Journal of Cell Science o:10.1242/jcs.102178 doi: 3813–3826 125, Science Cell of Journal 2012 April 10 Accepted nvriyo ina a .Prt aoaois etro oeua ilg,Dprmn fBohmsr n elBooy r-orGse9, Dr.-Bohr-Gasse Biology, Cell and Biochemistry of Department Biology, Molecular of Center Laboratories, Perutz F. Max Vienna, of University Ce and Koch* Johannes to Fis1 fission human peroxisomal and coordinate Mff attract proteins PEX11 Article Research eoioe.Teepoen,cle eois r noe by encoded of are and function been peroxins, and mechanisms called have assembly proteins, Proteins the underlying These in understood. peroxisomes. the participate poorly (Kim that However, only characterized novo are de 2006). regulations arose protein al., prevalently an membrane peroxisomes et using analyses that peroxisomal vivo the suggested In replenish photoactivatable cells. continually human to Toro engineered in 2007; seem peroxisomes al., pathways of et Both pool Toro 2009). 2007; (Geuze Hettema, al., reticulum and et endoplasmic Motley or 2003; the organelles al., from pre-existing et biogenesis steady- of novo division their de and via insure growth Huybrechts that by 1974; either exist state al., under mechanisms peroxisomes et of conditions, maintenance Duve the varying (de guarantee To a 2010). days 2009). revealed al., Sakai, two et cells about and mammalian of in Oku half-life turnover 2007; peroxisome specific al., on Studies 2006). and et Waterham, (Fagarasanu proliferation degradation and through in in changes microenvironments Wanders to and adapts cellular 2007; ageing compartment peroxisomal al., of the Accordingly, et process Koepke the 2010; with (Angermu immunity associated innate crucial antiviral been function a Their also assaults. harbor stress has organelles to response round in function These organelles detoxifying the lipids. with small associated of mainly peroxisomes, metabolism functions cellular contain several for cells essential eukaryotic Most Introduction provides proteins PEX11 PEX11 (2) of membrane; interaction peroxisomal concerted the the (3) protrude and to action; proteins words: of peroxisomes. Key PEX11 site of other their PEX11 division the to (1) and cells: (hFis1) of growth mammalian Fis1 one for in human least control proliferation PEX11 and spatiotemporal at with peroxisome Mff interaction of with both its mechanism coordination attract and molecular peroxisomes proteins in the on acts Mff of factor and features fission several required mitochondrial reveal the data of interaction presence Our PEX11 The the elongation. proteins. the demonstrate its PEX11 also regulatory that regulating We mutant thereby and establish factors. proliferation. membrane and region fission we peroxisomal peroxisome the wild-type amphipathic studies, onto an of vivo region during including amphipathic profile in an factors elongation dock and membrane which elongation assays domains, for anchored pegylation membrane biochemical domains Through a essential as Using thereof. revealed scission. act sequences PEX11 peroxisomes, membrane of scission to to of topology the progression localize shown membrane of coordinate which the proteins assembly were determined these the factors we family facilitate fission approach, and PEX11 with enable interaction the to through processes Furthermore, of remodeling membrane Proteins requires machinery. organelles membrane-bound of Fission Summary Austria Donau, der an Krems 3500 1, Magnesitstrasse Biosciences, Med ` Tissue address: *Current Austria 1030, Vienna uhrfrcrepnec ( correspondence for Author 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. ebaeeogto,PX1,Prxsm rlfrto,Pegylation proliferation, Peroxisome PEX11G, elongation, Membrane [email protected] cl Brocard* ´cile le ta. 09 ii tal., et Dixit 2009; al., et ¨ller ) c n eel eragmn ewe oo n eeoieiainadascainwith association and heterodimerization and homo- between rearrangement a reveals and , ` c n ftetrehmnPX1poen.Aayi fPEX11 of Analysis proteins. PEX11 human three the of one , PEX oe o he ifrn E1 rtis PEX11 proteins, this PEX11 PEX11 genome different in mammalian three The shared involved for roles. factors codes factors fission essential and play the family mitochondria PEX11 Within with the organelle Brocard, 2010). of and daughter proteins al., Koch process, new 2007; et al., a Koch et formation Fagarasanu 2011; 2010; the al., et to This (Delille rise scission. of membrane gives (5) import (2) and process (4) proteins; polarization; matrix elongation, peroxisome and membrane membrane (1) (3) protrusion; including: membrane steps several into 2006). al., or genetic et disorders lethal (Steinberg syndrome biogenesis of Zellweger peroxisomal e.g. development PBDs, as the characterized with diseases associated been have h alacoe rti ua i1(Fs)a ela the as well as (hFis1) Fis1 human protein tail-anchored the mice. in symptoms PEX11 Zellweger-like essential expressed of constitutively not development absence otherwise contrast, is In the protein peroxisomes. of functional this Taken of PEX11 2002). and formation the Gould, environment for of and the Li of expression PEX11 2002b; variations al., lack the et Li that Gould, together, 2002a; and studied al., Li et mice been 2002a; (Li Knock-out al., 2003). have al., et et Tanaka Li models 1998; 1998; al., et al., Schrader et 2002; Abe 1998; Fujiki, h eoioegot n iiinmdlcnb divided be can model division and growth peroxisome The neetnl,poen ftemtcodilfsinmachinery, fission mitochondrial the of proteins Interestingly, ee Dse ta. 96.Mttosi several in Mutations 1996). al., et (Distel genes c hc xii ifrn xrsinpten Aeand (Abe patterns expression different exhibit which , c euneicue w membrane- two includes sequence a sidcbethrough inducible is a a b PEX11 , n PEX11 and ed othe to leads PEX c mutants, 3813 b genes c and is b Journal of Cell Science ir saso iooe ihppie from peptides with liposomes on cerevisiae assays and proteins vitro PEX11 yeast that PEX11 finding mammalian the through provided recently the of details proliferation. the peroxisome of of analyze in to formation involved tool disappearance mechanism useful the molecular a the represents following cells to Thus, in JEPs peroxisomes. and the round the (JEPs) juxtaposed distinct of in affected composed peroxisomes proteins structures causing of PEX11 elongated overexpression formation cells the of to human leads excess Their cells cultured Ultimately, elongation. 2010). in proteins their peroxisomes of al., of class a morphology such et represent facilitate family (Koch PEX11 that to showed tight the previously of factors in We proteins division. act elongation and the constriction must membrane Indeed, membrane rather peroxisomes 2010). with as to al., a coordination et acted recruited hFis1 (Otera machinery Mff assigning DRP1 fission factor, on the factor, function fission by recruitment regulatory challenged mitochondrial DRP1 was 2007; the interpretation efficient al., membrane This that et the 2010). finding al., (Kobayashi of et peroxisomes recruitment Koch onto the machinery in fission suggesting participates hFis1 and PEX11 proteins analyses interaction that PEX11 Protein of 2005). association al., the et revealed Koch 2008; 2003; Bliek, al., der et the van Koch at and function (Gandre-Babbe their membrane execute peroxisomal also DRP1, protein dynamin-related 3814 rtisadislclzto tteprxsmlmmrn.We membrane. peroxisomal the PEX11 at other the machinery. localization with the its fission Mff and of the with proteins interaction of the interacts establish members we with protein Especially, and this proteins how PEX11 PEX11 elucidate influences of further directly targeting Based cells. domain peroxisomal human in on amphipathic membrane peroxisomal the the for of an elongation of modulating requirement that including activity show and the sequence the elongation membrane demonstrate for its region We this in domain. motifs amphipathic functional PEX11 identified of three topology how cells. mammalian question each in influence the proliferation or concert peroxisome raises in during act other This proteins similar to Pex25p. but related independent to closely more than sequence are acid they Pex11p amino altogether high and proteins share (83%) PEX11 they homology mammalian and Indeed, Pex11p related. three proteins closely the yeast are of the sequences to 2012; the contrast al., In Pex25p, et 2011). (Huber al., peroxisomes et generate Saraya to unable were Pex3p yeast the in membrane peroxisomal helix, amphipathic (Opalin membranes sapiens Homo e1pi novdi rwhaddvso fpre-existing of division ER, While the and from peroxisomes. biogenesis growth novo of different as de in a formation regulates Pex25p in involved the peroxisomes, involved is to is family leads Pex11p PEX11 that the pathway of that demonstrated member studies species yeast each Recent different proliferation factors. from the redundant proteins PEX11 in represent on role these different that a or plays process protein each either that ehnsi nihsit eoioa ebaeeogto were elongation membrane peroxisomal into insights Mechanistic sn iceia prah edtrie h detailed the determined we approach, biochemical a Using otognsscnansvrlPX1poen.Ti implies This proteins. PEX11 several contain organisms Most pex3 D pex25 ora fCl cec 2 (16) 125 Science Cell of Journal ( Sc Pex11p), D ( Hs a uatyatclsepesn plasmid-born expressing cells yeast mutant sie l,21) nteasneo its of absence the In 2011). al., et ´ski PEX11 n PEX11 and Hp c asnl polymorpha Hansenula e1pwsual opord the protrude to unable was Pex11p tteprxsmlmmrn and membrane peroxisomal the at a hwdteraiiyt elongate to ability their showed ) b abra amphipathic an harbor .polymorpha. H. c uae esos we versions, mutated ( Hp Saccharomyces e1p and Pex11p) a hlx In -helix. usin hte h w yrpoi tece identified whether or bilayer. PEX11 stretches membrane phospholipid for anchors the hydrophobic as the cross stand raises entirely they two This that 1B). domains the (Fig. represent protein whether membrane integral that question, indicating true pellet, a membrane is the it in detected exclusively was h o cr ugssta hs ein ersn membrane-buried represent regions 209–227. these aa that and suggests 127–149 score aa low segments: The hydrophobic two reveals algorithm a rsn nS,idctn h utr fperoxisomes. of rupture the EGFP–Px indicating contrast, S1, In in peroxisomes. present of was protein membrane integral and an (S2) PEX11 PEX14, loaded. supernatant were carbonate (MP) (S1), pellet supernatant membrane high-salt (I), input of score. higher PEX11 a yield would domains ( transmembrane true because domains yrpoi segments. hydrophobic PEX11 1. Fig. elsuidprxsmlmmrn rti E1 Wl tal., et (Will on PEX11 PEX14 1999), extraction protein PEX11 membrane carbonate peroxisomal expressing a well-studied protein– lysates out through cell carried it human the we to into attached interactions, integrated only it PEX11 protein 209– whether or aa determine membrane and of to peroxisomal Then, 127–149 1A]. (aa) analysis Fig. acids 227; first membrane, [amino silico we stretches issue, peroxisomal this hydrophobic al., in tackle the et To in Tanaka elucidated. performed be 1998; topology to al., remains the et exact however, the Schrader to Their 1998; termini al., that 2003). both et their (Abe showed exposed cytosol proteins 1996), experiments PEX11 al., mammalian et permeabilization (Marshall membrane However, peroxisomal differential the 2003). of side al., inner for et proposed were While al., Tanaka topologies proteins. PEX11 et various 1998; eukaryotic at organism Abe the localize al., on 1998; most depending to et Fujiki, shown and in Schrader were (Abe 1998; identified membrane studied peroxisomal proteins been the all and have organisms proteins PEX11 PEX11 Results in proliferation peroxisome for cells. model mammalian mechanistic a propose B abnt xrcinwspromdo E23 el expressing cells HEK293T on performed was extraction Carbonate ) c FA n GPP,aprxsmlmti akr qa fractions Equal marker. matrix peroxisomal a EGFP–Px, and –FLAG c net noteprxsmlmembrane peroxisomal the into inserts c c sa nerlmmrn rti abrn two harboring protein membrane integral an is a o xrcal ihsdu abnt and carbonate sodium with extractable not was ( A nslc nlsso PEX11 of analysis silico In ) Sc e1pwssgetdt oaiei the in localize to suggested was Pex11p c hc r nybre nothe into buried only are which , c hwdtesm itiuinas distribution same the showed c FA.Smlrt the to Similar –FLAG. c c eeln two revealing sn h TMpred the using Journal of Cell Science xetdy PEX11 Expectedly, PEX11 several ihasnl ytiewsmdfe ntepeec fdetergent of PEX11 presence the again, in Yet modified 2D). was (Fig. cysteine single a with -emnlhl,w bevdol n la adsitafter band-shift clear one freely only observed are are we two two half, N-terminal bridge, and 2010). al., et disulfide Schmidt domains 2010; al., et a (Sato cytosol helical the from accessible form six between the two Among one constrained matrix. only cysteines peroxisomal that the showing cytosolic faces analyses PEX3 structural of with seven cysteine This agreement added. whereas was in two X-100 detergent Triton is assay the without when pegylation accessible modified PEX3 were our cysteines be protein detergent In could membrane residues. the cysteines of cysteine from peroxisomal PEX14 seven addition The of contains 2B). part without (Fig. this pegylated of cytosol accessibility (C362) fully the cysteine demonstrating single Will its almost 2002; assay C- al., our was et in Accordingly, the (Oliveira 1999). cytosol al., that the expressed et faces showed PEX14 ectopically of experiments and terminus Accessibility the PEX14 genuine to the protein PEX3–EGFP. regard tested with membrane peroxisomes. we assay proteins, to integral our membrane peroxisomal route of of reliability en or study the lysates proteins insure cell cytosolic to the Further, of of peroxisomal preparation minor amounts the the proteinsmall during indicate occurs the might that to detergent leakage in of EGFP–C4-Px added modified absence of was portion the small X-100 The was 2B). 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(Fig. domain -helical neetnl,w dniidargo otiigfu prolines four containing region a identified we Interestingly, iial oEGFP–PEX11 to Similarly EGFP–PEX11 P165/167, motif, proline-rich identified the in prolines Two E1 rtisadprxsm iso 3817 fission peroxisome and proteins PEX11 c yial nue togmmrn lnainand elongation membrane strong induced typically c P152A,A182P c akn a1517 EGFP–PEX11 165–167, aa lacking i o omJP.Ti eosrtsthe demonstrates This JEPs. form not did c A182P EGFP–PEX11 , c c P158A P158A c c ntemolecular the in c c .Atlatertime c P158A o t ability its for , ucinand, function uain on mutations a trigger can c scapable is localized noPLP c c c , , Journal of Cell Science biul,i hscs h ocmtn xrsino Fs a a had hFis1 of expression 5E,F). concomitant (Fig. the case peroxisomes this in elongated Obviously, significantly of appearance xrsino y–Fs n EGFP–PEX11 simultaneous and the myc–hFis1 expected, as of peroxisomes expression round small, many EGFP–PEX11 While 5E,F). co-expressing (Fig. transfection after cells hours 48 and 24 morphology mut fPX1poen noeepesn el.I h L motif PLP the If PEX11 cells. high of overexpressing the in for proteins be compensated PEX11 of could and amounts hFis1 JEPs of Accordingly, overexpression 2010). by al., dissolved et (Koch machinery fission 3818 ec-xrse y–Fs n ihrwl-yeEGFP– wild-type either and myc–hFis1 cell. the PEX11 in co-expressed present already focused structures JEP we We the elongation, of dissolution peroxisome the on of subjective analysis to through experiment. reverse version prone a perform to is sought we observation thus and such interpretation However, formation. PEX11 ahrta olwn h ciiyo h uae PEX11 mutated the of activity the following than Rather c c noPLP rEGFP–PEX11 or c ora fCl cec 2 (16) 125 Science Cell of Journal sivle nhi1rcutette,oeepeso of overexpression then, recruitment hFis1 in involved is rti hudimdaeyla oetnieJEP extensive to lead immediately should protein c noPLP n vlae h peroxisome the evaluated and c n y–Fs presented myc–hFis1 and c noPLP e othe to led c ol o eetasgiiatcag nhi1pl onusing down pull hFis1 in we change However, significant proteins. a PEX11 detect two not the could between interaction to due be weakened could which morphology, peroxisome on effect mediocre h el.I ir xeiet hwdta PEX11 that showed experiments of vitro requirement metabolic In the cells. to the adapts the that ensure peroxisomes to of together cells, act number factors, human fission in and proliferation proteins peroxisome PEX11 in involved factors The PEX11 proliferation around peroxisome network interaction complex A omto fatraycmlxbtenPEX11 between complex ternary a of PEX11 formation that previously showed PEX11 we and and 2007) al., et (Kobayashi PEX11 between novdi Fs idn nlecsteatvt fhFis1. of activity results the influences Our binding PEX11 division. hFis1 in in peroxisome motif involved PLP for the that essential suggest be might hFis1 c noPLP c neatdi io(ohe l,21) Hence, 2010). al., et (Koch vivo in interacted sbi rslsntson.Drc interaction Direct shown). not (results bait as b n Fs a eosrtdi vitro in demonstrated was hFis1 and lnaino h eoioa membrane. peroxisomal the of and elongation bending the promoting of thereby leaflet membrane, outer the in anchored PEX11 PEX11 in ( buried membrane. are the residues these that X-100 Triton suggesting without modified and not 134 were position 219 at acids amino the The face cytosol. residues these X-100 that Triton indicating of addition were without shift observed mobility electrophoretic an asterisk) with (red for bands accessible Additional were pegylation. 238 and 206 positions 160, at introduced Cysteines as indicated. introduced were mutations PEX11 pegylation, of by region C-terminal the of ( amphipathic (blue). predicted helix a by separated (red) regions hydrophobic helical two the showing PEX11 the membrane. PEX11 domains anchor membrane-buried Two 3. Fig. fe rnfcin fe iain h cells the fixation, After days transfection. 2 after morphology peroxisome in changes ebaefo h yooi ie The amphipathic side. predicted cytosolic the from peroxisomal membrane the into (brown) regions PEX11 depicted red. are in residues in buried shown whereas are green, cytosol the from Cysteines accessible assays. pegylation and extraction 5 bar: Scale blue. in shown mutated or PEX11 wild-type whether of morphology regardless peroxisomal in changes displayed similar Cells channel). (green antibodies anti-FLAG using immunostained were PEX11 ( ( transfection 2 after types days peroxisome the of Quantification D umr hwn h oooyof topology the showing Summary ) c c c c FA,PEX11 –FLAG, FA a xrse.Nce are Nuclei expressed. was –FLAG A206C sdmntae hog carbonate through demonstrated as c c eunea ulndi i.2A, Fig. in outlined as sequence ( A FA eeaaye for analyzed were –FLAG B ahrta en directly being than rather c ceai ersnainof representation Schematic ) odtrietetopology the determine To ) c net t hydrophobic its inserts C nteperoxisome the in el co-expressing Cells ) a n hlx(lehlx is helix) (blue -helix c 5 A134C b 0cells). 20 PEX11 , c m .(Below) m. regulates FA or –FLAG b c directly –FLAG c and b Journal of Cell Science hc rastehlclsrcue(right). 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This that their 2010). idea execute al., the et (Koch favors hFis1 three strongly with all co-purified purification reported proteins affinity in PEX11 We Moreover, co-precipitate. not 2007). did PEX11 al., homodimerization, PEX11 both et its with beside (Kobayashi that hFis1 previously with interacts PEX11 4. Fig. fiiyprfctoswt l uae esoso PEX11 of EGFP–PEX11 versions mutated Although all PEX11 with 6B). (Fig. purifications affinity PEX11 binding PEX11 the analyzed of we se. possibilities, per these between binding binding. differentiate hFis1 hFis1 To influence for could required protein PEX11 not another is Alternatively, PEX11 proline non-mutated this with that obtained indicating those from undistinguishable oeua egt h GPtge rtiscnb iulzdas visualized be different can a proteins have EGFP-tagged the they weight, Because molecular S2). Fig. PEX11 material of (supplementary version non-mutated or proteins, three mutated co-expressing a cells namely, from originating lysates from PEX11 PEX11 via and suggests hFis1 finding This of blotting. co-precipitation western that by visualized as lower much e,teaonso GPmchi1poen bandusing obtained proteins EGFP–myc–hFis1 of PEX11 PEX11 amounts tested the all Yet, with The digitonin. co-precipitated 0.2% hFis1 containing appropriate buffer protein in the lysed expressing were co-precipitating pairs cells plasmid for HEK293T tested 6A,B). (Fig. and proteins purifications affinity performed GPmchi1adete EGFP–PEX11 either and EGFP–myc–hFis1 B h mn cd 7–9 fPEX11 of 176–193 acids amino The ) oaayewehrteitoue uain nPEX11 in mutations introduced the whether analyze To er-vlae hsfnigtruhimmunoprecipitations through finding this re-evaluated We c c c c FA,teaonso EGFP–PEX11 of amounts the –FLAG, A182P eietfe EGFP–PEX11 identified We . A182P c b uat ihwl-yevrin fete PEX11 either of versions wild-type with mutants u ahrta PEX11 that rather but c FA oadge iia otewild-type the to similar degree a to –FLAG FA sbi,wr infcnl ihrta those than higher significantly were bait, as –FLAG otisa mhpti einpeee yapoierc motif. proline-rich a by preceded region amphipathic an contains a n PEX11 and b oee,telte w proteins two latter the However, . c b c c sebeit eia ein lutae sawel(et.Tehdohbcsd smre nylo.Pymol-rendered yellow. in marked is side hydrophobic The (left). wheel a as illustrated region, helical a into assemble ietyitrcswt hFis1. with interacts directly Fg A.Itrsigy the Interestingly, 6A). (Fig. rwl-yePEX11 wild-type or c b c sntdet association to due not is n EGFP–PEX11 and opeiiae with co-precipitated b rEGFP–PEX11 or b dniidwere identified c P158A c c mutations. c interacts –FLAG, c c mdl)adamttdvrini hc rln a nrdcda oiin182, position at introduced was proline a which in version mutated a and (middle) ,we were c b or in c c c c , oee,tec-uiidaonswr ihrwt PEX11 with higher PEX11 were When amounts 6C). (Fig. co-purified the however, non- the to similar amounts in PEX11 mutated hFis1 co-precipitated FLAG xetdfo h oimnpeiiaineprmns we experiments, co-immunoprecipitation determine as the membrane peroxisomal unambiguously from the at To resides expected results Mff 6A). of the (Fig. portion to a opposite whether hFis1 in with is This PEX11 obtained 6C). with (Fig. purifications compared affinity as Mff of oehi1wsvsaie nteafnt uiidfractions. EGFP–PEX11 purified of affinity presence the the in in visualized However, was hFis1 more niois u eut hwta pnoeepeso fEGFP– PEX11 of anti-GFP overexpression with upon that PEX11 analysis show blot results western Our antibodies. through bands distinct two hte h E1 rtisas neatdwt f to Mff experiments PEX11 with both pull-down with interacted co-precipitated Our tested Mff also we that fission. show Hence, proteins 2008). peroxisomal studied. PEX11 been Bliek, coordinate not the der has cooperates whether van and proteins peroxisomes PEX11 and the to with localizes (Gandre-Babbe Knock- Mff to whether Mff 2010). However, DRP1 the al., of in elongated et peroxisomes recruits absence that showed (Otera also Mff, experiments membrane down that outer factor, to mitochondrial fission similar mitochondrial disulfide mechanism of a formation Hence, the for proposed require 6A,B). (Fig. not bridges did interactions these PEX11 contrast, PEX11 hFis1, In precipitated human 1996). al., of et version bridge (Marshall a 3 disulfide position a at of cysteine presence through the on with depends homodimerization interacts directly PEX11 indeed of interaction hFis1 that PEX11 demonstrate data PEX11 nvv swl si ir nlssetbihdta the that established analyses vitro in as well as vivo In its that suggested have Pex11p yeast on studies Previous ( E1 rtisadprxsm iso 3819 fission peroxisome and proteins PEX11 A ceai iwo h PEX11 the of view Schematic ) c b c c A182P iteaonso Fs opeiiae ihwild-type with co-precipitated hFis1 of amounts little , n htti neato togydpnso the on depends strongly interaction this that and u hs eeicesduo xrsinof expression upon increased were these but ncnrs,we EGFP–PEX11 when contrast, In . Sc c e1pi nieyfrtehmnPEX11 human the for unlikely is Pex11p FA splmnaymtra i.S) These S2). Fig. material (supplementary –FLAG c ihohrPX1proteins. PEX11 other with c A182P b c n idtp PEX11 wild-type and akn ytiersde C)co- (C0) residues cysteine lacking a sda at qa amounts equal bait, as used was c c eunea ulndi i.2A Fig. in outlined as sequence ol evsaie nthe in visualized be could c PEX11 , c b c a expressed was n PEX11 and hwn that showing c c ed. A182P . c d. b – , Journal of Cell Science 3820 ora fCl cec 2 (16) 125 Science Cell of Journal nispoierc oi,PEX11 motif, proline-rich its in hne)wr nlzdfrteraiiyto ability their for analyzed were channel) rdcanl.Clsepesn PEX11 expressing Cells channel). (red antibodies anti-PEX14 with visualized were Peroxisomes elongation. peroxisome induce myc–hFis1). cells EGFP–PEX11 expressing in category same the of peroxisomes ubri el xrsigPEX11 expressing cells in number Scale 5 blue. bar: in shown are Nuclei channel). 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(green either (red EGFP–Mff using antibodies immunostained anti-FLAG were or Cells anti-PEX14 morphology. peroxisome in changes EGFP–PEX11 of amounts PEX11 iulzdi h fiiyprfctos PEX11 purifications, affinity the in visualized h lae.Hge mut fEF–y–Fs eeotie with obtained were EGFP–myc–hFis1 in of PEX14 PEX11 protein amounts membrane Higher peroxisomal eluates. genuine the the by of separated demonstrated absence were was the (E) solubilization through peptides membrane 3xFLAG of of of Specificity 5% excess SDS-PAGE. and with (I) obtained material starting eluates of the 2% were antibodies. Immunoprecipitations anti-FLAG digitonin. using 0.2% performed containing buffer factors. in fission transfection the of assembly through ( PEX11 division around peroxisome network of interaction rate complex A 6. Fig. Mff when expression, PEX11 hFis1 overexpressed of was effect the to similar for that peroxisomes.Interestingly, Mff, isolated considered exclude contain only peroxisomes we cannot also study co-localization mitochondria mitochondrial our we and near Although present contained Mff 6D). peroxisomes (Fig. the also between visible made that first intersections were we signals Then, overlap signals, Mff an staining. mitochondrial to all and object due removed peroxisomal 3D co-localization on the through unclear exclusively between avoid confirmed present To was was analyses. finding portion 2010). This significant al., et peroxisomes. a that Otera 2008; addition, mitochondria Bliek, der In to van and of localized (Gandre-Babbe overexpression Mitotracker for Mff reported signal previously with marker, as EGFP fragmented mitochondria appeared matrix Most the 6D). peroxisomal stained (Fig. a and and mCherry–Px, EGFP–Mff co-expressed 3822 C A m oimnpeiiain eepromda ecie nA PEX11 A. in described as performed were Co-immunoprecipitations ) E23 el xrsigteidctdpoen eelsd4 after h 48 lysed were proteins indicated the expressing cells HEK293T ) .(ih)Qatfcto fprxsms2dy fe rnfcin( transfection after days 2 peroxisomes of Quantification (Right) m. c b c c P FA rPEX11 or –FLAG A182P , esos ( versions. n EGFP–PEX11 and .0 oprdwt eoioe ftesm aeoyi cells in category same the of peroxisomes with compared 0.001 ora fCl cec 2 (16) 125 Science Cell of Journal m n oprdwt idtp PEX11 wild-type with compared .(ih)Qatfcto fteprxsms2dy after days 2 peroxisomes the of Quantification (Right) m. 5 c FA n PEX11 and –FLAG 0cls * cells; 20 B c oimnpeiiain eepromda described as performed were Co-immunoprecipitations ) n PEX11 and D c c b el oepesn Cer–x(ht channel) (white mCherry–Px co-expressing Cells ) c P FA rPEX11 or –FLAG c c noPLP GPepy.( EGFP-empty). + , eercvrdi h precipitates. the in recovered were oee,uigPEX11 using However, . a niiulrudprxsms indicating peroxisomes, round individual had m .0 oprdwt eoioe ftesame the of peroxisomes with compared 0.001 .( m. c c epesn el i o oti JEP contain not did cells -expressing FA n GPMfwr nlzdfor analyzed were EGFP–Mff and –FLAG idcdprxsm lnain cl bar: Scale elongation. peroxisome -induced c E c A182P c c E23 el otasetdwith co-transfected cells HEK293T ) FA EGFP-empty). + –FLAG A182P rdcanl n GP EGFP–myc– EGFP, and channel) (red c FA opeiiae ohEGFP– both co-precipitated –FLAG oprbeaonso f were Mff of amounts comparable FA rcpttdEGFP–Mff. precipitated –FLAG c F b noPLP c el co-expressing Cells ) rcpttdsubstantially precipitated rwt te mutated other with or FA a expressed. was –FLAG c c e oeogtdand elongated to led A182P c eemnsthe determines FA,smaller –FLAG, c n hFis1 and n 5 b 20 – rmtcices ntenme fsalprxsms(see peroxisomes small a of observed we number Rather, the 7A). (Fig. in JEPs increase of formation dramatic the to nor omto fJP.Atog nbet rtueteperoxisomal (PEX11 the PEX11 of protrude time PEX11 to a number unable of membrane, Although transfection high JEPs. overexpression after of a which h formation 72 of at observed point manifestation also The was peroxisomes 7A). Fig. statistics ce nteprxsmlmmrnssmlrt PEX11 to for required region similar the that membranes implies finding peroxisomal our Thus, the 2E). (Fig. and on peroxisomes to localized acted still version mutated This cysteines. ugs lgtydfeetrl o PEX11 for role and different fission slightly peroxisomal a PEX11-driven suggest in Mff of involvement the eivle natrir tutr rtcigte from them protecting PEX11 structure engineered tertiary we control, a As in might pegylation. others the involved position pegylation, for at accessible be cysteine freely be only to Interestingly, seemed 39 PEX11 2). of (Fig. we cysteines the half reagent, six N-terminal all cysteine-selective of on a accessibility C-termini mPEG, the and action mapped Using N- cytosol. both the of that face confirming mode membrane, peroxisomal their 2011). for al., et (Opalinski some mechanism membrane in peroxisomal 2012). amphipathic a machinery, al., an fission et provide the Huber proteins, 2006; with PEX11 Trelease, interaction and (Koch their cells Lingard the Besides human 2010; to and lead al., plant that yeast, et the in pathways of peroxisomes in members of involved various formation are the that family and shown protein and (Oku been PEX11 pexophagy has growth by It Lazarow, 2009), organelles 2010). and al., the Sakai, of et (Purdue degradation Toro peroxisomes and 2006; 2001) pre-existing al., from et maintained biogenesis (Kim novo division rigorously de ER is via the proliferation cell from of per coordination the peroxisomes through of number The Discussion to fission leading events the of interactions and proliferation. sequence family peroxisome the molecular coordinate protein to of PEX11 seems the the machinery cascade of of regulated members of members between A maintenance between proper organelle. for interplay the machinery subtle proliferation a peroxisome for requirement f oeogtdprxsmswr noe yteepeso of expression the of by co-expression invoked PEX11 upon were hFis1, peroxisomes elongated to no contrast Mff in Moreover, 6E). (Fig. emdt ifrnilyifunetebnigcpblte of capabilities binding the influence PEX11 differentially to seemed is ed oa nrae ubro ml n round of mRFP– and of formation overexpression the small upon Here, to 2010). of PEX11 point, al., et number time Koch PEX11 2010; later to increased similar a clusters an peroxisome at and, to peroxisomes leads PEX11 first of Overproduction peroxisomes. on co-expression ocmtn xrsino mRFP–PEX11 contrast, of In process. 7A). this PEX11 expression (Fig. in role many important concomitant transfection contained an play cells proteins after PEX11 both the of activities h JEPs the PEX11 that to indicating 24 peroxisomes, addition small even in Remarkably, observed were ee epeetadtie nlsso PEX11 of analysis detailed a present we Here, eas ocmtn xrsino PEX11 of expression concomitant Because c c b c c c noPLP A182P otefsinmciey eaaye h fet ftheir of effects the analyzed we machinery, fission the to nprxsm rlfrtosaedfeetadta these that and different are proliferations peroxisome on A182P n EGFP–PEX11 and Fg E i.6) vrl,teefnig confirm findings these Overall, 6F). Fig. 5E, (Fig. neatdwt Fs ugsigthe suggesting hFis1 with interacted ) i ete edt togprxsm elongation peroxisome strong to lead neither did c akn t rln tpsto 182 position at proline its lacking c lnae eoioe n JEPs and peroxisomes elongated , a c hlxwssgetdto suggested was -helix Fg A Dllee al., et (Delille 7A) (Fig. b b n PEX11 and ln e othe to led alone b c b c oooya the at topology C0 n EGFP– and n PEX11 and c eodof devoid c ln its along . b b alone and c c Journal of Cell Science rdea ytie3(asale l,19) oee,our However, 1996). al., et PEX11 (Marshall PEX11 type that 3 confirmed experiments cysteine immunoprecipitation to at similar the protein bridge the of of structure could half the Sc moiety maintaining C-terminal N-terminal in the involved the be in cysteines in Nevertheless, resides protein. elongation membrane xeiet esoe httehdohbcrgosof regions hydrophobic the previous that in showed PEX11 Indeed, we immunoprecipitations. detergent experiments membrane-preserving during mild, the implied use digitonin been to necessity already the required had by is which bond interactions cysteine protein–protein covalent for no that shows this Ultimately, PEX11 PEX11 eesr o rtuinadsbeun iso fthe of fission subsequent curvature membrane and positive inserted protrusion generate is membrane an helix for to amphipathic peroxisomal span cytosol the necessary the Herein, the and 5A). in Fig. from anchor 4, region 3, that (Figs helical helices amphipathic membrane-inserted protein two strengthen or bridges. protein disulfide the transient through of interactions part N-terminal cytosolic e1pwihwspooe ohmdmrz i disulfide a via homodimerize to proposed was which Pex11p ls npcino h -emnlhl fPEX11 of half C-terminal the of inspection Close b c c n Fs Kc ta. 00.I tal h ytie of cysteines the all, at If 2010). al., et (Koch hFis1 and n loPEX11 also and ih ahrsaiieteoealsrcueo the of structure overall the stabilize rather might c n Fs oprfe ihPEX11 with co-purified hFis1 and a eerqie o neato with interaction for required were c C0 Fg 6A,B). (Fig. c revealed b wild- , rtisata ifrn eesi h rcs fperoxisome of process the in levels different at act proteins atr Fg DF.Orfnig htc-rdcino PEX11 fission of to co-production susceptibility PEX11 that their findings and PEX11 Our to 5D–F). in regard (Fig. with factors motif JEPs PLP Moreover, of 5B,C). properties the (Fig. points of time deletion early in at PEX11 increase number an of cis/trans with 158 peroxisome correlated position where and at elongation proline peroxisome motif, the delayed of mechanism. proline-rich mutation regulatory fact, plausible a In a by represent of could regulated isomerization domain be amphipathic might PEX11 the with that interaction show the we in PEX11 controlled Here spatially membrane. PEX11 and interaction region amphipathic of protein–protein its position maintain regions properly to membrane-bound protein the al., enable two et (Koch localization The proper their 2010). for required be to in shown whereas and family segments, protein membrane-buried PEX11 PEX11 two PEX11 the features of membrane-anchored members it other the within because out to Besides, stands PEX11 1) C-terminus. (Fig. of contrast region its amphipathic in at the Noteworthy, proteins PEX11 membrane. peroxisomal ihrEGFP–PEX11 either iso 3.Sl-ciae R1plmrzsadsiso fthe of (4). scission occurs and membrane for polymerizes peroxisomal assembles DRP1 DRP1 Self-activated and (3). elongates fission DRP1 membrane protein peroxisomal membrane dynamin-related The of the (2). site of the recruitment machinery, at allowing fission accumulates elongation, The Mff, (1). and fission hFis1 for including it remodel and membrane mRFP–PEX11 ( expressing cells in category rnfcin pnepeso fEGFP–PEX11 of after expression hours 24 Upon by transfection. 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Fig. B n E1 rtisadprxsm iso 3823 fission peroxisome and proteins PEX11 5 m oe fPEX11 of model A ) .(eo)Qatfcto fprxsms2dy fe transfection after days 2 peroxisomes of Quantification (Below) m. c a 0cls * cells; 20 n PEX11 and side eesr o ohmmrn lnainand elongation membrane both for necessary indeed is b n EGFP–PEX11 and b e oada hntp ugs htteetwo these that suggest phenotype dual a to led b P , rc-xrsigmRFP–PEX11 co-expressing or .0 oprdwt eoioe ftesame the of peroxisomes with compared 0.001 b c igehdohbcrgo a predicted, was region hydrophobic single a c rEGFP–PEX11 or b cigwt PEX11 with acting n Fs Fg AC i.6,) It 6A,B). 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(Fig. PEX11 wild-type PEX11 peroxisomes by to found formation clearly on JEPs only we counteracted which not PEX11-controlled present Mff do with of also be proteins scheme but hFis1, PEX11 the with interact Obviously, in fission. included peroxisomal be to now should mechanism proliferation. a peroxisomal of represent progress the might control of motifs adjustment oligomerization steric with even membrane-bending synchronized to membrane hFis1 or unlikely with of di- interaction are and and events Differential coordinated simultaneously. the be occur must since fission, plausible and elongation is explanation case, Such first PEX11 the case In second PEX11 hFis1. (2) binds PEX11 or efficiently hFis1, and with heterodimerizes, interact cannot and homodimerizes, adequate mutually ensure to not other each are regulation. influence likely mechanisms most two and exclusive these However, membrane. PEX11 PEX11 peroxisomes, While 7A). 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Probes). out Probes). (Molecular (Molecular 33342 al., carried FM Hoechst DABCO et Red were mg/ml Deep 25 Immunofluorescences (Koch Mitotracker with Germany). previously supplemented in Mowiol Karlsruhe, described formaldehyde in 3.7% Roth, embedded with and (Carl fixed min) were (15 cells PBS analysis, microscopic For (Amaxa). . al.Imnpeiiae eeeue rmteclmsuig3 using columns the from ng/ eluted (150 were peptides Immunoprecipitates NaCl). M 1.5 es Wimn nttt fSine sal.Mttoswr nrdcdi the in introduced were mRFP– Mutations Israel). For EGFP– Science, 2006). Jeffrey of original from Institute al., obtained (Weizmann (EcoRI/XhoI) et pcDNA3.1-mRFP Gerst into (Stanley inserted and before amplified described BglII/ was Hs (Clontech, EGFP–C1 EGFP–Px the in EcoRI). of CB396/397) insertion oligonucleotides by (annealed engineered linker EGFP–Mff was a 2010). 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DpnI by pairs by verified followed oligonucleotide S1), Table material with supplementary PCR via rcino P 1 2adM eelae noa D e o etr blot western for gel SDS an onto loaded (100,000 were centrifugation MP Equal and stored. and were min S2 (MP) 30 pellet S1, analysis. membrane for IP, final of and wheel (S2) fraction rotating supernatant the a min), 60 on mixing After ecpotao otesmls qa rcin eelae noa D e and gel SDS an onto loaded blotting. western were by fractions analyzed Equal samples. the to mercaptoethanol PEX11 2 b .Clswr rnfce sn uee Rce rnucleofected or (Roche) FuGene6 using transfected were Cells ). n –PEX11 and Hs E1 nioiswr idgf rmRl rmn (Ruhr Erdmann Ralf from gift kind a were antibodies PEX14 Hs m ;SgaAdih o 0mntsa 4 at minutes 30 for Sigma-Aldrich) l; PEX11 9 c mtyplehln lcl500 im-lrc]or Sigma-Aldrich] 5.000; glycol -methylpolyethylene c h oigsqec fPEX11 of sequence coding the , ecdn lsi hog iedrce mutagenesis site-directed through plasmid -encoding Hs ˚ )adwse xesvl 05MTi-C H7.4, pH Tris-HCl M (0.5 extensively washed and C) PEX11 c , Hs PEX11 ˚ .I oto xeiet,DTT experiments, control In C. g a 0mn,tespraat(S1) supernatant the min), 60 , hlclcnetwspredicted was content -helical c FA,EGFP–myc–hFis1, –FLAG, ˚ .Aiut rmipt (I) inputs from Aliquots C. b n PEX11 and g g 0mn.The min). 60 , i)cells min) 5 , g 0min). 10 , 6 c 2 FLAG CO m were l 3 ˚ b g C ). - , Journal of Cell Science eg . u . a . ag . i,H,Hn . a,J,Ln,Y n Mi, and Y. Long, J., Gao, R., Han, H., Liu, Y., Wang, S., Ma, C., A. Gu, M., R. Feng, Rachubinski, and M. Fagarasanu, A., Fagarasanu, ii,E,Buat . hn,Y,Le .S,Oedl,C,Su,B,Hchn N., Hacohen, B., Shum, C., Odendall, S., A. Lee, Y., Zhang, S., Boulant, E., Dixit, L60 o eprdde.Clswr admycoe,addtco anand gain detector and chosen, laser randomly nm were 633 Cells and for mCherry/mRFP dyes. (BP500–550) for red laser 575–615) deep BP nm or for 488 (LP585 (LP650) staining, laser Hoechst nm for 561 GFP, (BP420–480) 100 laser (Neofluar nm Zeiss 405 LSM510META, 45 a size on pixel acquired were images Confocal analysis image and Microscopy Healthcare. GE from purchased were antibodies anti-rabbit anti-mouse donkey sheep and HRP-conjugated Sigma-Aldrich. from purchased was conjugated) itl . rmn,R,Gud .J,Boe,G,Cae .I,Ceg .M,Dd,G., Dodt, M., J. Cregg, I., D. Crane, G., Blobel, J., S. Gould, R., Erdmann, B., Distel, B. Poole, and B. P. J. Lazarow, G. C., Duve, Barton, de and D. J. Barber, C., Cole, M. R. Epand, and R. Brasseur, M., Decaffmeyer, A., Thomas, S., Aoki, Y. Fujiki, and S. Tamura, K., Okumoto, I., Abe, Y. Fujiki, and I. Abe, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.102178/-/DC1 online and available material Science Supplementary for [grant Ministry C.B.]. to Federal Fund V39-B09 Austrian number Science [grant the Austrian Austrian Research and the the of Fund Elise-Richter-Program by the Science C.B.]; supported to P-20803 was number work This Funding pEGFP-N1-PEX3. plasmid the for David Christine thank We Acknowledgements (area normal either (ij-Plugins.sourceforge.net/plugins/ as clustering classified , were k-means Peroxisomes using Mff segmentation/index.html). segmented residual thresholded was peroxisome projection intensity the of maximum and Quantifications A of ImageJ. calculated. in Intersections performed were were analysis. number objects peroxisomal further with from was objects Mff excluded staining mitochondrial and and contained FM) also removed that Red colocalization Mff-object for Deep each Object created Then (Mitotracker Netherlands), were (EGFP–Mff). ImageJ. objects mitochondria The Briefly, in (mCherry–Px), Professional. (SVI, Huygens peroxisomes adjusted in deconvolved Professional performed and were Huygens was images analysis intensity) of All (maximum algorithm clipping. projected QMLE avoid to the adjusted using were offset amplifier niae sn h icxnrn u et(http://faculty.vassar.edu/lowry/ test sum as mean rank evaluated as was Wilcoxon significance presented statistical the VassarStats.html). The are (s.e.m.). using data mean indicated the The of analyzed. error standard were experiments independent eoioe ( peroxisomes eil,H . gioa . umre,S . ot,H,Lu H., Borta, C., S. Guimaraes, B., Agricola, K., H. Delille, K. J. Hiltunen, T., R. Sormunen, D., V. Antonenkov, Angermu ee’ diameter Feret’s 1 537-540. aayigPo1 i/rn isomerization. cis/trans Pro117 catalyzing H. inheritance. and division of story Biol. a populations: peroxisome hn .J,Wea,S . rne,M tal. et M. immunity. Fransen, innate P., antiviral for S. platforms Whelan, J., Z. Chen, rdcinserver. prediction caveolin-1. of helix re-entrant membrane the 33371-33380. in proline of role uii . oda,J . ut .W tal. et W. factors. W. Just, biogenesis M., peroxisome J. Goodman, Y., Fujiki, amla eoioe olw auainpathway. maturation a M. follows peroxisomes Schrader, mammalian and M. peroxisomes. particles. sensitive osmotically Physiol. are Cell Physiol. peroxisomes J. mammalian Am. vitro: in peroxisomes PEX11. by encoded is 472. protein membrane integral peroxisomal Pex11p. peroxin human 252 the of isoform expressed pce,alsigchallenge. lasting a species, m m 529-533. , 21) os KP3mdae ofre-pcfcfntoso i by BiP of functions conformer-specific mediates FKBP23 Mouse (2011). 2 23 le,S,Ilne,M n Vo and M. Islinger, S., ¨ller, ee’ diameter Feret’s ; 321-344. , 6 . 5n,zsak 0 m 1.6 nm, 200 z-stacks nm, 45 d.Cytopharmacol. Adv. 1 . m 0.75 uli cd Res. Acids Nucleic m 2 19) DAcoigadcaatrzto faconstitutively a of characterization and cloning cDNA (1998). .A es wnyrnol hsnclsfo three from cells chosen randomly twenty least At ). m , ;circularity m; 21) e1peamdae rwhaddvso of division and growth Pex11pbeta-mediated (2010). 0.75 itce.Cl Biol. Cell Histochem. 287 .Cl Biol. Cell J. m 1623-1635. , ;circularity m; ll A. ¨lkl, 2 219-223. , 36 W197-W201. , , Cell 17) ignssadtroe frat-liver of turnover and Biogenesis (1974). .)o Esbsdo h rao the of area the on based JEPs or 0.8) 20) eoioe n ecieoxygen reactive and Peroxisomes (2009). 20) h pe eodr structure secondary 3 Jpred The (2008). ice.Bohs e.Commun. Res. Biophys. Biochem. 135 m 141 ie wl ie 2bt sn a using 12-bit) time, dwell pixel s 19) lfbaeidcbe 28-kDa Clofibrate-inducible, (1998). 1-3. , . 668-681. , 19) nfe oecauefor nomenclature unified A (1996). 21) eoioe r signaling are Peroxisomes (2010). ice.Bohs e.Commun. Res. Biophys. 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Geuze, akr . ae,T,Teg .R,Bre .B n aoio,C G. C. Kalodimos, and B. R. Birge, R., S. Tzeng, T., Saleh, P., Sarkar, Saraya,R.,Krikken,A.M.,Veenhuis,M.andvanderKlei,I.J. B. P. Lazarow, and E. P. Purdue, H. E. Hettema, and M. A. Motley, oe,D T. D. Jones, ats .J,Qitnla .A,Tr,A,Gad,R,Dnmra .C,Godoy, C., M. Dinamarca, R., Grandy, A., Toro, A., R. Quintanilla, J., M. Santos, D. Baker, and J. Meiler, ubehs .J,VnVlhvn .P,Bes . anet,G . o,G .and V. G. Los, P., G. Mannaerts, C., Brees, P., P. Veldhoven, Van J., S. Huybrechts, estv ooieiaino Pex11 of homodimerization sensitive smrzto samlclrtimer. molecular a as isomerization aggregation. or duplication 119 elongation, peroxisome promote individually 20b.PX1bt eiinyi ehladipisnuoa irto u osnot does but migration neuronal impairs function. and peroxisome lethal abrogate is deficiency beta peroxisome PEX11 (2002b). for dispensable is proliferation. but peroxisome Biol. 4-phenylbutyrate alpha-mediated to receptor J. proliferator-activated response S. 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