eteIsiuefrCiia ri eerh 27 Tu 72076 Research, Brain Clinical for Institute Hertie Atosfrcrepnec [email protected] philipp.kahle@uni- tuebingen.de) ([email protected]; correspondence for *Authors Tu 72076 Diseases, Neurodegenerative for Center vnGeisler mitophagy Sven Parkin-dependent for essential and are UBE2L3 UBE2D2/3 UBE2N, enzymes ubiquitin-conjugating The ARTICLE RESEARCH ß 3280 2014 April 29 Accepted 2013; November 8 Received K63 whereas degradation, 1 the proteasomal to linked drives molecules ubiquitin residue four least the K48 at example, of of For each molecule. attachment at ubiquitin the the occur within can residues chains lysine seven polyubiquitin of E2 elongation The polyubiquitylated. The (ubiquitin-activating), or enzymes. (multi-)monoubiquitylated ligase) be E1 can (ubiquitin substrates by E3 multi-step and mediated a (ubiquitin-conjugating) in occurs reaction ubiquitin molecule catalytic small the substrate with of modification proteins The homeostasis 2002). of cellular Ciechanover, variety and and (Glickman regulation, biosynthesis a cycle organelle cell in responses, turnover, roles stress protein as regulatory such processes, has cellular system ubiquitylation The INTRODUCTION UBE2 Mitophagy, Proteasome, Ubiquitin, Parkin, synergistically WORDS: We KEY UBE2D2/3 UBE2s. and these of mitophagy. UBE2L3 Parkin-mediated ubiquitylate presence to UBE2N, contribute to a the with failed that in Parkin (C431S) conclude of even site version substrates voltage- A active in mitochondrial UBE2s. the the observed these was in of TOM70, Parkin mutation all and for and 1 silenced protein cells TOM20 channel anion subunits mitochondrial dependent recruitment. the receptor mitofusins, p62 of import and ubiquitylation reduced polyubiquitylation Moreover, UBE2 significantly mitochondrial together as individual UBE2s these reduced after known of mitochondria all (officially of on knockdown Knockdown PINK1 knockdown. found p62 still and mitochondrial UBE2N were mitochondrial SQSTM1) polyubiquitin at with translocation. Nevertheless, ubiquitylation sites. K63-linked interfere Parkin mitochondria. specifically not prevented depolarized and significantly did of (UBE2D2/3) stabilization this UBE2D3 clearance UBE2N, However, and enzymes autophagic Parkin- E2 UBE2D2 regulate reduced the might or of that UBE2L3 enzymes Knockdown we UBE2 Here, mitophagy. understood. of several mediated ubiquitin-conjugating poorly set of are a E2 mitophagy ubiquitylation investigate of in by roles (UBE2) PINK1 accompanied enzymes The products is substrates. gene This mitochondrial disease Parkinson’s Parkin. the and process on a in depends mitophagy that by degraded are mitochondria Depolarized ABSTRACT aoaoyo ucinlNuoeeis eateto Neurodegeneration, of Department Neurogenetics, Functional of Laboratory 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,38–23doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1, ,Seai Vollmer Stefanie *, 1 ¨ ign Germany. bingen, oi Golombek Sonia , ¨ ign Germany. bingen, 2 German 1 n hlp .Kahle J. Philipp and te ellrptwy,Pri a nesnilfnto in function essential as Mitochondria an control. has in quality roles and Parkin Besides homeostasis 2000). pathways, mitochondrial al., cellular et (Shimura (RING)-other ligase gene’ ubiquitin new al., interesting E3 ‘really et a type (Kitada for codes disease gene Parkinson’s The 1998). recessive autosomal of (David cause clear less 2012). Rape, is and linkages for Komander of 2010; whereas, selection al., et chains, the enzymes, ubiquitin E2 K63-linked other forms UBE2V, heterodimerizes which with Different Ubc13), as known 2012). (also The UBE2N Rape, linkages. enzyme ubiquitin E2 of and types distinct Komander such influence enzymes 2008; E2 Dikic, and trafficking subcellular (Ikeda regulate monoubiquitylations and linkages ih lo h neato fdfeetE nye,sc as such enzymes, E2 different of interaction This 2011). the al., the allow et of (Chaugule release might domain the in UBL (Iguchi resulting Parkin 2012), S65 al., autoinhibitory residue et Kondapalli crucial 2013; the al., et at that suggested Parkin is phosphorylates it PINK1 stimulus, 2013). the The depolarization Komander, mitochondrial and that auto- Wauer a 2011). an 2013; After shows al., in et buried al., (Trempe Parkin subsequent is state domain inhibited et of RING1 and the of structure (Wenzel surface UbcH7) UBE2-binding crystal substrates as published is to recently known C431 transfer cysteine E2 (also HECT-specific ubiquitin conserved the UBE2L3 Kumar, the with and activity Parkin, enzyme ligase (Rotin of ubiquitin bond for case required thioester the a In to through cysteine 2009). active-site ubiquitin internal to 2011). an al., use bind that ligases et ubiquitin carboxyl (Wenzel shows E3 E6AP family evidence HECT the ligase to ubiquitin recent ‘homologous (HECT) but the terminus’ of 2000), features al., shares Parkin et (Shimura Rakovic 2011). al., 2012; ligase et al., Yoshii et 2013; al., autophagic Okatsu et 2013; Sarraf the al., 2013; al., et Geisler in et Lazarou 2011; 2010a; al., results al., et finally (Chan et mitochondria which damaged of (VDAC1), elimination 1 channelprotein anion-selective voltage-dependent the and II, protein 1 hexokinase fission mitochondrial TOM70, GTPases, Rho mitochondrial import and members, family mitochondrial TOM40 Bcl-2 the TOM20, 2010a; Mfn2, subunits and receptor al., al., Mfn1 proteins, et mitochondrial mitofusins et Vives-Bauza the several including 2010a; ubiquitylates (Geisler al., Parkin to et Then, Parkin Narendra 2010). leads 2010; of al., which et activation Matsuda mitochondria, is autophagic and (PINK1) depolarized 1 recruitment kinase an on putative PTEN-induced al., stabilized the et by brief, of Twig 2008; In degradation al., 2008). et and mitochondria (Narendra oxidative selection mitophagy termed of the mechanism non-functional accumulation by oxidative prevented The fissioned faulty be environmental 2006). several can al., by by damage et or the species (Schapira 2001) by al., toxins oxygen either et damaged (Han reactive phosphorylation be of can organelle production producing energy the uain ntegn noigPri r h otcommon most the are Parkin encoding gene the in Mutations aknwspooe ofnto saRN-yeE ubiquitin E3 RING-type a as function to proposed was Parkin 1,2, *

Journal of Cell Science ta. 00.Pri loexerts also Shimura Parkin 2007; 2000). al., et al., (Olzmann et Parkin with UBE2N, or UBE2L3 ARTICLE RESEARCH eaclsslne o ahE nyewr rnfce with transfected were enzyme E2 cross- each minimal for and silenced Subsequently, 1A). cells siRNAs, (Fig. HeLa detected specific was siRNAs their between reactivity by down knocked Parkin- the on in cells. mitochondria enzymes HeLa depolarized of E2 degradation autophagic these dependent of downregulation siRNA- the of influence mediated the investigated on We al., 2011). et formation al., Lazarou et ubiquitin-ester Wenzel 2013; 2013; al., in et (Iguchi role C431 cysteine active-site important Parkin enzyme Parkin-interacting an established plays was an UBE2L3 as that Finally, study linkage 2010). our al., of in et K48, included (David types K11, linkages conjugate K27 other to and predicted potentially is K33 UBE2D3 and 2010). al., 2011) chains et (David K11-linked al., forms et UBE2S preferentially 2010). (Wickliffe E2-EPF) an al., et as in (David known least linkages (also ubiquitin at of UBE2T conjugate, types 2007). can all is al., assay, HSPC150) UBE2N et as chains. (Olzmann known linkages such (also K63 forming mediate of to study, capable known this potentially enzymes in ubiquitin-conjugating are Thus, E2 K27-linked 2010a; 2010). that following al., al., the et and investigated et Okatsu we (Geisler 2010b; whereas K63- al., detectable sites, et hardly Narendra mitochondrial mitophagy, are at chains of found K48-linked are process chains polyubiquitin the mitophagy to During contribute enzymes E2-conjugating Distinct RESULTS DC,idctn rca oprtv tpi mitophagy. in on step cooperative and E2 crucial chains TOM70 a TOM20, these Mfn2, indicating ubiquitin Mfn1, VDAC1, of proteins ubiquitylation mitochondrial K63-linked all Parkin-dependent the reduce of of to of required knockdown was amount enzymes simultaneous a the reduced mitochondria, led prevent a UBE2N of not depletion surprisingly, to the or, did Although mitochondria ubiquitylation. siRNA)] global damaged UBE2s same their to these Parkin the of of of recruitment targets knockdown both However, are individual elimination. mitochondrial enzymes Parkin-mediated two ubiquitin-conjugating prevented on these (UBE2D2/ UBE2D3 E2 as – and 3 well UBE2D2 several UBE2L3, as [UBE2N, of enzymes mitochondria Knockdown depolarized mitophagy. of influence the ubiquitylation investigated of on and knockdown UBE2s siRNA-mediated Parkin-interacting several membrane performed Parkin after with We substrates cooperate mitochondrial depolarization. that of 2013). enzymes Parkin- ubiquitylation al., E2 et the identify for (Haddad in to mitophagy aim in factor that we role a coenzymes Here, key disorder play E2 to a cell-type-specific a proposed other were cause as but which mitophagy, disability, mediated in intellectual that mutations events (also involves Rad6a), UBE2A ubiquitylation identified as report diverse recent known A the mitophagy. for during occur responsible are autophagosomal that Olzmann for 2005; al., substrates et 2007). Lim al., 2005; on et al., et 2005), chains (Doss-Pepe for al., degradation K63- et ubiquitin of chains conjugation Lim to leads 2000; linked ubiquitin UBE2N with al., interaction the et K48-linked whereas to (Imai thought with degradation is Parkin proteasomal Functionally, as substrates 2011). known al., Matsuda et designate 2013; (also Shin al., 2006; et UBE2D al., Lazarou et 2014; including al., et enzymes, (Kazlauskaite UbcH5) E2 several with muoltigrvae htteE nye eesignificantly were enzymes E2 the that revealed Immunoblotting itei nw bu h 2uiutncnuaigenzymes ubiquitin-conjugating E2 the about known is Little nvitro in bqiyainactivity ubiquitylation nvitro in 6 r rca o h iohnra elimination. mitochondrial respective the for the crucial are UBE2D2/ Thus, 3 or UBE2L3 2C,D). UBE2N, (Fig. the of respective activities by ubiquitin-conjugating their mutants after not rescued cysteine but significantly mitophagy enzymes active-site was E2 impaired wild-type enzyme of not E2 the overexpression each was Importantly, of treatment mutant depletion CCCP or 2B). (Fig. wild-type and of overexpressing h UBE2 h cells re- 24 2 of in or and altered after translocation h significantly silenced mitochondria The 2 to The for staining. Parkin CCCP immunofluorescent 2A). (Fig. with to treated enzyme subjected E2 were wild- E2 cells each respective mutant transfected the for of or knockdown overexpression prominent type efficient a an and enzyme confirmed Western analysis constructs. enzyme blot E2 with were mutant transfected or then wild-type cells were vector, and empty enzyme HeLa E2 single UBE2D3-C85A. each for silenced and UBE2L3-C86A Myc-tagged UBE2N- C87A, mutants active-site of non-functional the reintroduction or UBE2 by wild-type experiments rescue performed 1G). (Fig. Pa clearance affect (32 significantly not cells did control UBE2T of that with B2.B otat h ncdw fUEN B23or UBE2L3 UBE2N, At 1D). complete of and (Fig. knockdown mitophagy UBE2S a Parkin-dependent the against diminished administration, siRNA contrast, UBE2D2/3 and By CCCP and siRNA UBE2T. control after ubiquitylation m with depolarized h transfected connecting 24 of adaptor At elimination o an 1C). was (Fig. is machinery, autophagic which the to p62, of Parkin of of site of administration recruitment a the prominent at Moreover, The signal knockdown. a polyubiquitin UBE2 in 1B). in despite Fig. resulted mitochondria, undisturbed h depolarized CCCP, 2 was h for (0 basal CCCP network under cells mitochondrial polyubiquitin untreated the and alter The not Parkin did conditions. enzymes cytosolic a E2 of of amounts provide distribution the showed in execute staining they reduction immunofluorescent the cells 2003), that of Parkin-transfected analysis al., only The lack mitophagy. where cells et HeLa background (Denison As clean elimination. Parkin h) mitochondrial (24 of endogenous time-point study late a the and for mitochondria to SQSTM1) (officially polyubiquitin- as p62 translocation, known of recruitment Parkin and mitochondria of on investigation formation chain the time-point the early for an h) hydrazone with – (2 investigated m-chlorophenyl were mitochondria time-points cyanide Two of (CCCP). carbonyl depolarization agent by uncoupling followed Parkin infcnl mardatrkokono B2 (71 UBE2N of knockdown after impaired significantly contra By 1E,F). the (Fig. the mitochond recruitment of global Parkin, of of of effect translocation formation the quantification the on significant found enzymes E2 The no was individual of signal knockdown 1D). indicated (Fig. p62 data perinuclear as mitochondria immunofluorescence well at residual as clustered polyubiquitin were on some mitochondria Also, time-point, sites. h 24 this nmtpay l fteeE nye eekokddown knocked were enzymes western E2 by examined these was efficiency of Knockdown UBE2D2/3 simultaneously. and all UBE2L3 UBE2N, mitophagy, of in role the evaluate further To mitophagy proteins and mitochondrial UBE2D2/3 of and ubiquitylation UBE2L3 impaired UBE2N, of knockdown Simultaneous ..,UEL (58 UBE2L3 s.d.), oecueoftre fet fteUE ncdw,we knockdown, UBE2 the of effects off-target exclude To ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal 6 % n B223(66 UBE2D2/3 and 4%) 6 tcodi a bevdi cells in observed was itochondria 4) ncdw fUESand UBE2S of Knockdown 14%). iohnraadterecruitment the and mitochondria t iohnra lmnto was elimination mitochondrial st, ilpluiutnsga n p62 and signal polyubiquitin rial sre nPri-oiiecells Parkin-positive in bserved kndpnetmitochondrial rkin-dependent 6 2)compared 12%) 6 3281 28%,

Journal of Cell Science EERHARTICLE RESEARCH 3282 ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal iN-rnfce cells. siRNA-transfected ihtercrepnigantibody. probing corresponding specific their by with verified strongly as were down, enzymes knocked E2 by All analyzed blotting. buffer were western lysis lysates NP-40 total-cell in the lysed and were cells consecutive The three days. or on control siRNAs nM UBE2 5 indicated with transfected were cells mitophagy. inhibits Parkin but with translocation interfere not E2 does of enzymes knockdown siRNA-mediated 1. Fig. aknpstv el.(–) aarpeetthe in represent mean scored Data was (E–G). signal, cells. HtrA2 Parkin-positive of loss the visualized as by elimination, Mitochondrial (G) cells. hw.Pri-oiiecls()aeoutlined. are (D) cells are Parkin-positive experiments shown. independent three than from more images Representative in images). (blue merged 33342 were Hoechst nuclei with The counterstained green). the (Parkin, of tag epifluorescence EGFP the using was by distribution visualized Parkin (p62, The p62 right). adaptor turquoise, autophagic the or left) turquoise, either (Poly-Ub, and proteins red) polyubiquitylated Omi, HtrA2 as fixed marker known were mitochondrial (also cells the The (D,G). for h stained 24 and or (C,E,F) h 2 the for 10 or with B) depolarized CCCP, were h mitochondria (0 cells untreated The left h. were 24 for EGFP–Parkin further with were transfected and coverslips were onto siRNAs plated indicated cells the HeLa with (B–G) transfected control. loading a as iohnrawssoe in scored on was signal mitochondria p62 an or of polyubiquitin appearance additional the and mitochondria to Parkin 10 bars: Scale 6 .. * s.d.; P , m .(,)Tetasoainof translocation The (E,F) m. .5cmae ihcontrol- with compared 0.05 . 0 Parkin-positive 200 b atnserved -actin m A HeLa (A) CCCP M . 200

Journal of Cell Science EERHARTICLE RESEARCH ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal ihwl-yeUBE2. wild-type with * osdrd aarpeettemean the represent were Data mutant) considered. or type (wild Myc-E2 enzymes and EGFP–Parkin positive wild-type were this for that For cells treatment. only CCCP of quantification, h 24 after cells oiiecls C Immunofluorescent (C) cells. positive in scored CCCP was of treatment h 2 after to mitochondria Parkin depolarized of translocation staining. The immunofluorescent (B) to prior h 24 cells 10 These with h. treated 24 were for enzyme E2 mutant ( vector wild-type and of EGFP–Parkin combination a with transfected subsequently were onto and plated coverslips were cells HeLa siRNA. Silenced control (B–D) Ctr, control. loading a antibody. anti-Myc probing an by with detected was enzyme re-introduced Myc–E2 the their and with antibody, probing specific by verified E2 was each enzyme of knockdown blot The NP-40 western analysis. in to lysed subjected were and cells buffer The (A) h. for 24 constructs enzyme E2 UBE2D3-C85A) and UBE2L3-C86A (UBE2N-C87A, mutant ( empty vector were either cells with These transfected 1. subsequently Fig. in HeLa described in as downregulated were enzymes E2 mitophagy. mutants restores cysteine active-site not UBE2 but wild-type of Re-introduction 2. Fig. inl a crdin HtrA2 scored of was loss signal, a by visualized Mitophagy, shown. (D) are experiments independent of three images Representative outlined. are 10 cells bars: Scale in 1. described Fig. as performed was staining P , .5cmae ihclstransfected cells with compared 0.05 2 rmctge idtp W)or (WT) wild-type myc-tagged or ) m . AD h indicated The (A–D) m CPfr2hor h 2 for CCCP M .Parkin-positive m. 0 Parkin-positive 200 . b 2 0 Parkin- 200 atnsre as served -actin ,wl-yeor wild-type ), 6 3283 s.d.;

Journal of Cell Science oprdwt oto-iN-rnfce cells. control-siRNA-transfected with compared 2ezms hl eea elvaiiy iulzdby 3A). visualized (Fig. of affected viability, expression not cell the was general in probing, while reduction enzymes, substantial a E2 displayed cells depolarization. membrane The mitochondrial without or with blotting, ARTICLE RESEARCH 3284 10 with revealed treatment UBE2D3 to and prior UBE2L3 h UBE2N, 24 cells. for for control EGFP–Parkin antibodies of wild-type The with that blotting. control-siRNA- transfected with 10 western subsequently cells, compared with by were (UTF) enzymes treated and analyzed Untransfected E2 were coverslips were (A) of or onto amounts amount days. plated untreated protein the were consecutive left equal in were three and reduction (2N+2L3+2D2/3) on buffer, substantial siRNAs siRNA lysis a three UBE2D2/3 NP-40 all and in with UBE2L3 lysed transfected UBE2N, were cells of mitophagy. and nM Parkin-dependent (control) 5 reduces cells containing transfected enzymes mixture E2 a of or knockdown siRNA simultaneous The 3. Fig. niae ytels fteHr2sga fe 4ho CPtetet a one in counted was treatment, CCCP of h 24 Pa for after shown. quantified signal are was HtrA2 experiments mitochondria th the independent on and of three cells antibodies, loss from Parkin-positive specific the images in with by Representative signal stained indicated green). p62 were (Parkin, 10 or (red) tag bars: polyubiquitin p62 EGFP Scale additional and the outlined. red) using are (Poly-Ub, by cells proteins visualized positive polyubiquitylated was blue), Parkin (HtrA2/Omi, of mitochondria distribution The (F). h 24 m .(,)TePri rnlcto odplrzdmtcodi fe fCC ramn swl san as well as treatment CCCP of h 2 after mitochondria depolarized to translocation Parkin The (D,E) m. b -actin 4h eeivsiae.Udrbslcniin,tetriple- the conditions, basal Under (at mitophagy investigated. and h) were 2 (at h) translocation depolarized 24 Parkin were and CCCP, Mitochondria with EGFP–Parkin. with transfected el eltdo B2,UEL n B223were UBE2D2/3 and UBE2L3 UBE2N, of depleted Cells . b 0 aknpstv el.Dt ersn h mean the represent Data cells. Parkin-positive 200 ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal atnsre salaigcnrl BG iN-rae eacells HeLa siRNA-treated (B–G) control. loading a as served -actin . 0 el ntreidpneteprmns G Mitophagy, (G) experiments. independent three in cells 200 AG eaclswr rnfce ih5n fcontrol of nM 5 with transfected were cells HeLa (A–G) m CPfr0h() C or (C) h 2 (B), h 0 for CCCP M m CPfr2h h cells The h. 2 for CCCP M 6 .. * s.d.; e P , 0.05 rkin-

Journal of Cell Science n CPtetdcls eosre lgtuiutlto of ubiquitylation slight a observed We cells. CCCP-treated and 2hCC,Fg C.I oePri-oiieclsmc overlap much cells cells Parkin-positive control-siRNA-transfected some In in 3C). triple-siRNA- Fig. as resulted CCCP, in well h h mitochondria (2 2 and h as to (0 for cells Parkin CCCP of Parkin transfected cells of translocation administration control-siRNA-transfected the cytosolic The in of 3B). network Fig. of mitochondrial that CCCP, the with distribution of no disturbance compared changes, no the and morphological polyubiquitin obvious in no differences showed cells silenced ARTICLE RESEARCH uiiaino 6 proteasome- of with purification modified mitophagy. during are chains indicates these TOM70 ubiquitin possibly K48-linked and in This sensitive Mfn2 4A). changes (Fig. Mfn1, prominent observed that were no levels cells protein Moreover, triple-siRNA-treated MG-132. UBE2S- by and reverted in be control the could in which TOM70 cells, and reduced knockdown of h Mfn2 in Mfn1, (0 resulted of CCCP enzyme levels levels of were protein UBE2 administration The knocked-down steady-state cells 4A). the Fig. non-treated CCCP, of The regardless in altered 4A). proteins (Fig. not mitochondrial cells UBE2S levels investigated the as control with well compared as in cells UBE2D2/3 UBE2-siRNA-treated and in UBE2L3 proteins the UBE2N, in reduction of substantial beads a levels revealed Ni–NTA The lysates proteins. total-cell with ubiquitylated of pulldown covalently analysis only subsequent of 2010), in binding and the Lysis ensured buffer al., MG-132. urea rapidly inhibitor et stabilized M proteasome were 8 the particularly Tanaka proteins of those administration 2013; be of by al., species to ubiquitylated after et potential cells shown (Rakovic Parkin-overexpressing are in depolarization degraded TOM70 and Mfn1, ubiquitylated Because and h. 2 for Mfn2 CCCP with and depolarization FLAG–Parkin mitochondrial with of transfected knockdown of were single 6 cells knockdown as HeLa ubiquitylation well UBE2S. simultaneous as of UBE2D2/3, Parkin-dependent and after UBE2L3 the UBE2N, substrates analyzed mitochondrial next ubiquitylation protein We mitochondrial active for catalytically necessary as are well Parkin as UBE2D2/3 and UBE2L3 UBE2N, proteins. ubiquitylation mitochondrial 21 Parkin-dependent of the mediate siRNA together enzymes (control E2 depolarized enzymes 79 of E2 siRNA elimination of investigated knockdown these The triple of the after 3F). absence reduced Fig. CCCP significantly complete was CCCP, of or mitochondria p62 h mitochondria h and (24 of 24 polyubiquitin site signals after little the very even at either signal Notably, transfected observed cells siRNA. we for triple-siRNA- treatment, single seen as in each impaired, with mitophagy also was Parkin-dependent siRNA cells transfected The (control 3E). (Fig. polyubiquitin 3C), Fig. mitochondrial 79 CCCP, h with (2 82 mitochondria cells number the of reduced significantly site of siRNAs three the as the polyubiquitin of at for transfection p62 signal clear as a siRNA well showed (control cells control altered of although significantly h 2 not after 86 was mitochondria to treatment Parkin CCCP cytosol. of the translocation throughout in the dispersed more whereas Overall, still observed, was was Parkin cells mitochondria other clustered with Parkin of 6 6 6 6 odtrieteuiutlto tts epromdNi–NTA performed we states, ubiquitylation the determine To i-agduiutnepeso osrcsfr2 before h 24 for constructs expression ubiquitin His-tagged % rpesRA17 siRNA 21 triple siRNA 7%; triple 6%; 80 siRNA triple 6%; 6 9)(i.3) tapasta l fteinvestigated the of all that appears It 3G). (Fig. 19%) 6 i-agduiutltdpoen nuntreated in proteins ubiquitylated His-tagged 6 7%, 6 6 % n 6 cnrlsiRNA (control p62 and 9%) % nPri-oiiecells Parkin-positive in 7%) 6 .. Fg D.Nevertheless, 3D). (Fig. s.d.) 6 % triple 9%; bqiyainuigtectltclyiatv aknvariant Parkin inactive catalytically the using mitochondria ubiquitylation on signals ubiquitin This data faint detected. observed. immunofluorescence only were be where the 3C), could of supports (Fig. stabilization MG-132 approach no therefore with biochemical and species enzymes, E2 ubiquitylated these Apparently, of 4B). combined the Fig. absence in CCCP, proteins mitochondrial h these modify the as (2 cannot Parkin Parkin proteins, reduced ubiquitylated mitochondrial of greatly all as of transfection well species siRNA ubiquitylated system. of triple proteasome amount the ubiquitin the the 4B). contrast, through chains Fig. when By ubiquitin degradation CCCP, K48-linked fast down of h a attachment (2 pulled and the MG-132 reflect only might with were This and inhibited TOM70 of control was and proteasome species in Mfn2 ubiquitylated Parkin CCCP Mfn1, The and Mfn1, of of cells. VDAC1 species administration UBE2S-siRNA-transfected TOM20, ubiquitylated The TOM70, of 4B). amount Mfn2, not Fig. the were increases CCCP, Parkin and greatly h and Mfn2 (0 VDAC1 Mfn1, TOM20, detected of However, of events. species ubiquitylation fission and ubiquitylated fusion of mitochondrial possibly to level due This TOM70 basal treatment. the MG-132 after reflects membrane only mitochondrial without but even depolarization, TOM70 and Mfn2 Mfn1, o erddb h rtaoeudrteeconditions. these are under proteins proteasome these the that by seems degraded not it Thus, even treatment. with ubiquitylated MG-132 are inhibition without VDAC1 proteasome and TOM20 after contrast, these detected By of MG-132. be species only ubiquitylated and could 2013) degraded proteins al., and ubiquitylated et rapidly (Rakovic investigated are proteins TOM70 of and Mfn2, functions Mfn1, the activity. that Parkin on appears dependent are it UBE2s Thus, chains 4D). ubiquitin (Fig. proteasome-sensitive without possible modified of additionally stabilization contrast, were whereas the MG-132, Parkin of By and presence TOM20 in treatment. only VDAC1, TOM70 and CCCP polyubiquitylation Mfn2 in Mfn1, after resulted of Parkin itself wild-type of Parkin overexpression or proteins on Based C431S. usrtsi h iNAprfcto sa.Hr,HL cells HeLa Here, 6 with ( assay. vector empty purification either Ni–NTA with transfected the We mitochondrial in 4C). of ubiquitylation Fig. substrates of lack CCCP, the confirmed h additionally (24 cells 4C) in degraded Fig. Parkin-C431S-overexpressing not CCCP, were h mitochondria depolarized (2 consequently, 2013; observed and, chain al., was polyubiquitin recruitment et p62 neither (Lazarou nor Moreover, 4C) formation 2013). Fig. Hunter, CCCP, and h Zheng shown (2 as others mitochondria, by depolarized previously to as translocate ubiquitin not bound C431S did Parkin the inactive variant the trap Parkin, wild-type to to contrast C431S 2011). In variant al., oxyester. Parkin et the (Wenzel used 431 We cysteine active-site its thioester on a intermediate forms Parkin ligase ubiquitin E3 (RBR) between-RING’ epnil o hstp flnae epromdknockdown performed we linkage, to of indeed is, type of is this UBE2N formation UBE2N whether for the 2010). determine mediates responsible To mitochondria that al., linkages. enzyme et K63 E2 depolarized ubiquitin known Okatsu chains only on 2010b; the polyubiquitin date, al., K63 of et ubiquitin appearance (Narendra through the show linked reports Recent involved are mitophagy chains in ubiquitin K48-linked and K63-linked enx nlzdtePri eednyo mitochondrial of dependency Parkin the analyzed next We neetnl,mtcodilsbtae alit w groups. two into fall substrates mitochondrial Interestingly, 6 ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal i–bqii eeldn bqiyaino mitochondrial of ubiquitylation no revealed His–ubiquitin nvitro in aa twssgetdta h ‘RING- the that suggested was it data, 2 rPri 41 together C431S Parkin or ) 3285

Journal of Cell Science EERHARTICLE RESEARCH ela nUE-iN-rnfce el,irsetv of 5A; Fig. irrespective 3286 CCCP, cells, h (0 UBE2-siRNA-transfected depolarization membrane in mitochondrial ubiquitin as as general control a K63-linked in cytosol well displayed the that chains or throughout K48-linked distributed antibodies immunoreactivity for K48- antibody used and The either chains. We CCCP specifically with immunofluorescence. were recognize depolarized cells by these EGFP–Parkin, analyzed knockdown, with After cells. transfected HeLa in experiments 4. Fig. e etpg o legend. for page next See B2 togyrdcdti tiigpten hs nthese in of present Thus, knockdown are mitochondria pattern. The at chains staining ubiquitin and 5B). K63-linked this Fig. less UBE2L3 cells, reduced CCCP, control, strongly h with UBE2N (2 transfected siRNA depolarized CCCP, Parkin-positive cells UBE2D2/3 h on (0 appeared in cells signal mitochondria clear untreated a in 5A), 2010b; Fig. chains immunoreactivity al., K63-linked et for no (Narendra antibody others the displayed Although by 2010). shown al., as et 5B), Okatsu Fig. CCCP, h 2 ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal

Journal of Cell Science niae iN sdsrbdi i.3 olwdb rnfcinwt wild- with transfection 6 by with ( followed combination 3, in Fig. FLAG–Parkin in type triple described as after siRNA impaired is indicated enzymes. substrates E2 Parkin of of knockdown ubiquitylation The 4. Fig. ARTICLE RESEARCH a nce on niaigta aknclclzswith and enzyme colocalizes UBE2N E2 Parkin of which signals that Additionally, of above). indicating irrespective (see semi- down, mitochondria cells, knocked For all the was in 6). detect (Fig. observed to was CCCP scan immunoreactivity. line anti-UBE2 with respective a the E2 h performed of 2 intensities we against EGFP–Parkin imaging, for siRNA with quantitative transfected or depolarized further siRNA then and that were control cells and HeLa with enzymes, of in transfected appearance mitochondria the were on analyzed We immunoreactivities enzymes. translocation UBE2 biochemical E2 mitochondrial endogenous and the detect of approach to immunofluorescence experiments We fractionation an depolarization. mitochondria both membrane the exert on used mitochondrial directly enzymes reside of E2 or cytosol independently different the the in actions whether their investigated we is Next, mitochondria to Parkin UBE2L3 on and dependent UBE2N of translocation The Parkin-dependent in on participate present are mitophagy. and linkages we ubiquitin mitochondria Thus, K63 5C). their and depolarized Fig. and K48 CCCP, se, both h that per (6 suggest time MG-132 over K63-linked by increased affected also Notably, intensity not intensity mitochondria. were The on chains K48- ubiquitin 5C). of chains Fig. stabilization ubiquitin the CCCP, indicating linked time, h on over (2 increase cells to added immunoreactivity seemed Parkin-overexpressing was specific in MG-132 antibody additional chains, when the ubiquitin an K48-linked with for detected observed mitochondria, depolarized might we proteasome. mitochondria the depolarized inhibiting Indeed, by that chains degradation. ubiquitin possibility K48-linked proteasomal display the for tested substrates total We well-known that ubiquitin are the chains K48-linked with chains ubiquitin antibody modified with affect Proteins linkages. an modified different of be not by that might suggests proteins This detected did 1C,F). mitochondrial (Fig. was chains UBE2N polyubiquitin all that recognizes of signal Silencing polyubiquitin 5B). (Fig. iohnra ebaedplrzto ih10 with depolarization membrane mitochondrial B o h uiiaino rtismdfe ih6 with (500 modified amount proteins protein of equal purification the For Mfn2, Parkin. (B) and Mfn1, and buffer, UBE2S, VDAC1 urea with UBE2D3, TOM70, M blotting UBE2L3, TOM20, 8 western UBE2N, in by for lysed analyzed antibodies were and specific cells prepared The were (A) lysates CCCP). total-cell h (0 untreated left 6 tag EGFP ( the vector of 10 epifluorescence bars: the Scale Parkin using (green). The by (red). visualized p62 (HtrA2/ or was mitochondria red) distribution (Poly-Ub, for proteins staining polyubiquitylated immunofluorescent blue), to Omi, either 10 subjected with with and transfected treated h Parkin, were 24 C431S cells mutant the HeLa or and (C) (WT) VDAC1 control. wild-type TOM70, Ctr., TOM20, (Parkin). blotting Mfn2, tag western Mfn1, FLAG by for analyzed antibodies and specific eluted using were proteins ubiquitylated bound ftreidpneteprmnsaeson b,mnuiutltd Ub2, monoubiquitylated; polyubiquitylated. Ub1, Ubn, images shown. diubiquitylated; Representative are were B. experiments and independent for h three described 2 of as for purification depolarization Ni–NTA membrane to mitochondrial subjected to prior min 30 2 6 o 4h h el eetetdwt 10 with treated were cells The h. 24 for ) i-bqii osrc o 6h eetetdwt 10 with treated were h, 36 for construct His-ubiquitin togoelpo iohnra n GPPri signal EGFP–Parkin and mitochondrial of overlap strong A 2 ,wl-yeo 41 akncntut ncmiainwt the with combination in constructs Parkin C431S or wild-type ), m .()HL el eetasetdwt empty with transfected were cells HeLa (D) m. m )wsicbtdwt iNAbas The beads. Ni–NTA with incubated was g) AB eaclswr rnfce ihthe with transfected were cells HeLa (A,B) b atnsre slaigcontrol. loading as served -actin 6 i–bqii + repyvector empty or (+) His–ubiquitin m G12fr3 i ro to prior min 30 for MG-132 M m 6 CPfr2ho were or h 2 for CCCP M i-agduiutn an ubiquitin, His-tagged m m CPfr2hor h 2 for CCCP M G12for MG-132 M ta. 00 aedae l,21a.Ipraty Parkin Importantly, 2010a). al., et Matsuda Narendra 2013; al., 2010; et (Iguchi untreated al., others depolarized in by the shown et than to as in treatment 7A), 6), (Fig. abundant translocate CCCP (Fig. cells more after not fraction was approach mitochondrial did Parkin which, Consistently, immunofluorescence UBE2D3 fractions, mitochondria. mitochondrial that the in indicates did signal with UBE2D3 specific for together a analysis blot show western not The CCCP 7A). after fractions (Fig. mitochondrial treatment in enriched UBE2L3 the were enzymes UBE2N Supporting E2 and knockdown. endogenous enzymes the their E2-conjugating mitochondrial data, after all immunofluorescence enriched a of diminished levels into an greatly The separated and were 7A). were Fig. 7A) cells (M, Fig. The fraction h. (C, 2 in fraction for cytosolic UBE2s CCCP FLAG–Parkin with with of treated transfected were and UBE2s, Cells appearance indicated cells. the the for HeLa silenced CCCP-treated investigated of fractions we mitochondrial results, these was Parkin or 6). a (Fig. mitochondria detected with overlap not immunoreactivity specific indicating UBE2D3 no did because of or mitochondria, UBE2D3 to intensities, UBE2N translocate Interestingly, to when appear downregulated. these absent were completely UBE2L3 was with which colocalization, correlated UBE2L3 2ezmswr nce onsmlaeul,the simultaneously, down large a to knocked reduced was proteins were mitochondrial of these ubiquitylation enzymes When h. 24 after E2 (mitophagy) degradation mitochondria the of depolarized with ubiquitylation of interfered greatly Parkin-dependent but protein, prevent mitochondrial of not did lack UBE2D2/3 the enzymes. from that UBE2 arise Parkin-interacting might suggest of mitochondria UBE2– absence results depolarized the the on our of signals recruitment Thus, ubiquitin panel, depolarized to complex. on lead (right stabilized Parkin is might treatment PINK1 which that mitochondria, CCCP indicates This in 7B). after found additionally not Fig. were fractions enzymes is downregulation UBE2 the PINK1 as mitochondrial This well to after as that, Parkin mitochondria. siRNA, complex observation translocate by to a the the form that by not translocates Parkin supported argue ligase can then ubiquitin did E3 one that the 4C), C431S and (Fig. enzymes panel, above E2 Parkin (left seen as present and Because mitochondria, was Parkin CCCP UBE2N 7B). after wild-type Fig. fractions variant. additionally when mitochondrial only C431S the was treatment in cysteine found or and were control fraction active-site UBE2L3 wild-type UBE2L3 with Parkin mitochondrial transfected UBE2N, the cells HeLa the of in investigated presence in the active UBE2D3 of Therefore, independently mitochondria Parkin. depolarized to translocate that likely accumulation is PINK1 mitochondria. it of on downstream 2013), function is al., enzymes et PINK1 UBE2 (Iguchi the mitochondrial activation Because Parkin mitochondrial 7A). enzyme for Parkin the (Fig. E2 crucial which down in of for knocked irrespective PINK1 was treatment, prerequisite We CCCP of 2010a). after a al., stabilization fraction et is strong Narendra 2010; observed al., sites et (Matsuda mitochondrial translocation at kinase mitochondrial PINK1 the PINK1 with of stabilization interferes the UBE2 because stabilization, individual each of UBE2s. the downregulation of knockdown after abolished not was translocation opoieamtoooial needn ofrainof confirmation independent methodologically a provide To ae oehr h oneuaino B2,UEL or UBE2L3 UBE2N, of downregulation the together, Taken might enzymes E2 the that possible is it contrast, By the or knockdown triple the whether determined we Moreover, ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal 3287

Journal of Cell Science EERHARTICLE RESEARCH iohnra hc sPN1dpnet nconclusion, In depolarized dependent. 3288 to PINK1 UBE2L3 is and C431 which UBE2N residue active-site with Moreover, mitochondria, crucial the translocation inhibition. at ubiquitin activated proteasomal for be to K48-linked after has detected Parkin whereas were chains mitochondria, chains ubiquitin depolarized K63-linked of on formation UBE2N the mitophagy. mediated of attenuation possibly strong with correlating extent, EGFP–Parki wild-type with transfected 10 further with were depolarized cells MG-132. were The by mitochondria 1. stabilized the Fig. are or in chains (A) described ubiquitin as untreated K48-linked siRNAs left and UBE2 were indicated cells, cells the UBE2N-depleted The with in h. transfected reduced 24 were are cells chains HeLa ubiquitin (A–C) K63-linked Mitochondrial 5. Fig. rd a efre.TePri itiuinwsvsaie yuigteeiloecneo h GPtg(re) aknpstv el r outline are cells Parkin-positive (green). tag ubi EGFP Lys63-linked the or of Lys48- epifluorescence for the specific using by antibodies visualized the was and distribution 10 (blue) 10 Parkin TOM20 with The marker performed. treatment mitochondrial was to the (red) with prior staining cells Immunofluorescent the time. to incubation administered was MG-132 inhibitor m .Rpeettv mgso he needn xeiet r shown. are experiments independent three of images Representative m. m CPfr2ho .M-3 a anandi h eimdrn h entire the during medium the in maintained was MG-132 h. 6 or h 2 for CCCP M B2,UEL n B22 okn nacooperative a in mitophagy. Parkin-dependent working of regulators UBE2D2, key are and manner, UBE2L3 UBE2N, h ehns ftePN1 n Parkin-dependent mediating and is for mitochondria cofactors PINK1- the damaged Nevertheless, the of studied. degradation extensively of and ubiquitylation mechanism The DISCUSSION m CPfr2h()pirt iain C 10 (C) fixation. to prior (B) h 2 for CCCP M ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal m h proteasomal the M .Saebars: Scale d. utnchains quitin for n

Journal of Cell Science EERHARTICLE RESEARCH ee erpr htPri tlzsdfeetE ubiquitin- E2 on chains ubiquitin different K63-linked and mitochondrial utilizes K48- several K27-, Because Parkin of proteins. ubiquitylation that for enzymes report conjugating we known. well not Here, are ubiquitylation protein mitochondrial different represen 2 was line whereas distribution cells, 10 Parkin Parkin-positive bars: The in Scale mitochondrial intensities (red). fluorescent units). for UBE2 relevant arbitrary used endogenous the of (a.u., was shows the line 1 cells (blue) detect 1 Line A TOM20 untransfected to diagram. with (green). against in corresponding used tag depleted the Antibody were EGFP was in analysis. the UBE2D3 depicted potential is immunofluorescence of and membrane trace epifluorescence to UBE2L3 mitochondrial the subjected UBE2N, The using were h. for by cells 24 (Ab) visualized The for antibodies fixation. EGFP–Parkin specific to wild-type and prior with visualization, h transfected 2 were cells for The CCCP days. UBE2L3. and consecutive UBE2N three endogenous for of translocation Parkin-dependent 6. Fig. m .Rpeettv mgso he needn xeiet r shown. are experiments independent three of images Representative m. , 16 m a rw,adterltv nest fec hne vrteinvestigated the over channel each of intensity relative the and drawn, was m eaclswr iecdwt oto iN rsRAaantaseii UBE2 specific a against siRNA or siRNA control with silenced were cells HeLa ta. 00.UEN hc sawl-tde 2ezm that enzyme E2 well-studied a is (David which chains UBE2N, ubiquitin 2010). K63-linked capable are al., and that et K48- enzymes K27-, E2 forming analyzed we of group 2010a), our al., by et previously (Geisler detected were mitochondria depolarized ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal steintensities the ts 3289 0 m M

Journal of Cell Science EERHARTICLE RESEARCH B2 n B2 a enrpre.UETi novdin involved is 3290 UBE2T reported. 1 enzymes been E2 GTPase has the with UBE2T Rho Parkin an Mfn1 and of mitochondrial in interaction UBE2S monoubiquitylate no least the contrast, at polyubiquitylate to By or 2014) assay. to or al., 2013) et Parkin 2011) al., (Kazlauskaite with al., et interact et UBE2D (Lazarou 2010). to (Shin al., et shown substrates Tanaka 2007; been al., et has Olzmann 2006; al., Matsuda might 2006; et and al., al., Parkin et et for (Fallon enzyme (Olzmann (multi-)monoubiquitylation E2 promote responsible DJ-1 well-known can a is mutant is and chains, of UBE2L3 2007). 2005) degradation ubiquitin al., autophagic et the K63-linked for (Doss-Pepe Parkin of with formation interact the mediates show experiments. blots independent fraction Western three mitochondrial enzymes. of the UBE2 images in the representative appear co-translocate C431S not to mutant did failed of ligase and overexpression ubiquitin and E3 depletion the PINK1 Parkin, protein with PINK1 cells respective reduced In their strongly levels. with siRNA analyzed PINK1-specific was The proteins antibodies. The indicated (B) the enzymes. of UBE2 CCCP of distribution after downregulation the amount fraction of the mitochondrial irrespective in the administration, increase in strong PINK1 a endogenous the showed of within antibody cross-contamination anti-PINK1 minimal The MAPK or fractions. p38 no and revealed the marker) marker), in (mitochondrial (cytosolic FLAG–Parkin anti-VDAC1 wild-type with of Probing anti- appearance fractions. the the with shows Probing with UBE2D3. antibody knockdown and FLAG enzyme UBE2N UBE2L3, E2 antibodies and for specific (C) analyzed the cytosolic were obtained fractions The (M) (A) mitochondrial 10 assay. with fractionation treated mitochondrial then the were cells The transfection h. the 24 by ( followed vector days, empty consecutive with three on in siRNAs mitochondria indicated depolarized to manner. translocate Parkin-dependent UBE2L3 a and UBE2N 7. Fig. 2 ,wl-ye(T LGPri rC3SPri for Parkin C431S or FLAG–Parkin (WT) wild-type ), AB eaclswr rnfce ih5nM 5 with transfected were cells HeLa (A,B) m CPfr2hadsbetdto subjected and h 2 for CCCP M nvitro in ubiquitylation epnil o akndpnetmtpay u approach Our mitophagy. be might Parkin-dependent UBE2D2/3 other and UBE2A for UBE2L3 besides that, UBE2N), responsible likely (like is enzymes it Thus, E2 (de 2010). syndromes al., Parkinsonian show et from not Leeuw suffer substantia do who function the patients UBE2A in that of and expressed loss 2010) highly al., is et Note (Kupershmidt UBE2A PINK1. nigra not by but activation Parkin Parkin of that upstream even defect initial ikduiutno akna noyse.DfeetE enzymes, E2 Different oxyester. thioester- an this as trap Parkin to By possible on 2011). is ubiquitin al., it linked et C431S Wenzel mutant 2013; Parkin al., an the et using as Lazarou substrate 2013; C431 the al., cysteine to et passed (Iguchi active-site then Parkin’s is and transferred to intermediate is ubiquitin-thioester enzyme ubiquitin E2 where an ligase, from ubiquitin E3 2010). al., hybrid et HECT Parkin- Tanaka of 2010; dependency al., et proteasome (Gegg a in mitophagy is showing mediated This reports mitophagy. of with steps short-living early line at quickly these proteasome were the chains these by that ubiquitin removed to K48-linked indicates with chains might modified This ubiquitin proteins stabilization K48-linked proteins. This of Parkin. mitochondrial attachment of the presence reflect the on dependent after modified species 6 TOM70 cells, chains and with Mfn2 MG-132-treated ubiquitin Mfn1, of in amounts K48-linked significant Additionally, stabilized and inhibition. UBE2N, of proteasomal of presence presence the on a the dependent chains, strictly ubiquitin is K63-linked approach, that of immunofluorescence process 2011; formation the our al., confirm Using could et we 2013). (Chan al., the et depolarization in Haddad chains after ubiquitin 2010) K48-linked al., fraction of et amount mitochondrial Okatsu the in 2010b; increase al., an a at Contrary et or chains (Narendra in treatment. ubiquitin level K48-linked single-cell CCCP proteasome of the absence after the mitochondrial the either Parkin by show outer reports on degraded Moreover, dependent be 2010). manner on can al., proteins chains al., et et membrane polyubiquitin Narendra Okatsu 2010a; K63-linked of al., 2010b; et and (Geisler qualities mitochondria the K27- K48- mediates depolarized Parkin different of that that shown formation or evidence was depolarized it on provide Previously, present redundant enzyme are mitochondria. chains we ubiquitin to K63-linked E2 and Here, linked due on chains. each be formation polyubiquitin might of chain Parkin polyubiquitin This functions stabilization, global mitochondria. PINK1 and E2 depolarized with investigated to interfere the recruitment not of knockdown does individual enzymes the that revealed odplrzdmtcodi n usqetmtpayin mitophagy subsequent and mitochondria UBE2A depolarized translocation Parkin impairs to significantly UBE2A enzyme chains E2 far. ubiquitin the thus K11-linked mitochondria depolarized no on included because found was been control UBE2S ubiquitin have study, negative this K11-linked a In mediates 2010). as al., preferentially et and (Bremm chains 2009) al., (Garnett complex/cyclosome et promoting anaphase the of for types cofactor all perform might linkages and 2006) ubiquitin al., et (Machida repair DNA bqiyae.Ipraty aknwsnttasoae to less translocated also not CCCP was UBE2A was Parkin after global in K63-linked addition, mitochondria Parkin a fractions and depolarized In Importantly, of K48- mitochondrial observed. accumulation ubiquitylated. particular, was in no in treatment cells, signals) (and, these ubiquitin In signal 2013). ubiquitin al., et mrigeiec hw htPri ucin sRING/ as functions Parkin that shows evidence Emerging eety addadclege eotdta h osof loss the that reported colleagues and Haddad Recently, 6 ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal 2 i-agduiutnwr ulddw namanner a in down pulled were ubiquitin His-tagged / 2 os mroi irbat MF)(Haddad (MEFs) fibroblasts embryonic mouse nvitro in Dvde l,21) B2 sa is UBE2S 2010). al., et (David 2 / 2 Es onigt an to pointing MEFs,

Journal of Cell Science o nefr ihtetasoaino idtp aknto Parkin wild-type of the translocation contrast, the By in did with enzymes. UBE2D2/3 even and interfere UBE2 mitochondria, UBE2L3 However, not UBE2N, and 2013). to of knockdown al., PINK1 translocate combined et not of Lazarou did an presence 2013; C431S in al., of this Parkin least activity et HECT-like form at (Iguchi the Parkin C431S, can promotes which Parkin UBE2D3, assay, ubiquitylation on and thioester UBE2D2 ubiquitin UBE2L3, including ARTICLE RESEARCH B23adUED/.Mroe,tersimultaneous their Moreover, UBE2D2/3. and UBE2N, – enzymes after UBE2L3 E2 several mitophagy the requires mitochondria of depolarized out affected. process rule are the UBE2s cannot investigated in the we steps of knockdown Thus, other function. that possibility lysosomal the maintenance autophagosomes, proper the of and/or of lysosomes formation and the autophagosomes as of fusion such be might steps still recruitment – possible p62 are each disturbed is of It p62 downstream of steps abolished. be and E2-dependent is case that mitophagy cannot ubiquitin but the mitochondria knockdown, In at mitochondria present enzyme manner. signal, UBE2 of Parkin-dependent and, single lack degradation a recruited the in a to be degraded due approach. as Thus, cannot pull-down impaired. ubiquitin p62 is a mitophagy modifications, using consequently, these VDAC1 Mfn2, strong Mfn1, and Without a on chains TOM70 and polyubiquitin of level TOM20, amount single-cell the on in the formation three reduction chain at all ubiquitin of mitochondria less depleted both depolarized cells show In we mitophagy. enzymes, end-point UBE2 significantly h interferes 24 Parkin enzymes the E2 the this with the and in of individual knockdown the least combined both of at al., Nevertheless, type mitochondria, model. the et to overexpression on (Raasi insensitive linkage be detected might polyubiquitin 2008; recruitment lesser was p62 isolated monoubiquitin a al., Thus, the 2005). with of to et interaction domain weak and, (Long a UBA Also, K63-linked ubiquitin-associated 2004). chains al., to et The polyubiquitin Seibenhener binds 2007). K48-linked isolation p62 to extent, of al., bound (UBA) which is et domain p62, that (Pankiv LC3 protein with membranes adaptor signals ubiquitin-binding ubiquitin connects the recruit UBE2N, still through mitophagy. in chains participate K48- might forming polyubiquitin UBE2D2/3 of or that ability K63-linked UBE2L3 possibility the the or have out that linked rule ligases Smit ubiquitin cannot in E3 we (reviewed not other Thus, ligases is E3 2014). ligase RBR Sixma, E3 other or and final and E2 Parkin bound the whether for the of from resolved determines question arises specificity The itself chain reaction. the ligase ubiquitylation the the of of case outcome ligases, the in E3 that, believed is HECT-type It mitochondrial linkages. of different polyubiquitylation utilize with the substrates can for family, enzymes ligase E2 E3 different RING/HECT-hybrid the be of member to remain enzymes E2 other the shown. of of formation contributions The the substrates. the individual mitochondrial with that on interferes chains evidence polyubiquitin indeed K63-linked provide UBE2N al., we of of enzymes, et function knockdown UBE2 redundant (Haddad investigated possibly translocation the the besides in Parkin Nevertheless, which of abrogates 2013). knockout the largely not Parkin. UBE2A, of enzyme be MEFs activation could initial E2 candidate the another for likely necessary One that is study possible this in is tested it Thus, mitochondria. ti o e salse hte n o akn sanew a as Parkin, how and whether established yet not is It ncnlso,hr,w hwta h ia lmnto of elimination final the that show we here, conclusion, In mitochondria on linkages distinct of chains ubiquitin However, nvitro in 5 GGCAGCATTTGT-3 lrc,M)3 i eoeCC diitain G12wslf on time. left incubation was entire MG-132 the administration. during CCCP cells before the min 30 MO) Aldrich, 5 UBE2D2/3, Germany); 1027020-89072484, (Qiagen, nyecntut o 4huigXrmGn9(oh,Basel, or blot (Roche, purification western affinity XtremeGene9 to assay. Ni-NTA subjected fractionation using mitochondrial assay, subsequently immunofluorescence h were 24 analysis, cells The for Switzerland). constructs enzyme usrts tlzn h 2ezmsUEN B23and UBE2L3 UBE2N, enzymes mitochondrially E2 mitochondrial that of the UBE2D2/3. polyubiquitylation not evidence utilizing The was performs substrates, provide mitochondria Parkin proteins. we to depolarized targeted Thus, owing to mitochondrial p62 Parkin disturbed. of of of recruitment translocation ubiquitylation the with diminished interferes knockdown lrc,M)i B o 0mn ahdtretmswt PBS, PBS with in FBS times 10% with three blocked for and PBS washed PBS 4 with in times min, at MO) (Sigma- three 20 (Sigma-Aldrich, paraformaldehyde washed X-100 min, 4% for in 5 Triton fixed 1% PBS h, with 24 in permeabilized treated or were h MO) cells 6 The h, Aldrich, h. 2 24 for for CCCP C431S) with or type (wild EGFP–Parkin with eacls(TC A eemitie nDlec’ oiidEagle modified Dulbecco’s in maintained were VA) (ATCC, cells sequencing HeLa transfection as and by is culture trapped Cell verified C431S is were variant ubiquitin sequences Parkin attached the All The analysis. the intermediate. cysteine. because ubiquitin oxyester following active-site of inactive terms the catalytically in Site-Directed Germany), at non-functional GeneArt were UBE2L3- binding UBE2D3-C85A) (UBE2N-C87A, the (Invitrogen, and vector mutants using C86A UBE2N, These pCMV-Myc alanine instructions. enzymes System E2 the manufacturer’s to each E2 and into mutated (C431) the Mutagenesis Parkin cloned was of of cysteine previously was enzyme cDNA active-site The described The UBE2D3 CA). (Clontech, as 2010a). and al., were UBE2L3 et constructs (Geisler pCMV-6 expression FLAG-Parkin, EGFP-Parkin pcDNA3.1 The Constructs METHODS AND MATERIALS eaclstasetdwt iN eepae nocvrlp coated coverslips onto plated were poly- siRNA with with transfected cells HeLa microscopy and Immunocytochemistry 10 with inhibited was proteasome The h 2 h. for 24 10 MO) or with Sigma-Aldrich, depolarized (CCCP, were m-chlorophenylhydrazone cells cyanide HeLa of mitochondria The treatments and Inhibitor 5 UBE2S, Germany); 89072483, 5 UBE2N, GCGAGCUCAAAUAUUA-3 (AllStars 5 control UBE2L3, AAAGAAA-3 following three used: Germany); were on The Qiagen, sequences control, siRNA Germany). target negative indicated indicated (Qiagen, the the HiPerFect with of siRNAs nM using 5 days with consecutive transfected were cells The knockdown siRNA-mediated glucose, mM 4.5 Laboratories, PAA (FBS, with serum 37 bovine Germany) at fetal Germany) 10% Biochrom, with supplemented (DMEM, medium nioisi 0 B nPSfr2h ahdtretmswt B and with PBS conjugated with anti-rabbit-IgG times three or washed anti-mouse-IgG h, goat 2 for with PBS incubated in FBS 10% in antibodies eetasetytasetdwt h niae cN31FLAG-Parkin, pcDNA3.1 indicated the pCMV-6 with transfected transiently were 9 -GACGCTGTTCCTCGTTATGAA-3 ˚ vrih.Teclswr nuae ihteidctdprimary indicated the with incubated were cells The overnight. C ora fCl cec 21)17 2039 doi:10.1242/jcs.146035 3280–3293 127, (2014) Science Cell of Journal 6 i-bqii,pGPEF-akno CVMcE2 pCMV-Myc or pEGFP-EGFP-Parkin His-ubiquitin, D 9 lsn SgaAdih O n eefrhrtransfected further were and MO) (Sigma-Aldrich, -lysine -AACCAGATGATCCATTAGCAA-3 9 Damcn -1340,Gray;UET 5 UBE2T, Germany); D-010384-01, (Dharmacon, ˚ ne %CO 5% under C 9 Qae,12008028,Gray;PINK1, Germany); 1027020-89072486, (Qiagen, 9 Damcn -0880,Germany); D-004898-01, (Dharmacon, 9 2 -AAGGCACTGGGACCTGGATTT-3 n uiiidcniin.Tecells The conditions. humidified and 9 Qae,S0273,Germany). SI00287931, (Qiagen, 6 i-bqii n pEGFP- and His-ubiquitin 9 -UGAAGAGUUUAC- m 9 G12(Sigma- MG-132 M Qae,1027020- (Qiagen, 9 -AACAGTAAT- m carbonyl M 9 -CCU- 3291 9

Journal of Cell Science bqii xrsinvcosfr3 .Teclswr rae with treated were cells The h. 36 for vectors 10 expression ubiquitin 50 emn)i %FSi B o .Tence eecounterstained were nuclei The h. 1 for PBS (Invitrogen, in 2 647 FBS with Fluor Alexa 5% or in Germany) Germany) (Invitrogen, 568 Fluor Alexa ARTICLE RESEARCH os n abtat-O2 W,110;SnaCu,C) rabbit CA), 3292 Cruz, Santa 1:1000; IL), (WB, ProteinTechGroup, Abnova, anti-TOM20 1:1000; 1:8000; rabbit (WB, (WB, and anti-Mfn2 mouse anti-TOM70 mouse rabbit and Germany), anti-Mfn1 Sigma-Aldrich, 1:1000, 1:30,000; mouse WB, (WB, anti-Myc 1:750; MO), (WB, mouse (IF, MA), anti-UBE2S anti-UBE2L3 Biochem, rabbit rabbit Boston Germany), IL), NEB, ProteinTechGroup, (IF, 1:1000; anti-UBE2D3 1:3000; anti- rabbit rabbit WB, IL), CA), 1:750; ProteinTechGroup, Cruz, Santa Bioscience, 1:2000; MN), 1:1000; (WB, Systems, BD WB, UBE2T R&D 1:750; 1:1000; 1:1000; (IF, IF, WB, anti-UBE2N and goat (WB 1:750; Biomol, anti-HtrA2 (IF, rabbit 1:1000; Germany), anti-p62 WB, 1:750; mouse IF, Germany), mouse (FK1; analysis: (WB) proteins blot western anti-polyubiquitylated or (IF) immunofluorescence for either Hyperfilm UK). on Healthcare, (GE chemiluminescence primary Germany) high-performance ECL (Millipore, by substrate bound western antibodies Immobilon chemiluminescent-HRP proteins secondary the using HRP-coupled UK) Captured (Bio-Rad, Laboratory, using ImmunoResearch (Jackson method. by assay visualized transfer Germany) were Bradford antibodies wet protein (Millipore, membranes the the Equal Hybond- (PVDF) with onto P-40), buffer. difluoride using transferred polyvinylidene and Nonidet fractionation SDS-PAGE by P by or 1% separated determined were buffer pyrophosphate, Germany), as urea sodium amounts, M mM 8 10 7.6, denaturing NaCl, pH Tris-HCl mM mM (150 buffer 50 NP-40 in lysed and harvested were Cells antibodies and blotting Western Dounce The a needle. in gauge 860 strokes at 20 centrifugation a 20 by removed through by was buffer passages debris containing lysed cellular 20 EDTA) fractionation were mM by 3 in followed cells and homogenizer suspended sucrose The mM inhibitors. were 250 7.6, protease cells pH HEPES Briefly, mM (20 2010b). (Geisler previously al., described mitochondrial as enriched et centrifugation and stepwise (C) by cytosolic fractions into (M) separated were cells HeLa fractionation Mitochondrial 18,000 at centrifuged and needle 4 gauge at 20 a through NaH times mM 100 8.0, or pCMV-6 pH UBE2S empty for either then FLAG– silenced and or were FLAG–Parkin were and C431S wild-type UBE2D2/3 or control, Parkin and vector untreated with UBE2L3 co-transfected UBE2N, left for were simultaneously cells HeLa purification affinity Ni–NTA h eann yaewsfrhrcnrfgda 16,800 at centrifuged further was lysate remaining The 0mnt bantemtcodilyerce elt()adthe an and in resuspended (M) analyzed and were blotting. pellet fractions twice western Both enriched by washed buffer. fractionation was of mitochondrially amount pellet appropriate the The (C). obtain supernatant to min 10 ie ihlssbfe otiig2 Miiaoe n rtiswere three proteins washed and were imidazole, beads mM The 20 60 containing temperature. with eluted buffer room lysis at with h times 3 for buffer urea ooaiainwsasse vrteidctdln ln cn sn a using scan) 4.8.1. (line AxioVision line software Germany). indicated the Zeiss, the of (Carl over feature 63 software assessed 4.8.1 a was AxioVision Colocalization System using the Imaging using Germany), ApoTome processed Fluorescent an an Zeiss, with with using analyzed (Carl equipped were slides slides microscope The microscope AxioImager Germany). (Dako, onto Medium mounted Mounting were Coverslips fteeut eesbetdt etr ltanalysis. blot western to subjected were eluate the of le otiig50m mdzl o 0mna 95 at min 10 for imidazole mM 500 containing Blue) 15% glycerol, 30% SDS, h olwn nioiswr sda h niae concentrations indicated the at used were antibodies following The m m ˚ .Attlo 500 of total A C. CPfr2h ye ndntrn rabfe 1 MTris-HCl mM (10 buffer urea denaturing in lysed h, 2 for CCCP M fN–T ed Qae,Gray nattlvlm f1m of ml 1 of volume total a in Germany) (Qiagen, beads Ni–NTA of l m /lHeht332(oeua rbs emn)fr1 min. 10 for Germany) Probes, (Molecular 33342 Hoechst g/ml m lof3 6 2 m PO aml ufr(8. MTi-C H68 6% 6.8, pH Tris-HCl mM (187.5 buffer Laemmli fttlspraatpoenwsicbtdwith incubated was protein supernatant total of g 4 b raad1 Miiaoe,pse 20 passed imidazole), mM 10 and urea M 8 , mratehnlad003 Bromophenol 0.003% and -mercaptoethanol 6 betv.Teiae were images The objective. 6 I rpCMV-6 or HIS g ˚ .Attlo 10 of total A C. t4 at ˚ o 0min. 10 for C g g o 5min 15 for t4 at 6 ˚ for C HIS- m l eio,S . ag . ekr .A,Schu A., N. Becker, F., Wang, R., S. Denison, R., A. Zinn, A., L. Baker, L., Jaeckle-Santos, A., Green, S., Bulk, N., Leeuw, de h ttsia infcnewsasse yuigtetotie paired two-tailed the the using by represent assessed experiments. bars independent Student’s was three significance least error at statistical of The The mean the shown. of of deviation images are standard blot cells experiments Western only wild-type quantification. or 2, representative for mutant Fig. and considered in EGFP–Parkin were shown both Myc–UBE2 for data positive the doubly For were that experiment. each for condition ai,Y,Zv . do,A n ao,A. Navon, and A. Admon, T., Ziv, Y., David, ilgcl,Gray n abtat-3 AK(B :00 NEB, 1:1000; (WB, MAPK (WB, anti-p38 anti-FLAG Novus Germany). rabbit 1:2000; and mouse (WB, Germany) anti-PINK1 Germany), Biologicals, rabbit MO), Millipore, Lys63 Lys48 Sigma-Aldrich, anti-ubiquitin, 1:1000; 1:50,000; anti-ubiquitin, rabbit (IF, Germany), rabbit Millipore, specific MO), 1:1000; (IF, anti- Sigma-Aldrich, specific mouse 1:200,000; Germany), Millipore, (WB, 1:30,000; (WB, anti-VDAC1 etakRjoKru Rejko thank We Acknowledgements of h 24 after clearance mitochondrial the scoring by and assessed were after treatment treatment mitochondria CCCP CCCP and of p62 h polyubiquitin, 2 Parkin, of colocalization The analysis Statistical eg .E,Coe,J . hu .Y,Rj,M,Shpr,A .and H. A. Schapira, M., Rojo, Y., K. Chau, M., J. Cooper, P., E., Russell, J., M. Wu, Gegg, T., Matsusaka, C., Godwin, J., Mansfeld, J., M. Garnett, Be L., Fallon, K. Madura, and L. Chen, W., E. Doss-Pepe, S. G. Shaw, J., S. Leslie, A., Sidhu, R., K. J., Barber, L., N. Burchell, K., Kolawa, V. Chaugule, J., M. Sweredoski, H., A. Pham, M., A. Salazar, C., N. Chan, D. Komander, and V. M. S. Freund, A., Bremm, References Tu Medicine of fortu intramural Faculty an by supported also (MEFOPA). Parkinsonism was It of the Forms and Mendelian NGFNplus; on Consortium Network Research Research Education European of Genome Ministry National German (BMBF); the from Research P.J.K. and to grants by supported was work This Funding manuscript. the wrote and conceived project P.J.K. the the experiments. wrote supervised performed and experiments, Golombek experiments S. the and analyzed S.V. and manuscript. performed conceived, Geisler S. contributions Author interests. competing no declare authors The interests Competing rvdn h GPPri n LGPri xrsincntut n for and constructs expression discussions. FLAG–Parkin helpful Tu and Research, EGFP–Parkin Brain Wolfdieter the Clinical thank providing for We antibody. Institute anti-TOM20 (Hertie rabbit Springer the providing for Germany) copne ysiue,asn peh rgntl n knaoaisin anomalies skin and B. urogenital, B. speech, Vries, patients. absent de male M., seizures, A. by Morgante, accompanied Vianne J., J. al. Smagt, et der van T., Kleefstra, 8604. akni vra n te cancers. other and ovarian I. in D. Parkin Smith, and C. Klein, lysines. preferred to polyubiquitination direct enzymes ana,J W. J. Taanman, exit. mitotic promote to substrates R. APC/C A. on Venkitaraman, and J. in Pines, parkin implicates signalling. Eps15 al. PI(3)K-Akt protein et and UIM T. trafficking the Thorarinsdottir, receptor with G., EGF interaction Rouleau, regulated M., A Haber, (2006). J., chains. Voortman, F., multiubiquitin Moreau, 63-linked lysine ubiquitin of Chem. Biol. assembly J. the to contribute domain. H. mitophagy. Walden, and for critical C. is D. Chan, Parkin and Genet. by S. system Hess, J., ubiquitin-proteasome L. 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