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RESEARCH ARTICLE

The ubiquitin-conjugating enzymes UBE2N, UBE2L3 andUBE2D2/3 are essential for Parkin-dependent mitophagy

Sven Geisler1,*, Stefanie Vollmer1, Sonia Golombek1 and Philipp J. Kahle1,2,*

ABSTRACT

Depolarized mitochondria are degraded by mitophagy in a process

that depends on the Parkinson’s disease gene products PINK1

and Parkin. This is accompanied by ubiquitylation of several

mitochondrial substrates. The roles of E2 ubiquitin-conjugating

enzymes (UBE2) in mitophagy are poorly understood. Here, we

investigate a set of UBE2 enzymes that might regulate Parkin-

mediated mitophagy. Knockdown of the E2 enzymes UBE2N,

UBE2L3 or UBE2D2 and UBE2D3 (UBE2D2/3) significantly

reduced autophagic clearance of depolarized mitochondria.

However, this did not interfere with mitochondrial PINK1

stabilization and Parkin translocation. UBE2N knockdown

prevented specifically K63-linked ubiquitylation at mitochondrial

sites. Nevertheless, polyubiquitin and p62 (officially known as

SQSTM1) were still found on mitochondria after individual UBE2

knockdown. Knockdown of all of these UBE2s together significantly

reduced mitochondrial polyubiquitylation and p62 recruitment.

Moreover, reduced ubiquitylation of mitofusins, the mitochondrial

import receptor subunits TOM20 and TOM70, the voltage-

dependent anion channel protein 1 and Parkin was observed in

cells silenced for all of these UBE2s. A version of Parkin with a

mutation in the active site (C431S) failed to ubiquitylate these

mitochondrial substrates even in the presence of UBE2s. We

conclude that UBE2N, UBE2L3 and UBE2D2/3 synergistically

contribute to Parkin-mediated mitophagy.

KEY WORDS: Parkin, Ubiquitin, Proteasome, Mitophagy, UBE2

INTRODUCTIONThe ubiquitylation system has regulatory roles in a variety of

cellular processes, such as protein turnover, cell cycle regulation,

stress responses, organelle biosynthesis and cellular homeostasis

(Glickman and Ciechanover, 2002). The modification of substrateproteins with the small molecule ubiquitin occurs in a multi-step

catalytic reaction mediated by E1 (ubiquitin-activating), E2

(ubiquitin-conjugating) and E3 (ubiquitin ligase) enzymes. The

substrates can be (multi-)monoubiquitylated or polyubiquitylated.

The elongation of polyubiquitin chains can occur at each of theseven lysine residues within the ubiquitin molecule. For example,

the attachment of at least four ubiquitin molecules linked to the

K48 residue drives proteasomal degradation, whereas K63

linkages and monoubiquitylations regulate subcellular trafficking(Ikeda and Dikic, 2008; Komander and Rape, 2012). DifferentE2 enzymes influence such distinct types of ubiquitin linkages. The

E2 enzyme UBE2N (also known as Ubc13), which heterodimerizeswith UBE2V, forms K63-linked ubiquitin chains, whereas, forother E2 enzymes, the selection of linkages is less clear (Davidet al., 2010; Komander and Rape, 2012).

Mutations in the gene encoding Parkin are the most commoncause of autosomal recessive Parkinson’s disease (Kitada et al.,

1998). The gene codes for a ‘really interesting new gene’ (RING)-type E3 ubiquitin ligase (Shimura et al., 2000). Besides roles inother cellular pathways, Parkin has an essential function in

mitochondrial homeostasis and quality control. Mitochondria asthe energy producing organelle can be damaged either by theproduction of reactive oxygen species by faulty oxidative

phosphorylation (Han et al., 2001) or by several environmentaltoxins (Schapira et al., 2006). The accumulation of oxidativedamage can be prevented by the selection and degradation offissioned non-functional mitochondria by an autophagic

mechanism termed mitophagy (Narendra et al., 2008; Twig et al.,2008). In brief, the PTEN-induced putative kinase 1 (PINK1) isstabilized on depolarized mitochondria, which leads to the

recruitment and activation of Parkin (Geisler et al., 2010a;Matsuda et al., 2010; Narendra et al., 2010a; Vives-Bauza et al.,2010). Then, Parkin ubiquitylates several mitochondrial proteins,

including mitofusins Mfn1 and Mfn2, the mitochondrial importreceptor subunits TOM20, TOM40 and TOM70, hexokinase II,Bcl-2 family members, mitochondrial Rho GTPases, mitochondrial

fission 1 protein and the voltage-dependent anion-selective channelprotein 1 (VDAC1), which finally results in the autophagicelimination of damaged mitochondria (Chan et al., 2011; Geisleret al., 2010a; Lazarou et al., 2013; Okatsu et al., 2012; Rakovic

et al., 2013; Sarraf et al., 2013; Yoshii et al., 2011).

Parkin was proposed to function as a RING-type E3 ubiquitin

ligase (Shimura et al., 2000), but recent evidence shows thatParkin shares features of the ‘homologous to the E6AP carboxylterminus’ (HECT) ubiquitin ligase family (Wenzel et al., 2011).

HECT E3 ubiquitin ligases use an internal active-site cysteine tobind to ubiquitin through a thioester bond (Rotin and Kumar,2009). In the case of Parkin, the conserved cysteine C431 is

required for ubiquitin ligase activity with the HECT-specific E2enzyme UBE2L3 (also known as UbcH7) and subsequentubiquitin transfer to substrates (Wenzel et al., 2011). Therecently published crystal structure of Parkin shows that the

UBE2-binding surface of the RING1 domain is buried in an auto-inhibited state (Trempe et al., 2013; Wauer and Komander, 2013).After a mitochondrial depolarization stimulus, it is suggested that

PINK1 phosphorylates Parkin at the crucial residue S65 (Iguchiet al., 2013; Kondapalli et al., 2012), resulting in the release of theautoinhibitory Parkin UBL domain (Chaugule et al., 2011). This

might allow the interaction of different E2 enzymes, such as

1Laboratory of Functional Neurogenetics, Department of Neurodegeneration,Hertie Institute for Clinical Brain Research, 72076 Tubingen, Germany. 2GermanCenter for Neurodegenerative Diseases, 72076 Tubingen, Germany.

*Authors for correspondence (sven.geisler@uni-tuebingen.de; philipp.kahle@uni-tuebingen.de)

Received 8 November 2013; Accepted 29 April 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3280–3293 doi:10.1242/jcs.146035

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UBE2L3 or UBE2N, with Parkin (Olzmann et al., 2007; Shimuraet al., 2000). Parkin also exerts in vitro ubiquitylation activity

with several E2 enzymes, including UBE2D (also known asUbcH5) (Kazlauskaite et al., 2014; Lazarou et al., 2013; Matsudaet al., 2006; Shin et al., 2011). Functionally, Parkin is thought todesignate substrates with K48-linked ubiquitin chains for

proteasomal degradation (Imai et al., 2000; Lim et al., 2005),whereas the interaction with UBE2N leads to conjugation of K63-linked ubiquitin chains on substrates for autophagosomal

degradation (Doss-Pepe et al., 2005; Lim et al., 2005; Olzmannet al., 2007).

Little is known about the E2 ubiquitin-conjugating enzymes

that are responsible for the diverse ubiquitylation events thatoccur during mitophagy. A recent report identified UBE2A (alsoknown as Rad6a), mutations in which cause a disorder that

involves intellectual disability, as a key factor for Parkin-mediated mitophagy, but other cell-type-specific E2 coenzymeswere proposed to play a role in mitophagy (Haddad et al., 2013).Here, we aim to identify E2 enzymes that cooperate with Parkin

in the ubiquitylation of mitochondrial substrates after membranedepolarization. We performed siRNA-mediated knockdown ofseveral Parkin-interacting UBE2s and investigated the influence

on ubiquitylation of depolarized mitochondria as well as onmitophagy. Knockdown of several E2 ubiquitin-conjugatingenzymes [UBE2N, UBE2L3, UBE2D2 and UBE2D3 (UBE2D2/

3 – these two enzymes are both targets of the same siRNA)]prevented Parkin-mediated mitochondrial elimination. However,individual knockdown of these UBE2s did not prevent the

recruitment of Parkin to damaged mitochondria or, surprisingly,their global ubiquitylation. Although the depletion of UBE2N ledto a reduced amount of K63-linked ubiquitin chains onmitochondria, a simultaneous knockdown of all of these E2

enzymes was required to reduce Parkin-dependent ubiquitylationof the mitochondrial proteins Mfn1, Mfn2, TOM20, TOM70 andVDAC1, indicating a crucial cooperative step in mitophagy.

RESULTSDistinct E2-conjugating enzymes contribute to mitophagyDuring the process of mitophagy, K63- and K27-linkedpolyubiquitin chains are found at mitochondrial sites, whereasK48-linked chains are hardly detectable (Geisler et al., 2010a;Narendra et al., 2010b; Okatsu et al., 2010). Thus, in this study,

we investigated the following E2 ubiquitin-conjugating enzymesthat are potentially capable of forming such chains. UBE2N isknown to mediate K63 linkages (Olzmann et al., 2007). UBE2T

(also known as HSPC150) can conjugate, at least in an in vitro

assay, all types of ubiquitin linkages (David et al., 2010). UBE2S(also known as E2-EPF) preferentially forms K11-linked chains

(Wickliffe et al., 2011) and potentially other types of linkage(David et al., 2010). UBE2D3 is predicted to conjugate K11, K48,K33 and K27 linkages (David et al., 2010). Finally, UBE2L3 was

included in our study as an established Parkin-interacting enzymethat plays an important role in ubiquitin-ester formation on theParkin active-site cysteine C431 (Iguchi et al., 2013; Lazarou et al.,2013; Wenzel et al., 2011). We investigated the influence of siRNA-

mediated downregulation of these E2 enzymes on the Parkin-dependent autophagic degradation of depolarized mitochondria inHeLa cells.

Immunoblotting revealed that the E2 enzymes were significantlyknocked down by their specific siRNAs, and minimal cross-reactivity between siRNAs was detected (Fig. 1A). Subsequently,

HeLa cells silenced for each E2 enzyme were transfected with

Parkin followed by depolarization of mitochondria with theuncoupling agent carbonyl cyanide m-chlorophenyl hydrazone

(CCCP). Two time-points were investigated – an early time-point(2 h) for the investigation of Parkin translocation, polyubiquitin-chain formation on mitochondria and recruitment of p62 (officiallyknown as SQSTM1) to mitochondria and a late time-point (24 h)

for the study of mitochondrial elimination. As HeLa cells lackendogenous Parkin (Denison et al., 2003), they provide aclean background where only Parkin-transfected cells execute

mitophagy. The analysis of immunofluorescent staining showedthat the reduction in the amounts of E2 enzymes did not alter thedistribution of cytosolic Parkin and polyubiquitin under basal

conditions. The mitochondrial network was undisturbed inuntreated cells (0 h CCCP, Fig. 1B). The administration ofCCCP for 2 h resulted in a prominent recruitment of Parkin to

depolarized mitochondria, despite UBE2 knockdown. Moreover, apolyubiquitin signal at the site of mitochondria and the recruitmentof p62, which is an adaptor connecting ubiquitylation andthe autophagic machinery, was observed in Parkin-positive cells

(Fig. 1C). At 24 h after CCCP administration, a completeelimination of depolarized mitochondria was observed in cellstransfected with control siRNA and siRNA against UBE2S and

UBE2T. By contrast, the knockdown of UBE2N, UBE2L3 orUBE2D2/3 diminished Parkin-dependent mitophagy (Fig. 1D). Atthis 24 h time-point, mitochondria were clustered at perinuclear

sites. Also, some polyubiquitin as well as p62 signal was foundon residual mitochondria (Fig. 1D). The quantification of theimmunofluorescence data indicated no significant effect of the

knockdown of individual E2 enzymes on the translocation of Parkin,the formation of global mitochondrial polyubiquitin signal and p62recruitment (Fig. 1E,F). By contrast, mitochondrial elimination wassignificantly impaired after knockdown of UBE2N (71628%,

6s.d.), UBE2L3 (5864%) and UBE2D2/3 (66612%) comparedwith that of control cells (32614%). Knockdown of UBE2S andUBE2T did not significantly affect Parkin-dependent mitochondrial

clearance (Fig. 1G).To exclude off-target effects of the UBE2 knockdown, we

performed rescue experiments by reintroduction of Myc-tagged

wild-type UBE2 or the non-functional active-site mutants UBE2N-C87A, UBE2L3-C86A and UBE2D3-C85A. HeLa cells weresilenced for each single E2 enzyme and were then transfected withempty vector, wild-type or mutant E2 enzyme constructs. Western

blot analysis confirmed an efficient knockdown for each E2enzyme and a prominent overexpression of the respective wild-type or mutant E2 enzyme (Fig. 2A). The silenced and re-

transfected cells were treated with CCCP for 2 h or 24 h andsubjected to immunofluorescent staining. The translocation ofParkin to mitochondria after 2 h of CCCP treatment was not

significantly altered in cells overexpressing wild-type or mutantUBE2 (Fig. 2B). Importantly, the impaired mitophagy afterdepletion of each E2 enzyme was significantly rescued by the

overexpression of wild-type E2 enzymes but not their respectiveactive-site cysteine mutants (Fig. 2C,D). Thus, the respectiveubiquitin-conjugating activities of UBE2N, UBE2L3 or UBE2D2/3 are crucial for the mitochondrial elimination.

Simultaneous knockdown of UBE2N, UBE2L3 and UBE2D2/3impaired ubiquitylation of mitochondrial proteinsand mitophagyTo further evaluate the role of UBE2N, UBE2L3 and UBE2D2/3in mitophagy, all of these E2 enzymes were knocked down

simultaneously. Knockdown efficiency was examined by western

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Fig. 1. siRNA-mediated knockdown of E2enzymes does not interfere with Parkintranslocation but inhibits mitophagy. (A) HeLacells were transfected with 5 nM control orindicated UBE2 siRNAs on three consecutivedays. The cells were lysed in NP-40 lysis bufferand the total-cell lysates were analyzed bywestern blotting. All E2 enzymes were stronglyknocked down, as verified by specific probingwith their corresponding antibody. b-actin servedas a loading control. (B–G) HeLa cellstransfected with the indicated siRNAs wereplated onto coverslips and were furthertransfected with EGFP–Parkin for 24 h. The cellswere left untreated (0 h CCCP, B) or themitochondria were depolarized with 10 mM CCCPfor 2 h (C,E,F) or 24 h (D,G). The cells were fixedand stained for the mitochondrial marker HtrA2(also known as Omi, red) and eitherpolyubiquitylated proteins (Poly-Ub, turquoise,left) or the autophagic adaptor p62 (p62,turquoise, right). The Parkin distribution wasvisualized by using the epifluorescence of theEGFP tag (Parkin, green). The nuclei werecounterstained with Hoechst 33342 (blue inmerged images). Representative images frommore than three independent experiments areshown. Parkin-positive cells (D) are outlined.Scale bars: 10 mm. (E,F) The translocation ofParkin to mitochondria and the appearance of anadditional polyubiquitin or p62 signal onmitochondria was scored in .200 Parkin-positivecells. (G) Mitochondrial elimination, as visualizedby the loss of HtrA2 signal, was scored in .200Parkin-positive cells. (E–G). Data represent themean6s.d.; *P,0.05 compared with control-siRNA-transfected cells.

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Fig. 2. Re-introduction of wild-type UBE2but not active-site cysteine mutantsrestores mitophagy. (A–D) The indicatedE2 enzymes were downregulated in HeLaas described in Fig. 1. These cells weresubsequently transfected with either emptyvector (2) or myc-tagged wild-type (WT) ormutant (UBE2N-C87A, UBE2L3-C86A andUBE2D3-C85A) E2 enzyme constructs for24 h. (A) The cells were lysed in NP-40buffer and subjected to western blotanalysis. The knockdown of each E2enzyme was verified by probing with theirspecific antibody, and the re-introducedMyc–E2 enzyme was detected by probingwith an anti-Myc antibody. b-actin served asa loading control. Ctr, control siRNA.(B–D) Silenced HeLa cells were plated ontocoverslips and were subsequentlytransfected with a combination of wild-typeEGFP–Parkin and vector (2), wild-type ormutant E2 enzyme for 24 h. These cellswere treated with 10 mM CCCP for 2 h or24 h prior to immunofluorescent staining.(B) The translocation of Parkin todepolarized mitochondria after 2 h of CCCPtreatment was scored in .200 Parkin-positive cells. (C) Immunofluorescentstaining was performed as described inFig. 1. Scale bars: 10 mm. Parkin-positivecells are outlined. Representative images ofthree independent experiments are shown.(D) Mitophagy, visualized by a loss of HtrA2signal, was scored in .200 Parkin-positivecells after 24 h of CCCP treatment. For thisquantification, only cells that were positivefor wild-type EGFP–Parkin and Myc-E2enzymes (wild type or mutant) wereconsidered. Data represent the mean6s.d.;*P,0.05 compared with cells transfectedwith wild-type UBE2.

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blotting, with or without mitochondrial membrane depolarization.The cells displayed a substantial reduction in the expression of

E2 enzymes, while general cell viability, visualized by b-actinprobing, was not affected (Fig. 3A).

Cells depleted of UBE2N, UBE2L3 and UBE2D2/3 weretransfected with EGFP–Parkin. Mitochondria were depolarized

with CCCP, and Parkin translocation (at 2 h) and mitophagy (at24 h) were investigated. Under basal conditions, the triple-

Fig. 3. The simultaneous knockdown of E2 enzymes reduces Parkin-dependent mitophagy. (A–G) HeLa cells were transfected with 5 nM of controlsiRNA or a mixture containing 5 nM of UBE2N, UBE2L3 and UBE2D2/3 siRNA on three consecutive days. (A) Untransfected (UTF) cells, control-siRNA-transfected cells (control) and cells transfected with all three siRNAs (2N+2L3+2D2/3) were left untreated or were treated with 10 mM CCCP for 2 h. The cellswere lysed in NP-40 lysis buffer, and equal protein amounts were analyzed by western blotting. The antibodies for UBE2N, UBE2L3 and UBE2D3 revealeda substantial reduction in the amount of E2 enzymes compared with that of control cells. b-actin served as a loading control. (B–G) siRNA-treated HeLa cellswere plated onto coverslips and were subsequently transfected with wild-type EGFP–Parkin for 24 h prior to treatment with 10 mM CCCP for 0 h (B), 2 h (C) or24 h (F). The mitochondria (HtrA2/Omi, blue), polyubiquitylated proteins (Poly-Ub, red) and p62 (red) were stained with specific antibodies, and thedistribution of Parkin was visualized by using the EGFP tag (Parkin, green). Representative images from three independent experiments are shown. Parkin-positive cells are outlined. Scale bars: 10 mm. (D,E) The Parkin translocation to depolarized mitochondria after 2 h of CCCP treatment as well as anadditional polyubiquitin or p62 signal in Parkin-positive cells on mitochondria was quantified for .200 cells in three independent experiments. (G) Mitophagy,indicated by the loss of the HtrA2 signal after 24 h of CCCP treatment, was counted in .200 Parkin-positive cells. Data represent the mean6s.d.; *P,0.05compared with control-siRNA-transfected cells.

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silenced cells showed no obvious morphological changes, nodifferences in the distribution of cytosolic Parkin and

polyubiquitin and no disturbance of the mitochondrial networkcompared with that of control-siRNA-transfected cells (0 hCCCP, Fig. 3B). The administration of CCCP for 2 h resultedin the translocation of Parkin to mitochondria in triple-siRNA-

transfected cells as well as in control-siRNA-transfected cells(2 h CCCP, Fig. 3C). In some Parkin-positive cells much overlapof Parkin with clustered mitochondria was observed, whereas in

other cells Parkin was still more dispersed throughout the cytosol.Overall, the translocation of Parkin to mitochondria after 2 h ofCCCP treatment was not significantly altered (control siRNA

8666%; triple siRNA 8067%, 6s.d.) (Fig. 3D). Nevertheless,although control cells showed a clear signal for polyubiquitin aswell as p62 at the site of mitochondria (2 h CCCP, Fig. 3C),

transfection of the three siRNAs significantly reduced the numberof cells with mitochondrial polyubiquitin (control siRNA8266%; triple siRNA 2169%) and p62 (control siRNA7967%; triple siRNA 1767%) in Parkin-positive cells

(Fig. 3E). The Parkin-dependent mitophagy in triple-siRNA-transfected cells was also impaired, as seen for cells transfectedwith each single siRNA. Notably, even after 24 h of CCCP

treatment, we observed either very little polyubiquitin and p62signal at the site of mitochondria or complete absence of thesesignals (24 h CCCP, Fig. 3F). The elimination of depolarized

mitochondria was significantly reduced after triple knockdown ofthe investigated E2 enzymes (control siRNA 2169%; triplesiRNA 79619%) (Fig. 3G). It appears that all of the investigated

E2 enzymes together mediate the Parkin-dependent ubiquitylationof mitochondrial proteins.

UBE2N, UBE2L3 and UBE2D2/3 as well as catalytically activeParkin are necessary for mitochondrial protein ubiquitylationWe next analyzed the Parkin-dependent ubiquitylation ofmitochondrial substrates after simultaneous knockdown of

UBE2N, UBE2L3 and UBE2D2/3, as well as single knockdownof UBE2S. HeLa cells were transfected with FLAG–Parkin and66His-tagged ubiquitin expression constructs for 24 h before

mitochondrial depolarization with CCCP for 2 h. Because Mfn1,Mfn2 and TOM70 are shown to be particularly rapidlyubiquitylated and degraded in Parkin-overexpressing cells afterdepolarization (Rakovic et al., 2013; Tanaka et al., 2010),

potential ubiquitylated species of those proteins were stabilizedby administration of the proteasome inhibitor MG-132. Lysis in8 M urea buffer and subsequent pulldown with Ni–NTA beads

ensured the binding of only covalently ubiquitylated proteins. Theanalysis of total-cell lysates revealed a substantial reduction in thelevels of UBE2N, UBE2L3 and UBE2D2/3 as well as UBE2S

proteins in UBE2-siRNA-treated cells compared with the levelsin control cells (Fig. 4A). The steady-state levels of theinvestigated mitochondrial proteins in non-treated cells were

not altered regardless of the knocked-down UBE2 enzyme (0 hCCCP, Fig. 4A). The administration of CCCP resulted in reducedprotein levels of Mfn1, Mfn2 and TOM70 in control and UBE2S-knockdown cells, which could be reverted by MG-132. Moreover,

in triple-siRNA-treated cells no prominent changes in theseprotein levels were observed (Fig. 4A). This possibly indicatesthat Mfn1, Mfn2 and TOM70 are modified with proteasome-

sensitive K48-linked ubiquitin chains during mitophagy.To determine the ubiquitylation states, we performed Ni–NTA

purification of 66His-tagged ubiquitylated proteins in untreated

and CCCP-treated cells. We observed a slight ubiquitylation of

Mfn1, Mfn2 and TOM70 even without mitochondrial membranedepolarization, but only after MG-132 treatment. This possibly

reflects the basal level of ubiquitylation of Mfn1, Mfn2 andTOM70 due to mitochondrial fusion and fission events. However,ubiquitylated species of TOM20, VDAC1 and Parkin were notdetected (0 h CCCP, Fig. 4B). The administration of CCCP

greatly increases the amount of ubiquitylated species of Mfn1,Mfn2, TOM70, TOM20, VDAC1 and Parkin in control andUBE2S-siRNA-transfected cells. The ubiquitylated species of

Mfn1, Mfn2 and TOM70 were only pulled down when theproteasome was inhibited with MG-132 (2 h CCCP, Fig. 4B).This might reflect the attachment of K48-linked ubiquitin chains

and a fast degradation through the ubiquitin proteasome system.By contrast, the triple siRNA transfection greatly reduced theamount of ubiquitylated species of all mitochondrial proteins, as

well as of ubiquitylated Parkin (2 h CCCP, Fig. 4B). Apparently,Parkin cannot modify these mitochondrial proteins in the combinedabsence of these E2 enzymes, and therefore no stabilization ofubiquitylated species with MG-132 could be detected. This

biochemical approach supports the immunofluorescence data(Fig. 3C), where only faint ubiquitin signals on mitochondriawere observed.

We next analyzed the Parkin dependency of mitochondrialubiquitylation using the catalytically inactive Parkin variantC431S. Based on in vitro data, it was suggested that the ‘RING-

between-RING’ (RBR) E3 ubiquitin ligase Parkin forms a thioesterintermediate on its active-site cysteine 431 (Wenzel et al., 2011).We used the Parkin variant C431S to trap the bound ubiquitin as

oxyester. In contrast to wild-type Parkin, the inactive Parkin C431Svariant did not translocate to depolarized mitochondria, as shownpreviously by others (2 h CCCP, Fig. 4C) (Lazarou et al., 2013;Zheng and Hunter, 2013). Moreover, neither polyubiquitin chain

formation nor p62 recruitment was observed (2 h CCCP, Fig. 4C)and, consequently, depolarized mitochondria were not degraded inParkin-C431S-overexpressing cells (24 h CCCP, Fig. 4C). We

additionally confirmed the lack of ubiquitylation of mitochondrialsubstrates in the Ni–NTA purification assay. Here, HeLa cellstransfected with either empty vector (2) or Parkin C431S together

with 66His–ubiquitin revealed no ubiquitylation of mitochondrialproteins or Parkin itself after CCCP treatment. By contrast,overexpression of wild-type Parkin resulted in polyubiquitylationof Mfn1, Mfn2 and TOM70 only in presence of MG-132, whereas

VDAC1, TOM20 and Parkin were additionally modified withoutthe stabilization of possible proteasome-sensitive ubiquitin chains(Fig. 4D). Thus, it appears that the functions of investigated

UBE2s are dependent on Parkin activity.Interestingly, mitochondrial substrates fall into two groups.

Mfn1, Mfn2, and TOM70 are rapidly ubiquitylated and degraded

proteins (Rakovic et al., 2013) and ubiquitylated species of theseproteins could only be detected after proteasome inhibition withMG-132. By contrast, TOM20 and VDAC1 are ubiquitylated even

without MG-132 treatment. Thus, it seems that these proteins arenot degraded by the proteasome under these conditions.

K63-linked and K48-linked ubiquitin chains are involvedin mitophagyRecent reports show the appearance of polyubiquitin chainslinked through ubiquitin K63 on depolarized mitochondria

(Narendra et al., 2010b; Okatsu et al., 2010). UBE2N is, todate, the only known E2 enzyme that mediates the formation ofubiquitin K63 linkages. To determine whether UBE2N is indeed

responsible for this type of linkage, we performed knockdown

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experiments in HeLa cells. After knockdown, these cells weretransfected with EGFP–Parkin, depolarized with CCCP and

analyzed by immunofluorescence. We used antibodies thatrecognize specifically either K48- or K63-linked ubiquitinchains. The antibody for K48-linked chains displayed a general

immunoreactivity distributed throughout the cytosol in control aswell as in UBE2-siRNA-transfected cells, irrespective ofmitochondrial membrane depolarization (0 h CCCP, Fig. 5A;

2 h CCCP, Fig. 5B), as shown by others (Narendra et al., 2010b;Okatsu et al., 2010). Although the antibody for K63-linked chains

displayed no immunoreactivity in untreated cells (0 h CCCP,Fig. 5A), a clear signal appeared on Parkin-positive depolarizedmitochondria in cells transfected with control, UBE2L3 and

UBE2D2/3 siRNA (2 h CCCP, Fig. 5B). The knockdown ofUBE2N strongly reduced this staining pattern. Thus, in thesecells, less K63-linked ubiquitin chains at mitochondria are present

Fig. 4. See next page for legend.

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(Fig. 5B). Silencing of UBE2N did not affect the total

polyubiquitin signal that was detected by an antibody thatrecognizes all polyubiquitin chains (Fig. 1C,F). This suggests thatmitochondrial proteins might be modified with ubiquitin chainsof different linkages. Proteins modified with K48-linked ubiquitin

chains are well-known substrates for proteasomal degradation.We tested the possibility that depolarized mitochondria mightdisplay K48-linked ubiquitin chains by inhibiting the proteasome.

Indeed, we observed an additional immunoreactivity ondepolarized mitochondria, detected with the antibody specificfor K48-linked ubiquitin chains, in Parkin-overexpressing cells

when MG-132 was added (2 h CCCP, Fig. 5C). The intensityseemed to increase over time, indicating the stabilization of K48-linked ubiquitin chains on mitochondria. Notably, K63-linked

ubiquitin chains were not affected by MG-132 per se, and theirintensity also increased over time (6 h CCCP, Fig. 5C). Thus, wesuggest that both K48 and K63 ubiquitin linkages are present ondepolarized mitochondria and participate in Parkin-dependent

mitophagy.

The translocation of UBE2N and UBE2L3 to mitochondria isdependent on ParkinNext, we investigated whether the different E2 enzymes exerttheir actions in the cytosol or reside directly on the mitochondria

independently of mitochondrial membrane depolarization. Weused both an immunofluorescence approach and biochemicalfractionation experiments to detect mitochondrial translocation

of the endogenous E2 enzymes. We analyzed the appearance ofUBE2 immunoreactivities on mitochondria in HeLa cells thatwere transfected with control siRNA or siRNA against E2enzymes, and were then further transfected with EGFP–Parkin

and depolarized for 2 h with CCCP (Fig. 6). For semi-quantitative imaging, we performed a line scan to detect theintensities of the respective anti-UBE2 immunoreactivity.

A strong overlap of mitochondrial and EGFP–Parkin signalwas observed in all cells, irrespective of which E2 enzymewas knocked down, indicating that Parkin colocalizes with

mitochondria (see above). Additionally, signals of UBE2N and

UBE2L3 correlated with these intensities, indicating acolocalization, which was completely absent when UBE2N or

UBE2L3 were downregulated. Interestingly, UBE2D3 did notappear to translocate to mitochondria, because no specific overlapof UBE2D3 immunoreactivity with mitochondria or Parkin wasdetected (Fig. 6).

To provide a methodologically independent confirmation ofthese results, we investigated the appearance of UBE2s inmitochondrial fractions of CCCP-treated HeLa cells. Cells were

silenced for the indicated UBE2s, transfected with FLAG–Parkinand treated with CCCP for 2 h. The cells were separated into acytosolic fraction (C, Fig. 7A) and an enriched mitochondrial

fraction (M, Fig. 7A). The levels of all E2-conjugating enzymeswere greatly diminished after their knockdown. Supporting theimmunofluorescence data, the endogenous E2 enzymes UBE2L3

and UBE2N were enriched in mitochondrial fractions after CCCPtreatment (Fig. 7A). The western blot analysis for UBE2D3 didnot show a specific signal in mitochondrial fractions, which,together with the immunofluorescence approach (Fig. 6),

indicates that UBE2D3 did not translocate to depolarizedmitochondria. Consistently, Parkin was more abundant in themitochondrial fraction after CCCP treatment than in untreated

cells (Fig. 7A), as shown by others (Iguchi et al., 2013; Matsudaet al., 2010; Narendra et al., 2010a). Importantly, Parkintranslocation was not abolished after knockdown of the UBE2s.

Moreover, we determined whether the triple knockdown or thedownregulation of each individual UBE2 interferes with PINK1stabilization, because the stabilization of the mitochondrial kinase

PINK1 at mitochondrial sites is a prerequisite for Parkintranslocation (Matsuda et al., 2010; Narendra et al., 2010a). Weobserved strong stabilization of PINK1 in the mitochondrialfraction after CCCP treatment, irrespective of which E2 enzyme

was knocked down (Fig. 7A). Because mitochondrial PINK1 iscrucial for Parkin activation (Iguchi et al., 2013), it is likely thatthe UBE2 enzymes function downstream of PINK1 accumulation

on mitochondria.By contrast, it is possible that the E2 enzymes might

translocate to depolarized mitochondria independently of active

Parkin. Therefore, the presence of UBE2N, UBE2L3 andUBE2D3 in the mitochondrial fraction was additionallyinvestigated in HeLa cells transfected with wild-type control orthe Parkin active-site cysteine C431S variant. UBE2N and

UBE2L3 were found in the mitochondrial fractions after CCCPtreatment only when wild-type Parkin was present (left panel,Fig. 7B). Because Parkin C431S did not translocate to

mitochondria, as seen above (Fig. 4C), one can argue that theE2 enzymes and the E3 ubiquitin ligase Parkin form a complexthat then translocates to mitochondria. This is additionally

supported by the observation that, after PINK1 downregulationby siRNA, Parkin as well as the UBE2 enzymes were not found inmitochondrial fractions after CCCP treatment (right panel,

Fig. 7B). This indicates that PINK1 is stabilized on depolarizedmitochondria, which might lead to recruitment of the UBE2–Parkin complex. Thus, our results suggest that the lack ofubiquitin signals on depolarized mitochondria might arise from

the absence of Parkin-interacting UBE2 enzymes.Taken together, the downregulation of UBE2N, UBE2L3 or

UBE2D2/3 did not prevent Parkin-dependent ubiquitylation of

mitochondrial protein, but greatly interfered with the degradationof depolarized mitochondria (mitophagy) after 24 h. When theseE2 enzymes were knocked down simultaneously, the

ubiquitylation of mitochondrial proteins was reduced to a large

Fig. 4. The ubiquitylation of Parkin substrates is impaired after tripleknockdown of E2 enzymes. (A,B) HeLa cells were transfected with theindicated siRNA as described in Fig. 3, followed by transfection with wild-type FLAG–Parkin in combination with 66His–ubiquitin (+) or empty vector(2) for 24 h. The cells were treated with 10 mM MG-132 for 30 min prior tomitochondrial membrane depolarization with 10 mM CCCP for 2 h or wereleft untreated (0 h CCCP). (A) The cells were lysed in 8 M urea buffer, andtotal-cell lysates were prepared and analyzed by western blotting withspecific antibodies for UBE2N, UBE2L3, UBE2D3, UBE2S, Mfn1, Mfn2,TOM20, TOM70, VDAC1 and Parkin. b-actin served as loading control.(B) For the purification of proteins modified with 66His-tagged ubiquitin, anequal protein amount (500 mg) was incubated with Ni–NTA beads. Thebound ubiquitylated proteins were eluted and analyzed by western blottingusing specific antibodies for Mfn1, Mfn2, TOM20, TOM70, VDAC1 and theFLAG tag (Parkin). Ctr., control. (C) HeLa cells were transfected with eitherwild-type (WT) or mutant C431S Parkin, treated with 10 mM CCCP for 2 h or24 h and subjected to immunofluorescent staining for mitochondria (HtrA2/Omi, blue), polyubiquitylated proteins (Poly-Ub, red) or p62 (red). The Parkindistribution was visualized by using the epifluorescence of the EGFP tag(green). Scale bars: 10 mm. (D) HeLa cells were transfected with emptyvector (2), wild-type or C431S Parkin constructs in combination with the66His-ubiquitin construct for 36 h, were treated with 10 mM MG-132 for30 min prior to mitochondrial membrane depolarization for 2 h and weresubjected to Ni–NTA purification as described for B. Representative imagesof three independent experiments are shown. Ub1, monoubiquitylated; Ub2,diubiquitylated; Ubn, polyubiquitylated.

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extent, correlating with strong attenuation of mitophagy. UBE2Npossibly mediated the formation of K63-linked ubiquitin chains

on depolarized mitochondria, whereas K48-linked ubiquitinchains were detected after proteasomal inhibition. Moreover,Parkin has to be activated at the crucial active-site residue C431

for translocation with UBE2N and UBE2L3 to depolarizedmitochondria, which is PINK1 dependent. In conclusion,

UBE2N, UBE2L3 and UBE2D2, working in a cooperativemanner, are key regulators of Parkin-dependent mitophagy.

DISCUSSIONThe mechanism of the PINK1- and Parkin-dependent

ubiquitylation and degradation of damaged mitochondria isextensively studied. Nevertheless, the cofactors for mediating

Fig. 5. Mitochondrial K63-linked ubiquitin chains are reduced in UBE2N-depleted cells, and K48-linked ubiquitin chains are stabilized by MG-132.(A–C) HeLa cells were transfected with the indicated UBE2 siRNAs as described in Fig. 1. The cells were further transfected with wild-type EGFP–Parkin for24 h. The cells were left untreated (A) or the mitochondria were depolarized with 10 mM CCCP for 2 h (B) prior to fixation. (C) 10 mM the proteasomalinhibitor MG-132 was administered to the cells prior to treatment with 10 mM CCCP for 2 h or 6 h. MG-132 was maintained in the medium during the entireincubation time. Immunofluorescent staining with the mitochondrial marker TOM20 (blue) and the antibodies specific for Lys48- or Lys63-linked ubiquitin chains(red) was performed. The Parkin distribution was visualized by using the epifluorescence of the EGFP tag (green). Parkin-positive cells are outlined. Scale bars:10 mm. Representative images of three independent experiments are shown.

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different mitochondrial protein ubiquitylation are not well known.Here, we report that Parkin utilizes different E2 ubiquitin-

conjugating enzymes for ubiquitylation of several mitochondrialproteins. Because K27-, K48- and K63-linked ubiquitin chains on

depolarized mitochondria were detected previously by our group(Geisler et al., 2010a), we analyzed E2 enzymes that are capable

of forming K27-, K48- and K63-linked ubiquitin chains (Davidet al., 2010). UBE2N, which is a well-studied E2 enzyme that

Fig. 6. Parkin-dependent translocation of endogenous UBE2N and UBE2L3. HeLa cells were silenced with control siRNA or siRNA against a specific UBE2for three consecutive days. The cells were transfected with wild-type EGFP–Parkin for 24 h. The mitochondrial membrane potential was depleted with 10 mMCCCP for 2 h prior to fixation. The cells were subjected to immunofluorescence analysis. Antibody against TOM20 (blue) was used for mitochondrialvisualization, and specific antibodies (Ab) for UBE2N, UBE2L3 and UBE2D3 were used to detect the endogenous UBE2 (red). The Parkin distribution wasvisualized by using the epifluorescence of the EGFP tag (green). A line of ,16 mm was drawn, and the relative intensity of each channel over the investigatedtrace is depicted in the corresponding diagram. Line 1 shows the relevant fluorescent intensities in Parkin-positive cells, whereas line 2 represents the intensitiesin untransfected cells (a.u., arbitrary units). Scale bars: 10 mm. Representative images of three independent experiments are shown.

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mediates the formation of K63-linked ubiquitin chains, caninteract with Parkin (Doss-Pepe et al., 2005) and is responsiblefor the autophagic degradation of mutant DJ-1 (Olzmann et al.,2007). UBE2L3 is a well-known E2 enzyme for Parkin and might

promote (multi-)monoubiquitylation (Fallon et al., 2006; Matsudaet al., 2006; Olzmann et al., 2007; Tanaka et al., 2010). UBE2Dhas been shown to interact with Parkin to polyubiquitylate

substrates (Shin et al., 2011) or to monoubiquitylate Mfn1(Lazarou et al., 2013) or the mitochondrial Rho GTPase 1(Kazlauskaite et al., 2014) at least in an in vitro ubiquitylation

assay. By contrast, no interaction of Parkin with the E2 enzymesUBE2S and UBE2T has been reported. UBE2T is involved in

DNA repair (Machida et al., 2006) and might perform all types ofubiquitin linkages in vitro (David et al., 2010). UBE2S is a

cofactor for the anaphase promoting complex/cyclosome (Garnettet al., 2009) and preferentially mediates K11-linked ubiquitinchains (Bremm et al., 2010). In this study, UBE2S was includedas a negative control because no K11-linked ubiquitin chains

have been found on depolarized mitochondria thus far.Recently, Haddad and colleagues reported that the loss of

the E2 enzyme UBE2A significantly impairs Parkin translocation

to depolarized mitochondria and subsequent mitophagy inUBE2A2/2 mouse embryonic fibroblasts (MEFs) (Haddadet al., 2013). In these cells, no accumulation of a global

ubiquitin signal (and, in particular, K48- and K63-linkedubiquitin signals) in mitochondrial fractions after CCCPtreatment was observed. In addition, Parkin was also less

ubiquitylated. Importantly, Parkin was not translocated todepolarized mitochondria in UBE2A2/2 MEFs, pointing to aninitial defect even upstream of Parkin activation by PINK1. Notethat Parkin but not UBE2A is highly expressed in the substantia

nigra (Kupershmidt et al., 2010) and that patients who suffer fromloss of UBE2A function do not show Parkinsonian syndromes (deLeeuw et al., 2010). Thus, it is likely that, besides UBE2A other

E2 enzymes (like UBE2N), UBE2L3 and UBE2D2/3 might beresponsible for Parkin-dependent mitophagy. Our approachrevealed that the individual knockdown of the investigated E2

enzymes does not interfere with PINK1 stabilization, Parkinrecruitment to and global polyubiquitin chain formation ondepolarized mitochondria. This might be due to redundant

functions of each E2 enzyme or different qualities ofpolyubiquitin chains. Here, we provide evidence that K48-linked and K63-linked ubiquitin chains are present on depolarizedmitochondria. Previously, it was shown that Parkin mediates the

formation of K27- and K63-linked polyubiquitin chains ondepolarized mitochondria (Geisler et al., 2010a; Narendra et al.,2010b; Okatsu et al., 2010). Moreover, outer mitochondrial

membrane proteins can be degraded by the proteasome in amanner dependent on Parkin after CCCP treatment. Contraryreports show either the absence of K48-linked ubiquitin chains at

the single-cell level (Narendra et al., 2010b; Okatsu et al., 2010)or an increase in the amount of K48-linked ubiquitin chains in themitochondrial fraction after depolarization (Chan et al., 2011;Haddad et al., 2013). Using our immunofluorescence approach,

we could confirm the formation of K63-linked ubiquitin chains, aprocess that is strictly dependent on the presence of UBE2N, andthe presence of stabilized K48-linked ubiquitin chains after

proteasomal inhibition. Additionally, in MG-132-treated cells,significant amounts of Mfn1, Mfn2 and TOM70 species modifiedwith 66His-tagged ubiquitin were pulled down in a manner

dependent on the presence of Parkin. This stabilization mightreflect the attachment of K48-linked ubiquitin chains to thesemitochondrial proteins. This indicates that these short-living

proteins modified with K48-linked ubiquitin chains were quicklyremoved by the proteasome at early steps of mitophagy. This is inline with reports showing a proteasome dependency of Parkin-mediated mitophagy (Gegg et al., 2010; Tanaka et al., 2010).

Emerging evidence shows that Parkin functions as RING/HECT hybrid E3 ubiquitin ligase, where ubiquitin is transferredfrom an E2 enzyme to Parkin’s active-site cysteine C431 as an

ubiquitin-thioester intermediate and is then passed to the substrate(Iguchi et al., 2013; Lazarou et al., 2013; Wenzel et al., 2011). Byusing the Parkin mutant C431S it is possible to trap this thioester-

linked ubiquitin on Parkin as an oxyester. Different E2 enzymes,

Fig. 7. UBE2N and UBE2L3 translocate to depolarized mitochondria ina Parkin-dependent manner. (A,B) HeLa cells were transfected with 5 nMindicated siRNAs on three consecutive days, followed by the transfectionwith empty vector (2), wild-type (WT) FLAG–Parkin or C431S Parkin for24 h. The cells were then treated with 10 mM CCCP for 2 h and subjected tothe mitochondrial fractionation assay. (A) The obtained cytosolic (C) andmitochondrial (M) fractions were analyzed for E2 enzyme knockdown withthe specific antibodies UBE2L3, UBE2N and UBE2D3. Probing with the anti-FLAG antibody shows the appearance of wild-type FLAG–Parkin in thefractions. Probing with anti-VDAC1 (mitochondrial marker) and p38 MAPK(cytosolic marker), revealed no or minimal cross-contamination within thefractions. The anti-PINK1 antibody showed a strong increase in the amountof endogenous PINK1 in the mitochondrial fraction after CCCPadministration, irrespective of the downregulation of UBE2 enzymes. (B) Thedistribution of the indicated proteins was analyzed with their respectiveantibodies. The PINK1-specific siRNA strongly reduced PINK1 proteinlevels. In cells with PINK1 depletion and overexpression of mutant C431SParkin, the E3 ubiquitin ligase did not appear in the mitochondrial fractionand failed to co-translocate the UBE2 enzymes. Western blots showrepresentative images of three independent experiments.

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including UBE2L3, UBE2D2 and UBE2D3, can form thisubiquitin thioester on Parkin C431S, at least in an in vitro

ubiquitylation assay, which promotes the HECT-like activity ofParkin (Iguchi et al., 2013; Lazarou et al., 2013). However,Parkin C431S did not translocate to mitochondria, even inpresence of PINK1 and UBE2 enzymes. By contrast, the

combined knockdown of UBE2N, UBE2L3 and UBE2D2/3 didnot interfere with the translocation of wild-type Parkin tomitochondria. Thus, it is possible that another E2 enzyme not

tested in this study is necessary for the initial activation of Parkin.One likely candidate could be UBE2A, the knockout of which inMEFs largely abrogates Parkin translocation (Haddad et al.,

2013). Nevertheless, besides the possibly redundant function ofthe investigated UBE2 enzymes, we provide evidence that theknockdown of UBE2N indeed interferes with the formation of

K63-linked polyubiquitin chains on mitochondrial substrates. Theindividual contributions of the other E2 enzymes remain to beshown.

It is not yet established whether and how Parkin, as a new

member of the RING/HECT-hybrid E3 ligase family, can utilizedifferent E2 enzymes for the polyubiquitylation of mitochondrialsubstrates with different linkages. It is believed that, in the case of

HECT-type E3 ligases, the ligase itself determines the finaloutcome of the ubiquitylation reaction. The question of whetherthe chain specificity arises from the bound E2 or E3 ligase is not

resolved for Parkin and other RBR E3 ligases (reviewed in Smitand Sixma, 2014). Thus, we cannot rule out the possibility thatother E3 ubiquitin ligases that have the ability of forming K48-

linked or K63-linked polyubiquitin chains through UBE2N,UBE2L3 or UBE2D2/3 might participate in mitophagy.

However, ubiquitin chains of distinct linkages on mitochondriastill recruit the ubiquitin-binding adaptor protein p62, which

connects ubiquitin signals with LC3 that is bound to isolationmembranes (Pankiv et al., 2007). The ubiquitin-associateddomain (UBA) of p62 binds to K63-linked and, to a lesser

extent, K48-linked polyubiquitin chains (Long et al., 2008;Seibenhener et al., 2004). Also, a weak interaction of the isolatedUBA domain with monoubiquitin was detected (Raasi et al.,

2005). Thus, p62 recruitment might be insensitive to the type ofpolyubiquitin linkage on mitochondria, at least in this Parkinoverexpression model. Nevertheless, both the individual and thecombined knockdown of the E2 enzymes interferes significantly

with the 24 h end-point mitophagy. In cells depleted of all threeUBE2 enzymes, we show both less ubiquitin chain formation ondepolarized mitochondria at the single-cell level and a strong

reduction in the amount of polyubiquitin chains on Mfn1, Mfn2,TOM20, TOM70 and VDAC1 using a pull-down approach.Without these modifications, p62 cannot be recruited and,

consequently, mitophagy is impaired. Thus, due to the lack ofubiquitin as a degradation signal, mitochondria cannot bedegraded in a Parkin-dependent manner. In the case of each

single UBE2 enzyme knockdown, ubiquitin and p62 are stillpresent at mitochondria but mitophagy is abolished. It is possiblethat E2-dependent steps downstream of p62 recruitment might bedisturbed – steps such as the formation of autophagosomes, the

fusion of autophagosomes and lysosomes and/or the maintenanceof proper lysosomal function. Thus, we cannot rule out thepossibility that other steps in the process of mitophagy after

knockdown of the investigated UBE2s are affected.In conclusion, here, we show that the final elimination of

depolarized mitochondria requires several E2 enzymes – UBE2N,

UBE2L3 and UBE2D2/3. Moreover, their simultaneous

knockdown interferes with the recruitment of p62 owing todiminished ubiquitylation of mitochondrial proteins. The

translocation of Parkin to depolarized mitochondria was notdisturbed. Thus, we provide evidence that mitochondriallytargeted Parkin performs polyubiquitylation of mitochondrialsubstrates, utilizing the E2 enzymes UBE2N, UBE2L3 and

UBE2D2/3.

MATERIALS AND METHODSConstructsThe pcDNA3.1 FLAG-Parkin, pCMV-66His-ubiquitin and pEGFP-

EGFP-Parkin expression constructs were as described previously

(Geisler et al., 2010a). The cDNA of the E2 enzymes UBE2N,

UBE2L3 and UBE2D3 was cloned into the pCMV-Myc vector

(Clontech, CA). The active-site cysteine of Parkin (C431) and each E2

enzyme was mutated to alanine using the GeneArt Site-Directed

Mutagenesis System (Invitrogen, Germany), following the

manufacturer’s instructions. These mutants (UBE2N-C87A, UBE2L3-

C86A and UBE2D3-C85A) were non-functional in terms of ubiquitin

binding at the active-site cysteine. The Parkin variant C431S is

catalytically inactive because the attached ubiquitin is trapped as

oxyester intermediate. All sequences were verified by sequencing

analysis.

Cell culture and transfectionHeLa cells (ATCC, VA) were maintained in Dulbecco’s modified Eagle

medium (DMEM, Biochrom, Germany) with 4.5 mM glucose,

supplemented with 10% fetal bovine serum (FBS, PAA Laboratories,

Germany) at 37 C under 5% CO2 and humidified conditions. The cells

were transiently transfected with the indicated pcDNA3.1 FLAG-Parkin,

pCMV-66His-ubiquitin, pEGFP-EGFP-Parkin or pCMV-Myc E2

enzyme constructs for 24 h using XtremeGene9 (Roche, Basel,

Switzerland). The cells were subsequently subjected to western blot

analysis, immunofluorescence assay, Ni-NTA affinity purification or

mitochondrial fractionation assay.

siRNA-mediated knockdownThe cells were transfected with 5 nM of the indicated siRNA on three

consecutive days using HiPerFect (Qiagen, Germany). The following

siRNAs with the indicated target sequences were used: control (AllStars

negative control, Qiagen, Germany); UBE2L3, 59-UGAAGAGUUUAC-

AAAGAAA-39 (Dharmacon, D-010384-01, Germany); UBE2T, 59-CCU-

GCGAGCUCAAAUAUUA-39 (Dharmacon, D-004898-01, Germany);

UBE2N, 59-AACCAGATGATCCATTAGCAA-39 (Qiagen, 1027020-

89072483, Germany); UBE2S, 59-AAGGCACTGGGACCTGGATTT-39

(Qiagen, 1027020-89072484, Germany); UBE2D2/3, 59-AACAGTAAT-

GGCAGCATTTGT-39 (Qiagen, 1027020-89072486, Germany); PINK1,

59-GACGCTGTTCCTCGTTATGAA-39 (Qiagen, SI00287931, Germany).

Inhibitor and treatmentsThe mitochondria of HeLa cells were depolarized with 10 mM carbonyl

cyanide m-chlorophenylhydrazone (CCCP, Sigma-Aldrich, MO) for 2 h

or 24 h. The proteasome was inhibited with 10 mM MG-132 (Sigma-

Aldrich, MO) 30 min before CCCP administration. MG-132 was left on

the cells during the entire incubation time.

Immunocytochemistry and microscopyHeLa cells transfected with siRNA were plated onto coverslips coated

with poly-D-lysine (Sigma-Aldrich, MO) and were further transfected

with EGFP–Parkin (wild type or C431S) for 24 h. The cells were treated

with CCCP for 2 h, 6 h or 24 h, fixed in 4% paraformaldehyde (Sigma-

Aldrich, MO) in PBS for 20 min, washed three times with PBS,

permeabilized with 1% Triton X-100 (Sigma-Aldrich, MO) in PBS for

5 min, washed three times with PBS and blocked with 10% FBS in PBS

at 4 C overnight. The cells were incubated with the indicated primary

antibodies in 10% FBS in PBS for 2 h, washed three times with PBS and

incubated with goat anti-mouse-IgG or anti-rabbit-IgG conjugated with

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Alexa Fluor 568 (Invitrogen, Germany) or Alexa Fluor 647 (Invitrogen,

Germany) in 5% FBS in PBS for 1 h. The nuclei were counterstained

with 2 mg/ml Hoechst 33342 (Molecular Probes, Germany) for 10 min.

Coverslips were mounted onto microscope slides using Fluorescent

Mounting Medium (Dako, Germany). The slides were analyzed with an

AxioImager microscope equipped with an ApoTome Imaging System

(Carl Zeiss, Germany), using a 636 objective. The images were

processed using the AxioVision 4.8.1 software (Carl Zeiss, Germany).

Colocalization was assessed over the indicated line (line scan) using a

feature of the software AxioVision 4.8.1.

Ni–NTA affinity purificationHeLa cells were left untreated or were silenced for UBE2S or

simultaneously for UBE2N, UBE2L3 and UBE2D2/3 and were then

co-transfected with vector control, wild-type FLAG–Parkin or FLAG–

Parkin C431S and either empty pCMV-66HIS or pCMV-66HIS-

ubiquitin expression vectors for 36 h. The cells were treated with

10 mM CCCP for 2 h, lysed in denaturing urea buffer (10 mM Tris-HCl

pH 8.0, 100 mM NaH2PO4, 8 M urea and 10 mM imidazole), passed 20

times through a 20 gauge needle and centrifuged at 18,000 g for 15 min

at 4 C. A total of 500 mg of total supernatant protein was incubated with

50 ml of Ni–NTA beads (Qiagen, Germany) in a total volume of 1 ml of

urea buffer for 3 h at room temperature. The beads were washed three

times with lysis buffer containing 20 mM imidazole, and proteins were

eluted with 60 ml of 36Laemmli buffer (187.5 mM Tris-HCl pH 6.8, 6%

SDS, 30% glycerol, 15% b-mercaptoethanol and 0.003% Bromophenol

Blue) containing 500 mM imidazole for 10 min at 95 C. A total of 10 ml

of the eluate were subjected to western blot analysis.

Mitochondrial fractionationHeLa cells were separated into cytosolic (C) and enriched mitochondrial

(M) fractions by stepwise centrifugation as described previously (Geisler

et al., 2010b). Briefly, cells were suspended in fractionation buffer

(20 mM HEPES pH 7.6, 250 mM sucrose and 3 mM EDTA) containing

protease inhibitors. The cells were lysed by 20 strokes in a Dounce

homogenizer followed by 20 passages through a 20 gauge needle. The

cellular debris was removed by centrifugation at 860 g at 4 C for 10 min.

The remaining lysate was further centrifuged at 16,800 g at 4 C for

10 min to obtain the mitochondrially enriched pellet (M) and the

supernatant (C). The pellet was washed twice and resuspended in an

appropriate amount of fractionation buffer. Both fractions were analyzed

by western blotting.

Western blotting and antibodiesCells were harvested and lysed in NP-40 buffer (150 mM Tris-HCl pH 7.6,

50 mM NaCl, 10 mM sodium pyrophosphate, 1% Nonidet P-40),

denaturing 8 M urea buffer or fractionation buffer. Equal protein

amounts, as determined by using the Bradford assay (Bio-Rad,

Germany), were separated by SDS-PAGE and transferred onto Hybond-

P polyvinylidene difluoride (PVDF) membranes (Millipore, Germany)

with the wet transfer method. Captured proteins bound by primary

antibodies were visualized by using HRP-coupled secondary antibodies

(Jackson ImmunoResearch Laboratory, UK) using the Immobilon western

chemiluminescent-HRP substrate (Millipore, Germany) on Hyperfilm

ECL high-performance chemiluminescence (GE Healthcare, UK).

The following antibodies were used at the indicated concentrations

either for immunofluorescence (IF) or western blot (WB) analysis: mouse

anti-polyubiquitylated proteins (FK1; IF, 1:750; WB, 1:1000; Biomol,

Germany), mouse anti-p62 (IF, 1:750; WB, 1:1000; BD Bioscience,

Germany), rabbit anti-HtrA2 (WB and IF, 1:1000; R&D Systems, MN),

goat anti-UBE2N (IF, 1:750; WB, 1:1000; Santa Cruz, CA), rabbit anti-

UBE2T (WB, 1:2000; ProteinTechGroup, IL), rabbit anti-UBE2D3 (IF,

1:750; WB, 1:3000; ProteinTechGroup, IL), rabbit anti-UBE2S (WB,

1:1000; NEB, Germany), rabbit anti-UBE2L3 (IF, 1:750; WB, 1:1000,

Boston Biochem, MA), mouse anti-Myc (WB, 1:30,000; Sigma-Aldrich,

MO), mouse anti-Mfn1 and mouse anti-Mfn2 (WB, 1:8000; Abnova,

Germany), rabbit anti-TOM70 (WB, 1:1000; ProteinTechGroup, IL),

mouse and rabbit anti-TOM20 (WB, 1:1000; Santa Cruz, CA), rabbit

anti-VDAC1 (WB, 1:30,000; Millipore, Germany), mouse anti-b-actin

(WB, 1:200,000; Sigma-Aldrich, MO), rabbit anti-ubiquitin, Lys48

specific (IF, 1:1000; Millipore, Germany), rabbit anti-ubiquitin, Lys63

specific (IF, 1:1000; Millipore, Germany), mouse anti-FLAG (WB,

1:50,000; Sigma-Aldrich, MO), rabbit anti-PINK1 (WB, 1:2000; Novus

Biologicals, Germany) and rabbit anti-p38 MAPK (WB, 1:1000; NEB,

Germany).

Statistical analysisThe colocalization of Parkin, polyubiquitin, p62 and mitochondria after

2 h of CCCP treatment and the mitochondrial clearance after 24 h of

CCCP treatment were assessed by scoring >200 Parkin-positive cells per

condition for each experiment. For the data shown in Fig. 2, only cells

that were doubly positive for both EGFP–Parkin and mutant or wild-type

Myc–UBE2 were considered for quantification. Western blot images of

representative experiments are shown. The error bars represent the

standard deviation of the mean of at least three independent experiments.

The statistical significance was assessed by using the two-tailed paired

Student’s t-test. The significance is indicated by an asterisk, *P,0.05.

AcknowledgementsWe thank Rejko Kruger (Hertie Institute for Clinical Brain Research, Tubingen,Germany) for providing the rabbit anti-TOM20 antibody. We thank WolfdieterSpringer (Hertie Institute for Clinical Brain Research, Tubingen, Germany) forproviding the EGFP–Parkin and FLAG–Parkin expression constructs and forhelpful discussions.

Competing interestsThe authors declare no competing interests.

Author contributionsS. Geisler conceived, performed and analyzed the experiments and wrote themanuscript. S.V. and S. Golombek performed experiments. P.J.K. conceivedexperiments, supervised the project and wrote the manuscript.

FundingThis work was supported by grants to P.J.K. from the German Ministry of Educationand Research (BMBF); National Genome Research Network NGFNplus; and theEuropean Research Consortium on Mendelian Forms of Parkinsonism (MEFOPA).It was also supported by an intramural fortune 2072-0-0 grant to S. Geisler from theFaculty of Medicine Tubingen; as well as the Hertie Foundation.

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