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LAF1 ubiquitination by COP1 controls

LAF1 ubiquitination by COP1 controls
LAF1 ubiquitination by COP1 controls

potential of240mV tot40mV(each step10mV for100ms)17were recorded with the patch-clamp ampli?er.Data were?ltered at2kHz and collected at10kHz.Ca2tchannel activity was evaluated by measuring peak inward currents.

Received15January;accepted19May2003;doi:10.1038/nature01751.

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Supplementary Information accompanies the paper on https://www.wendangku.net/doc/ff6165506.html,/nature. Acknowledgements We thank K.Kato for technical assistance and W.R.Jelinek for critical comments on the manuscript.This work was supported by grants from NIH NINDS and a Whitehead Fellowship(K.H.).M.T.L.is an investigator of the Howard Hughes Medical Institute.

Competing interests statement The authors declare that they have no competing?nancial interests.

Correspondence and requests for materials should be addressed to K.H.

(hongk02@https://www.wendangku.net/doc/ff6165506.html,)............................................................... LAF1ubiquitination by COP1controls photomorphogenesis and is stimulated by SPA1

Hak Soo Seo,Jun-Yi Yang,Masaki Ishikawa,Cordelia Bolle,

Maria L.Ballesteros&Nam-Hai Chua

Laboratory of Plant Molecular Biology,Rockefeller University,1230York Avenue, New York,New York10021,USA ............................................................................................................................................................................. Far-red light regulates many aspects of seedling development, such as inhibition of hypocotyl elongation and the promotion of greening1,acting in part through phytochrome A(phyA).The RING motif protein COP1is also important because cop1 mutants exhibit constitutive photomorphogenesis in darkness2,3. COP1is present in the nucleus in darkness but is gradually relocated to the cytoplasm upon illumination4.Here we show that COP1functions as an E3ligase ubiquitinating both itself and the myb transcription activator LAF1,which is required for complete phyA responses5.In transgenic plants,inducible COP1overexpression leads to a decrease in LAF1concentrations, but is blocked by the proteasome inhibitor MG132.The coiled-coil domain of SPA1,a negative regulator of phyA signalling6,has no effect on COP1auto-ubiquitination but facilitates LAF1ubi-quitination at low COP1concentrations.These results indicate that,in darkness,COP1functions as a repressor of photomorpho-genesis by promoting the ubiquitin-mediated proteolysis of a subset of positive regulators,including LAF1.After the activation of phyA,SPA1stimulates the E3activity of residual nuclear COP1 to ubiquitinate LAF1,thereby desensitizing phyA signals.

In plants,the light signal is perceived by different photoreceptors that activate a network of signalling intermediates to control the expression of hundreds of genes7.The phytochrome photoreceptor family regulates downstream responses by switching between bio-logically inactive and active forms in response to absorbing red or far-red light,respectively8.Several Arabidopsis mutants de?cient in intermediates of the phyA signalling pathway have been identi?ed. Some of these show reduced responses,whereas others are hyper-sensitive9,10.The COP/DET/FUS proteins have been proposed to act as negative regulators of photomorphogenic development because their loss-of-function mutants develop as light-grown plants in darkness11.Among these repressors,the RING motif protein COP1 seems to be a key regulator that interacts with components of the phyA signalling pathway such as HY5(ref.12)and SPA1(ref.13). LAF1is a myb transcription activator that participates in the transmission of phyA signals to downstream responses;this acti-vator is localized in nuclear bodies5.Because COP1is also found in nuclear bodies14,we asked whether the two proteins are localized together.To this end,we transiently expressed LAF1tagged with yellow?uorescent protein(YFP)and COP1tagged with cyan ?uorescent protein(CFP)in onion epidermal cells.Figure1a shows that the two proteins were indeed localized in the same nuclear bodies,although COP1was also found in cytoplasmic regions not containing LAF1,which is strictly localized in the nucleus. Similar experiments showed that SPA1was distributed in the nucleus and the cytosol but it could be relocalized by COP1to the same LAF1/COP1nuclear bodies,which is consistent with an interaction between SPA1and COP1in vitro13.

The co-localization of LAF1and COP1indicates that the two proteins might interact.To study this,we performed a pull-down of products translated in vitro by using maltose-binding protein (MBP)and MBP–COP1.Control experiments showed that MBP alone did not interact with any of the products translated in vitro.As expected,COP1interacted with CIP8(ref.15)but not luciferase

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(Fig.1b)and,under the same conditions,LAF1translated in vitro

puri?ed together with COP1(Fig.1b).The interaction between

LAF1and COP1also occurred in vivo ,as con?rmed by co-puri?-

cation experiments (Fig.1c).Being a RING motif protein,COP1has been proposed to act as an E3ubiquitin protein ligase,with its interacting protein,HY5,as its putative substrate 16.Because no E3activity has been directly associated with COP1,it has not been possible to verify whether

the observed effect of COP1on HY5stability 16is direct or indirect.

Indeed,recent work showed that HY5can be ubiquitinated by CIP8,

a cytoplasmic RING motif protein,instead of COP1(ref.17).

Moreover,other than the putative substrate HY5,no substrate of

COP1has yet been identi?ed.To test whether COP1is indeed an E3

ligase,we produced the protein in Escherichia coli and assayed its E3

activity in vitro .Figure 2a shows that COP1was able to catalyse self-

ubiquitination in a reaction that required both E1and E2activities.

The reaction was dependent on Zn 2tions (Fig.2b)and required an

intact RING motif because Cys !Ser substitutions in residues 52

and 55of COP1abolished its E3activity (Fig.2c).Moreover,wild-

type COP1E3activity was blocked by a 2–10-fold excess of the

COP1mutant (C52S;C55S),whereas a 10-fold excess of MBP alone

had no inhibitory effect (Fig.2d).These results indicate that COP1

E3activity might require dimerization and that excess amounts of

the mutant (C52S;C55S)can inhibit E3activity by forming inactive

dimers with wild-type COP1,thus acting in a dominant-negative

manner.The coiled-coil region (amino acids 110–255)of COP1has

been shown to be involved in its dimerization 18.Our observation

that COP1dimerization activates its E3activity con?rms previous

results with another RING protein 19.

The interaction between LAF1and COP1prompted us to

investigate whether the former might be a substrate for the latter.

Indeed,LAF1was ubiquitinated by COP1in a reaction that was also

dependent on E1and E2activities (Fig.3a).This reaction was

speci?c,because no LAF1ubiquitination was detected with SINAT5

(Fig.3b),an Arabidopsis E3ligase that modi?es NAC1(ref.19).

LAF1ubiquitination was dependent on the RING motif but not the

WD40domain of COP1(Fig.3c).

Additional evidence of LAF1ubiquitination by COP1was

obtained by comparing reaction products with glutathione S

transferase (GST)-labelled ubiquitin and hexahistidine (His 6)-

labelled ubiquitin.Fig.3d shows that the mobility difference of

ubiquitinated LAF1products is consistent with the difference in

molecular mass between GST–ubiquitin and His 6–ubiquitin.

LAF1–COP1interaction and the ubiquitination of LAF1by

COP1predict that LAF1concentrations might be reduced by

COP1overexpression in vivo .We therefore determined LAF1

concentrations in transgenic plants carrying a 35S–LAF1–HA 3transgene (where HA encodes haemagglutinin)and an oestradiol-

inducible 20XVE–COP1–Myc 6transgene.In non-induced plants

without COP1overexpression,LAF1concentrations can be

increased about 3.5-fold by MG132,indicating possible proteaso-

mal degradation (Fig.3e).The induced expression of COP1

noticeably decreased LAF1concentrations;however,these concen-

trations could be elevated to the same as those in non-induced

plants treated with https://www.wendangku.net/doc/ff6165506.html,F1transcript concentrations were

comparable under these four conditions.Taken together,these

results show that LAF1stability in vivo can be regulated by COP1

through a proteasome-mediated process.Consistent with its self-

ubiquitination activity (Fig.2)was the observation that

COP1

Figure 1Interaction of LAF1,COP1and SPA1.a ,LAF1,COP1and SPA1localize together

to nuclear bodies in onion epidermal cells.Scale bar,25m m.Dic,differential interference

contrast.b ,LAF1binds to COP1in vitro .MBP–COP1was mixed with in vitro

translated luciferase (negative control),CIP8(positive control)and LAF1.Left panel,

autoradiogram of SDS gels of proteins translated in vitro ;middle panel,autoradiogram

after binding with MBP resin;right panel,autoradiogram after binding with MBP–COP1.

c ,LAF1interacts with COP1in vivo .Lane 1,transgenic plant expressing COP1–Myc 6;

lane 2,transgenic plant expressing LAF1–Flag 3;lane 3,transgenic plant expressing

COP1–Myc 6and His 6–LAF1–Flag 3.

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concentrations were similarly increased by MG132(Fig.3e),indi-cating possible proteasomal regulation as well.

Of the various loci required for phyA signalling,only EID1(ref.

21)and SPA1(ref.22)encode negative regulators.Because SPA1 interacts with COP1through its coiled-coil domain13,we speculated that this protein might inhibit COP1auto-ubiquitination or facili-tate LAF1ubiquitination by COP1,or both.To examine these possibilities,we produced a GST fusion protein containing the coiled-coil domain of SPA1(GST–SPA1cc).Figure4a shows that this fusion protein had no signi?cant effect on COP1auto-ubiqui-tination over a40-fold range of COP1concentrations.By contrast, at600ng COP1,increasing amounts of GST–SPA1cc(0–1.2m g) stimulated LAF1ubiquitination,whereas GST alone had no notice-able effect(Fig.4b).In this series of experiments,the stimulatory effect was saturated at300ng GST–SPA1cc.We next examined the effect of300ng GST–SPA1cc on LAF1ubiquitination over a range of COP1concentrations,using GST as a negative control.Whereas GST–SPA1cc had little effect on LAF1ubiquitination when COP1 was present in large amounts(0.6–1.2m g;Fig.4c and data not shown),it had a stimulatory effect that became more pronounced as the amount of COP1decreased below600ng(Fig.4c and data not shown).Similar results were obtained using His6–SPA1cc(data not shown).These results show that GST–SPA1cc stimulates LAF1 ubiquitination by COP1when the latter is present at low concentrations.

The in vitro and in vivo results described here provide a mechan-istic explanation of data obtained in vivo with Arabidopsis mutants and transgenic plants implicated in phyA signalling2,6.Previous work showed that cop1mutants display constitutive photomorpho-genesis in darkness,indicating that the COP1protein might act as a repressor of light responses.Here we show that COP1is an E3ligase and that it can polyubiquitinate LAF1,a transcription activator, presumably tagging the latter for subsequent proteolysis.This obser-vation is consistent with the notion that,in darkness,COP1might repress light responses by targeting a subset of positive regulators such as LAF1for destruction.On exposure to light,depletion of COP1in the nucleus4allows the positive regulators to accumulate and to promote downstream photomorphogenic responses.

Our results with SPA1cc indicate that,after the activation of phyA,residual nuclear COP1continues to have a role in regulating phyA responses,but now in concert with SPA1.SPA1is a nuclear protein synthesized in response to far-red light22.We show here that

Figure2COP1self-ubiquitination activity.a,MBP–COP1was assayed for E3activity by using32P-labelled ubiquitin(Ub)in the presence or absence of E1and E2.b,COP1E3 activity requires Zn2t.MBP–COP1was treated with TPEN and reconstituted with(t)or without(2)Zn2t.c,MBP–COP1E3activity is dependent on its RING motif.Wild-type (WT)MBP–COP1and MBP–COP1mutant(C52S,C55S)were assayed for self-ubiquitination.d,COP1mutant(C52S,C55S)blocks COP1E3activity.Numbers indicate the relative amounts of proteins present in the reaction,where1represents200ng MBP, MBP–COP1or MBP–COP1(C52S,C55S).Arrowheads indicate polyubiquitinated

COP1.Figure3Ubiquitination of LAF1by COP1.a,COP1E3activity was assayed in the presence or absence of rabbit E1,UbcH5b,His6–ubiquitin and His6–LAF1–Myc.b, Ubiquitination of LAF1by COP1but not SINAT5.Reaction mixtures contained500ng MBP–SINAT5.c,The WD40motif of COP1is dispensable for its E3activity and the MBP–COP1(C52S,C55S)mutant protein has no E3activity for LAF1.Reaction mixtures contained500ng COP1(wild-type,D WD40or C52S,C55S).Ub,ubiquitin.d,COP1E3 activity was assayed with GST–ubiquitin.e,Degradation of LAF1by COP1in vivo is mediated by the proteasome pathway.Double transgenic plants of35S–LAF1–HA3and XVE–COP1–Myc6were induced by b-oestradiol and then treated with or without MG132. After incubation overnight,COP1and LAF1concentrations were assessed by western blotting with tubulin concentrations as loading controls.RNA concentrations(5m g per lane)were determined by northern blotting,with18S rRNA as a loading control.In the top northern panel,asterisks indicate endogenous cop1transcript.Numbers under lanes indicate relative intensities.

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SPA1cc has no signi?cant effect on COP1auto-ubiquitination activity over a wide range of concentrations,but it stimulates LAF1ubiquitination at low,but not high,concentrations of the COP1E3ligase.These results indicate that at low nuclear COP1 concentrations,which is true shortly after phyA activation,newly synthesized SPA1might facilitate the ubiquitination of LAF1by COP1,and probably other positive signalling components,to desensitize the light signal.In spa1mutants,the absence of SPA1 is expected to impair COP1E3activity severely,resulting in higher concentrations of positive regulators for a given far-red light intensity,and hence the hypersensitive response6.It is also possible that in plants grown under light–dark conditions SPA1might synergize with COP1in the dark period to deplete transcription activators,like LAF1,more rapidly to prepare plants for the light signals of the following day.

RING motif E3ligases are known to catalyse both auto-ubiqui-tination and substrate ubiquitination.How these two opposing reactions are regulated in the cell during signalling is unclear and relatively unexplored.Several regulators have been shown to aid the auto-ubiquitination of DIAP1,a Drosophila E3ligase that inhibits apoptosis23–26,but their effects on substrate ubiquitination are unknown.By contrast,SPA1has little effect on COP1auto-ubiquitination but stimulates its E3activity towards substrates. The mechanism of this stimulation is not yet clear but it does not involve the interaction of SPA1with the coiled-coil domain of COP1monomer,because this would dissociate COP1dimers and inhibit E3activity.One possibility is that SPA1enhances the binding of LAF1to COP1at low concentrations of the latter.Subcellular localization experiments demonstrated that in darkness,LAF1and COP1are found in nuclear bodies5,14which also contained SPA1. On the basis of the ability of COP1to ubiquitinate LAF1,these nuclear bodies might be sites of protein degradation similar to the clastosomes of animal cells27.The characterization of additional components of plant clastosomes and the identi?cation of other substrates and regulators of COP1will lead to a better under-standing of the role of regulated proteolysis in phyA signalling.A Methods

Construction of expression vectors and preparation of recombinant proteins Sequences encoding full-length COP1and deletion mutants(WD40,amino acids1–255; RING,amino acids110–675;RING/coiled-coil,amino acids216–675)were ampli?ed by polymerase chain reaction(PCR)and cloned into pMal-c2(New England Biolabs)to generate complementary DNA molecules encoding fusions with MBP.Mutations in the RING motif(C52S,C55S)were generated with the Quick Change site-directed mutagenesis kit(Promega).Sequences for a carboxy-terminal Myc tag were appended to the full-length LAF1cDNA by PCR and cloned into pET-28a(t)(Novagen)to generate the coding sequence for His6–LAF1–Myc.The sequence encoding the coiled-coil domain of SPA1(SPA1cc;amino acids521–696)was ampli?ed by PCR and cloned into pGEX4T-1 (Pharmacia).

All recombinant proteins were expressed in E.coli strain BL21.For puri?cation of MBP–COP1,bacteria were lysed in50mM Tris-HCl pH7.5,200mM NaCl,1%Triton X-100,5mM dithiothreitol(DTT),2mM phenylmethylsulphonyl?uoride(PMSF)and a proteinase inhibitor cocktail(Promega)and puri?ed on amylose resins(New England Biolabs).For His6–LAF1–Myc puri?cation,bacteria were lysed in50mM NaH2PO4pH 8.0,300mM NaCl,1%Triton X-100,1mM imidazole,5mM DTT,2mM PMSF and a

Figure4SPA1cc modulates COP1E3activity.a,Effect of GST–SPA1cc on COP1self-ubiquitination activity.Reactions were performed with GST–SPA1cc(?led circles)or GST (open circles).The relative concentrations of ubiquitin n–COP1(Ub n–COP1)were measured by scanning the relevant regions of the X-ray?lm with the Scion image program.Percentages are relative to the amount of ubiquitin n–COP1at1.2m g MBP–COP1.Results are for n?3and are means^s.e.m.b,Effect of SPA1cc on LAF1ubiquitination at600ng COP1.c,Effect of COP1on LAF1ubiquitination at300ng SPA1cc.Reactions were performed with GST–SPA1cc(?lled circles)or GST(open circles). The relative concentrations of ubiquitin n–LAF1were measured by scanning regions of the X-ray?lm corresponding to the61–111-kDa range with the Scion image program. Percentages are relative to the amount of ubiquitin n–LAF1at1.2m g MBP–COP1.Results are for n?5and are means^s.e.m.

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proteinase inhibitor cocktail,and puri?ed on Ni2t-nitrilotriacetate(Ni2t-NTA)resins (Qiagen).For GST–SPA1cc puri?cation,bacteria were lysed in PBS pH7.5containing1% Triton X-100,2mM PMSF and a proteinase inhibitor cocktail,and puri?ed on glutathione resins(Pharmacia).Protein concentrations were determined by the Bradford assay (Bio-Rad).

Subcellular localization of LAF1,COP1and SPA1

The CFP and YFP coding sequences were fused in-frame to the50end of COP1cDNA and to the30end of SPA1cDNA,respectively,and cDNA sequences for LAF1–YFP and LAF1–CFP were constructed similarly.All fusion genes or genes for YFP and CFP were expressed from a35S promoter.Onion epidermal cells were bombarded with different combinations of plasmids with a helium biolistic gun28and analysed by confocal microscopy after incubation in the dark for12h.

In vitro and in vivo binding assays

T7-promoter-driven in vitro transcription/translation templates were derived from full-length LAF1and CIP8coding sequences5,15cloned into pRSETA(Invitrogen).

[35S]methionine-labelled luciferase,LAF1and CIP8were generated by transcription and translation in vitro with wheatgerm extracts by using a T7/T3coupled TnT kit(Promega). For binding in vitro,10m l of the translation mixture was added to500m l binding buffer (50mM HEPES pH7.5,1mM EDTA,150mM NaCl,10%glycerol,0.1%Tween20, 0.5mM DTT)and the mixture was incubated at258C for1h.The reaction mixture was incubated with amylose resin beads,which were then washed?ve times with washing buffer(50mM Tris-HCl pH7.5,150mM NaCl,0.2%Nonidet P40).In vitro translation products and COP1-interacting proteins were analysed by SDS–polyacrylamide-gel electrophoresis(SDS–PAGE).

For binding in vivo,LAF1–Flag3and His6–LAF1–Flag3cDNAs were cloned into pBI121(Clontech)and COP1-Myc6cDNA was cloned into pBA002(ref.29).Constructs were transformed into Arabidopsis thaliana(Landsberg erecta)by vacuum in?ltration30. Five-day-old light-grown plants(l6h light/8h dark)on MS medium were ground in liquid nitrogen and resuspended in an extraction buffer(50mM HEPES pH7.5,150mM NaCl, 0.5%Triton X-100,1mM DTT)containing1£complete protease inhibitor cocktail (Roche)and1mM imidazole(Sigma).After centrifugation,supernatants were puri?ed on Ni2t-NTA columns and eluted proteins were detected by western blotting with anti-Myc (Santa Cruz Biotechnology)or anti-Flag(Sigma)antibodies.

In vitro ubiquitination assay

32P-labelled ubiquitin was prepared with recombinant GST–ubiquitin20.Reaction mixtures(30m l)contained500ng COP1(E3),20ng each of rabbit E1and UbcH5B(both from Af?niti),and either2£104c.p.m.of32P-labelled ubiquitin or10m g unlabelled His6–ubiquitin(Sigma)or GST–ubiquitin(Boston Biochemical)in a buffer containing50mM Tris pH7.4,2mM ATP,5mM MgCl2and2mM DTT.After incubation at308C for2h, reaction mixtures were heated to958C in SDS–PAGE sample buffer containing2-mercaptoethanol,before electrophoresis on8%polyacrylamide SDS gels.

For cation chelation,MBP–COP1was immobilized on amylose resin and incubated overnight with2mM N,N,N0,N0-tetrakis(2-pyridylmethyl)-ethylenediamine(TPEN; Biotium)at48C.After extensive washing,the immobilized MBP–COP1was treated with 20m M ZnCl2for45min at228C before the ubiquitination assay.

For LAF1ubiquitination,each assay contained10ng puri?ed His6–LAF1–Myc. Ubiquitinated LAF1was detected by western blotting with anti-Myc antibody(Santa Cruz Biotechnology).

Effects of COP1overexpression on LAF1concentrations in vivo

Four-day-old light-grown(16h light/8h dark)plants carrying35S–LAF1–HA3and XVE–COP1–Myc6transgenes on MS medium were treated in the light with or without b-oestradiol for15h and then with or without50m M MG132(Calbiochem)for15h. Samples were ground in liquid nitrogen and equivalent amounts were analysed by western blotting with anti-HA or anti-Myc antibodies.Plant samples were also processed for northern blotting.

Received22October2002;accepted8April2003;doi:10.1038/nature01696.

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Acknowledgements We thank X.-W.Deng for COP1cDNA,and P.Hare for discussions.This work was supported by an NIH grant to N.-H.C.J.-Y.Y.is a graduate student on leave from Chung Hsing University,Taiwan.

Competing interests statement The authors declare that they have no competing?nancial interests.

Correspondence and requests for materials should be addressed to N.-H.C.

(chua@https://www.wendangku.net/doc/ff6165506.html,). .............................................................. Enhanced gravi-and phototropism in plant mdr mutants mislocalizing the auxin ef?ux protein PIN1

Bosl Noh*?,Anindita Bandyopadhyay??,Wendy Ann Peer?,

Edgar P.Spalding*&Angus S.Murphy?

*Department of Botany,University of Wisconsin,430Lincoln Drive,Madison, Wisconsin53706,USA

?Department of Horticulture and Landscape Architecture,625Agriculture Mall Drive,Purdue University,West Lafayette,Indiana47907,USA

?These authors contributed equally to this paper ............................................................................................................................................................................. Many aspects of plant growth and development are dependent on the?ow of the hormone auxin down the plant from the growing shoot tip where it is synthesized1,2.The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1membrane protein,which controls the ef?ux of the auxin anion3.Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are

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?2003 Nature Publishing Group

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