Ato o orsodne([email protected]) correspondence USA. for 32224, *Author FL Jacksonville, Disease, of Neurobiology School, Graduate aineC Fiesel C. Fabienne differently regulate mitophagy enzymes and ubiquitin-conjugating activation E2 Parkin of subset specific A ARTICLE RESEARCH ß 3488 2014 May 13 Accepted 2013; December 4 Received 1 genes, To parkinsonism 2011). degradation recessive potential al., protein three et into (Corti date, of dysfunctions insights failure mitochondrial and allow as pathways forms such mechanisms, familial The cases pathogenic rare disease nigra. Parkinson’s sporadic, most are Although substantia degeneration understood. neuronal poorly distinctive the are of this loss neurodegenerative for in mechanisms selective molecular common the neurons from most arise dopamine-producing the Symptoms disorder. is movement disease Parkinson’s INTRODUCTION enzymes, Mitophagy might E2 Autophagy, Ubiquitin, Proteasome, Mitochondria, that PINK1, Parkin, mitophagy WORDS: KEY and pathogenesis. E2 disease Parkin Parkinson’s in distinct of role putative of regulation a suggest functions the uncovered in antagonistic study enzymes or Our cooperative uncoupling. redundant, mitochondrial and upon translocation Parkin to clustering enhanced for in resulted prerequisite UBE2N, order UBE2R1 and of a UBE2L3 in depletion UBE2D, mitochondria, to contrast mechanism of in Strikingly, clustering different degradation. however, proper a the UBE2N, through mediate mitochondria. operated to primarily the translocation initial its charged and in resulting redundantly ubiquitin, activation with UBE2L3 Parkin ligase and hybrid control. RING-HECT members quality and mitochondrial family translocation during UBE2D Parkin activation, of the functions regulate enzymatic either negatively that E2s or four of total positively a not discovered have we Here, pathway investigated. ubiquitin-dependent been this in Parkin of ubiquitin co-enzymes E2 cognate E3 the selective However, the mitochondria. de-energized recruits to Parkin and ligase the activates PINK1 kinase mediate mitochondrial the The (mitophagy). via pathway autophagy-lysosome organelles together the whole and proteasome and proteins Both mitochondrial of recessive degradation of disease. causes common most the Parkinson’s are PARK2) Parkin as and known PINK1 (also encoding genes the in mutations Loss-of-function ABSTRACT e/h iaeta scevdi elh iohnra(i tal., et (Jin mitochondria healthy in cleaved is that Vives-Bauza kinase 2010b; 2013). Ser/Thr al., al., et et Burchell 2010; Narendra al., 2010; et al., et Matsuda al., et 2010; (Geisler control quality mitochondrial for pathway molecular FBXO7 2004), eateto ersine aoCii,Jcsnil,F 22,USA. 32224, FL Jacksonville, Clinic, Mayo Neuroscience, of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,38–54doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. PARKIN D oz ta. 09,hv enlne noasingle a into linked been have 2009), al., et Fonzo (Di as nw as known (also 1 lsbt .Moussaud-Lamodie L. Elisabeth , PARK2 PINK1 iaae l,19)and 1998) al., et Kitada ; noe mitochondrial a encodes PINK1 Vlnee al., et (Valente 2 Mayo `re 1 aaAndo Maya , nd-nrie raels IK cuuaino damaged process. on stabilized sequential pathway; accumulation is a PINK1 protective protein organelles. of de-energized PINK1 this steps on dysfunction, disrupt distinct mitochondrial affect gene Upon identify, they either to however, degradation. in FBXO7 selective cooperate for and mitochondria Mutations functionally damaged Parkin target functions and PINK1, and label ubiquitin-independent Strikingly, associate 2013). has physically al., also et ubiquitin but (Nelson E3 multi-protein complex of a ligase of product component recognition gene rapidly substrate proteins. and substrate The to 2012) ubiquitin 2013). protein al., Youle, et Greene and PARKIN 2011; (Yamano al., degraded et Meissner 2010; ta. 02.I at 4- 6-adK7lne bqii chains ubiquitin K27-linked (Sheng and K63- enzymes K48-, E2 fact, poly- In the growing 2012). of the al., case, of independent et this type largely In linkage thioester chain, protein. the dictates substrate ubiquitin a before itself a C431 ligase in of E3 site residue enzyme the lysine been active E2 a discovered has onto an recently transfer the Parkin from 2011). its ligases, on of ubiquitin al., intermediate ubiquitin accept members et E3 to linkages other ubiquitin shown HECT-type (Wenzel chain and to the family Similar ubiquitin challenged Parkin (RBR) has the for RING-between-RING data denominate mechanism recent transfer ligase However, domain E3 finger formed. RING substrate an the a to to bound of E2 enzymes the E3 E2 from Thereby, ubiquitin RING-type protein. of to a transfer enzymes direct conjugating as the ubiquitin regarded mediate E2 utilizes and been that C- ligase long its ubiquitin has between linkage Parkin intermolecular biological can N-termini. to by ubiquitin and chains used addition, linear In topologies be 2012). form unique Rape, can and all of (Komander functions that chains residues ubiquitin lysine generate internal characteristics Cedazo-Mı seven distinct mono- contains with and (multi-) chains of from (Sandebring poly-ubiquitin formation ranging numerous the to catalyze modifications ubiquitin modifies to ubiquitin shown and been various controlled has 2012). Parkin tightly Martinez-Torres, Moreover, and (Walden is proteins substrate that unrelated ligase E3 of removal the Parkin involved. of crucially activities facilitate ligase are ubiquitin E3 and to the case, co-recruited either tags In are substrates. ubiquitin 2010), al., respective al., et et decode adaptor (Lee (Geisler HDAC6 p62/SQSTM1 2013), mitochondria, al., al., or et et 2010) of (Kim the VCP/p97 Sarraf modification as by such 2011; ubiquitin organelles proteins, individual al., whole Upon of of et 2013). or (Chan degradation proteasome system the 26S mitochondrialautophagy-lysosomal for the facilitate by numerous to prerequisites to proteins localized proteins ubiquitylates Once are cytosol. substrate the activity Parkin from kinase Parkin mitochondria, of its translocation the and mitochondria akni nw sabodynuortcie multipurpose neuroprotective, broadly a as known is Parkin sactslcE iaeta tahstesalmodifier small the attaches that ligase E3 cytosolic a is 1 n ofitrSpringer Wolfdieter and ´ ge,21) bqii itself Ubiquitin 2012). nguez, FBXO7 1,2, noe putative a encodes *

Journal of Cell Science ossetwt hi eeal eylwezmtcactivity. enzymatic low conformation, very ‘closed’ generally auto-inhibited, their recently an with been have show consistent 2013), al., and et (Duda resolved ligases The RBR- 2013; other ubiquitin 2014). and al., E3 2013), al., et Komander, type course Spratt and et Wauer 2013; 2013; Birsa the al., al., 2011; et et Trempe along (Riley al., Parkin et of aspects Chan structures crystal certain 2010; quality al., facilitate et mitochondrial (Geisler might during formed and successively control be to appear ARTICLE RESEARCH uin h itiuino aknwti el a uniida a as quantified was cells within (EGFP)- Parkin that protein of distribution onto fluorescent cells the green fusion, cytosol enhanced HeLa an Using the expressed High stably mitochondria. from a de-energized established recruitment of chemically have and monitoring we unbiased Parkin allows 2014), UBE2S that PINK1-dependent al., assay (HCI) CDC34), et Lefebvre Imaging McCoy 2013; Content as al., 2013; et al., (Hasson known studies et UBE2L3, recent to (also distinct UBE2D4, Similar UBE2T]. of UBE2D3, UBE2R1 formation UBE2A, UBE2D2, UBE2N, [i.e. specificity the UBE2D1, for with controls in UBE2C, as association or roles types previous linkage well-defined their ubiquitin on their we small based 2010), Parkin, Timmers, cofactors and using Wijk 11 mitochondrial (van of by selected enzymes total E2 and a active cofactors human From 35 knockdown. activation select (siRNA)-mediated RNA screened its interfering we regulate translocation, or otherwise ubiquitin with Parkin charge might might that enzymes E2 identify To and Parkin activation of regulate translocation that mitochondrial enzymes E2 of Identification RESULTS Parkin. that of Parkinson’s functions details ligase molecular of E3 further and pathogenesis the activation the the of underlie determination crucial in a a enzymes and for quality disease E2 foundation the mitochondrial of establishes evaluation study Parkin-mediated this Thus, and differentiallycontrol. that PINK1- enzymes E2 distinct regulate of of translocation involvement crucial highlight the findings the These enhances mitochondria. of, UBE2R1 clustering and to, of We Parkin enzyme. depletion E2 acting that negatively a found mechanisms, as regulatory UBE2R1 positive identified also these we different to a is contrast through In which removal, peri- mechanism. autophagic mitochondria, subsequent their facilitate the ubiquitin-labeled to thought particular, of in clustering and, are the nuclear and modifies Parkin family however, ubiquitin of UBE2N, of with UBE2D activation. translocation Parkin initial the ‘charge’ translocation its to of for essential able mitochondrial members are that UBE2L3 and and demonstrate We activation four antagonistically Parkin. identified or the we cooperatively redundantly, Strikingly, regulate that 2010). cofactors enzymes Timmers, E2 E2 and active human, Wijk 35 of upon (van out we 11 total, Parkin analyzed In previously potential. of have membrane translocation mitochondrial the mitochondrial of uncoupling and activation co- the enzymatic E2 and of investigated. been to roles not have translocation the mitochondria is to activation, on 2013), actions UBE2A localize Parkin a al., suggests for onto to et that enzymes this order (Haddad study pass involved in recent and crucially itself, a enzyme include Besides E2 mitochondria. might an which from substrate, moiety al., receive ubiquitin et must Parkin Lazarou Accordingly, a through 2013). 2013; Hunter, Parkin and al., Zheng 2013; et of (Iguchi its activation and translocation activity(ies) enzymatic mitochondrial the its to coupled that is charging’ ‘ubiquitin suggest studies Recent nti td,w ie oietf 2c-nye htregulate that co-enzymes E2 identify to aimed we study, this In B2,UESo B2 hwdn infcn difference significant no UBE2R1 showed upon UBE2T of translocation or knockdown UBE2S measurements, Parkin UBE2A, all accelerated across as Consistent the UBE2N, knockdown. and as UBE2L3 inhibitory UBE2D2/3, the well against corroborated the siRNAs cells of individual of effects frequency the Parkin distribution among Analyses and 1D) 1C) mitochondria. with (Fig. (Fig. ratio to Cyto:Nuc cells GFP recruitment average of more against indicative accelerated cells, siRNA significantly an control-silenced an with in with compared translocation transfection resulted note, with Of treatment UBE2R1 of 1B). h (Fig. 2 UBE2D2/3) after CCCP Parkin-translocation- 50% each about UBE2N by as or percentage of that the UBE2L3 decreased of hereafter percentage siRNA knockdown Similarly, single cells. the (abbreviated positive a reduced genes with significantly both UBE2D3 and targeted UBE2D2 members modifiers negative and CCCP, positive with 1B). determine treatment (Fig. to of alike us h cells allowed 2 control-silenced after which the translocation of Parkin 50% showed With approximately 2.5. at threshold, cut-off of this a percentage used we the cells, m- calculate Parkin-translocation-positive carbonylcyanide To uncoupler the (CCCP). (Cyto:Nuc) with GFP chlorophenylhydrazone treatment nuclear upon and cytoplasmic 1A) of (Fig. intensity the of ratio B23adUEN n eaie(B21 euaosof regulators (UBE2D, (UBE2R1) uncoupling. positive negative mitochondrial identify upon and translocation to Parkin UBE2N) us and allowed UBE2L3 assay UBE2D HCI decrease sensitive of further shown). not knockdown not (data translocation Parkin did additional and levels UBE2D4 protein Consistently, an and/or of S1E). use UBE2D1 (supplemental in by proteins Fig. UBE2D judged resulted four material as all S1D) signal, recognized that Fig. UBE2D antibody of material reduction (supplemental complete UBE2D2/3 reverse of knockdown quantitative Combined 80– S1A–C). reduced by Fig. experiments material significantly and/or (supplementary all 90% throughout siRNAs levels analysis protein Most and/or transcript blot PCR. (qRT-) western transcriptase using by cells. control-silenced with compared iecn esse eodti ieon.Kokonof timepoints. analyzed translocation the Knockdown Parkin of any affect timepoint. at not UBE2N clustering did and mitochondrial this UBE2T or UBE2L3 or for beyond UBE2S trends UBE2A, 2B). CCCP, persisted (Fig. until with CCCP significant treatment silencing with of remained treatment h effects 2 of siRNA decrease h UBE2D2/3 1 significant Although after a colocalization and revealed (Parkin) of signal GFP (TOM20) between coefficient mitochondrial correlation 2A; Pearson’s of (Fig. calculations the treatment Consistently, CCCP S2B,C). reduced or Fig. of material UBE2L3 timepoints confirmed supplementary early UBE2D2/3, at inspection least of at not Manual silencing UBE2N, upon CCCP). did translocation h analyzed Parkin 0 magnification. enzymes S2A, material intracellular (supplementary Fig. Parkin E2 conditions high non-stress or the under morphology with localization mitochondrial of affect analyzed each overly of and Knockdown mitochondria TOM20) for stained were imaging. (against cells conventional HeLa-EGFP-Parkin employed this, we a For yield events, to of and resolution microscopy HCI higher automated from findings verify To differently and clustering translocation mitochondrial Parkin regulate enzymes E2 obndkokono h eudn B2 family UBE2D redundant the of knockdown Combined ae oehr cenn fE oezmsb highly a by co-enzymes E2 of screening together, Taken confirmed was enzymes E2 all for efficiency Knockdown ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal 3489

Journal of Cell Science 4wlswr esrd oto n IK-iecdsmlswr u nalpae o ult oto fsRAslnigadmtpayidcinupon induction mitophagy and silencing siRNA of control quality for Z plates high all received on analysis run ( of were wher CCCP types samples mitochondria, with Both PINK1-silenced to CCCP. treatment and Parkin with of Control of treatment h measured. translocation 2 were the after wells inhibited Parkin 24 significantly of UBE2N recruitment or the testing, UBE2L3 increased multiple UBE2D2/3, significantly of UBE2R1 Knockdown of shown. knockdown are experiments all across EERHARTICLE RESEARCH 3490 Cyto:Nuc GFP a with cells Parkin-translocation-positive of percentages The (B) h. ra 2 white Cyto:Nuc for GFP in CCCP resulting 10 (given with The bars: yellow). signal treated in Scale and GFP indicated UBE2T in merge. (Nuc) (border ratio or GFP the signal nuclear of UBE2S GFP with intensity and UBE2R1, nuclear The together the (Cyt) UBE2N, staining. of white UBE2L3, cytoplasmic Hoechst intensity in the of mean with cell the ratio together by each the shown, divided for schematically was as are pink) measured regions in was (indicated nuclear nucleus inner translocation the and Parkin ring blue. cytoplasmic The in cells). enzymes. (Hoechst) specific E2 nuclei of and knockdown green upon in translocation Parkin of 10 Imaging with Content High 1. Fig. vnulysqetrdit e igr‘mito-aggresomes’. bigger few a and regions into perinuclear sequestered around clustered eventually swiftly are organelles pntasoaino aknt eeegzdmitochondria, de-energized to Parkin of translocation Upon . . r hw ( shown are 2.5 m CPfr2h rlf nrae.Iae aebe curduigatmtdmcocp n hwmtcodi TM0 nrd EGFP–Parkin red, in (TOM20) mitochondria show and microscopy automated using acquired been have Images untreated. left or h 4 h, 2 for CCCP M P , 0.0001, n § 4 n-a NV,Bnern orce o utpetesting, multiple for corrected Bonferroni ANOVA, one-way 24, F 5 6.) D h itiuino h F yoNcrtoo l nlzdclsa rqec npreti hw.PrsRA tleast at siRNA, Per shown. is percent in frequency as cells analyzed all of ratio Cyto:Nuc GFP the of distribution The (D) 460.3). 9 m crs( scores .(–)HL-GPPri el eetetdwt iNsaantUEA UBE2D2/3, UBE2A, against siRNAs with treated were cells HeLa-EGFP-Parkin (B–D) m. . .)prpae niaieo necletasy *** assay. excellent an of indicative plate, per 0.5) ipre hogottecl eihr vralne iein time longer a over periphery cell the more throughout stayed Parkin) dispersed (and Mitochondria clustering cells. mitochondrial UBE2N-silenced altered in particularly a noted we However, P ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal , 0.0001, A eaclsta tbyepesdEF–aknwr treated were EGFP–Parkin expressed stably that cells HeLa (A) F 5 6.) C h vrgso h F yoNcratio Cyto:Nuc GFP the of averages The (C) 366.6). n § 4 n-a NV,Bnern orce for corrected Bonferroni ANOVA, one-way 24, P , .05 s o significant. not ns, 0.0005; igaround ring a i sgiven is tio eas for

Journal of Cell Science EERHARTICLE RESEARCH iohnrauo rnfcinwt B2 iN,a es at least at with siRNA, Parkin UBE2N of Pearson’s with colocalization transfection Nevertheless, upon reduced HCI. mitochondria in showed analyzed around also pronounced ring was correlation cytoplasmic less that a a nucleus in the in intensity EGFP–Parkin result in might increase This cells. UBE2N-silenced 2. Fig. e etpg o legend. for page next See akntasoaini C n napeibyenhanced appreciably did an correlation Pearson’s and However, HCI clustering. in mitochondrial translocation Parkin clustering. affect mitochondrial might UBE2N and of translocation silencing Parkin Therefore, 2B). both (Fig. CCCP h 1 ncdw fUER eutdi infcnl accelerated significantly in resulted UBE2R1 of Knockdown ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal 3491

Journal of Cell Science poieted en infcn o h iohnra omfco tlater at factor form mitochondrial the the *** in for timepoints. resulted significant UBE2N being of trend, and Knockdown opposite elongation compaction. decreased increased showed but UBE2R1 branching against siRNA with treated P opcinidxwscluae s[2 as calculated was index compaction eeslne ihsRA gis 2c-nye n rae ih10 with treated and co-enzymes E2 against siRNAs upon with altered silenced is were mitochondria enzymes. damaged E2 to of knockdown translocation Parkin 2. Fig. ARTICLE RESEARCH tal ncdw fUED n B23ol minimally only UBE2D3 If substrate 3492 and degradation. UBE2D2 their of monitor of species to knockdown time, ubiquitylated all, same the at of capture at exposures and, to different proteins analyzed blots we that (supplementary this cells western HeLa For (HeLa-Parkin) of S3A). CCCP timeline Fig. with Parkin treatment material upon h untagged Parkin 6 performed and expressed 4 profiling altered 2, blot 1, western degradation 0, we using at of by and substrates modification Parkin mitochondrial mitochondria, effects ubiquitin of monitor to the to experiments validate translocation biochemically To substrates and mitochondrial ubiquitylation of delays degradation enzymes E2 of Knockdown of recruitment delayed, clustering. analysis the UBE2N mitochondrial accelerated, secondary concomitant or and UBE2R1 UBE2L3 the Parkin of UBE2D, summary, reduction of In increase whereas loss of an UBE2R1. that decrease and of confirmed UBE2N a of loss with loss consistent upon upon are at clustering factor data form mitochondrial trend These increased opposite significantly timepoints. the a later mitochondria in with connectivity parameters resulted damaged both UBE2N decreased for of of Silencing in compaction controls, 2C,D). their (Fig. increased with resulted therefore strongly Compared UBE2R1 and and 2010a). of pool al., mitochondrial knockdown et network the (Narendra measures mitochondrial of clustering index a compaction circularity of mitochondrial the of measure whereas the 2005), and branching a of al., the is et 2C) (Koopman read-outs (Fig. factor and form elongation factor as in The the form compaction. knockdown 2D) and (Fig. the UBE2R1 with morphology calculated index To and controls. Parkin we UBE2N compaction in already CCCP of detail, of with had effects greater treatment correlation of the colocalization h maximal 2 analyze after A enhanced reached 2B). been an (Fig. mitochondria reveal not P CCCP: NV,Tkysps-o;0hCCCP: h 0 post-hoc; Tukey’s ANOVA, factor form The (C) (Pm). perimeter or [(Pm outline as the calculated of was length mitochondrial CCCP. the measure with and to (Am) ImageJ treatment using area of binarized h were images 2 TOM20 Parkin until (C,D) reduced only significantly mitochondria UBE2L3 with analyzed, or colocalization timepoints UBE2D2/3 all of at silencing translocation whereas Parkin inhibited strongly PINK1 were 10 bars: cells see Scale control, PINK1. CCCP; positive against h siRNA a 4 with of As or transfected h 2 S2). 1 0, Fig. after after information images blue supplementary corresponding in (for (Hoechst) CCCP nuclei with and treatment red are in Shown (TOM20) HCI. mitochondria automated (63 using magnification used by high was observed representative translocation effects Parkin the of validate inspection to Manual (A) indicated. as CCCP CCCP: h 0 P of colocalization the of ( measure mitochondria a with as Parkin calculated was coefficient correlation CCCP: , 5 , 0.0001, 0.0003, 0.0001, P P , 5 0.0001, 0.0005, F F F P 5 5 5 5 P 88) oprdwt otos(tl,clsta a been had that cells (ctrl), controls with Compared 38.80). 31;2hCCCP: h 2 23.18; .4;4hCCCP: h 4 9.443; 0.0858, , .05 ** 0.0005; F F 5 5 94;4hCCCP: h 4 69.42; .2;1hCCCP: h 1 3.821; 2 F )/(4 5 n .7;1hCCCP: h 1 3.172; p § P m]( Am)] , eaclsta tbyepesdEGFP–Parkin expressed stably that cells HeLa ,oewyAOA ue’ othc h 0 post-hoc; Tukey’s ANOVA, one-way 4, .0;* 0.005; P P n 5 , § 6 0.0007, 0.0001, ,oewyAOA ue’ post-hoc; Tukey’s ANOVA, one-way 4, mgs GPPri ngreen, in EGFP–Parkin images: ) P p P 6 , 5 P P ((Am/ .5 s o significant. not ns, 0.05; 0.0909, , , 0.0001, 0.0001, F F P 5 5 5 p ) 78;4hCCCP: h 4 17.88; 19) ncdw of Knockdown 41.94). 0.0053, 2 2 F )P]( ))/Pm] 5 F F m .7;1hCCCP: h 1 3.078; 5 5 .()TePearson’s The (B) m. 07) D The (D) 40.74). 43;2hCCCP: h 2 24.36; F 5 n .3;2h 2 9.936; § ,one-way 4, m M npriua,frVA1 entdacnitn nraeof increase consistent a noted we and VDAC1, enhanced Parkin for overly timepoints. analyzed particular, an the of In reveal at and rate not degradation translocation and/or did ubiquitylation ubiquitylation mitochondria, accelerates of substrate of clustering which Knockdown exposures). or delayed different UBE2R1, UBE2L3 by revealed in UBE2D2/3, (as of degradation resulted Knockdown are 3A–C. UBE2N images Fig. the Representative in analyze detail. more to shown substrates in decided model timepoints as We VDAC1 different CCCP). and at with TOM70 MFN1, treatment of of also levels 2, (CypD), protein (1, h timepoints were 6 immediate D the and clearance, at 4 Cyclophilin cells autophagic control in actual or unaffected enzymes (yet) of matrix (SOD2) substrates the 2 probable as adenine well dismutase the Inner as as superoxide 2013). (ANT1), al., such degradation et transporter proteins, Sarraf nucleotide 2011; proteasomal (IMM) al., membrane et for Yoshii mitochondrial 2011; substrates al., outer et described Parkin these least, (Chan been of the as have All at proteins previously delayed. (OMM) or was the membrane degraded, proteins mitochondrial contrast, and these of By ubiquitylated modification VDAC1 less siRNA. and clearly TOM20 control were TOM70, with substrates mitophagy immediate transfected that subsequent the cells been with of compared MFN2 had degradation and MFN1 and substrates mitophagy ubiquitylation the affected rayo h nlzdE bqii ojgtn enzymes conjugating ubiquitin E2 analyzed without S3C). any the Fig. or enzyme observe material of activating (supplementary not with ubiquitin E1 any did the cells experiment. of or we levels HeLa timecourse protein However, in in a alteration Parkin. the levels of performed for protein overexpression we of analyzed responsible levels, We hundreds solely protein are a E2 be that substrate. there might particular exclude a enzyme that of cannot ubiquitylation we E2 our (given substrates), Because upon certain observed. limited Parkin not substrates mitochondrial was extent, is E2 distinct particular minor analysis a between An a of determined. be knockdown difference to to translocation. remains Parkin specific unambiguous (and, affect are not effects did these knockdown unexpected Whether that revealed UBE2T) UBE2S of also knockdown VDAC1, of substrates of factor. For correction of silencing for the effects profiling 3E,F). applying upon trend (Fig. blot after TOM70 Western significant a UBE2N all not and 3D). were or noted for (Fig. means MFN1 UBE2L3 we significant CCCP) of UBE2D2/3, not analyzed, h levels (0 were substrates increased effects levels and observed steady-state timepoints factor’ the proteins their ‘correction Although a substrate on introduced model we based CCCP, of express with levels not treatment Parkin, did upon the To that functional S3B). quantify cells Fig. material HeLa appropriately of (supplementary in Parkin of presence observed levels effect not detectable the was this it Interestingly, upon because 3D). dependent (Fig. silenced on seemed depending enzyme increase twofold UBE2 upon Quantification to the CCCP up 3A). an with (Fig. revealed treatment enzymes densitometry by UBE2 of several h 0 of knockdown at levels protein steady-state ofcltt hi lseigadatpai erdto.p2is p62 degradation. autophagic adaptor and mitochondria clustering ubiquitin damaged their to facilitate the co-recruited to is mitophagy, p62/SQSTM1 protein Parkin-mediated During adaptor the of p62/SQSTM1 recruitment protein delays enzymes E2 of Knockdown oivsiaeapsil feto ramn ihCC on CCCP with treatment of effect possible a investigate To ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal

Journal of Cell Science EERHARTICLE RESEARCH nce onE ee n efre rpelbln fParkin, we of labeling step, triple subsequent performed this and genes on E2 up down LC3 follow knocked cellular recruit To both to systems. bridging and degradation membranes, chains autophagosomal ubiquitin thereby K63-linked and bind to known 3. Fig. e etpg o legend. for page next See euaeatvto fPri n/rtasoainto translocation and/or after Parkin translocation Parkin that of of inhibition enzymes Although E2 activation mitochondria. analyze among to and differences regulate recruitment its morphological monitor potential to p62 and mitochondria ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal 3493

Journal of Cell Science *** P F CP(TOM70): CCCP aknaeculd(gcie l,21;Lzrue l,2013; al., et Lazarou 2013; al., 2013). et Hunter, and (Iguchi Zheng UBE2L3 coupled of translocation are mitochondrial and finding and thereby Parkin the function(s) supports members and enzymatic also This the Parkin activation. family that initial charge’ its UBE2D in ‘ubiquitin involved are to that consistent function is idea This redundantly 5A). the (Fig. knockdown with PINK1 upon observed (VDAC1): oatr,a niae.Clswr etutetdo rae ih10 with treated E2 or against untreated siRNA left or were siRNA Cells indicated. control as with cofactors, either treated were treatment Parkin proteins. upon untagged E2 quantification against and siRNAs profiling with substrate Parkin 3. Fig. ARTICLE RESEARCH 3494 Parkin in reduced Simultaneous further combinations HCI. to UBE2L3 UBE2D2/3, triple and translocation performed down UBE2D2/3 and of and double knocked knockdown cells in effects We UBE2N HeLa-EGFP-Parkin cooperative enzymes. and and E2 UBE2L3 redundant these clustering, potential morphological among mitochondrial investigated and different Parkin by the we of to respect translocation given knockdown with However, phenotypes Parkin individual siRNA. earlier, reduced UBE2N shown UBE2L3 , As and (Wenzel 2011). discharge proteins UBE2D2/3 substrate to al., of able residues et are lysine onto isoforms also to UBE2D ubiquitin reactive only, is UBE2L3 residues Although are ligases. UBE2L3 cysteine E3 and HECT members charge family to UBE2D able that shown been has Parkin It of UBE2N functions and/or impairs UBE2L3 strongly UBE2D2/3, of knockdown Combined compared 4B). enhanced (Fig. significantly cells control not with on by was measured accumulation correlation as p62 colocalization Pearson’s However, strong 4A). revealed (Fig. mitochondria UBE2R1 no of translocation, Parkin showed knockdown accelerated an By progression UBE2T with normal Consistent 4B). a mitophagy. or (Fig. suggesting of UBE2S conditions, coefficient control UBE2A, to Pearson’s difference UBE2N of the silencing mitochondria, or using with contrast, by 4). UBE2L3 p62 (Fig. measured of PINK1 UBE2D2/3, as colocalization to major reduced mitochondria of Similar without significantly PINK1 mitochondria. knockdown diffuse and upon to remained silencing, Parkin and signal strongly to recruitment p62 colocalization 2) siRNA Fig. the UBE2N earlier p62 Consistently, see at or observed CCCP, UBE2L3 that diminished as h UBE2D2/3, prominent 1 as silencing, not (for was timepoints CCCP of h 4 xeiet eeaaye,wihhdbe otoldfrknockdown P for independent controlled five been least had efficiency. At which TOM70. analyzed, and were MFN1 UBE2N experiments of or levels UBE2L3 protein Compared UBE2D2/3, CCCP the samples. with of increased untreated treatment knockdown of of cells, h levels control-silenced 2 respective protein with and the to h above according 1 shown adjusted at are were quantification CCCP) further h for factors’ of (0 and ‘correction Results state VDAC1) D, bars. steady and In at CCCP. TOM70 siRNA with (MFN1, E2 treatment substrates each of model h h, the 2 0 for (D) (F) calculated after used and proteins were h quantify GAPDH 1 to or used (E) Vinculin the was levels C. of Densitometry the as controls. form monitor is loading to ubiquitylated blots as used all the was of denotes blotting Labeling Western -Ub protein. h. suffix indicated 4 The (C) substrates. or h Parkin 1 of (B) h, 0 (A) for 5 , 5 0 fcnrllvl,a esrdb C,smlrto similar HCI, by measured as levels, control of 50% P .8;2hCC (MFN1): CCCP h 2 1.384; 0.0001, 0.1920, , .05 ** 0.0005; P n F F 5 § 5 5 0.0212, ,oewyAOA ue’ othc CP(MFN1): CCCP h 0 post-hoc; Tukey’s ANOVA, one-way 5, .2;0hCC (TOM70): CCCP h 0 1.528; .3;2hCC (VDAC1): CCCP h 2 9.338; P P , 5 .0;* 0.005; , 0.0002, F %o l el,smlrt h togeffect strong the to similar cells, all of 4% 5 .9;1hCC (MFN1): CCCP h 1 2.791; P , F P 5 .5 s o significant. not ns, 0.05; , .1;1hCC (VDAC1): CCCP h 1 7.712; 0.0001, eaclsta tbyexpressed stably that cells HeLa F P 5 P 5 , 15;2hCC (TOM70): CCCP h 2 21.59; 0.9220, 0.0001, P , F 0.0001, F 5 5 .28 CCCP h 0 0.3228; 5.672. P 5 F 5 0.2742, m 32;1h 1 13.21; CCCP M vnatr4ho ramn ihCC Fg DE o other for 5D,E; (Fig. CCCP with treatment of h timepoints, 4 all after at colocalization even additive and greatly translocation a the in Parkin resulted confirmed reduced that siRNAs UBE2L3 mitochondria and UBE2D2/3 and of enzymes effect Parkin E2 of PINK1 correlation three to all similar of translocation, Parkin loss knockdown. abolish complete entirely a might Fig. material Therefore, triple (supplementary a but S1F). efficient had knockdown, less knockdown individual appeared to knockdown Double efficiency E2s. silencing of similar knockdown distributions combined ratio upon Cyto:Nuc GFP respective 5C). complete their (Fig. almost an of with almost and effects further overlap and the any UBE2D2/3 UBE2L3 cause not UBE2D2/3, to of did of UBE2N transfection Compared knockdown siRNA triple effect. upon UBE2L3, show additive not translocation Combined did or abolished significant UBE2N transfections. synergistic with not single siRNA together any was with UBE2D2/3 single this of compared with knockdown however, 5A,B) compared UBE2s; (Fig. when these ratio of Cyto:Nuc of silencing average GFP the and the cells Parkin-translocation-positive of number fadtoa easi akntasoaincmae ihthose with alone. UBE2N compared of translocation silencing Parkin because upon clustering in overall delays of less additional a knockdown from of result might with clustering, This material S2C). supplementary combination Fig. 5D; mitochondrial Fig. in (compare UBE2L3 particular or prominent intermediateUBE2D2/3 the less an as UBE2N appeared in of such distinctions resulted morphological knockdown, of UBE2L3 the knockdown However, with simultaneous phenotype. assay, together translocation the UBE2N S2). in Fig. material with seen supplementary 2A; compared As Fig. S4), (see alone Fig. siRNA either material supplementary see timepoints oPN1kokon ramn iha iN gis a against siRNA this an strongly of with linkage. Similar UBE2N treatment formation chains. K63 of knockdown, poly-ubiquitin the towards PINK1 K63-linked knockdown in levels to shift role type, the possible unique reduced linkage its a particular with with ubiquitin Consistent K48 but total affecting (compared overly CCCP reduced levels, without with knockdown) appreciably treatment knockdown single upon more with combined levels UBE2L3 expected, ubiquitin and cellular of As a UBE2D2/3 6B). amounts with (Fig. of treatment increased upon siRNA that and single with significantly unmodified double compared of substrate showed yet analysis unmodified densitometry silencing and this, with triple ubiquitylated in this line in degraded In resulted of At siRNA proteins. mostly CCCP. single amounts a h been with 4 intermediate treatment have whereas at cells, proteins degradation control substrate delay and knockdown a siRNA timepoint, modification indicating upon proteins, triple both substrate in unmodified those Furthermore, ubiquitylated of of and levels 6A). with species (Fig. appreciable showed substrates compared clearly siRNAs with combinations protein single UBE2L3 treatment OMM UBE2D2/ using and of upon of knockdown combined 3 Levels upon substrates stabilized greatly siRNAs. of were E2 profiling performed we combined blot mechanisms, underlying the western elucidate further To mechanisms Parkin regulatory with distinct cooperate through UBE2N and UBE2L3 UBE2D2/3, nln ihHI ihrrslto muoloecneand immunofluorescence resolution higher HCI, with line In efficiency silencing the monitor to qRT-PCR used We the reduced siRNA UBE2N with UBE2L3 of Combination ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal

Journal of Cell Science EERHARTICLE RESEARCH rae ihrwt oto iN rsRAaantE oatr,a niae.Clswr etutetdo rae ih10 with treated or mitochondria. untreated damaged left were to Cells p62/SQSTM1 indicated. of as cofactors, recruitment E2 the against reduces siRNA or UBE2N siRNA or control UBE2L3 with either UBE2D, treated of Knockdown 4. Fig. B2,kokono B223 B23o B2 hwdsgiiatyls 6 inlo aae iohnra *** mitochondria. damaged on signal p62 less significantly 10 showed bars: significant. UBE2N Scale not or shown. ns, UBE2L3 are UBE2D2/3, images of Representative knockdown (A) UBE2T, red). (TOM20, mitochondria and cyan) in (shown p62 a nlzd( analyzed was n § 0 n-a NV,Tkyspost-hoc, Tukey’s ANOVA, one-way 10, P , 0.0001, F 5 68) ncnrs ihcnrl n iN gis B2,UER,UESor UBE2S UBE2R1, UBE2A, against siRNA and controls with contrast In 16.80). ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal m .()Pasnscreaino 6 n TOM20 and p62 of correlation Pearson’s (B) m. m CPfr4h ie n tie for stained and fixed h, 4 for CCCP M P , .05 ** 0.0005; eaEF-aknclswere cells HeLa-EGFP-Parkin P , 0.005; 3495

Journal of Cell Science EERHARTICLE RESEARCH 3496 be 6C,D). knockdown not Pearson’s (Fig. combinatorial could enzymes triple the of colocalization or E2 h double in of the 4 by Thus, at decreased reduction further CCCP. (TOM20) maximal mitochondria with and a treatment p62 between showed correlation E2 single 10 bars: Scale PINK1. ( given are PINK1- knockdown with compared single values for graph, values the of comparison, top For on bars. indicated Parkin-translocation-positi is of respective controls percentage the with the above compared (A) Significance given and shown. are performed is CCCP. was cells experiments with HCI all treatment Automated across h. ratio upon Cyto:Nuc ratio 2 Parkin GFP Cyto:Nuc for of GFP CCCP the translocation with as treated blocks calculated and UBE2L3 was indicated and as UBE2D siRNA with of transfected knockdown Combined 5. Fig. ero’ orlto ewe O2 n F E.Frcmaio,vle fsnl iN ramnsaeson oprdwt igeE knockdown, E2 single with Compared shown. are ( treatments effect siRNA inhibitory single increased of an values showed observe comparison, combinations CCCP: effects For the siRNA (E). validate triple to GFP and used (C) and was double phenotype. translocation TOM20 intermediate Parkin between an of inspection showed correlation Manual (L+N) Pearson’s (D) knockdown cells. UBE2N (63 analyzed and all high-magnification UBE2L3 for Representative of ratio Cyto:Nuc Combination GFP PINK1. the of of silencing distribution to similar translocation, Parkin blocked testing, P P , , 0.0001, 0.0001, F F 5 5 43;2hCCCP: h 2 24.36; 6.) iutnoskokono B223adUEL DL rUED/ ihUEL n B2 DLN lotcompletely almost (D+L+N) UBE2N and UBE2L3 with UBE2D2/3 or (D+L) UBE2L3 and UBE2D2/3 of knockdown Simultaneous 460.3). m .*** m. P , .05 ** 0.0005; 6 mgsaeson GPPri ngen iohnra(O2)i e n uli(ocs)i le swl as well as blue, in (Hoechst) nuclei and red in (TOM20) mitochondria green, in EGFP–Parkin shown: are images ) P . , . ( 2.5 P 0.0001, , .0;* 0.005; n § 4 n-a NV,Bnern orce o utpetesting, multiple for corrected Bonferroni ANOVA, one-way 24, F 5 P .4;4hCCCP: h 4 9.443; , .5 s o significant. not ns, 0.05; P , 0.0001, n 5 0 n-a NV,Tkysps-o;0hCCCP: h 0 post-hoc; Tukey’s ANOVA, one-way 10, eue akntasoainadclclzto with colocalization of degradation and and/or recruitment. ubiquitylation p62 as and translocation substrates significantly well UBE2N) as Parkin of mitochondria, silencing additional reduced without or (with nsmay obekokono B223adUBE2L3 and UBE2D2/3 of knockdown double summary, In F 5 19) sapstv oto,clswr rnfce ihsRAagainst siRNA with transfected were cells control, positive a As 41.94). ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal n § 4 n-a NV,Bnern orce o multiple for corrected Bonferroni ANOVA, one-way 24, P , 0.0001, eaEF-aknclswere cells HeLa-EGFP-Parkin F 5 6.) B h vrg fthe of average The (B) 366.6). P 5 0.0005, yuigHCI. using by d F 5 .2;1h 1 3.821; silenced ecells ve The

Journal of Cell Science r hw.Cmie iecn boae ooaiaino 6 oasmlretn ota pnslnigo B223 B23 B2 rPN1( PINK1 or UBE2N UBE2L3, UBE2D2/3, of silencing upon that to extent similar a siRNAs. blu to single (Hoechst, p62 respective staining post-hoc, of the nuclear Tukey’s colocalization include ANOVA, of images abrogated effects one-way Merged silencing the cyan). 10 Combined in enhanced bars: (shown shown. (D+L+N) Scale p62 are UBE2N right. and the and red) to (TOM20, UBE2L3 mitochondria shown UBE2D2/3, for are or stained were (D+L) cells UBE2L3 HeLa-EGFP-Parkin with (C) UBE2D2/3 of silencing Combined EERHARTICLE RESEARCH bqiyae omo h niae rti.()Rpeettv etr lt r hw.()Dnioer a sdt uniytepoen ( proteins the quantify the to denotes used -Ub was suffix Densitometry The (B) indicated. shown. those are 10 (MFN1): and blots with CCCP VDAC1 western treated h and Representative or 4 TOM70 (A) untreated post-hoc; MFN1, protein. Tukey’s left substrates indicated ANOVA, and Parkin the siRNAs the of CCCP. UBE2 against form treatment of antibodies upon ubiquitylated combination recruitment with p62 a probed and or and degradation single substrate prepared blocks control, UBE2L3 with and transfected UBE2D of were knockdown Combined 6. Fig. P m , .()Pasnscreainbtenp2adteTM0sga a acltd o oprsn igesRAvalues siRNA single comparison, For calculated. was signal TOM20 the and p62 between correlation Pearson’s (D) m. 0.0001, P F , 5 0.0001, 68) *** 16.80). F 5 P 36;4hCC (TOM70): CCCP h 4 13.67; , .05 ** 0.0005; P , .0;* 0.005; P , 0.05. ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal P , 0.0001, F 5 17;4hCC (VDAC1): CCCP h 4 31.70; m CP sidctd etr lt were blots Western indicated. as CCCP, M AB eaPri cells HeLa-Parkin (A,B) P 5 0.0347, n § ,one-way 5, F 5 n 2.952). § )and e) 3497 10,

Journal of Cell Science IGhbi ehns Wne ta. 01.Acrigt this to According 2011). the HECT- al., novel et as a (Wenzel by mechanism ubiquitin well hybrid charging’. transfer RING ‘ubiquitin to as active suggested its been measure an has Parkin, to and employed Parkin C431S, Parkin, we of of UBE2D2/3 UBE2N, mutant activation of site of role initial cooperative functions the potentially redundant in probably UBE2L3 the Given functionally Parkin and with physically interact UBE2N and UBE2L3 UBE2D2/3, ARTICLE RESEARCH om fUE nye eevsbea -D shifted less 8-kDa found 3498 as we CCCP, visible when UBE2N with or UBE2L3 were treatment UBE2D3, of Upon species enzymes ubiquitin-charged 7C). (Fig. UBE2 ubiquitin-loaded E2 bands conditions, of to gel bound was forms non-reducing that Using ubiquitin of enzymes. analyses CCCP. thioester with performed treated been had or untreated left samples been between by had observed judged the that be in (as could differences Parkin discernable mutation of No C431S auto-ubiquitylation Flag). the against horseradish- antibody of an using not by of but auto-ubiquitylation obvious Parkin, judged catalyze wild-type to able that [as was noteworthy UBE2L3 is only Parkin It Streptavidin]. wild-type (HRP)-conjugated peroxidase modifications modifications by ubiquitin of ubiquitin catalyzed formation with the these treatment increased slightly Consistently, generate CCCP S3D). Fig. to C431S enzymes. material whereas unable (supplementary E2 UBE2D2, ubiquitylation, was different enzymes facilitated E2 Parkin as UBE2N the well or with Parkin UBE2L3 as wild-type recombinant enzyme, of ATP, Incubation E1 of mix Affinity and untreated. complete left a ubiquitin or with h incubated 1 were for CCCP beads with treated cells been from had Flag that of immunoprecipitation by Parkin purified affinity expressed that 3 cells HEK293E wild-type stable an Using to assay. coupled ubiquitylation Parkin of immunoprecipitation probably performed is which efficiency. knockdown significance, limited statistical a observed to reach obvious related and an not despite did UBE2D2/3 alone, UBE2D2/ UBE2L3 trend, of of of knockdown silencing single knockdown gene to 3, NaOH, contrast In combined with 7B). (Fig. or treatment UBE2L3 upon siRNA reduction levels PINK1 significant from C431S–ubiquitin highly quantification Parkin the Indeed, in confirmed ubiquitin. experiments with several the Parkin. Parkin on effect of of discernable no loading charging had UBE2N ubiquitin UBE2D2/3 of of diminished knockdown C431S– However, knockdown strongly Parkin combined UBE2L3 of degree, and levels some reduced of to knockdown both ubiquitin Although UBE2L3 Parkin. Parkin or of bonded UBE2D2/3 activation of thus oxyester treatment. and shift ubiquitin NaOH an the charging blocked to band as completely sensitive knockdown PINK1 confirmed was a Importantly, that was down in C431S–ubiquitin This combination. Parkin knocked resulted 7A). in (Fig. and CCCP and signal alone with mutation UBE2N, Treatment C431S and we Parkin, UBE2L3 Parkin of UBE2D2/3, the co-enzymes the resolve E2 oxyester To the expressed an transfer. of further roles as differential disables although obviously and type, ubiquitin can wild traps mutant to that Parkin E3 C431S similar other a for ubiquitin However, have family. receive they RBR yet the Parkin, of for ligases detected been instable not and protein, have transient substrate and are a intermediates of Thioester residue residue itself. lysine cysteine including a center onto catalytic transfer its further on before bond E2 thioester ‘charged’ a a in from moiety enzyme ubiquitin a accepts Parkin hypothesis, oso htE nye r tlzdb akni el,we cells, in Parkin by utilized are enzymes E2 that show To we enzymes, E2 of roles differential the prove further To 6 lgPri risiatv 41 uain we mutation, C431S inactive its or Flag–Parkin nvitro in ifrn ehns,poal ae ntepoesatrthe after process the initial Parkin. in thus of later loading and ubiquitin through probably functions initial mechanism, UBE2N charging, this, different with in line a In roles Parkin. of redundant activation, play the of UBE2L3 steps different with at are purposes treatment enzymes process. E2 distinct mitophagy an after select for sequential whether confirm h Parkin unclear to 3 by is it utilized unable UBE2D3 point, were this with At we CCCP. Parkin that CCCP, of noteworthy with interaction or is basal UBE2L3 treatment with It under co-purified UBE2N. upon robustly Parkin and specifically with However, was interaction Parkin 7D). an (Fig. UBE2 show Myc-tagged conditions not transfected and did Transiently Parkin enzymes enzymes. of E2 interactions select physical demonstrate analyzed. unequivocally observe enzymes not E2 could other for We the case cells mutant. of HeLa discharging C431S the for specific Parkin or neither the Parkin expressed was exogenous that without This cells present. HeLa parental was Parkin functional nsmay ehv hrceie h oe fseii E2 to specific translocation of and roles activation Parkin the for characterized cofactors have as enzymes we summary, In DISCUSSION hc a led enietfe na ale eott interact to UBE2L3, report 2011). earlier an al., in et cysteine identified Wenzel been and already 2009; lysine has Rape, both linkages which onto and various ubiquitin have (Ye members of transfer residues UBE2D to chains addition, shown generate In been 1990). can Jentsch, enzymes that and E2 (Seufert Ubc5 yeast stress-induced and the Ubc4 to closely E2 homologous are human and UBE2D4 The translocation, and related PINK1. UBE2D3 against Parkin UBE2D2, siRNA UBE2D1, an delayed enzymes of effects depletion the to Hunter, UBE2L3. simultaneous similar with and UBE2D fact, ‘charging’ of Zheng silencing showed In initial 2013; combined experiments of its al., double-knockdown effects this, et additive in with Lazarou line Parkin involved In 2013; that 2013). are al., idea enzymes et coupled the are (Iguchi both translocation with that mitochondrial consistent and and is actions UBE2L3 enzymatic or UBE2D E2s. related be closely and molecules among might remaining redundancy greatly few enzymes partial by even the time, conjugating over that least ubiquitin conceivable at compensated, of is it amounts dynamic pairs, reduced and proteins, E2–E3 facilitates transient of stable certainly the association PINK1 Given more incomplete knockdown. of their siRNA-mediated with instability to well-documented due Compared robust the be at (80–90%). most might However, which CCCP. reduction the obvious, enzymes with E2 less individual showed of treatment became effects knockdown after enzymes the timepoints, is h shown), later E2 2 not and until single (data Parkin phenotype analyzed damaged completely of timepoint of to any depletion PINK1 translocation at p62 significant of mitochondrial of highly recruitment the knockdown and the abrogated Although ubiquitylation as substrate well mitochondria. translocation, as UBE2N Parkin degradation, and UBE2L3 delay members, each family UBE2D profiling. the these blot of of western validated Knockdown and have regulators microscopy conventional We positive using Parkin. results are of substrate that UBE2N translocation indicate subsequent mitochondrial and data HCI UBE2L3 as Our UBE2D, recruitment. well p62 and as ubiquitylations mitochondria, de-energized ae oehr u eut enoc h data B2 and UBE2D that idea the reinforce results our together, Taken to co-immunoprecipitations performed we addition, In h iiaiyosre ewe niiulkokonof knockdown individual between observed similarity The ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal

Journal of Cell Science ihCC o olwdb muorcptto o y.Uo CPtetet aknseiial n togyc-muorcpttdwt Myc–UB with co-immunoprecipitated strongly and specifically treate Parkin or treatment, untreated CCCP left were Upon Cells Myc. (Ø). for control immunoprecipitation vector *** by empty Myc–UBE2N. followed an and h or Myc–UBE2N) 3 Myc–UBE2L3, for (Myc–UBE2D3, CCCP enzymes with E2 Myc-tagged with transfected were aknddntso lee hose eesuo ramn ihCC.N biu hne nti bqii-icag eefudfrteohrE enzy E2 3 other expressed the stably for that found cells were HeLa ubiquitin-discharge CCCP. this with in treatment changes after obvious enzymes No non-functional CCCP. UBE2 expressed that with with cells treatment Parkin HeLa upon and of cells levels Co-immunoprecipitation HeLa thioester (D) parental contrast, altered analyzed. By show UBE2N. not and UBE2L3 did UBE2D3, Parkin for species ‘charged’ ubiquitin of decrease n o-euigcniin.Tersligwsenboswr rbdwt nioisaantedgnu B2ezmst oio noiidUBE2 unmodified 3 monitor HeLa to arrowheads). enzymes (open UBE2 enzymes endogenous UBE2 against ubiquitin-bound antibodies ‘charged’ with shifted probed 8-kDa were and blots arrowheads) western (closed resulting enzymes 10 The with conditions. treated were non-reducing or and untreated left were C431S or (WT) EERHARTICLE RESEARCH hc a oprbet h fet fslnigPN1 hsis formation oxyester This Parkin enzymes PINK1. recombinant for utilized silencing that combined of report previous effects a effect, with Parkin, the consistent this to enhanced ubiquitin-charged comparable was greatly which UBE2L3 of with together amount knockdown oxyester significantly the C431S–ubiquitin alone knockdown Parkin reduced UBE2D2/3 of Although discharges analysis formation. specifically al., by further et 2000), are (Wenzel UBE2L3 corroborated ligases and UBE2D E3 of al., functions HECT redundant The of 2011). sites et active the (Shimura onto ubiquitin Parkin with ( formation oxyester on effect no had combined UBE2N siRNAs, of two Knockd Silencing B. of PINK1. in effects against shown stronger siRNA is the blots to oxyes western with similar Parkin-C431S–ubiquitin of post-hoc, formation, Consistent a analysis Tukey’s the oxyester. of Densitometric ANOVA, reduced C431S–ubiquitin formation treatment. one-way UBE2L3 the NaOH Parkin and to monitor the sensitive to UBE2D2/3 of is used of formation and was knockdown shift blotting the band Western reduced a harvested. significantly as and appears UBE2D2/3 h which 4 arrow), mitophagy. for an during CCCP by Parkin with (indicated by treated utilized were are Cells UBE2N enzymes. and UBE2 UBE2L3 UBE2D, 7. Fig. P , .05 s o significant. not ns, 0.0005; nvitro in P , 0.0001, Lzrue l,2013). al., et (Lazarou F 5 .1) C aetlHL el n eaclsta tbyoeepesd3 overexpressed stably that cells HeLa and cells HeLa Parental (C) 9.911). m CPfr2h el eelsdi o HMSbfe n ujce oSSPG ne reducing under SDS-PAGE to subjected and buffer MES pH low in lysed were Cells h. 2 for CCCP M A eaclswr rnfce ihFa–aknC3Suo ncdw of knockdown upon C431S Flag–Parkin with transfected were cells HeLa (A) aedae l,21a ktue l,21) l fthese of All 2010). 2010; al., al., et et by (Geisler Okatsu p62 2010a; phenocopied knockdown adaptor al., by indeed ubiquitin et or is cognate variant Narendra ubiquitin the UBE2N K63R of a of with of treatment overexpression knockdown distribution upon and morphology and levels upon mitochondrial clustering observed poly-ubiquitin The mitochondrial K63 CCCP. primarily, total Rape, Knockdown inhibited, reduced Parkin. and of UBE2N charging (Komander ubiquitin of on ubiquitylation effect no had K63-linked 2012), mediate to ncnrs oUEDadUEL,UEN hc sknown is which UBE2N, UBE2L3, and UBE2D to contrast In ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal 6 lgPri Tclssoe a showed cells WT Flag–Parkin 6 lgtge aknwl type wild Parkin Flag-tagged 6 LGPri idtype wild FLAG–Parkin C431S w of own 3499 n E2L3 mes § ter 6, d

Journal of Cell Science rvossuyta ugssahge idn fiiyo aknto Parkin of affinity binding a higher with a line suggests in be that also study would to previous This interesting resolution. temporal be greater depolarization would with mitochondrial it upon interaction Nevertheless, of Parkin–E2 utilization unclear. measure treatment the is co- in of enzymes change to h time-dependent E2 3 inability a reflects after our CCCP Parkin from with Whether with Parkin CCCP. UBE2D3 with with immunoprecipitate treatment interacted after specifically and cells UBE2L3 and Moreover, strongly Parkin. UBE2N of auto-ubiquitylation obvious erdto fMNpoen eutn nmitochondrial in resulting a towards Therefore, proteins balance stress. the MFN upon Poole shift 2011) 2010; might of al., HUWE1 al., levels et degradation et UBE2R1 through (Gegg Glauser of Parkin reduction mediated 2010; or al., 2012) are destabilizing et al., generate that et by activating (Leboucher to These counterbalanced events 2013). shown are al., ubiquitylation result been et however 2003). that (Anton has modifications al., proteins fusion MFN et complex mitochondrial of (Staropoli in SCF modifications complex ubiquitin particular earlier a SCF stabilizing an of a addition, in in is described In been in function This has to complex. itself implicated study substrate Parkin SCF that the is a given as interest function in that can adaptor 2013), protein disease recruitment al., third et the Parkinson’s (Burchell FBXO7, mitophagy that with noteworthy progression. is approximately associated with It cycle compared consistently, control. knockdown cell UBE2R1 of had, upon those for we wells in assay, important cells less HCI 35% be our 2013). to in al., Indeed, known et the (Lydeard is promotes chains Cdc34 Skp–Cullin–Fbox ubiquitin and K48-linked for ligases In of enzyme ubiquitin siRNA. synthesis E2 E3 control dedicated a multiprotein the using unequivocally (SCF)-type is that not to cdc34 Parkin difference yeast, did significant a enhanced profiling show However, substrate clustering. UBE2R1 interference mitochondrial western-blot-based increased RNA of and mitophagy. translocation silencing Parkin-mediated (RNAi)-mediated of regulator E2 three all utilizes indeed Parkin functional enzymes. 2010). cells, in al., that, show et in demonstrated experiments be (Sha E2-discharging not experiments, could co-immunoprecipitation PINK1 UBE2D3 of interaction by an Although phosphorylation upon UBE2N B23 u ahrfntosa ifrn tg.Ti sfurther is and This Parkin and between stage. UBE2D measurements different colocalization additive a to by at strong redundant corroborated functions of not no rather is but showed knockdown UBE2N UBE2L3, UBE2L3 that suggesting or Combined effect, UBE2D clearance. with UBE2N this autophagic mito-aggresomes of of formation preceding the recognition in for chains role or important ubiquitin an K63-linked formation corroborates and reduced linkage ubiquitin particular in result conditions ARTICLE RESEARCH 3500 its not but ubiquitin Parkin, of wild-type in formation mutant with the inactive catalyzed together UBE2N modifications UBE2D, Although substrate and events. and linked UBE2L3 clustering yet separate, mitochondrial and mitochondrial are activation as ubiquitylation to with Parkin well that translocation as interference indicates translocation, this Parkin to summary, In delay addition by clustering. might chains in K63-linked UBE2N mitochondria reducing of Therefore, knockdown 2013). (Zheng Hunter, overexpressed to is Parkin and PINK1 recruit cytoplasmic to Parkin when sufficient that are mitochondria which suggested chains, recently K63-linked and was binds morphology it organelle Interestingly, of distribution. independent are that mitochondria neetnl,w icvrdUER/D3 sanegative a as UBE2R1/CDC34 discovered we Interestingly, nvitro in sas nyUEL facilitated UBE2L3 only assays, nlsst h noiidseiso usrt rtisonly. our proteins restricted substrate of thus species We unmodified the proteins. as to determined. extent unmodified analysis same not be the respective do to that to species antibodies the weight by remains molecular detection higher Whether on specific recognize relies TOM70. are assay the the of indeed we in However, levels UBE2T, effects turnover and enhanced Parkin- UBE2S basal these significantly of constant noted silencing out VDAC1 its Upon also Parkin. endogenous rule counteract of of of to presence levels cannot upregulation high serve we of an might presence effects, Although the CCCP). independent in Parkin. of visible absence only enzymes exogenous was (the E2 effect conditions several of This levels of steady-state increased knockdown al., repair under noted upon damage already an et we particular DNA However, in (Williamson in has 2006). VDAC1 involved regulation al., is which et cycle which (Machida UBE2S, UBE2T, cell and for specific 2009), This role K11-linkage recruitment. important p62 the or Parkin includes of translocation mitochondrial or direct and other out form rule this not activation effects. mitochondrial does indirect Although reduced it silencing, slightly cause 2008). UBE2R1 a upon al., factor with could et consistent be (Narendra turn would Parkin in of translocation which fragmentation, prt tdsic tp uigti rcs n rbbywith probably and Parkin-mediated process this enzymes during E2 during purposes. specific steps different that roles distinct show at findings important operate our fact, play In additional mitophagy. that enzymes possible knockdown thus is but E2 It screen, the monitored. this not nor however from were UBE2A efficiencies recovered regulators, neither been Parkin have Nevertheless, UBE2R1 as elusive. UBE2O/E2- identified remain E2 mechanisms also orphan the co- were and the 230K ER-associated pathway, to in (ERAD) implicated addition degradation been In (ER)-anchored has reticulum which study. endoplasmic UBE2J2/NCUBE2, the present here, the UBE2L3 studied enzymes and an to in UBE2D3 translocation similar Parkin Parkin data, of approach regulators our positive of (Hasson with as identified approach Consistent regulators were siRNA 2013). HCI negative al., genome-wide et and a by positive translocation identified study enzymes. the E2 on the depending of mitophagy type context Parkin cell cellular of of of UBE2B/ importance regulation knockdown the and related the underlines specificity this for closely case compensate either to the In able UBE2A. cells, be also HeLa might RAD6B human versus knockdown In non-complete different from knockout. or from timepoints CCCP different with no result treatment cells), of human apparent might but versus The (murine studies retardation, models 2010). cellular both al., mental between et causes Leeuw discrepancy (de UBE2A remained upon syndromes mechanisms of parkinsonian underlying translocation the Loss fibroblasts but Parkin unclear. embryonic 2013), rather mouse al., inhibit be et knockout (Haddad in to to CCCP with appeared reported treatment UBE2A of this UBE2A recently Loss Parkin. of yet functional was of discharge presence CCCP, slight the of in with a independent recruitment noted treatment we p62 upon However, or cells. clustering HeLa mitochondrial proteins. translocation, might substrate of and levels proteins remaining respective the the underestimate of thus weight molecular species higher ubiquitylated) account into (i.e. take not does this However, iecn fohrE nye a oefc nthe on effect no had enzymes E2 other of Silencing neetnl,drn h rprto fti aucit novel a manuscript, this of preparation the during Interestingly, Parkin affect not did UBE2A of knockdown Surprisingly, ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal

Journal of Cell Science EERHARTICLE RESEARCH upeetdwt 0 ea oiesrm(A)a 37 (DMEM) at medium (PAA) serum Eagle’s bovine modified fetal Dulbecco’s 10% with in supplemented grown or 3 or were cells EGFP- (HeLa-GFP-Parkin) (HeLa-Parkin), HeLa Parkin Parkin and untagged Myc expressed HEK293E stably Parental that cells Invitrogen. clonal cells from (HEK293E) American obtained kidney the embryonic were from human and obtained Collection, were Culture (HeLa) Type cells cancer epithelial silencing Human RNA and culture Cell request. (Applied on Analyzer obtained Genetic be 3100 can ABI sequences an Primer Biosystems). and v.3.1 Terminator into BigDye using mutagenesis cloned al., site-directed were et and constructs using pCMV-Myc-E2 (Geisler cloned The was previously pcDNA3-Flag. C431S described Parkin been 2010). has Flag–Parkin Wild-type mutagenesis and Cloning METHODS AND MATERIALS hi seby(eadRp,20)ta a eselectively be can that ubiquitin 2009) of Rape, mediators and key active are (Ye their enzymes assembly be of E2 might modification chain that enzymes by Given E2 disease center. 2014), sporadic similar Dawson, Thus, in 2012). and inactivated al., (Dawson et stress (Doris Parkin stress to 1997) 2001), cellular oxidative al., al., to et sensitive et Moreover, (Jahngen-Hodge or (Takada redox-regulated E2s 2013). are some others whereas of al., charging been enzymes ubiquitin et recently increases E2 have (Wiener uncharged cells and particular suggested in the charged UBE2N) of surprising its of versus 2007), ratio (UBE2D2 al., roles and the et in biological (Jin Parkin differences steady-state the at of to ubiquitin-charged believed and as were be activation E2s Wauer Although well generated. the modifications 2013; as ubiquitin understand certainly al., functions, al., will better et E2s enzymatic et cognate Trempe to (Riley the structure 2013; of help knowledge resolved al., (Wenzel 2013), recently et Komander, its transfer Spratt and ubiquitin 2013; 2011) al., Parkin-mediated et for mechanism that their enzymes conjugation. E2 as its roles the in the identify well involved dissect to are further and as to ubiquitin PINK1-dependent interesting modifications, phosphorylated be of thus involved the the the will It emphasizes for the complexity. with Parkin), cofactor itself of of domain a (together importance UBL discovery as ubiquitin the Parkin acts of striking phosphorylation which of and of 2014), recent activation al., phosphorylation very et The (Kazlauskaite PINK1-dependent 2014). select 2011; al., on al., of et K27) Chan et 2010; and al., et Birsa of K63 (Geisler course appearance Parkin-mediated (K48, the sequential along types in substrates the linkage enzymes with ubiquitin consistent E2 distinct also several is mitophagy. of mitophagy and Parkin E2 involvement of distinct regulators Hasson of The indirect enzymes. 2013; importance or the direct al., underscores as E2 et study enzymes our (Haddad among 2013), reports have al., recent et functions, two we with antagonistic and redundant, Together Moreover, or to including mechanisms, mitochondria. translocation cooperative molecular its at distinct and identified Parkin activities of enzymatic activation the regulate lcdt hi eeac n otiuint ies pathogenesis. their disease to to of as contribution well understanding and as relevance our modifiers, their and elucidate expand cofactors it Parkin future further essential in as to functions 2011), important al., et be (Ceccarelli will molecules small by targeted uiiid5 CO 5% humidified nlgto h el dniidRING-HECT-hybrid identified newly the of light In that enzymes E2 four uncovered study our summary, In Not .Oeepeso osrcswr eunevrfe using verified sequence were constructs Overexpression I. 2 ar N rnfcin eepromdusing performed were transfections DNA /air. 6 lgPri idtp rC431S or type wild Flag–Parkin ˚ under C Bgl II o obndkokonwt w rtresRA,teaon feach of amount nmol). the (2.5 siRNAs, halved three was or siRNA two with knockdown combined For 3 ACCUGGAUUU-3 AACAAGUUCCCCAUCGACUAC-3 3 AGUCCUUACCAA-3 AACAGUAAUGGCAGCAUUUGU-3 B215 UBE2D1 AGUAUU-3 rUE-pcfcsRA alfo ign:PN15 PINK1 Qiagen): from PINK1- control, (all nmol 5 siRNAs with TGTTCCTCGTTATGAA-3 HiPerfect UBE2-specific using performed or days were according consecutive transfections two (Roche) siRNA on instructions. XtremeGene9 manufacturers’ or the to (Invitrogen) 2000 Lipofectamine o C eaGPPri el eesee ih40 cells/100 4000 with seeded were cells HeLa-GFP-Parkin HCI For Imaging Content High el eepae nogascvrlp otdwt poly- with coated coverslips glass onto plated were Cells Immunostaining 4 at overnight antibodies onto primary transferred Millipore). with (both and incubated membranes (Invitrogen) were nitrocellulose gels Membranes or NuPAGE fluoride Biotechnology). 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Journal of Cell Science eecnetdit -i i ie n nlzduigIae n the and the of ImageJ analysis using For analyzed 1993). [(Pm al., factor and et form files (Manders Plugin tif Coefficients 8-bit Manders’ into images raw converted correlation, Pearson’s were of analysis ApoTome For an (Zeiss). with System equipped Imaging microscope AxioObserver an images slides with fluorescent taken Hoechst microscope Confocal were (Dako). onto with medium mounted mounting stained fluorescent were were using Coverslips Nuclei 1:5000. diluted 1:2000. 33342 diluted Probes) (Molecular ARTICLE RESEARCH EE H75 0m C,10m al MET,05mM 0.5 EDTA, mM 1 NaCl, 3502 mM 150 KCl, mM mM (50 10 buffer immunoprecipitation 1 7.5, in pH lysed cold and in HEPES NaCl) mM 10 once 150 without washed 7.4, were pH or Cells with lysis. incubated before 3 were expressed mutant stably C431S that cells HEK293E vitro In dilutions 0.2 10 with in 1 triplicate cDNA primer. in (Qiagen) Fidelity oligo-dT used High anchored Kit were and Transcriptor Mini (Roche) using Kit RNeasy transcribed total Synthesis of reverse ng the 1000 was qRT-PCR, using RNA For instruction. extracted manufacturer’s the was following RNA Cellular sc- Cruz Santa qRT-PCR 1:2000; (WB, UBE2T against Santa 103913). 1:2000; goat (WB, (WB, sc-131354), UBE2S UBE2R2 against Cruz goat against sc-167440), goat Cruz Santa UBE2N 10964-2-AP), 1:5000; against Group (WB, ProteinTech UBE2R1 mouse against 1:1000; rabbit 6999), 37-1100), Invitrogen no. 1:1000; against (WB, Technology (Ubc13) Signaling rabbit against (WB, Cell goat UBE2N 4330), against 1:5000; rabbit 14415-1-AP), sc-47549), Cruz no. 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UBE2:PBGD obtained the as to calculated were normalized were and HMBS) levels transcript as Relative UBE2D2, and method. known UBE2T UBE2D1, UBE2S, (also UBE2C, UBE2R1, PBGD UBE2N, UBE2A, UBE2L3, (Roche). for UBE2D4, system 480 UBE2D3, levels LightCycler a transcript on block Absolute 384-well a in executed were bqiyainassay ubiquitylation p 6 ((Am/ 2 )/(4 p ) 2 p 2 )P](aedae l,21a,iae were images 2010a), al., et (Narendra ))/Pm] m](opa ta. 05 n h compaction the and 2005) al., et (Koopman Am)] m rmrad5 and primer M 6 m lgPri idtp rthe or type wild Flag–Parkin ecin n PRanalyses qPCR and reaction, l 6 B 2 MHEPES mM (20 HBS m m CPfr1h 1 for CCCP M TqUniversal iTaq l 11,0;SgaA52 n rMc(:00 oh 11667149001). Roche (1:5000; Flag–HRP Myc against or antibodies and mouse A8592) with Sigma (1:10,000; blotting western by analyzed GA .%N-0 lsCmlt rtiaeihbtr.Poen(750 Protein inhibitors. proteinase Complete plus NP-40) 0.2% EGTA, lgPri 41 sn ioetmn 00acrigt the (95 The preheated later. to 10 in h without 4 or according harvested with replaced treated were was 2000 were with cells medium transfected day, Lipofectamine the were next and cells At using protocol, siRNA. transfection, manufacturer’s with siRNA C431S days second consecutive the Flag–Parkin two after on h transfected 24 were cells HeLa analysis Oxyester 0N057] h ihe .FxFudto;My lncFudto n the and Foundation Clinic Mayo Foundation; - number Fox Health [grant J. of Stroke Michael Institutes and the National Disorders R01NS085070]; the Neurological from of grants Institute by National supported was work This Funding manuscript. the analyzed wrote W.S. W.S. and and F.C.F. E.L.M.-L. F.C.F., F.C.F., data. experiments. experiments. the the performed designed M.A. W.S. and and E.L.M.-L. F.C.F. study. the conceived W.S. contributions Author interests. competing no declare authors The interests Competing Tu of constructs. (University UBE2 Geisler Myc-tagged data Sven providing HCI and generously consultation. with Kahle statistical help Philipp for for Florida) thank Clinic, Florida) We (Mayo Clinic, Heckman (Mayo Michael Younkin and Curtis extraction to grateful are We as all are Acknowledgements levels for Significance ANOVA s.e.m. one-way the *** indicate using indicated: bars performed Error was experiments. analysis Statistical analysis Statistical mM 1 NaCl, mM 150 KCl, mM MgCl 10 mM 1.5 7.5, EGTA, pH mM 0.5 HEPES EDTA, mM (50 buffer 3 expressed stably that cells HeLa Co-immunoprecipitation omto,aiut flstswr rae iho ihu NaOH without oxyester or by 37 with at shift h treated band 1 the for ten were concentration) by verify lysates final homogenized To mM of (100 were needle. Lysates aliquots G 20 SDS). formation, a 1% through NaCl, strokes mM 150 7.6, pH odc-muorcptto ufrbfr h uentn was supernatant 50 95 in the at eluted boiled before were and Beads buffer removed. completely co-immunoprecipitation cold gels. Bis-Tris 4–12% eobnn rtiswr ucae rmBso Biochem. Boston from no. (GST-Ube1, enzyme purchased E1 1 nM E306), 100 were contained reactions Ubiquitylation proteins Recombinant ed SgaE64 t4 20 at E6654) (700 using (Sigma lysate beads immunoprecipitated Protein was inhibitors. condition proteinase Complete plus ape eerno e,botdadpoe ihHRP-coupled with probed and Flag. against blotted antibody 016-030- an gel, and Immunoresearch 084) a Jackson on (1:100,000–1:250,000; Streptavidin run were Samples a muorcpttdfr4huig20 using h 4 for immunoprecipitated was h auatrrsisrcin o as el eelf nrae or untreated left were Cells days. 10 2 with treated for UBE2D3, to according instructions Myc-tagged (Invitrogen) manufacturer’s 2000 or the Lipofectamine using vector, UBE2N empty or UBE2L3 pCMV-Myc with transfected 37 20 1 a in in UB-560) 5 (no. ubiquitin DTT, biotinylated terminally mM 2 ATP, al MMgCl buffer mM immunoprecipitation 5 1 cold NaCl, in in once twice washed and were Beads (Sigma). ˚ .Lemibfe a de n ape oldfr1 i t95 at min 10 for boiled samples and added was buffer Laemmli C. m ora fCl cec 21)17 4830 doi:10.1242/jcs.147520 3488–3504 127, (2014) Science Cell of Journal ecin bqiyainratoswr are u o . at h 1.5 for out carried were reactions Ubiquitylation reaction. l m 2ezm n.E-2,n.E-4 rn.E-6) . mM 2.5 E2-664), no. or E2-640 no. E2-622, (no. enzyme E2 M P 6 , m bqiyainbfe 2 MHPSp .,5 mM 50 7.4, pH HEPES mM (20 buffer ubiquitylation CPfr3hadlsdi co-immunoprecipitation in lysed and h 3 for CCCP M .05 ** 0.0005; ˚ .Imnpeiiae n nu ellstswere lysates cell input and Immunoprecipitates C. 2 eoetespraatwscmltl removed. completely was supernatant the before ) m nagduiutn(o -0H,1 U-100H), (no. ubiquitin untagged g ˚ o .Baswr ahdtretmsin times three washed were Beads h. 3 for C P , .0;* 0.005; ˚ )SSlssbfe 5 MTris mM (50 buffer lysis SDS C) 2 6 0 lcrlad02 NP-40) 0.2% and Glycerol 10% , P lgPri idtp were type wild Flag–Parkin , m ˚ .5 s o significant. not ns, 0.05; fat-lgEve beads EZview anti-Flag of l eoelaigte onto them loading before C m m fat-y EZview anti-Myc of l CPfr4h Cells h. 4 for CCCP M m 6 l2 bne,Gray for Germany) ¨bingen, bqiyainbuffer ubiquitylation 6 aml buffer Laemmli m )freach for g) m gN- m ˚ C. g)

Journal of Cell Science lue,L,Sna,S,Saa .adMoe .J. D. Moore, and K. Stafa, S., Sonnay, L., Glauser, and H. A. Schapira, M., Holmstro Rojo, S., Geisler, Y., K. Chau, M., J. Cooper, E., M. Gegg, i,S . aao,M,Wn,C,Kn,L . aeda .P n ol,R J. R. Youle, and P. D. Narendra, A., L. Kane, C., Wang, M., W. Lazarou, J. M., Harper, S. Jin, and P. S. Gygi, X., Li, J., Jin, Gong, Jr, R., T. Nowell, F., Shang, X., Gong, S., M. Obin, J., Jahngen-Hodge, Suzuki, M., Kimura, H., Kosako, F., Koyano, K., Okatsu, Y., Kujuro, M., Iguchi, ua .M,Osesi .L,Shemn,J . uio,I,Mle,D J., D. Miller, I., Kurinov, P., J. Schuermann, L., A. J. B. Olszewski, M., Morgan, D. and Duda, L. E. Rumsby, S., K. Doris, asn .A,Kn,L . aao . un,C . ltr .A,Belr E., Buehler, A., D. Sliter, H., C. Huang, K., Yamano, A., L. Kane, A., S. Hasson, iFno . ekr .C,Mnan,P,Brzi . ooa .H,Correia H., E. Yonova, A., Baruzzi, P., Montagna, C., M. Dekker, A., Fonzo, Di R., A. Zinn, A., L. Baker, L., Jaeckle-Santos, A., Green, S., L. Bulk, N., V. 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