A role for the anaphase-promoting complex inhibitor Emi2XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of Xenopus eggs Jeffrey J. Tung* †, David V. Hansen*†, Kenneth H. Ban* †, Alexander V. Loktev*, Matthew K. Summers*, John R. Adler III*, and Peter K. Jackson* †‡ *Department of Pathology and †Program in Cancer Biology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305 Communicated by Marc W. Kirschner, Harvard Medical School, Boston, MA, February 9, 2005 (received for review November 30, 2004)
Unfertilized vertebrate eggs are arrested in metaphase of meiosis II wi with th hig high h cyc cyclinB linB Cdc2activity to prev prevent ent part partheno henogene genesis. sis. Unti Untill fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous en ous Emi Emi1 1 is un unsta stable ble in CSF CSF-ar -arres rested ted Xenopus eg eggs gs an and d is TrCP destroyed by the SCF ubiquitin ligase, suggesting that endogenouss Emi1, an appa enou apparent rent 44-kDa prot protein, ein, requires requires a stab stabilizi ilizing ng factor. facto r. Howe However, ver, anti anti-Emi1 -Emi1 anti antibodi bodies es cross crossreact react withnative Emi2 Erp1FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner man ner in resp response onse to calciu calcium-me m-mediat diated ed egg activ activatio ation. n. Thes These e results identify Emi2 as a candidate CSF maintenance protein.
Upon fertilization of Xenopus eggs, calcium signaling inactivates CSF arrest, which requires the Xenopus Polo-like kinase 1 (Plx1). Thee tar Th targe gett of Pl Plx1 x1 in thi thiss pa pathw thway ay re rema main inss un unkno known wn (1 (13).In 3).In hu huma man n somatic cells, MPF and human Polo-like kinase 1 (Plk1) target Emi1 for degradation by the Skpl Cullin F-box protein (SCF)TrCP ubiquitin ligase (14–17). Specifically, Plk1 phosphorylates Emi1 on its DSGxxS sequence, creating a consensus degron recognized by TrCP (17). Thus, Xenopus Emi1 (xEmi1) could be a Plx1 target downstr dow nstream eam of cal calciu cium m sig signal naling.An ing.An app appare arent nt para paradox dox is how Emi Emi1 1 levels are sustained in the CSF-arrested egg amid high MPF and Plx1 Plx 1 act activit ivitie ies. s. In lin linee with thisparado thisparadox, x, a rec recent ent rep report ort sug sugges gests ts tha thatt Emi1 is unstable and undetectable in Xenopus eggs (18). On the other hand, Emi1 appears to be prese present nt in mouse eggs (10). In this study, we want to clarify our understanding of Emi1 regulation in eggs gs an and d fi find nd tha thatt Em Emi2 i2,, an Em Emi1 i1 ho homol molog,may og,may con contr trib ibut utee Xenopus eg to CSF arrest.
cyclin B meiosis maturation-pro maturation-promoting moting factor oocyte maturation
Methods
T
o prev prevent ent part partheno henogene genesis, sis, unfe unfertil rtilize ized d eggs from many animals anima ls arrest in metap metaphase hase of mei meiosis osis II (MII (MII). ). Sperm penetration triggers the release from metaphase arrest and the commencem comme ncement ent of alte alternat rnating ing cycle cycless of DNA repl replica ication tion and cell division in the embryo. The regulatory basis for metaphase II arrest was first characterized in frog eggs 30 years ago and termed cytostatic factor (CSF) (1). CSF is operationally defined as an activity, rather than a single molecule, present in unfertilized eggs that blocks cleavage of dividing blastomeres upon injection (reviewed in ref. 2). Mos, an activator of the mitogenactivated protein kinaseRsk pathway, is a key component of CSFthat app appear earss at theonset of me meios iosis is I (MI (MI)) andacti andactivat vates es CSF to block cleavage of blastomeres (3). The anaphase-promoting complex (APC) is an E3 ubiquitin ligas li gasee tha thatt tri trigge ggers rs MM-pha phase se exit by dir direc ectin tingg pro protea teasom someedependent cyclin B destruction (4), resulting in the swift inactivation of the cyclin B Cdc2 kinase, or maturation- promoting factor (MPF) (5, 6). A rise in intracellular calcium after fertilization induces metaphase II release by relieving the APC from repression. Early mitotic inhibitor 1 (Emi1), originally cloned from a Xenopus oocyte cDNA library, blocks the cleavage of injected blastomeres similar to CSF (7) and efficiently inhibits the APC in vitro (8). Recently, Emi1 was shown to be required for maintenance of CSF arrest in frog and mouse eggs. Immunodepletion nodepleti on of Emi1 from Xenopus CSF egg extract causes rapid cyclin B proteolysis and exit from metaphase arrest independent of calcium mobilization, mobilization, and ablation of Emi1 by small interfering RNA in mous mousee oocyte oocytess indu induces ces parthenoge parthenogenes nesis is (9, 10). Recent work has shown that the Mos mitogen-activated protein kinaseRsk pathway establishes, establishes, but is not required to maintain, CSF arrest (11, 12). Therefore, CSF arrest is a complex process established by the mitogen-activated protein kinase pathway and maintained through inhibition of the APC. 4318–4323 PNAS March 22, 2005 vol. 102 no. 12
Reagents. Sera from four rabbits immunized with maltose binding
protein (MBP)protein (MBP)-Emi1 Emi1 fusion protein were affinity-purified by f lowing over a column of GST-Emi1 immobilized on CNBr-Sepharose resin resi n with acid eluti elution. on. Other antibo antibodies dies used were against -catenin, cyclin B2, Plx1, Plk1 (Zymed), myc epitope, and actin (Santa Cruz Bio Biotec technol hnology). ogy). xEm xEmi2 i2 was PCRPCR-clo cloned ned from an oocyte cDNA library, and a human Emi2 (hEmi2) clone was purchased from Invitrogen. pCS2-cDNA constructs were linearized and in -trans anscri cribed bed to gen genera erate te mRN mRNA A by usi using ng a mMe mMessa ssage ge Mac Machin hinee vitro-tr kit (Ambion, Austin, TX). pCS2-cDNA constructs were in vitrotranslated transl ated (IVT) in rabbit reticulocyte lysate (TNT, Promega) and labeled with 35S-methionine. All Emi1 and Emi2 experiments used sequen uence cess unl unles esss othe otherwise rwise note noted d as hEm hEmi1 i1 and hEm hEmi2 i2 for Xenopus seq human sequences. MBP-fusion proteins and GST-Plk1 were expressed in Escherichia coli and purified by batch binding bacterial protein lysate to affinity resin and elution with maltose or glutathione, then dialyzed into XB buffer (20 mM Hepes, pH 7.7 100 mM KC KCl) l).. Po Poin intt mu mutat tatio ions ns we were re en engi gine neer ered ed wit with h a Qu Quik ikCha Change nge kit (Stratagene). Handling of Xenopus Oocytes. Oocyte Oocytess wer weree obtai obtained ned and pro proces cessed sed
for H1 kinase activity and immunoblot as described (19). Oocytes were injected with 30 ng of MBP-Emi1 fusion protein or 10 ng of various mRNA in total volumes not exceeding 50 nl. Maturation Freely available online through the PNAS open access option. Abbreviations: APC, anaphase-promoting complex; CHX, cycloheximide; CSF, cytostatic factor; Emi, early mitotic inhibitor; hEmi, human Emi; xEmi, Xenopus Emi; GVBD, germinal vesicle breakdown; IVT, in vitro-translated; MI, meiosis I; MII, meiosis II; MBP, Maltose binding protein; MPF, mitosis-promoting factor; Plk1, human Polo-like kinase 1; Plx1, Xenopus Polo-like kinase 1; SCF, Skpl Cullin F-box protein. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AY928267). ‡To whom correspondence should be addressed. E-mail:
[email protected].
© 2005 by The National Academy of Sciences of the USA
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Fig. 1. Characterization of anti-Emi1 antibodies. ( A) A 44-kDa 44-k Da prot protein ein is reco recognize gnized d by affin affinityity-purifi purified ed anti anti-Emi1 antibodies antibodies in CSFCSF-arres arrested ted eggs eggs.. CSF extr extract act was immunoblotted with affinity-purified antibodies from four rabbits immunized against Emi1. ( B) Anti-Emi1 antibodies bodi es immu immunopr noprecip ecipitateexpresse itateexpressed d Emi1 Emi1.. IVT mycmyc-Emi1 Emi1 wasimmunopreci wasimmunop recipita pitated(IP) ted(IP) by reac reactivesera tivesera andaffinity andaffinity-purified antibodies, but not by preimmune (PI) sera. ( C ) Emi1 antibody antibody recog recognitio nition n of the 44-kD 44-kDa a speci species es is blocke blo cked d wit with h ant antige igen. n. CSFextra CSFextract ct wasblott wasblotted ed wit with h affi affinnity-purifi itypurified ed anti antibodythat bodythat was unbl unblocke ocked d or bloc blocked ked with increasin incr easing g purifi purified ed MBP-E MBP-Emi1fusionproteinup mi1fusionproteinup to 10-f 10-fold old molarr exce mola excess ss overantibod overantibody y or bloc blocked ked with20-fold mola molarr excess of MBP protein over antibody. ( D) Each of the anti-Emi1 antibodies detects the same 44-kDa protein. Immunopr Immu noprecipi ecipitatesfrom tatesfrom CSF extra extract ct with two anti anti-Emi1 -Emi1 antibodi anti bodies es orcontrolIgG wereimmun wereimmunoblo oblottedwiththree ttedwiththree anti-Emi anti -Emi1 1 anti antibodi bodies. es. ( E ) Anti Anti-Emi -Emi1 1 anti antibodydetects bodydetects conserved Emi1 localization to the spindle poles. Metaphase chromosomes, spindles, and Emi1 were visualized in Xenopus somatic XTC cells, human U2OS cells, and human HCT116 HCT1 16 cell cellss by fluor fluoresce escence nce micr microsco oscopy. py. The merg merged ed images show show DNA (blue), (blue), -tub -tubulin ulin (red (red), ), and Emi1 (green (green). ). (Magnification: 63.) (F ) Addition of anti-Emi1 antibody to CSF extract induces chromatin decondensation, MPF inactivat inac tivation, ion, and cycli cyclin n B dest destruct ruction ion with without out calc calcium ium addition.CSF diti on.CSF extr extractwas actwas supp suppleme lementedwithCHX ntedwithCHX andsperm and treated with anti anti-Emi -Emi1 1 anti antibodi bodies es or cont control rol IgG. After 60 min, sperm chromatin was stained with Hoechst and visualized by epifluorescenc epifluorescence e microscopy. Similar extract tra ct wa wass inc incuba ubatedwith tedwith ant anti-E i-Emi1antib mi1antibodi odies es or IgGand incubate incu bated d for60 minbeforeadditio minbeforeaddition n of calci calcium um to trig trigger ger MII exit exit.. Timepointswere proc processedfor essedfor hist histoneH1 oneH1 kina kinase se activity and immunoblot analysis. A nonspecific band ( *) recogniz reco gnized ed by the anti anti-cyc -cyclin lin B2 anti antibodyserves bodyserves as a load load-ing control.
was induced by treating oocytes with 10 gml progesterone. Eggs were activated with A23187 ionophore (Sigma). Destructi Dest ruction on and APC Ubiqui Ubiquitina tination tion Assays Assays.. Egg ext extrac ractt was pre prepar pared ed
as described (20). Destruction assays and in vitro APC ubiqu ubiquitinaitination reactions were performed as described (8). Immunodepletion and in Vitro Phosphorylation Assays. Plx1 immunodepletion, Plk1 in vitro kinase reactions, and TrCP binding
assays were performed as described (17). Immunofluorescence Microscopy. Staining of Emi1 in a Xenopus
cell line (XTC) and human cell lines was performed as described (7, 21). Results Characterization of Anti-Emi1 Antibodies. To examine Emi1 expres-
sion levels, levels, high titer sera selected selected f rom the best four of six rabbits immuniz imm unized ed with reco recombi mbinan nantt MBP MBP-Em -Emi1 i1 fus fusion ion pro protei tein n wer weree purified against immobilized GST-Emi1 by affinity chromatography. These four affinity-purified affinity-purified antibodies antibodies (ab1–4) (ab1– 4) vary in affinity and an d spe speci cifi fici city ty bu butt ea each ch de dete tect ctss a ba band nd cor corre resp spond ondin ingg to th thee cor corre rect ct molecular mass of 44-kDa Emi1 in CSF extract (Fig. 1 A). All four antibodies, using crude serum or affinity-purified antibodies, immunoprecipitate IVT myc-Emi1 (Fig. 1 B). We tested whether the 44-kDa band in CSF extract recognized by ab1, the most specific and highest-affinity antibody of the four in hand, is indeed Emi1. Preincubating ab1 with increasing MBPEmi1 protein almost completely blocked detection of the 44-kDa band (Fig. 1C). Incubating ab1 with MBP at twice the blocking concentr conc entrati ation on of MBP MBP-Em -Emi1 i1 did not blo block ck reco recogniti gnition on of the 44-kDa species. Moreover, blotting ab1 and ab2 immunoprecipitates from egg extract with ab1–3 showed that all three antibodies Tung et al .
recognize the same 44-kDa band (Fig. 1 D). Using ab1, we estimate the concentration of 44-kDa Emi1 in CSF extract to be 50 nM (Fig. 7, which is publi published shed as supporting information on the PNAS web site), somewhat lower than our previous estimate of 300 nM (9). Additional validation demonstrated that these Emi1 antibodies detect overexpressed Emi1 and the endogenous 44-kDa protein in oocytes, embryos, and XTC cells (Fig. 7; see also Fig. 8, which is published as supporting information on the PNAS web site). To val valida idate te the anti antibody body ab1 fur further ther,, we exa examin mined ed the sub subcel cellul lular ar localizati local ization on of Emi1 in XTC cells by immun immunofluorescen ofluorescence ce microscopy. hEmi1 localizes specifically to the spindle poles in a variety of human cell lines (Fig. 1 E and ref. 21). Importantly, this conserved and specific localization of Emi1 at the spindle poles is observed by ab1 stai staining ning in mit mitoti oticc XTC cel cells ls in agre agreeme ement nt with pre previou viouss stu studie diess (7). Emi1 depletion in human cell lines by small interfering RNA abolishes aboli shes the detection of Emi1 at spindl spindlee poles (data not shown). However, we could not validate ab1 in a similar fashion because we have found that XTC cells are refractory to small interfering interfering RNA delivery. To fun functi ctional onally ly val valida idate te the ant anti-E i-Emi1 mi1 anti antibodi bodies es,, we dete determin rmined ed whethe whetherr neu neutral tralizi izing ng Emi Emi1 1 in CSF extr extract act tri trigge ggers rs cal calciu ciummindependent metaphase release. Addition of ab1, but not control IgG, to CSF extract triggered rapid decline of cyclin B2 levels and Cdc2 activity and induced morphological decondensation of sperm nuclei nuc lei (Fi (Fig. g. 1 F ). ). Thi Thiss effe effect ct is spe specif cific ic bec becaus ausee MBP MBP-Em -Emi1 i1 can bloc block k the effect of ab1 on meiotic progression (27). Taken together, the above immunological evidence and conserved localization of Emi1 suggest that these antibodies most likely detect Emi1 or a highly related relat ed protein in the oocyte, CSF-arrested CSF-arrested egg, embryo, and XTC cells. Exogenous Emi1 Is Destroyed in CSF-Arrested Eggs. In mitotic egg
extract extr act prep prepare ared d by addi adding ng nonde nondestruc structibl tiblee
90
cyclin B to
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kinetics in mitotic and CSF extract ( t1/2 15 min; Fig. 2 A). In contrast, IVT Emi1 is stable in interphase extract for 120 min (data not shown). hEmi1 is also destroyed in CSF extract (Fig. 9, which is published as supporting information on the PNAS web site). IVT Emi1 mutated in a critical serine residue residue (Ser-95) of the consensus TrCP recognition degron is stable in CSF extract (Fig. 2 B), indicating that CSF extract contains the factors required to ubiquitinate and destroy exogenous Emi1 through its DSGxxS sequence sequence.. Emi1 Emi 1 lev levels els in the egg may reflect the steady-state steady-state accumuaccumulation lat ion of uns unstabl tablee Emi Emi1 1 pro protei tein. n. The Theref refore ore,, we exa examin mined ed whether translation of myc-Emi1 mRNA in CSF would allow Emi1 Em i1 to acc accum umul ulate ate.. Tra Transl nslat ated ed WT Em Emi1 i1 acc accum umul ulate atess to modest steady-state levels (Fig. 2 C), whereas nondestructible Emi1 Emi 1 S95N mutant accum accumula ulates tes to incr increas easingl inglyy high leve levels, ls, sugge su ggesti sting ng tha thatt WT Emi Emi1 1 is sim simul ultane taneou ousl slyy tra transl nslate ated d and destroyed. To assess the stability of translated exogenous Emi1 directly, cycloheximide cycloheximide (CHX) was added to the CSF extract 2 h after transcript addition. WT Emi1 is rapidly destroyed ( 15 min) in contrast to stable Emi1 S95N (Fig. 2 C). Thus, newly synthesized Emi1 appears to be dynamically accumulated by a balance bal ance of tran translat slation ion and des destruct truction, ion, but could pote potentia ntially lly accumulate to higher levels if a pool of the protein was sequestered from destruction. G2 oocytes contain a stockpile of inactive MPF that is robustly activated at the onset of germinal vesicle breakdown (GVBD) MI in response to hormonal stimulation (22). Thus, Emi1 may have important functions functions in G2-MI oocytes but could be destr destroyed oyed after MPF activation after MI. We injected WT Emi1 or Emi1 S95N mRNA mR NA in into to G2 oocyte oocytess and indu induced ced mat matura uration tion with prog proges ester terone. one. Both Bo th WT an and d mu mutan tantt S95 S95N N ec ectop topic ic Em Emi1 i1 ar aree sta stabl blyy exp expre resse ssed d in the G2 oocyte oocyte,, alt althou hough gh Emi Emi1 1 S95N accumulat accumulates es at muc much h high higher er steady ste ady-sta -state te lev levelsthan elsthan WT Emi Emi1 1 in the CSFCSF-arr arres ested ted egg (Fi (Fig. g. 2 D). Expression of dominant negative TrCP (TrCPF) missing the F-box domain enables exogenous WT Emi1 protein to accumulate to similar levels as Emi1 S95N in the MII egg (Fig. 2 D). Thus, SCFTrCP is act active ive in the egg and directs directs exo exogeno genous us Emi1 for proteolysis. proteol ysis. The stabili stability ty of both endoge endogenous nous and exogeno exogenous us Emi1 in the G2 oocyte would be consistent with a role in stabilizing stabilizing APC substrates substr ates during G2-MI or possibly for the MI–MII transition, as seen in mouse (12). Fig. 2. Exog Fig. Exogenou enouss Emi1 prot protein ein is dest destroyedthrough royedthrough SCF TrCP in CSFextra CSFextract; ct; nonetheless, Emi1 can accumulate by de novo translation. ( A) IVT Emi1 is destroyed in mitotic extract and CSF extract. IVT, radiolabeled Emi1 was incubated in mitotic 90 cyclin B extract or CSF extract and processed for autoradi auto radiogra ography phy at the indic indicatedtimes. atedtimes. ( B) Dest Destruct ruction ion of exog exogenou enouss Emi1in CSF extract is conserved and DSGxxS sequence-dependent. sequence-dependent. IVT [ 35S]Met Emi1 or Emi Emi1 1 S95 S95N N wasadde wasadded d to CSFextra CSFextract ct andproce andprocesse ssed d forautor forautoradi adiogr ograph aphy y at the indicated times. ( C ) Accumulation of exogenous Emi1 protein translated in CSF egg extract. myc-Emi1 mRNA (WT or S95N mutant) was added to CSF extract with or without CHX and processed for immunoblot at the indicated times. time s. ( D) Exoge ExogenousEmi1 nousEmi1 dest destructi ruction on in meta metaphas phase e II eggsis medi mediatedby atedby the SCFTrCP ligas ligase. e. Stag Stage e VI oocy oocytes tes wereinjecte wereinjected d withmyc-Emi withmyc-Emi1, 1, mycmyc-Emi1S95N, Emi1S95N, or simultaneously with myc-Emi1 and TrCP F mRNA. Injected oocytes were left at G 2 arrest or matured by progesterone stimulation and processed for immunoblot. ( E ) Endogenous Emi1 is protected from destruction in CSF extract. trac t. Puri PurifiedMBP-Emi1proteinwas fiedMBP-Emi1proteinwas adde added d to CSFextractsupplem CSFextractsupplemente ented d with CHX and prepared for immunoblot at the indicated times after additions. ( F ) Endogenous Emi1 is stable in the maturing oocyte. Stage VI oocytes were injected with purified MBP-Emi1 protein and induced to mature by progesterone treatment. Emi1 was detected by immunoblotting lysates from immature oocytes, MI (GVBD) oocytes, and metaphase II eggs.
interphase extract, IVT Emi1 requires MPF for destruction (7). We suspected that although CSF extract contains high MPF activity IVT Emi1 would be refractory to destruction because Emi1 is required to maintain the CSF-arrested state. Instead, we found foun d that radiolabe radiolabeled led I VT Emi Emi1 1 is degra degraded ded w ith similar similar 4320 www.pnas.orgcgidoi10.1073pnas.0501108102
Endogenous Emi1 Is Stabilized in CSF-Arrested Eggs. Next, we deter-
mined whe mined whethe therr en endog dogeno enous us Em Emi1 i1 is sta stabl blee in MI MIII eg eggs gs wit with h charac cha racteri terized zed ant antibo ibodie dies. s. CSF ext extrac ractt was tre treate ated d with CHX CHX,, incubated with MBP-Emi1 protein (300 nM final concentration), and processed for immunoblotting at the indicated times postadditions. dition s. MBP-Emi1 is destroyed with similar kinetics as I VT Emi1 (Fig. (Fi g. 2 E). Stri Striking kingly, ly, levels levels of the 44-kDa protein protein det detect ected ed by anti-Emi1 antibodies remain unchanged for up to 120 min in this destru de structi ction on ass assay. ay. More Moreove over, r, in CHX CHX-tr -treat eated ed CSF ext extrac ract, t, the 44-kDa protein is extremely stable, with no apparent degradation in 48 h (Fig. 9 B). Emi1 stability is also observed in vivo, as the endoge end ogenous nous 4444-kDa kDa ban band d is pre presen sentt thro through ughout out oocyte mat matura uratio tion. n. On the oth other er han hand, d, reco recombi mbinantMBP-E nantMBP-Emi1 mi1 is de destro stroyed yed at GVBD GVBD,, when MPF first appears in MI (Fig. 2 F ). ). Differential stability of endogenous and exogenous forms of proteins is not uncommon. For exa exampl mple, e, IVT -ca -cateni tenin n is comp complet letely ely de destro stroyed yed withi within n 120 min in CSF extract by SCFTrCP, yet endogenous -catenin remains stable (Fig. 10, which is published as supporting information on the PNAS PN AS we web b si site te). ). If the44-k the44-kDa Da ba band nd de dete tect cted ed by an anti ti-E -Emi mi1 1 is in inde deed ed Emi1, these results results suggest that Emi1 is a stable protei protein n in the egg and a mechanism exists to protect it from SCF TrCP. Emi2 Is a Homolog of Emi1 and Crossreacts with Anti-Emi1 Antibodies.
Given that our anti-Emi1 antibodies dramatically inactivate CSF maintenance in egg extract, we considered the possibility that a previously unidentified Emi1 homolog could be immunologically Tung et al .
Fig. 3. Emi2, a homolog of Emi1, is recognized by anti-Emi1 antibodies. ( A) Emi2 is an Emi1-related protein conserved in vertebrate species. A schematic of Emi1 and Emi2 orthologs from human (H.s.), mouse (M.m.), and frog (X.l.) is show shown. n. The cons conserve erved d C-ter C-termina minall F-box and zinczinc-bind binding ing ‘‘in‘‘in-betw betweeneenregion’’ (IBR) domains are boxed. The identified TrCP degrons in hEmi1 and xEmi1 and candidate degrons (DSG A-X2-3-SDE) in Emi2 orthologs orthologs are shown.. ( B) Antishown Anti-xEmi1xEmi1-specifi specificc antibo antibodies dies can immuno immunoprecip precipitate itate native xEmi2, but do not recognize denatured protein. HEK 293T cells were transfected with pCS2 myc-xEmi1 or pCS2 myc-xEmi2. Lysates were prepared after 48 h and either directly blotted with anti-myc antibodies or immunoprecipitated with anti-myc, anti-xEmi1 (Ab1), or control antibodies and then immunoblotted. The band indicated by * is an unknown anti-Emi1 crossreactive species. The band indicated by ** is IgG heavy chain.
crossreactive. A search of TBLASTN for xEmi1 identified a homologous ORF in Xenopus Fbx26. We originally identified Fbx26 from a Xenopus oocyte cDNA library as a highly abundant Skp1 interactor (384 of 444 clones isolated) in the same yeast two-hybrid screen scr een that ide identi ntifie fied d Emi Emi1. 1. Howe However ver,, fram framesh eshift ift err errors ors in the Fbx26 Fbx 26 cDN cDNA A seq sequen uence ce gen genera erated ted an inco incorre rrect ct ORF tha thatt mas masked ked its similarity to Emi1. The correct Fbx26 ORF is entered into the database as Emi2 Erp1 (GenBank accession no. AY928267), a 651-aa protein that is 25% identical to Emi1 that we refer to as Emi2. Human and mouse Emi2 orthologs are given the systema systematic tic namee FBX nam FBXO43 O43 (23) (23).. In ourfollowi ourfollowing ng stu studie dies, s, we focu focuss our att attenti ention on on xEmi2 unless noted otherwise. An alignment of Emi1 and Emi2 orthologs is shown in Fig. 3 A. Residues 434–651 of Emi2 are 35% identic ide ntical al to Emi Emi1, 1, sha sharin ringg conse conserved rved F-b F-box ox and IBR domains. domains. Furthermore, Emi2 has conserved DSG-sequence degrons. To determine whether anti-Emi1 antibodies detect denatured Emi2 in addition to Emi1, anti-myc immunoprecipitates from 293T cells transfected with pCS2 myc-Emi2 or pCS2 myc-Emi1 were processed for immunoblotting with anti-Emi1 antibodies (Fig. 3 B). Although anti-Emi1 antibodies recognize a robust Emi1 band, no signal sig nal was det detect ected ed for Emi Emi2. 2. Equ Equiva ivalen lentt amo amounts unts of myc myc-Em -Emi1 i1 and myc-Em myc -Emi2 i2 wer weree imm immuno unoprec precipi ipitate tated d bec becaus ausee both wer weree eas easily ily detected in a blot with anti-myc antibodies. We concluded that anti-E ant i-Emi1 mi1 ant antibo ibodie diess do not det detect ect dena denatur tured ed Emi Emi2 2 by imm immunob unoblot lot.. On this basis, the 44-kDa band detected in egg extract is most likely not a form of Emi2. Next, we asked whether anti-Emi1 antibodies crossreact with native Emi2. To test this idea, lysates from 293T cells transfected with myc-Emi1 or myc-Emi2 were immunoprecipitated with antiEmi1 antibodies. antibodies. Blotti Blotting ng the antianti-Emi1 Emi1 immunoprecipitates immunoprecipitates with w ith anti-m ant i-myc yc anti antibodi bodies es show showss tha thatt anti anti-Em -Emi1 i1 ant antibod ibodie iess reco recogniz gnizee both nati na tive ve Em Emi1 i1 an and d Em Emi2 i2 (Fi (Fig. g. 3 B). Additi Additionall onally, y, anti-E anti-Emi1 mi1 antibo antibodies dies Tung et al .
Fig.4. Emi2is modi modified,appare fied,apparentlyubiquit ntlyubiquitinate inated d by SCF TrCP, anddest anddestroy royedin edin CSF extract after calcium addition. ( A) Emi2 is destroyed during CSF release. Full-length, radiolabeled IVT Emi2 was incubated in CSF extract with or without Ca2 addition or in interphase extract for the indicated times. ( B) Emi2 is destroyed stro yed thro throughits ughits cons conserved erved TrCPrecogni TrCPrecognitiondegron.Emi2 tiondegron.Emi2 or muta mutants nts in one (DS33AAor (DS3 3AAor DS28 DS283AA)or 3AA)or both(2xDS-A both(2xDS-AA) A) cand candidat idate e degr degron on site sitess wereincubat wereincubated ed in CSF extract and destruction was assayed at the indicated times after calcium addition.( C ) Addi Additionof tionof calc calciumtrigger iumtriggerss Emi2bindi Emi2bindingto ngto TrCPin CSFextract CSFextract.. IVT myc-Emi2 and radiolabeled IVT TrCP were incubated in CSF extract with proteasome inhibitors, with or without calcium addition, for the indicated times. Anti-myc immunoprecipitates immunoprecipitates were analyzed for bound TrCP.
immunoprecip immun oprecipitate itate an IVT C-termin C-terminal al fragment of Emi2 (Fig. 11, which is published as supporting information on the PNAS web site). These results suggest that one feasible explanation for the ability abili ty of the anti-E anti-Emi1 mi1 antibodies antibodies to cause CSF relea release se is through neutralization of Emi2. Emi2 Is Destroyed upon Egg Activation. The immun immunologic ological al cross-
reactivity of Emi1 and Emi2 prompted us to explore whether Emi2 exhibits properties consistent with a candidate CSF maintenance protein. One of Masui and Markert’s (1) original postulates for the identity ide ntity of CSF is tha thatt it is ina inacti ctivat vated ed upo upon n egg activati activation on in response to calcium signaling. To test whether Emi2 fulfills this criterion, we incubated radiolabeled IVT myc-Emi2 in CSF extract in the absence or presence of calcium. The autoradiogram shows that Emi2 is stable in CSF extract but, upon calcium addition, becomes rapidly converted to an electrophoretically retarded form consistent with ubiquitination and is subsequently destroyed (Fig. 4 A). Fur Furtherm thermore ore,, Emi Emi2 2 app appear earss to be phos phosphoryl phorylate ated d by an M-phase kinase, judging by its reduced electrophoretic mobility in CSF extract but not in interphase extract. Emi2 Em i2 has two pot potent entia iall se sequ quenc encee deg degron ronss re recogni cognize zed d by TrCP SCF , one DS 34GxxDS39 at the N terminus and a centrally located DS284 AxxS288 sequence. We asked whether these two sequencess contributed to Emi2 destruction upon CSF release by sequence calc ca lciu ium. m. Whe Wherea reass WT Em Emi2 i2 is rap rapidl idlyy pho phosph sphoryl orylate ated d and ubiquitinated after calcium addition, mutating the N-terminal DSGxxDS degron (DS33A A) prevents Emi2 ubiquitination and PNAS March 22, 2005 vol. 102 no. 12 4321
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destruction destruct ion (Fig (Fig.. 4 B). An Em Emi2 i2 mut mutant ant la lacki cking ng the central central DSAxxS sequence (DS283AA) is ubiquitinated and degraded with with si simil milar ar kin kineti etics cs as WT Emi Emi2 2 in re respo sponse nse to cal calci cium um addition. Consistently, Consistently, the stability of Emi2 mutant lacking both candidat cand idatee degro degrons ns (2xD (2xDS-AA) S-AA) is indi indistin stinguis guishabl hablee from the single sing le DS33 DS33AA AA mutan mutant, t, indi indicati cating ng that DS 34GxxDS39 is the primary degron involved in Emi2 destruction during egg acti vation. Human IVT myc-Emi2 stability is regulated essentially the same way with one obvious exception: hEmi2 contains two destruction motifs that both contribute to calcium sensitivity (Fig. 12 A and B, which is published as supporting information on thePNAS website website). ). Be Becau cause se xE xEmi2appea mi2appears rs to us usee a pot potent ential ial TrCP degron, we asked whether calcium triggers TrCP binding to Emi2. Indeed, calcium addition to CSF extract promoted the binding of radiolabeled TrCP to myc-Emi2 within 5 min (Fig. 4C), consistent with the kinetics of Emi2 ubiquitination. Thiss fin Thi findin dingg sug sugge gests sts tha thatt Emi Emi2 2 req requi uire ress SCFTrCP fo forr ubiquitination. Emi2 Emi 2 Inh Inhibi ibits ts the APC and Is Suf Suffic ficien ientt to Pre Preven ventt CSF Rel Releas ease. e.
Becausee Emi Becaus Emi2 2 is a hom homolog olog of Emi Emi1, 1, a wel welll esta establi blished shed APC inhibitor, inhibi tor, we tested the likely activity of Emi2 as an APC inhibi inhibitor. tor. Full-length Emi2 (MBP-Emi2) or a C-terminal fragment (MBPEmi2 CT) blocks the ability of the APC Cdc20 to polyubiquitinate radiolabeled securin substrate in vitro as effectively as Emi1 (Fig. 5 A). Similar results results were obtained with Cdh1 as the APC A PC activator (Fig. 12C). These results indicate that the C terminus of Emi2, which bears the most identity with Emi1, is sufficient to inhibit the APC. We determined whether Emi2 is sufficient to prevent calciuminduced CSF release. Addition of 2 M MBP-Emi2 to CSF extract inhibi inh ibited ted cycl cyclin in B degr degrada adatio tion n in re respon sponse se to cal calciu cium m and prev prevente ented d sperm nuclei decondensation decondensation (Fig. 5 B). The slow partial decline in cyclin B levels is most likely the result of calcium-induced Emi2 destruction. The 2 M conce concentra ntration tion of MBP MBP-Em -Emi2 i2 use used d her heree appears to saturate the destruction machinery and suffices to block CSF release in respo response nse to calci calcium. um. Finally, we examin examined ed whether Emi2 destruction is a prerequisite for CSF release in vivo. Calcium ionophore A23187 triggers the decline in cyclin B2 levels and H1 kinase activity in matured eggs injected at G 2 with water or WT Emi2 Emi 2 mRN mRNA, A, but not nond nondest estruct ructibl iblee Emi Emi2 2 2xD 2xDS-AA S-AA mRN mRNA A (Fi (Fig. g. 5C). Together with the finding that Emi2 inhibits the APC in vitro, these results suggest that Emi2 is an APC inhibitor that must be destroyed for CSF release. Emi2 Is a Plk1 Target During CSF Release. Another potential parallel
between Emi1 and Emi2 is regulation by Plk1. Given that Plx1 is requir req uired ed for Emi Emi1 1 pro proteol teolysi ysiss in mit mitoti oticc egg ext extrac ractt (17 (17), ), we supposed that Emi2 destruction during egg activation is a Plx1dependent process. Two rounds of immunodepletion using antiPlx1 antibodies effectively removed Plx1 from CSF extract (Fig. 6 A). Consistent with prior work (13), calcium addition to Plx1depleted, but not mock IgG-depleted, CSF extract failed to trigger cyclin B2 destruction and H1 kinase inactivation inactivation (Fig. 6 B). Radiolabeled label ed I VT myc-Emi2 is rapidl rapidlyy destro destroyed yed upon calci calcium um addition to mock-depleted CSF extract, but remains stable in Plx1-depleted CSF CS F tr trea eate ted d wit with h ca calc lciu ium m (F (Fig.6 ig.6C). Fin Finall ally, y, we det determi ermined ned whe whether ther Plk1 Plk 1 pro promote motess Emi Emi2 2 bind binding ing to TrCP in vi Autora oradio diograp graphy hy of vitr tro o. Aut anti-MB anti -MBP P immu immunopr nopreci ecipitat pitates es show showss that const constitu itutive tively ly acti active ve GST-Plk1 GST-Pl k1 (T210D mutant) protei protein n enhance enhancess the binding of radiolabeled TrCP to full-length MBP-Emi2 fusion protein but not to the C terminus of Emi2 lacking TrCP degrons (Fig. 6 D). These resul re sults ts sug sugges gestt thatPlx1 is req requir uired ed for Emi Emi2 2 des destruc tructio tion n in re respon sponse se to calcium signaling by stimulating Emi2 and TrCP binding. Discussion During our studies we learned that Ohsumi et al. (18) raised an antibo ant ibody dy aga agains instt Emi Emi1 1 and failed failed to dete detect ct a 4444-kDa kDa band by 4322 www.pnas.orgcgidoi10.1073pnas.0501108102
Fig. 5. Emi2 inhibits the APC Cdc20 complex and blocks exit from CSF arrest. ( A) Emi2 is an APC inhibitor. inhibitor. Recombinant Recombinant hEmi1, hEmi2, an hEmi hEmi2 2 C-te C-termin rminal al fragment (residues 541–708), or control (MBP) proteins (2 M) were tested for theirr abili thei ability ty to inhi inhibitthe bitthe in vitro ubiq ubiquiti uitinatio nation n of huma human n secu securinby rinby theAPC Cdc20 complex. ( B) Emi2 is sufficient to prevent CSF release. Addition of excess hEmi2 protein blocks the calcium-induced exit from MII in CSF extract. CSF extract was initiated to exit MII by calcium addition and assayed for cyclin B2 destruction (above)or (abo ve)or theformati theformation on of inte interpha rphase se nucl nuclei ei (bel (below).Additio ow).Addition n of MBP-h MBP-hEmi2to Emi2to extract blocked MII exit. ( C ) CSF release requires Emi2 destruction. Injection of nondestructible xEmi2, but not WT, into maturing oocytes prevents MII exit. In vitro-transcribed xEmi2 (WT or nondestructable 2x DS-AA mutant) was injected into oocytes. Oocytes were matured with progesterone and harvested at GVBD and various times afterward. At 3 h post-GVBD, oocytes were released from MII arrest by addition of calcium ionophore A23187. Samples were immunoblotted for cyclin B2 and assayed for H1 kinase activity.
immunoblot in the egg or developing embryo until gastrulation. Consistent with our results, Ohsumi et al. observed the destruction of exogenous Emi1 in CSF extract and maturing oocytes. However, they concluded from the inability to detect endogenous Emi1 and the observation that exogenous Emi1 is unstable in the egg that Emi1 does not and cannot possibly exist in Xenopus until gastrulation. However, no evidence is provided that the 44-kDa band, which Ohsumi et al. only see appearing at 10 –12 h postfert postfertiliza ilization tion in the embryo, is indeed Emi1. The species they see is induced at gastrulation, shortly after zygotic transcription is activated (24), but no specific validation or blocking experiment of the endogenous band shows this species is Emi1. To shed light on this discrepancy in greater detail, we characterized four antibodies raised against Emi1 and present evidence that the 44-kDa species detected by these antibodies, presumably Emi1, is present in the egg. Importantly, another research group Tung et al .
Fig.6. Plx Plx1 1 isrequ isrequire ired d forthedest forthedestruc ructio tion n ofEmi2 ofEmi2.. ( A) Deple Depletionof tionof Plx1from eggextract.CSF extr extract act was eith either er mock mock-dep -deplete leted d withIgG bead beadss or depl depleted eted with Plx1 antibodies for two rounds of depletion. Depleted extract or beads were immunoblotted for Plx1 protein. ( B) Depletion of Plx1 prevents CSF release. CSF extract depleted as above was assayed for cyclin B2 destruction andH1 kin kinaseinact aseinactiva ivatio tion n wit with h or wit withou houtt cal calciu cium m add additi ition.( on.( C ) Dep Deplet letionof ionof Plx1 stabilizes Emi2 in calcium-stimulated CSF extract. CSF extract depleted as above was incubated with IVT Emi2 with or without calcium addition, and samples were collected for autoradiography at the indicated times. ( D) Plk1 stimulates the binding of hEmi2 to TrCP.
readily detects Emi1 at constant levels during oocyte maturation with an independently raised antibody (T. Lorca, personal communication). Ohsumi et al. propose that Emi1 cannot exist in the egg because exogenous Emi1 is destroyed. However, we show here that, although unstable, newly synthesized Emi1 can accumulate to detectable steady-state levels. During its synthesis, Emi1 could be sequestered by some cellular structure or stabilizing factor that would would all allow ow hig higher her lev levels els of accu accumul mulati ation. on. The ins instabi tability lity of exogenous -catenin in egg extract provides an example of an unstable protein that is sequestered in a stable complex (organized 1. 2. 3. 4.
Masui, Y. & Markert, Masui, Markert, C. L. (1971 (1971)) J. Exp. Zool. 177, 129–145. Tunquist, Tunqui st, B. J. & Mall Maller, er, J. L. (2003) Genes Dev. 17, 683–710. Sagata,, N., Watanabe, N., Vande Woude, Sagata Woude, G. F. & Ikawa, Y. (1989) Nature 342, 512–518. King, R. W., Peters, J. M., Tugendreich, S., Rolfe, M., Hieter, P. & Kirschner, M. W. (1995) Cell 81, 279–288. 5. Gautie Gautier, r, J., Norbury, C., Lohka, M., Nurse Nurse,, P. & Maller, J. (1988) Cell 54, 433–439. 6. Gau Gautie tier,J., r,J., Min Minshul shull,J., l,J., Lohka Lohka,, M.,Glot M.,Glotzer zer,, M.,Hunt M.,Hunt,, T.& Mal Maller ler,, J.L. (19 (1990) 90) Cell 60, 487–494. 7. Reimann, J. D., Freed, E., Hsu, J. Y., Kramer, E. R., Peters, J. M. & Jackson, P. K. (2001) Cell 105, 645–655. 8. Reimann, J. D., Gardner, B. E., Margottin-Goguet, F. & Jackson, P. K. (2001) Genes Dev. 15, 3278–3285. 9. Reim Reimann, ann, J. D. & Jackso Jackson, n, P. K. (2002) Nature 416, 850–854. 10. Paronetto, M. P., Giorda, E., Carsetti, R., Rossi, P., Geremia, R. & Sette, C. (2004) EMBO J. 23, 4649–4659. 11. Bhatt Bhatt,, R. R. & Ferrell, Ferrell, J. E., Jr. (1999) Science 286, 1362–1365. 12. Tunquis Tunquist, t, B. J., Schwab, M. S., Chen, L. G. & Maller, J. L. (2002) Curr. Biol. 12, 1027–1033. 13. Descom Descombes, bes, P. & Nigg, E. A. (1998) EMBO J. 17, 1328–1335. 14. Margottin-Goguet, F., Hsu, J. Y., Loktev, A., Hsieh, H. H. M., Reimann, J. D. & Jackson, P. K. (2003) Dev. Cell 4, 813–826.
Tung et al .
at the ce cell ll cor corte tex) x).. Th Thee si simpl mples estt exp expla lana nati tion on for Oh Ohsu sumi mi e data ta ett al.’s da is that the antibody used in the study failed to detect endogenous Emi1 and these negative data by themselves are insufficient evidence to support the idea that the protein does not exist. On the other hand hand,, we rem remain ain cautious cautiously ly ske skeptic ptical al tha thatt the 44-kDa species detected by our antibodies is unambiguously Emi1 for the sim simple ple reason reason tha thatt Xeno organi anism sm Xenopus pus laev laevis is is not an org allowing a direct gene knockout strategy to definitively settle this matter. Nonetheless, our functional evidence strongly suggests that Emi1 Emi 1 andor an imm immuno unolog logica ically lly cro crossr ssreac eactiv tivee prot protein ein is imp importa ortant nt in CSF arrest. Bearing in mind that new synthesis of B-type cyclins was originally nal ly thou thought ght to be none nonesse ssenti ntial al for Xenopus oocytematura oocytematuratio tion n (25) until the discovery of three additional cyclin B members a decade later lat er (26) (26),, we cons conside idered red the exist existenc encee of unid unident entifi ified ed Emi Emi1 1 homologs highly plausible. In this study, we identify Emi2 as an Emi1 Emi 1 hom homolo ologg that cros crossre sreact actss with ant antibod ibodie iess rai raised sed aga agains instt full-length Emi1 in immunoprecipitation experiments but not in immunoblots. Thus, we conclude that the 44-kDa band detected by anti-Emi1 antibodies in eggs is unlikely to be a form of Emi2, but loss-of-func lossof-function tion experime experiments nts using anti-E anti-Emi1 mi1 antibod antibodies ies could have simulta sim ultaneo neousl uslyy or exc exclus lusive ively ly inhi inhibite bited d Emi Emi2. 2. Thi Thiss concl conclusi usion on rai raise sess the possibility that the calcium-independent CSF release caused by Emi1 immunodepletion in our previous work (9) and antibody additi add ition on expe experim riment ent (Fig (Fig.. 1 F ) is a re resu sult lt of in inac acti tiva vati ting ng Em Emi2 i2,, wh whic ich h is a candidate CSF maintenance protein. In support for a role of Emi2 in CSF arrest, we show here that ( i) Emi2 is an A PC inhibitor sufficient suffici ent to prevent CSF release, (ii) Emi2 destru destruction ction through its SCFTrCP recognition sequence is a requirement for CSF release in response to calcium signaling, and ( iii) Emi2 is targeted for destruction by Plk1 upon CSF release. These observations strongly sugge su ggest st th that at Em Emi2 i2 ha hass a ro role le in CSF ar arre rest st.. It re rema main inss to be fo forma rmall llyy demonstrated that disabling either Emi1 or Emi2 function without perturbing the other causes loss of CSF maintenance. Do the negative data from Ohsumi et al. (18) and the identification of Emi2 justify the dismissal of the role of Emi1 in maintainin tai ningg CSF ar arre rest st?? Re Rece cent nt wo work rk in mo mous usee ooc oocyte ytess sho shows ws th that at Em Emi1 i1 ablation ablat ion by small interfering RNA causes spontaneous egg activa activa-tion, tio n, provi providin dingg pla plausi usible ble gene genetic tic evide evidence nce tha thatt Emi Emi1 1 is ind indeed eed essential for CSF arrest (10). As was the case for B-type cyclins, witho without ut th thee lu luxu xury ry of a com compl plet etee X. lae sequence nce laevis vis genome seque database, there may be additional members of the early mitotic inhibitor inhibi tor family of protei proteins ns awaiti awaiting ng identi identificati fication. on. As our current knowled know ledge ge stan stands, ds, the prod product uction ion of Emi Emi1 1 / , Em Emi2 i2 / , and double homozygous null mice will most constructively resolve the relative importance of these two homologs in CSF arrest. We thank James Nelson (Stanford University, Stanford, CA) for -catenin antibody, William Dunphy (California Institute of Technology, Pasadena) for Plx Plx1 1 ant antibo ibody, dy, Tim Hun Huntt (Imp (Imperi erial al Can Cancer cer Re Resear search ch Fun Fund, d, Lond London) on) for cyclin B2 antibody, and Thierry Lorca for communicating unpublished results. This work was supported by Public Health Service Grants 5T32 CA09302-27 (to J.J.T.) and RO1 GM60439 and GM54811 (to P.K.J.). 15. Guardavaccaro, D., Kudo, Y., Boulaire, J., Barchi, M., Busino, L., Donzelli, M., MargottinMargottinGoguet, F., Jackson, P. K., Yamasaki, L. & Pagano, M. (2003) Dev. Cell 4, 799–812. 16. Moshe Moshe,, Y., Boulaire, J., Pagano, M. & Hershko, A. (2004) Proc. Natl. Acad. Sci. USA 101, 7937–7942. 17. Hansen, D. V., Loktev, A. V., Ban, K. H. & Jackson, P. K. (2004) Mol.Biol. Cel Cell. l. 15, 5623–5634. 18. Ohsumi, K., Koyanagi, A., A., Yamamoto, T. M., Gotoh, T. & Kishimoto, T. (2004) Proc. Natl. Acad. Sci. USA 101, 12531–12536. 19. Furuno, N., Nishizawa, M., Okazaki, K., Tanaka, H., Iwashita, J., Nakajo, Nakajo, N., Ogawa, Y. & Sagata, N. (1994) EMBO J. 13, 2399–2410. 20. Murra Murray, y, A. W., Solomo Solomon, n, M. J. & Kirschner, M. W. (1989) Nature 339, 280–286. 21. Hsu, J. Y., Reimann, J. D., Sorensen, C. S., Lukas, J. & Jackson, P. K. (2002) Nat. Cell Biol. 4, 358–366. 22. Ferr Ferrell, ell, J. E., Jr. (1999) BioEssays 21, 833–842. 23. Jin, J., Cardozo, T., Lovering, R. C., Elledge, S. J., Pagano, M. & Harper, J. W. (2004) Genes Dev. 18, 2573–2580. 24. Howe, J. A., Howell, M., Hunt, T. & Newport, J. W. (1995) Genes Dev. 9, 1164–1176. 25. Minsh Minshull, ull, J., Murray, Murray, A., Colman, A. & Hunt, T. (1991) J. Cell Biol. 114, 767–772. 26. 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