Mascot’s C13 setting was set to 1. Only peptides that

were ranked one and had an ion score at least equal to the corresponding identity threshold at

99% confidence were withheld and further data handling was done in the ms_lims database(34).

Respiratory Assays

Oxidative phosphorylation complex measurements performed on mitochondrial homogenates

from fibroblast cells were analysed by spectrophotometric assays as previously described (25).

Briefly, measurements of Complex I were performed with either coenzyme Q1 (CoQ1) or

decylubiquinone (NADH:ubiquinone oxidoreductase, rotenone sensitive) or with

hexammineruthenium (HAR). The protein concentration was in the range of 2–4 mg/ml. Values

were plotted according to the ratio between the specific complex’s activity and citrate synthase

activity.

ATP determination

ATP levels measured in lysates from cell lines or adult flies were determined as described in (19)

using a luminescent solution (ATP Determination Kit, Invitrogen) according to the supplier’s

protocol. Luminescence values was measured on an EnVision Multilabel Reader (Perkin Elmer)

and luminescence ATP (nmol) was determined using a standard curve and normalized to total

protein content (mg) measured by BCA assay (Pierce).

Proteinase K accessibility assay and Sodium Carbonate extraction

Isolated mitochondria (0.1 mg/ml) from HeLa cells were treated with 100 µg/ml Proteinase K

(PK) in mitochondria Isolation buffer at 4°C for 30 min. A hypotonic rupture of the outer

membrane (OM) was achieved by diluting the mitochondrial suspension 1:40 in 2 mm

HEPES/KOH pH 7.4. Disruption of the inner mitochondrial membrane (IM) was achieved by

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adding Triton X-100 at a final concentration of 0.3% (V/V). PK was inactivated by incubation

with 1 mm PMSF at 4°C for 5 min. Samples were prepared for SDS-PAGE analysis by

precipitation with 10% trichloroacetic acid (TCA), followed by cold acetone washes, and

resuspended in NuPAGE loading buffer (Invitrogen). For the Sodium Carbonate (Na2CO3)

extraction, 500µg of isolated mitochondria were treated with 100 mM Na2CO3 pH 11.5 for 30

min on ice, followed by centrifugation at 100.000 x g for 60 min. Supernatant is enriched for

soluble IMS and matrix proteins and pellet is enriched for OM and IM integral and associated

proteins. Samples were prepared for SDS-PAGE analysis and resuspended in NuPAGE loading

buffer (Invitrogen).

Immunoblot analyses

Proteins were separated by SDS-PAGE on 10% NuPAGE Bis-Tris gels (Invitrogen) in MOPS

buffer and electrophoretically transferred onto nitrocellulose membranes. Membranes were

blocked with 5% milk powder in Tris-buffered saline with 0.5% Tween (TBS-T) for 1 h. Primary

antibodies used: mouse anti-FLAG M2 1:5000 (Sigma), mouse anti-Hsp60 1:5000 (BD

laboratories), rabbit anti-NdufA10 1:1000 (ab96464; ABCAM), mouse anti-Tom20 1:1000 (BD

laboratories), goat anti-Tim23 1:500 (Santa Cruz), rabbit anti-PINK1 1:500 (Novus Biologicals

BC100-494), mouse anti-V5 tag 1:5000 (Invitrogen), rabbit anti-Omi 1:1000 (R&D). Secondary

antibodies: anti-rabbit HRP conjugated and anti-mouse HRP conjugated (Bio-Rad) at 1:10000,

anti-goat HRP conjugated (Sigma) at 1:2000. Detection was done using the ECL-Plus detection

kit (Amersham) and imaged on a Fuji-Film imaging system.

Mitophagy assay

To assess CCCP-mediated Parkin recruitment, MEF cells were treated with the mitochondrial

uncoupler CCCP at 25 μM for 8 h. Cells were fixed in 4% paraformaldehyde in PBS for 20 min

at room temperature. Cells were permeabilized with 0.1% Triton X-100 for 10 min, followed by

1h blocking with BSA supplemented with 5% goat serum. Primary antibodies used: rabbit anti-

TurboGFP 1:1000 (Evrogen), mouse anti-Hsp60 1:250 (BD BioSciences). Secondary antibodies:

Alexa-488 or -555 conjugated antibodies (Invitrogen) were used at 1:1000. Preparations were

mounted in Moviol and visualized with a Zeiss LAS510 confocal microscope and a 63X NA 1.4

oil lens.

6

H202-induced apoptosis

To measure H202-induced apoptosis, Pink1+/+, Pink1-/- and Pink1-/- fibroblasts expressing the

phosphomimetic NdufA10 mutants were treated with increasing concentrations of H2O2 and after

4 h apoptosis was determined using the luminescent Cleaved Caspase-Glo 3/7 assay (Promega)

according to the protocol supplied by the manufacturer.

Drosophila Genetics

Flies were kept on standard molasses medium. w pink1REV controls and w pink1B9 flies were

kindly provided by Jeehye Park and Jongkyeong Chung (Korea Advanced Institute of Science

and Technology)(12) and mutant larvae were selected using GFP balancers.

Generation of UAS-CG6343 transgenic flies

dNDUFA10 (CG6343) cDNA was ordered from DGRC (Drosophila Genomics Resource Center)

DGC (Drosophila Gold Collection) clone LD29280 and cloned with 5’-

CAGAATTCCAAAATGACCGCCGTGTTCCGCG-3’ and 5’ -

GTGCGGCCGCCTAGTGGTGATGGTGATGATGGATGCCCTGGTTGATGCCTATTTTC-3’

using 2x BIO-X-ACT Short Mix (BIOLINE), cloned in the EcoR1 and Not1 site of pUAST-Attb

(PMID: 17360644) and sequenced (A10wt). UAS-CG6343S-A (A10SA) and UAS-CG6343S-D

(A10SD) were generated similarly, with the following primers to introduce point mutations for

S-A: 5’-CGGCATGGGTGGCGATGTCCTTG-3’ and

5’-CAAGGACATCGCCACCCATGCCG-3’ and for S-D:

5’-CGGCATGGGTGTCGATGTCCTTG-3’ and 5’-CAAGGACATCGACACCCATGCCG-3’.

Transgenic flies were created at GenetiVision Inc. (Houston, USA) using PhiC31 mediated

transgenesis in the VK1 docking site (2R, 59D3) (PMID: 17138868).

For Drosophila genetics, pink1B9 null and pink1rev mutants were crossed with transgenic UAS-

(CG6343) (wild type; A10wt). UAS-CG6343S-A (phospho-deficient; A10SA) and UAS-CG6343S-D

(phosphomimetic; A10SD) flies (CG6343; dNdufA10; ND-42). Experiments were performed on

the following genotype: w pink1B9 / Y; UAS-CG6343 / +; daGal4 / + and pink1rev / Y; UAS-

CG6343 / +; daGal4 / +.

7

Imaging of mitochondrial membrane potential

The dye TMRE was used to evaluate mitochondrial membrane potential. Human fibroblasts and

MEFs were grown in 3 cm plastic dishes with glass coverslips (Nunc) and treated with 10 nM

TMRE for 10 min at 37 °C as previously described(35). TMRE fluorescence intensity was

measured using the ImageJ software.

For live imaging of membrane potential, fibroblast cells were grown in 3cm plastic dishes with

glass coverslips (Nunc) and after 24h loaded with 10nM TMRM (Molecular Probes) in the

presence of 2µg/ml cyclosporine H (Sigma) for 30 min at 37°C. Subsequently cells were placed

on the stage of an Olympus IX81 inverted microscope equipped with a CellR Imaging system.

Sequential images of TMRM fluorescence were acquired every 60s using exposure times of 40ms

with a 40x, 1.4 NA Plan Apo objective (Olympus), a 525 ± 20 excitation filter and an emission

570 LP filter. TMRM fluorescence over mitochondrial regions of interest was measured. When

indicated (arrows), 2 µg/ml oligomycin and 2 µM FCCP were added.

Third instar larval fillets were labelled with JC-1 (Molecular Probes) as described (22). Briefly,

larval fillets were incubated for 90 sec with 4 mM JC-1 in HL-3 buffer, washed in HL-3 buffer

and red and green fluorescence was imaged. Images were captured on a Nikon FN-1 microscope

with a DS-2MBWc digital camera, 63x water objective, NA 0.8 and quantification of labelling

intensity (red to green intensity in the red labelled areas within the boutons) was performed using

NIS-Elements BR 3.1 software.

Immunolabeling

Immunolabeling was performed as previously described in (19). Briefly, Drosophila larval fillets

were fixed using 3.7% formaldehyde in PBS for 20 min at room temperature, and permeabilized

with 0.4% Triton X-100 for 10 min, followed by 1h blocking with BSA supplemented with 5%

goat serum. As primary antibody mouse anti-Complex V subunit beta (MitoSciences) at 1:250

was used. For secondary antibody Alexa-488 conjugated antibodies (Invitrogen) at 1:1000 was

used. Preparations were mounted in Vectashield (Vector Laboratories) and visualized with a

Leica DMRXA confocal microscope and a 63X NA 1.4 oil lens.

8

Flight assay

Batches of five male flies were used to conduct the flight assays. Briefly, flies were placed in an

empty vial, gently tapped and flies that were able to fly were given a score of 1 while those that

did not were given a score of 0.

Electrophysiology

Larval electrophysiological recordings were performed as described (22, 36). Data was recorded

with a Multiclamp 700B amplifier (Molecular Devices) and stored using pClamp 10; 2 mM Ca2+

concentration was used. EJP amplitudes were binned per 30 s and normalized to the average

amplitude of the first 15s of recordings.

FM1-43

FM 1-43 labelling and unloading were performed as described (22). Briefly, both the exo/endo

cycling pool (ECP) and RP were labelled by electrically stimulating motor neurons of third instar

filets in the presence of 2 mM Ca2+ for 10 min and then leaving the dye with the preparation for 5

min. ECP and RP vesicles are labelled. Depolarisation with 90 mM KCl, 2 mM Ca2+ and

following washing in Ca2+ free medium results in unloading of the ECP vesicles but not RP

vesicles. Images were captured on a Nikon FN-1 microscope with a DS-2MBWc digital camera,

63x water objective, NA 0.8 and quantification of labelling intensity was performed using NIS-

Elements software.

Electron microscopy

Adult fly thoraxes were processed for electron microscopy as previously described (37). Briefly,

thoraxes were fixed in paraformaldehyde/glutaraldehyde, post-fixed in osmium tetroxide,

dehydrated and embedded in Epon. Sections 80 nm thick were stained with uranyl acetate and

lead citrate and subjected to electron microscopy analysis. The morphology of the mitochondria

were blindly scored as 1) normal mitochondria with organized cristae; 2) swollen mitochondria

with organized or partially-organized cristae and 3) swollen mitochondria devoid of cristae.

9

Statistical analysis

The statistical significance of differences between a set of two groups was evaluated using

student two-tailed unpaired t-tests (*, p<0.05; **, p<0.01; ***, p<0.001) in GraphPad Prism5.

Mean was calculated using standard deviation (s.d.) or standard error (s.e.m.).

10

Fig. S1.

Fig. S1. Human PINK1 patient derived fibroblasts present mitochondrial membrane

potential deficits.

A and B, Mitochondrial morphology analysis and quantification in fibroblasts of PINK1 patients

(L1703 and L2122) and age-matched controls (L2134 and L2132). Following electroporation of

cells with mitochondrial targeted RFP, fluorescent visualization and corresponding morphometric

analysis was performed using ImageJ. Absence of PINK1 does not lead to mitochondrial

morphological changes.

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C and D, Analysis and quantification of the mitochondrial membrane potential in these

fibroblasts using the potentiometric dye TMRE. Quantification of TMRE fluorescence was

performed using ImageJ software. The mitochondrial membrane potential is decreased in the

patient derived fibroblasts.

E, Analysis of ATP levels in these fibroblasts where a decrease in overall ATP content is

observed in the patient derived fibroblasts.

F,G and H, Analysis of iPS cell derived neurons from a PD patient harboring a PINK1 mutation

and a healthy control individual. IPS cells were established from two PD patient with mutant

PINK1 (c.1366C>T) and from a healthy family member (H). Analysis and quantification of the

mitochondrial membrane potential in iPS cell derived neurons of PINK1 patients (L2124 and

L2122) and age-matched controls (L2134 and L2135) using the potentiometric dye TMRE (F).

Quantification of TMRE fluorescence was performed using ImageJ software. The mitochondrial

membrane potential is decreased in the patient derived iPS cells. Analysis of ATP levels in these

patient derived iPS cells shows a decrease in overall ATP content in the patient derived cells (G).

Statistical analysis: student t-test; **, p<0.01; *, p<0.05; mean ± s.d.; n = 100. Scale bar, 10 µm.

12

Fig. S2.

A) Non-phosphorylated peptide

13

B) Phosphorylated peptide

Fig. S2. CID-MS/MS spectra of the NdufA10 S250 non-phosphorylated (A) and

phosphorylated (B) peptide.

The tryptic peptide 249-MSEMCEVLVYDSWEAEDPTK-268 was identified in both Pink1+/+ as

well as Pink1-/- tissue samples, but was only found phosphorylated in Pink1+/+ tissue samples.

Representative and annotated MS/MS spectra for the non-phosphorylated (A) and the

14

phosphorylated peptide (B) are shown together with tables that indicate which b and y type of

fragment ions were covered (shown in red). Note that the Mascot search algorithm was used at

99% confidence settings and the non-phosphorylated peptide in panel (A) was identified with a

Mascot score of 95 (threshold score of 34), whereas the phosphorylated peptide in panel (B) was

identified with a score of 97 versus a threshold score of 32. Results indicate that phosphorylation

of Serine250 in NdufA10 is dependent on the presence of PINK1.

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Fig. S3.

Fig. S3. Expression of phosphomimetic NdufA10 restores Complex I activity in down

regulated NdufA10 HeLa cells.

A, HeLa cells were transfected with an siRNA against NdufA10 or a control scrambled-siRNA

followed by reintegration of the 3xFLAG-tagged wild type NdufA10wt, phosphorylation-deficient

NdufA10S250A and phosphomimetic NdufA10S250D. Expression levels were analysed by

Immunoblotting of mitochondria-enriched fractions using anti-NdufA10 antibody that recognizes

both endogenous and exogenous NdufA10. Efficiency of downregulated is observed when

comparing expression levels between siRNA and scramble control for the endogenous protein.

H, homogenate; C, cytosol; M, mitochondria; exo, exogenous; endo, endogenous.

B, Analysis of enzymatic function of Complex I. Spectrophotometric assays were performed to

measure Complex I (NADH:ubiquinone oxidoreductase) and citrate synthase activities on

mitochondria homogenates from HeLa cells expressing NdufA10 mutants as indicated. In (B),

NADH:ubiquinone reduction (rotenone sensitive) was measured. Values were normalized to

citrate synthase acitivity. The enzymatic activity of Complex I is reduced in the down-regulated

NdufA10 cell line transfected with the phosphomimetic mutant NdufA10S250A.

16

Fig. S4.

Fig. S4. Restoration of synaptic defects in Drosophila pink1B9 null mutants by expressing

phosphomimetic NdufA10

A, Schematic representation of a neuromuscular junction (NMJ) from a Drosophila third instar

larva. The position of the stimulating electrode and nerves, and the recording electrode in the

muscle is illustrated.

B, Imaging of Reserve pool (RP) labelling in controls (pink1REV) and pink1 mutants (pink1B9).

Both the exo/endo cycling pool (ECP) and RP were labelled with FM1-43, after depolarization

only ECP vesicles but not RP vesicles were unloaded. Synapses were imaged after this unloading

procedure. Note that the loading defect in pink1B9 is restored upon expression of A10SD.

17

C, Imaging of mitochondrial membrane potential in third instar Drosophila larval NMJs in

controls (pink1REV) and pink1 mutants (pink1B9) using the ratiometric dye JC-1. The

mitochondrial membrane potential was restored upon expression of A10SD.

Scale bar, 4.5 µm.

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Fig. S5

Fig. S5. Phosphomimetic NdufA10SD does not restore muscle morphology or flight defects in

pink1B9 flies, nor does it rescue CCCP-induced Parkin recruitments in MEF cells.

A and B, Imaging of the mitochondrial membrane potential in third instar Drosophila larval

NMJs in controls (pink1REV) and pink1 mutants (pink1B9), expressing NdufA10wt, NdufA10SA and

NdufA10SD using the ratiometric dye JC-1 . For quantification (B), the red JC-1 fluorescence

emission to green emission (in the same area) is compared. The mitochondrial membrane

potential was not altered upon expression of NdufA10 mutants in pinkREV.

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Statistical analysis: student t-test; ns, not significant; mean ±s.d.; n = 6 animals. Scale bar, 4.5

µm.

C, Mitochondria morphology from third instar larval body wall muscles. Larval filets from

pink1rev, pink1B9 and in pink1B9 expressing NdufA10wt, NdufA10SA and NdufA10SD were stained

with Complex V antibody (labels mitochondria) and imaged. Defects in mitochondria

morphology observed in pink1B9 muscle tissue are not rescued with NdufA10 mutants. n = 5

animals.

D, Quantification of flight in pink1rev, pink1B9 and in pink1B9 expressing NdufA10wt, NdufA10SA

and NdufA10SD. Reduced flight observed in pink1B9 mutant flies is not restored with

phosphomimetic mutants of NdufA10.

Statistical analysis: student t-test; ns, not significant; mean ± s.e.m.; n = 10 experiments with 5

flies each.

E, CCCP-induced Parkin recruitment is not restored in Pink1-/- cells expressing the

phosphomimetic NdufA10 mutant. Pink1+/+, Pink1-/- and Pink1-/- cells expressing the

phosphomimetic NdufA10 mutants transfected with Parkin-GFP were treated with CCCP for 8h,

after which they were further processed for immunostaining.Anti-GFP and anti-HSP60 were used

to visualize Parkin and the mitochondrial matrix protein HSP60, respectively. Parkin

translocation to the mitochondria was assessed. As previously reported, Parkin fails to be

recruited to CCCP-treated mitochondria in the absence of Pink1, presenting a cytosolic

distribution pattern in the cells. Additionally, the phosphomimetic NdufA10 mutant does not

restore this defect either.

20

Fig. S6.

Fig. S6: Proteinase K accessibility assay peformed on HeLa cells indicates that PINK1 is

translocated to the Inner Mitochondrial Membrane.

A, Purified mitochondria from HeLa cells expressing endogenous hPINK1 were treated with 100

µg/ml Proteinase K (PK). Where indicated, hypotonic rupture of the outer mitochondrial

membrane (OM) was achieved by diluting the mitochondrial suspension 1:40 in 2 mm

HEPES/KOH pH 7.4; Triton X-100 was added where indicated at a final concentration of 0.3%

(V/V). PK was inactivated by incubation with 1 mm PMSF at 4°C for 5 min. Tom20 is an OM

protein, Tim23 is an integral Inner Mitochondrial Membrane (IM) protein. Results indicate that

PINK1 is a mitochondrial inner membrane integral or membrane-associated protein. The double

band is probably reflecting full length PINK1 and PINK1 processed by MPPβ (protease involved

in the cleaving of mitochondrial targeting sequences) or by AFG3L2 (a subunit of the mAAA

protease), based on comparison of these results with those described by Greene and co-workers

(40). SW, swelling.

21

Fig. S7.

Fig. S7: Proteinase K accessibility assay and Sodium carbonate extraction performed on

MEF cells expressing hPINK1 indicates that PINK1 is translocated to the Inner

Mitochondrial Membrane.

A, Purified mitochondria from MEF cells expressing hPINK1 with a V5 tag were treated with

100 µg/ml Proteinase K (PK). Where indicated, hypotonic rupture of the outer mitochondrial

membrane (OM) was achieved by diluting the mitochondrial suspension 1:40 in 2 mm

HEPES/KOH pH 7.4; Triton X-100 was added where indicated at a final concentration of 0.3%

(V/V). PK was inactivated by incubation with 1 mm PMSF at 4°C for 5 min. Tom20 is an OM

protein, Omi is a intermembrane space (IMS) protein and HSP60 is a mitochondrial matrix

protein. Results indicate that PINK1 is a mitochondrial inner membrane integral or membrane-

associated protein. The double band is probably reflecting full length PINK1 and PINK1

processed by MPPβ (protease involved in the cleaving of mitochondrial targeting sequences) or

by AFG3L2 (a subunit of the mAAA protease), and lower band to the PARL-cleavage product,

based on comparison of these results with those described by Greene and co-workers (40). SW,

swelling.

B, Sodium carbonate (Na2CO3) extraction was performed using 100 mM Na2CO3 on 500µg of

isolated mitochondria from MEF cells expressing hPINK1 with a V5 tag. Supernatant is enriched

22

for soluble IMS and matrix proteins (sup Na2CO3) and pellet is enriched for OM and IM integral

and associated proteins (pellet Na2CO3).

23

Fig. S8.

Fig. S8. Expression of phosphomimetic NdufA10 in Pink1 deficient cells expressing PINK1

PD-causing mutants.

A, Pink1-/- MEFs expressing human wildtype (hPINK1wt), PINK1 containing PD-causing

mutations (hPINK1G309D, hPINK1W437X) and a kinase inactive form of PINK1 (hPINK1KD) were

stably transduced with 3xFLAG-tagged wild type NdufA10wt, phosphorylation-deficient

NdufA10S250A and phosphomimetic NdufA10S250D. Expression levels were analysed by

Immunoblotting of mitochondria-enriched fractions using anti-FLAG and anti-Hsp60 (loading

control) antibody.

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Table SI - Protein coverage of immunocaptured Complex I subunits.

Complex I was immunocaptured from Pink1+/+ and Pink1-/- mouse brain and liver. The peptide

sequences obtained are linked to a specific protein with a 99% confidence settings (p ≤ 0.01), and

proteins identified in at least two out of the three independent experiments in each tissue

performed are shown (+; a positive identification). Previously reported human subunits NdufA4L

and NdufB1 have no known mouse homologue. Notice that MS analysis covered 40 out of the 46

Complex I subunits.

Accession n° Brain LiverNADH dehydrogenase (ubiquinone) 1 alpha subcomplex O35683 NdufA1 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5kDa + +Q9CQ75 NdufA2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2, 8kDa + +Q9CQ91 NdufA3 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3, 9kDa + +Q62425 NdufA4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa + +Q4FZG9 NdufA4L2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 not identified not identifiedQ9CPP6 NdufA5 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5, 13kDa + +Q9CQZ5 NdufA6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14kDa + +Q9Z1P6 NdufA7 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7, 14.5kDa + +Q9DCJ5 NdufA8 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, 19kDa + +Q9DC69 NdufA9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39kDa + +Q99LC3 NdufA10 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42kDa + +Q9D8B4 NdufA11 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 11, 14.7kDa + +Q7TMF3 NdufA12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 + +Q9ERS2 NdufA13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 + +NADH dehydrogenase (ubiquinone) 1 beta subcomplex Q9CPU2 NdufB2 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2, 8kDa + +Q9CQZ6 NdufB3 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3, 12kDa + +Q9CQC7 NdufB4 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 4, 15kDa + +Q9CQH3 NdufB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa + +Q3UIU2 NdufB6 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6, 17kDa + +Q9CR61 NdufB7 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7, 18kDa + +Q9D6J5 NdufB8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19kDa + +Q9CQJ8 NdufB9 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9, 22kDa + +Q9DCS9 NdufB10 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10, 22kDa + +O09111 NdufB11 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 11, 17,3kDa + +NADH dehydrogenase (ubiquinone) 1, subcomplex unknown Q9CQY9 NdufC1 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1, 6kDa not identified not identifiedQ9CQ54 NdufC2 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2, 14,5kDa + +NADH dehydrogenase (ubiquinone) Fe-S protein Q91VD9 NdufS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa + not identifiedQ91WD5 NdufS2 NADH dehydrogenase (ubiquinone) Fe-S protein 2, 49kDa + +Q9DCT2 NdufS3 NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30kDa + +Q9CXZ1 NdufS4 NADH dehydrogenase (ubiquinone) Fe-S protein 4, 18kDa + +Q99LY9 NdufS5 NADH dehydrogenase (ubiquinone) Fe-S protein 5, 15kDa + +P52503 NdufS6 NADH dehydrogenase (ubiquinone) Fe-S protein 6, 13kDa + +Q9DC70 NdufS7 NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa + +Q8K3J1 NdufS8 NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23kDa + +NADH dehydrogenase (ubiquinone) flavoprotein 1 Q91YT0 NdufV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa + +Q9D6J6 NdufV2 NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa + +Q8BK30 NdufV3 NADH dehydrogenase (ubiquinone) flavoprotein 3, 10kDa + +Mitochondrially encoded NADH dehydrogenase subunit P03888 ND-1 mitochondrially encoded NADH dehydrogenase subunit 1 + +P03893 ND-2 mitochondrially encoded NADH dehydrogenase subunit 2 + +P03899 ND-3 mitochondrially encoded NADH dehydrogenase subunit 3 + +P03911 ND-4 mitochondrially encoded NADH dehydrogenase subunit 4 + +P03903 ND-4L mitochondrially encoded NADH dehydrogenase subunit 4L not identified not identifiedP03921 ND-5 mitochondrially encoded NADH dehydrogenase subunit 5 + +P03925 ND-6 mitochondrially encoded NADH dehydrogenase subunit 6 not identified not identified

25

Table SII – Phosphorylation of Complex I subunit NdufA10 is dependent on the presence of

PINK1.

Phosphoproteome of Complex I from Pink1+/+ and Pink1-/- tissues was performed by LC-MS/MS

analysis. Identified phosphorylation sites are indicated in the table, where “-” corresponds to the

identification of the non-phosphorylated peptide sequence. Phosphopeptides that scored below

the threshold set by the 99% confidence interval are indicated in “±”. We identified phosphosite

Serine250 in Complex I subunit NdufA10 to be dependent on the presence of PINK1.

Additionally, nine novel Complex I phosphosites, that are independent of PINK1, were

identified; and previously described NdufA2-S78, NdufB4-S26 and NdufB10-S21 (38, 39) were

confirmed.

Pink1+/+ Pink1-/- Pink1+/+ Pink1-/-

Q9CQ75 NdufA2 ± ± S78 S78

Q9CPP6 NdufA5 ± T9/T8 T9 T9

± ± S68 S68

S250 - S250 -

Q9CQC7 NdufB4 ± ± S26 S26

Q9CQJ8 NdufB9 ± T144 T144 ±

Q9DCS9 NdufB10 S21 ± S21 S21

Q9CQ54 NdufC2 T41 T41 T41 T41

Q91VD9 NdufS1 / ± ± S216

Q9CXZ1 NdufS4 ± S116 ± ±

± Y112 Y112 Y112

± ± S189 S189

Brain LiverProtein

Q91YT0 NdufV1

Q99LC3 NdufA10

Accession n°

26

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