Determinants of the Ubiquitin-mediated Degradation of theMet4 Transcription Factor*

Received for publication, January 3, 2006, and in revised form, February 23, 2006 Published, JBC Papers in Press, February 23, 2006, DOI 10.1074/jbc.M600037200

Alexandra Menant‡1, Peggy Baudouin-Cornu§2, Caroline Peyraud‡3, Mike Tyers§4, and Dominique Thomas‡¶5

From the ‡Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvettte, France, ¶CytomicsSystems SA, Ba� timent 5, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France, and §Samuel Lunenfeld Research Institute, MountSinai Hospital, Toronto, Canada M5G 1X5

In yeast, the Met4 transcription factor and its cofactors Cbf1,Met28, Met31, andMet32 control the expression of sulfur metabo-lism and oxidative stress response genes. Met4 activity is tuned tonutrient and oxidative stress conditions by the SCFMet30 ubiquitinligase. The mechanism whereby SCFMet30-dependent ubiquityla-tion of Met4 controls Met4 activity remains contentious. Here, wehave demonstrated that intracellular cysteine levels dictate the deg-radation ofMet4 in vivo, as shown by the ability of cysteine, but notmethionine or S-adenosylmethionine (AdoMet), to trigger Met4degradation in an str4� strain, which lacks the ability to producecysteine from methionine or AdoMet. Met4 degradation requiresits nuclear localization and activity of the 26 S proteasome. Analysisof the regulated degradation of a fully functional Met4-Cbf1 chi-mera, in which Met4 is fused to the DNA binding domain of Cbf1,demonstrates that elimination of Met4 in vivo can be triggeredindependently of both its normal protein interactions. Strains thatharbor the Met4-Cbf1 fusion as the only source of Cbf1 activityneeded for proper kinetochore function exhibit high rates ofmethi-onine-dependent chromosomal instability. We suggest thatSCFMet30 activity or Met4 utilization as a substrate may be directlyregulated by intracellular cysteine concentrations.

The temporal and spatial control of transcription requires a battery ofdifferent factors, including transcription activators/repressors, co-acti-vators/repressors, general transcription factors, chromatin-modifyingand -remodeling enzymes, and the multisubunit enzyme RNA polym-erase II. Diverse biochemical events dictate interaction of RNA polym-erase II with gene regulatory sequences. Protein phosphorylation con-trols interactions between many factors (1), while a complex code ofhistone acetylation, methylation, and ubiquitylation modifies chroma-tin accessibility (2). Finally, the abundance and/or activity of many tran-scription-associated factors is dynamically controlled by the ubiquitin-proteasome system (3, 4).Conjugation of ubiquitin to substrate proteins is achieved through a

cascade of E1,6 E2, and E3 enzymes, which activate and then seriallytransfer ubiquitin onto substrate lysine residues (5). The best under-stood function of ubiquitylation is to target proteins for destruction bythe 26 S proteasome, a large compartmentalized protease particle thatrecognizes the polyubiquitin tag (6). More recently, non-proteolyticfunctions have been ascribed to ubiquitin conjugation, including target-ing to different subcellular compartments and allosteric control of enzy-matic events (7). How ubiquitylation leads to different outcomes indifferent contexts is not fully understood.The pervasive role of the ubiquitin proteasome pathway in the regu-

lation of transcription has gradually emerged from numerous studies.RNA polymerase II is polyubiquitylated in response to DNA damage,whereas histone monoubiquitylation is associated with activation oftranscription (8, 9). Surprisingly, the proteasome itself regulates differ-ent steps in transcription and is recruited to active gene regions (10).Finally, the regulated ubiquitin-dependent degradation of transcrip-tional activators allows the cells to dynamically control the expression oftarget genes (3). Moreover, an intriguing correlation between activatorpotency and instability suggests that ubiquitylation of transcription fac-torsmay be directly linked to transcriptional activation (4, 11). Themostpotent class of transcriptional activators, those containing transcriptionactivation domains rich in acidic residues, are by far the most unstable;strikingly, such transcription activation domains can function asdegrons that target heterologous proteins for degradation (12, 13). Deg-radation of transcription factors may thus be coupled to assembly ofactive transcriptional complexes on promoter DNA (14, 15).In addition to proteasomal-dependent degradation, non-proteolytic

regulation of transcription factor activity by ubiquitylation has recentlybeen described. The first and best documented example of this mecha-nism is the yeast transcriptional activatorMet4, which governs theMETgene network responsible for the biosynthesis of the sulfur-containingamino acids, methionine and cysteine (16). Met4 is the essential sub-strate of the SCFMet30 ubiquitin ligase, a multiprotein E3 complex thatuses the F-box protein subunitMet30 to recruitMet4 for ubiquitylationby the core SCF complex, in conjunctionwith the E2 enzymeCdc34 (17,18). Met4 is recruited to promoter DNA by one of two distinct sets ofcofactors that bind different elements inMET gene regulatory regions,either the Met28-Cbf1 complex or the complex Met31-Met32 (19, 20).In addition to its role in facilitating activation of someMET genes, Cbf1is a component of the core kinetochore complex that binds the CDEI ofthe yeast centromere (21, 22).Upon environmental changes, Met4 can be either rapidly ubiquity-

lated and degraded (23) or ubiquitylated and inactivated in the absenceof degradation (24). These alternative fates of ubiquitylatedMet4 forms

* This work was supported in part by funds from the Centre National de la RechercheScientifique and the Association de la Recherche sur le Cancer (to D. T.) and by grantsfrom the National Cancer Institute of Canada and the Canadian Institutes of HealthResearch (CIHR) (to M. T.). The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a thesis fellowship from the Ministere de la Recherche et del’Enseignement Superieur and by the Fondation pour la Recherche Medicale.

2 Supported by the Leukemia and Lymphoma Society of America. Present address: Ser-vice de Biochimie et Genetique Moleculaire, CEA/Saclay, 91191 Gif-sur-Yvette,France.

3 Present address: Institut Pasteur, 25 Rue du Docteur Roux, 75015 Paris, France.4 Supported by a Canada Research Chair in Functional Genomics and Bioinformatics.5 To whom correspondence should be addressed. Tel.: 33-1-69-82-42-66; Fax: 33-1-69-

82-42-38; E-mail: dthomas@cytomics.fr.

6 The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier pro-tein; E3, ubiquitin-protein isopeptide ligase; GFP, green fluorescence protein; HA,hemagglutinin; AdoMet, adenosylmethionine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 17, pp. 11744 –11754, April 28, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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depend upon the growth conditions: in minimal media Met4 is ubiqui-tylated and degraded in response to methionine excess, whereas in richmedia Met4 is oligo-ubiquitylated but remains stable (25). In the lattergrowth condition, oligo-ubiquitylated Met4 is not recruited to METgene promoters but is recruited to the SAM genes, which are requiredfor production of S-adenosylmethionine, an unstable metabolite that isnot present in rich medium. Thus, ubiquitylation appears to not onlyregulateMet4 by distinctmechanisms but also to control the differentialrecruitment ofMet4 to distinct target promoters. Despitemultiple linesof evidence that SCFMet30-dependent ubiquitylation of Met4 leads toMet4 degradation specifically in minimal media (23, 25), the degrada-tion ofMet4 under minimal conditions has recently been disputed (26).To settle this discrepancy, we have investigated the determinants ofMet4 degradation in yeast grown in minimal media in the presence ofhigh extracellular methionine levels. We find that Met4 instability isultimately dictated by intracellular cysteine levels and that degradationdepends on both proper localization of Met4 in the nucleus and on anactive proteasome. Through the use of chimericMet4-Cbf1 fusion pro-tein we have shown that destruction of ubiquitylatedMet4 is independ-ent of the means by which it is tethered to DNA. These results furtherconfirm that the transcriptional activator Met4 is indeed regulated bydegradation-dependent and -independent mechanisms.

EXPERIMENTAL PROCEDURES

Yeast Culture—Saccharomyces cerevisiae strains used in this studyare listed in Table 1. All strains are isogenic with the wild-type strainW303 (provided by R. Rothstein). Standard yeast media and minimal Bmediumwere prepared as described by Cherest and Surdin-Kerjan (27).Minimal B medium is a synthetic medium without sulfur sources (27).

For methionine repression, cells were grown in B medium supple-mentedwith 0.05mM L-methionine to early log phase and transferred toa Bmediumwithout sulfur source for 1 h before a high concentration ofL-methionine (1 mM) was added. For derepression, cells were firstgrown in B medium containing a repressive amount of L-methionine toearly log phase and then transferred to B medium without sulfur aminoacids. Transformation was by the lithium acetate method (28).

Plasmid Construction—The plasmid pGAL1-GFPMET4�i thatencodes different GFP-Met4 derivatives expressed from theGAL1 pro-moter was created by cloning the fragments EcoRI-BamHI of plasmidpLexMet4�i (29) into the plasmid pGAL306GFP (23) that had beendigested by EcoRI and BamHI. The plasmid pGAL1-GFPMET4�2NLSwas constructed by amplifyingMET4 nucleotides 548–1200 from plas-mid pMET4–1 (29) with oligonucleotide primers containing XhoIrestriction sites and cloning into the XhoI site of pLexMET4�11. TheEcoRI-BamHI fragment of the resulting plasmid was then cloned intothe corresponding sites of pGAL306GFP. The above plasmids were lin-earized by StuI to direct integration into the chromosomal URA3 locusin amet4::TRP1 strain (CC849–1A).The Met4-Cbf1 chimera comprises the entire Met4 protein with the

exception of the basic leucine zipper domain fused to the basic helix-loop-helix DNA binding domain of Cbf1 (residues 210–351). Plasmidswere constructed as follows: pGAL316MET4-CBF1, which allows theexpression of the Met4-Cbf1 chimera from the GAL1 promoter, wascreated by inserting the ClaI-ClaI fragment of pMET4–4 (30) treatedwith Klenow enzyme into pGAL316 (23) that had been digestedby BamHI and EcoRI, treated with Klenow, and dephosphorylated.The resulting plasmid was digested with SpeI and NotI and in-frameligated with a Cbf1-encoding EcoRI-NotI fragment from plasmid

TABLE 1Yeast strains

Strain Genotype SourceC301 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4::URA3 Ref. 23C313 Mata, his3, leu2, ura3, trp1, ura3::pGAL1-GFP-MET4�12::URA3 This studyC314 Mat-a, his3, leu2, ura3, trp1, ura3::pGAL-GFP-MET4�30::URA3 This studyC315 Mat-a, his3, leu2, ura3, trp1, ura3::pGAL-GFP-MET4�37::URA3 This studyC323 Mata, his3, leu2, ura3, trp1, ura3::pGAL1-HA3MET4�12::URA3 This studyCC849–8A Mat�, his3, leu2, ura3, trp1, met4::TRP1 Ref. 23CC857–2A Mata, his3, leu2, ura3, ade2, trp1, cbf1::TRP1 This studyCC874–18D Mata, his3, leu2, ura3, ade2, trp1, lys2, str4::URA3 This studyCD233 Mat�, his3, leu2, ura3, trp1, met4::HA3MET4 This studyCD303 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�2::URA3 This studyCD304 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�3::URA3 This studyCD305 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�4::URA3 This studyCD306 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�6::URA3 This studyCD307 Mat����, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�7::URA3 This studyCD308 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�8::URA3 This studyCD309 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�9::URA3 This studyCD310 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�39::URA3 This studyCD311 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�5::URA3 This studyCD312 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�10::URA3 This studyCD313 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�11::URA3 This studyCD327 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�4–4::URA3 This studyCD328 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�18::URA3 This studyCD329 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�34::URA3 This studyCD330 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGAL1-GFP-MET4�35::URA3 This studyCD385 Mat�, his3, leu2, ura3, trp1, met4::TRP1, ura3::pGal1-GFP-MET4�2NLS::URA3 This studyCY110–11B Mata, his3, leu2, ura3, ade2, trp1, met4::TRP1, cbf1::URA3 This studyCY155–1C Mat�, his3, leu2, ura3, trp1, met4::HA3MET4 This studyCY169–5B Mata, his3, leu2, ura3, ade2, trp1, lys2, str4::URA3, met4::HA3MET4 This studyCY172–3D Mata, his3, leu2, ura3, ade2, trp1, met4::MET4-CBF1 This studyCY172–4C Mat�, his3, leu2, ura3, ade2, trp1, met4::MET4-CBF1, cbf1::URA3 This studyCY187–1D Mat�, his3, leu2, ura3, trp1, met4::GFP-MET4, gsh1::HIS3 This studyCY194–4A Mata, his3, leu2, ura3, trp1, met4::GFP-MET4 This studyCY194–2A Mat�, his3, leu2, ura3, ade2, trp1, met4::GFP-MET4, str4::URA3 This studyCY202–1B Mata, his3, leu2, ura3, ade2, trp1, met4::HA3MET4-CBF1 This studyCY202–4B Mat�, his3, leu2, ura3, ade2, trp1, met4::HA3MET4-CBF1, cbf1::URA3 This studyCYS151 Mata, his3, leu2, ura3, trp1,ura3::pGAL1-GFP-MET4::URA3, pdr1�, pdr3� P. ZarzovW303–1A Mata, ade2, his3, leu2, ura3, trp1 R.Rothstein

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pET28Cbf1�1-209 (19) via a 16-base pair linker with SpeI and EcoRIcompatible ends. pProMET4-CBF1, which allows the expression of theMet4-Cbf1 chimera from the endogenousMET4 promoter, was createdby inserting the EcoRI-NotI fragment from pGAL316MET4-CBF1 intothe pProMET4 plasmid (containing the MET4 promoter region, (29))that had been digested by EcoR1/NotI and dephosphorylated. To directthe homologous replacement of the MET4 chromosomal locus, wecloned a HindIII-HindIII fragment that contains theMET4 terminatorregion of pMET4–4 into pProMET4-CBF1 that had been digested byHindIII and dephosphorylated. The ClaI-NotI fragment of the resultingplasmid pProMet4-Cbf1 was isolated, non-yeast nucleotides wereremoved by brief Bal31 digestion, and the resulting purified fragmentwas used to transform the CC849–8A strain.

Chromatin Immunoprecipitation—Cross-linked chromatin prepara-tion and immunoprecipitation were performed as described previously(25), except that cells were fixed with 1.4% formaldehyde and disruptedusing the Fastprep apparatus (Qbiogene) instead of vortexing. Rabbitpolyclonal Met4 antiserum against full-length Met4 produced in insectcells was used for immunoprecipitation (25). Immunoprecipitated andtotal DNA samples were purified using the Quiaquick PCR purificationkit (Qiagen) and analyzed by quantitative real-time PCRperformedwiththe Light Cycler System (Roche Applied Science). Input DNA wasdiluted to 1/5 and 1/25. Immunoprecipitated and total DNAs were sub-jected to 40 cycles of PCR using the FastStart DNAMaster SYBRGreenI reaction mix (Roche Applied Science). The occupancy level at a givenpromoter was defined as the ratio of immunoprecipitated DNA overtotal DNA for each PCR product. The higher occupancy level measuredwas arbitrarily set to 100, and all other values were represented relativeto this standard.

Protein and RNA Analysis—Total proteins were extracted by a 20%cold trichloroacetic acid procedure (23) using a Fastprep apparatus(Qbiogen). Samples were separated on a 7.5% acrylamide gel, trans-ferred to a nitrocellulose membrane (Optitran; Schleicher & Schuell),and probed. Anti-HA antibody (12CA5; Roche Applied Science) andperoxidase-conjugated anti-mouse antibody (Sigma) were used at a1:1,000 dilution. Anti-Met4 polyclonal antibody was used at 1:2,000 andperoxidase-conjugated anti-rabbit antibody (Sigma) was used at1:10,000 dilution. Detection was by Supersignal chemiluminescent sub-strate (Pierce). Equal loading was established by detection with a lysyl-tRNA synthetase antibody. Total cellular RNAwas extracted from yeastby the hot phenol method, separated, and probed as described (23).

Fluorescent Microscopy and Cytometry—GFP-Met4 fusion proteinfluorescence was monitored in living cells on a Zeiss Axioplan 2 fluo-rescence microscope using a Zeiss GFP filter. All the images were col-lected with a Roper Photometrics CoolSNAP HQ CCD camera usingidentical settings and analyzedwith theMetamorph software (UniversalImaging Corp., Downingtown, PA). Immediately before observation,cells were harvested by a brief centrifugation, washed, and suspended inDabco (Sigma) buffer. Nuclei were stained using the dye HOECHSTnumber 33342 (Sigma). Levels of the GFP-Met4 fusion protein wereassessed by quantitative fluorescence of live cells using a BDBiosciencesFacsScalibur flow cytometer.

Metabolic Sulfur Analysis—Yeast cells were grown at a cell density of107cells/ml in B medium containing 0.2 mM homocysteine as sulfursource. After filtration, cells were transferred to B medium containing0.05 mM sulfate and 4 �Cie/ml of radioactive sulfate. At the indicatedtime, a 5-ml aliquot of cells was harvested by a brief centrifugation,washed two times with 1 mM cold sulfate, boiled, and analyzed by thinlayer chromatography.

Plasmid Loss Assay—Plasmid loss assays were performed asdescribed in Ref. 31. Plasmid pRS313 carrying the selective markerURA3 and the CEN6 centromere was transformed into yeast. Transfor-mants were grown in a synthetic medium without histidine and dilutedin a medium containing histidine and either repressive or non-repres-sive concentration of L-methionine. This step corresponds to the begin-ning of the experiment (0). Cells were plated on a synthetic mediumcontaining or not histidine and either a repressive or a non-repressiveconcentration of L-methionine at 0 and 8 (T1), 24 (T2), 32 (T3), 48 (T4)h. The number of cells harboring the plasmid at each time point wascalculated by counting the total colonies formed in the presence orabsence of histidine.

RESULTS

Met4 Elimination Depends on the Trans-sulfuration Pathway—Toelucidate the mechanisms that underlie the proteasome-dependentdegradation of Met4, we first investigated the cellular signal that mighttrigger the elimination of Met4. High concentrations of the three pre-dominant sulfur-containing compounds (1 mM L-methionine, 0.2 mM

S-adenosylmethionine (AdoMet), and 1 mM L-cysteine; Fig. 1A) wereequally capable of repressing MET gene expression in wild-type cellsgrown in minimal medium (Fig. 1B). In contrast, in str4� mutants thatlack a functional cystathionine-� synthase and are thus unable to con-vert either methionine or AdoMet into cysteine, only high levels ofcysteine were able to cause fullMET gene repression (Fig. 1B). To deter-mine whether cysteine causedMet4 elimination, we monitored the sta-bility of a GFP-Met4 fusion protein expressed from the endogenousMET4 promoter in cells exposed to either methionine or cysteine. Inwild-type cells, the GFP-Met4 fluorescence signal disappeared withsimilar kinetics in response to either amino acid, whereas in str4� cellsGFP-Met4 disappeared only in response to cysteine (Fig. 1C). To cor-roborate these results, we determined the stability of an HA epitope-tagged version of Met4 expressed from the endogenous MET4 pro-moter. The changes in abundance of HA3Met4 in wild-type versus str4�cells exposed to eithermethionine or cysteine paralleled effects onMETgene expression and GFP-Met4 (Fig. 1D). The SCFMet30-dependentelimination ofMet4 in response tomethionine thus requires an increasein intracellular cysteine, which arises from the conversion of methio-nine into AdoMet and subsequent metabolism by the methyl cycle andthe trans-sulfuration pathway (Fig. 1A). Glutathione, the most abun-dant cysteine-containing peptide in the cell, was not required for Met4regulation because cysteine was able to promote rapid GFP-Met4 elim-ination in a gsh1� mutant that lacks �-glutamylcysteine synthase and isthus unable to convert cysteine into glutathione (Fig. 1E).

Met4DegradationRequiresNuclear Localization—Met30 is localizedwithin the nucleus, and its correct compartmentalization is required forits function (32). Nuclear localization of Met4 should thus be requiredfor its degradation. To identify nuclear localization signals in Met4, weconstructed several Met4 deletion derivatives and monitored the sub-cellular localization of each derivative fused to GFP. This analysis iden-tified three sequences within Met4 that were important for its subcel-lular localization (Fig. 2,A and B, data not shown). Deletion of theMet4carboxyl-terminal region (404–589; deletions �7 and �8) caused cyto-plasmic localization, implying the existence of an NLS in this region.Unexpectedly, larger deletions that removed additional adjacent resi-dues (189–589; deletions �9 and �10) caused relocalization to thenucleus, suggesting that the 309–404 region of Met4 contains residuesthat help restrictMet4 to the cytoplasm. The cytoplasmic localization ofseverely truncated derivative GFP-Met4�11 suggested that Met4 con-tains a second NLS in the 78–189 region, which is close to the Met4

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activation domain. A final GFP-Met4 derivative that was devoid of bothpotential NLS sequences, Met4�2NLS, displayed mainly cytoplasmiclocalization (Fig. 2C). Interestingly, the sequence that helps restrict

Met4 to the nucleus perfectly overlaps with the Met4 auxiliary domain,a sequence previously identified as being required for the full transcrip-tion activation potency of Met4 (29).

FIGURE 1. Methionine-induced Met4 degradation is dependent on the intracellular conversion of methionine into cysteine. A, S. cerevisiae interconversion pathways betweenmethionine, AdoMet, and cysteine. B, cysteine-mediated repression of MET genes. Expression of MET genes was assessed by Northern blot analysis in W303–1A (wild-type) andCC874 –18D (str4�) cells before and after addition of either 1 mM L-methionine, 0.2 mM AdoMet, or 1 mM L-cysteine. C, methionine-induced Met4 degradation in live cells requires afunctional STR4 gene. The GFP-Met4 fusion protein was expressed from the MET4 promoter in CY194 – 4A (wild-type) and CY194 –2A (str4�) cells in the absence or presence of either1 mM L-methionine or 1 mM L-cysteine. Fluorescence images were acquired at the indicated times. D, methionine-induced Met4 protein degradation requires a functional STR4 gene.CY155–1C (met4::HA3MET4) and CY169 –5B (met4::HA3MET4, str4::URA3) cells were treated as in panel C. At the indicated times, proteins were extracted using the trichloroacetic acidprocedure and immunoblotted with anti-HA antibody and polyclonal antibody to the yeast lysyl-tRNA synthetase as a control for equal loading. E, destabilization of the fusion proteinGFP-Met4 in a gsh1� strain. CY187–1D (gsh1�) cells harboring a GFP-Met4 construct were grown in the absence (�) or presence (�) of 1 mM L-cysteine and fluorescence imagesacquired at the indicated times.

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FIGURE 2. Met4 degradation is dependent upon its nuclear localization. A, schematic representation of the internal in-frame deletions of Met4. Plasmids encoding the differentGFP-Met4 derivatives were integrated at the URA3 locus in a met4::TRP1 strain. B, localization of the GFP-Met4-derivative proteins. GFP-Met4 derivatives were expressed from the GAL1promoter, nuclei stained with the HOECHST 33342 dye (Hoe), and fluorescent images acquired. C, Met4 that lacks both NLS sequences is localized in the cytoplasm. The fusion proteinGFP-Met4�2NLS was expressed from the GAL1 promoter and fluorescent images acquired. D, methionine triggers destabilization of a GFP-Met4 fusion protein localized within thenucleus. GFP-Met4 (strain C301; met4::TRP1, ura3::pGAL1-GFP-MET4::URA3) and GFP-Met�7 (strain CD307; met4::TRP1, ura3::pGAL1-GFPMET4�7::URA3) fusion proteins were expressedfrom the GAL1 promoter and then repressed by the addition of glucose, either in the absence (�Met) or presence of 1 mM L-methionine (�Met). Fluorescence images were acquiredat the indicated times.

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To determine whether Met4 degradation was dependent on itsnuclear localization, we investigated the stability of the GFP-Met4�7derivative (deletion of residues 470–589), which leaves both theMet30-interacting region and the known ubiquitylation site (lysine 163) intact(26). The stability of GFP-Met4�7 and wild-type GFP-Met4 fusion pro-teins was analyzed in the presence and the absence of high methionineafter repression of theGAL1 promoter from which each was expressed.Wild-type GFP-Met4 was rapidly depleted in the presence of methio-nine, whereas much of the GFP-Met4�7 derivative was still present 60min after methionine addition (Fig. 2D). Close inspection revealed thatGFP-Met4�7 fluorescence was restricted to the cytoplasm in cellsexposed to highmethioninewhile it was present in both the nucleus andthe cytoplasm in the absence of methionine. This result suggested thatonly nuclear localized GFP-Met4�7 was actively degraded. Immuno-blot analysis revealed that the Met4�7 protein was less destabilizedupon methionine exposure than wild-type Met4 (data not shown).

Met4 Degradation Is Dependent on Proteasome Activity—We nextdemonstrated that the loss of GFP-Met4 fluorescence signal in responsetomethionine indeed resulted from the proteasome-mediated degrada-tion of the fusion protein and not, for instance, from nonspecific dis-ruption of the GFP moiety. To this end, the stability of the wild-typeGFP-Met4 expressed from the GAL1 promoter was analyzed in thepresence of Mg132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), a pep-tide aldehyde inhibitor of proteasome (33). The concurrent addition ofMG132 and methionine to minimal medium prevented the disappear-ance of GFP-Met4 that was observed when promoter shutoff was per-formed in the presence ofmethionine alone (Fig. 3,A and B). Moreover,both fluorescence microscopy and quantitative flow cytometry assaysshowed that the inhibition of fluorescence depletion was proportionalto the concentration of added MG132 (Fig. 3, A and B).We further ascertained that the disappearance of GFP-Met4 fluores-

cence was a result of ubiquitin-mediated degradation by analysis of aGFP-Met4�12 fusion protein, which lacks residues 79–180, a regionthat spans the activation domain and includes lysine 163, the sole ubiq-uitylation site on Met4 (26). The fluorescent signal emanating fromGFP-Met4�12 was stable in the presence of methionine (Fig. 3C). Ananalogous Ha3Met4�12 derivative was also not significantly destabilizedupon methionine exposure as compared with wild-typeMet4 (Fig. 3D).The HA3Met4�12 proteinwas only faintly post-translationallymodified,consistent with a primary defect in ubiquitylation. Based on theseresults, we conclude that the rapid depletion of GFP-Met4 in live cellsexposed to high methionine indeed arises from its ubiquitin-dependentelimination by the proteasome.

AMet4-Cbf1 Chimera Protein Drives Methionine Synthesis—Met4 isrecruited to DNA by two distinct sets of cofactors, Cbf1-Met28 andMet31-Met32 (19) (20). In contrast to Met4, the Cbf1, Met28, Met31,and Met32 DNA-binding proteins were shown to be stable proteins(23). Rather than proteasome-mediated elimination ofMet4, it has beenpostulated that Met4 ubiquitylation causes ejection of the Cbf1 DNAbinding subunit from promoter DNA without apparently affectingMet4 occupancy (24). To test this model and assess the possible effectsof local protein interactions on Met4 degradation, we constructed aMet4-Cbf1 chimera protein in which the basic leucine zipper domain ofMet4 that tethers it to other factors was replaced by the basic helix-loop-helix DNA binding domain of Cbf1 (see “Experimental Proce-dures”). The functionality of this Met4-Cbf1 chimera was assayed bytesting its ability to relieve the methionine auxotrophy of singlemet4�and cbf1� and double met4�,cbf1� mutant strains. The expression ofthe Met4-Cbf1 chimera from a GAL1 promoter restored the methio-nine prototrophy of both the single and the doublemet4�,cbf1�mutant

cells (Fig. 4, A and B). We then constructed strains in which theMET4-CBF1 chimera was expressed from the endogenous MET4 promoter.Expression of chimera from the chromosomalMET4 locus rescued themethionine auxotrophy of the double met4�,cbf1� deletion (data notshown). The kinetics of sulfate reduction was then assessed in wild-typeand met4::MET4-CBF1 cells by measuring the incorporation of radio-active sulfate into methionine and glutathione. Both compounds accu-mulated at similar rates in both strains (Fig. 4C). The Met4-Cbf1 chi-mera thus recapitulates the collective activities of Met4 and Cbf1 inmethionine synthesis.

Methionine-triggered Elimination of a Met4-Cbf1 Chimera—Toaddress how the activity of the Met4-Cbf1 chimera is regulated in cellsexposed to highmethionine, we analyzed the kinetics of bothMET genederepression and repression in cells whose MET4 chromosomal genewas replaced by theMET4-CBF1 allele. Cells were grown in minimal Bmedium in the presence of highmethionine, shifted tomediumwithoutmethionine, and the kinetics ofMET gene derepression was followed byNorthern blot analysis. The derepression kinetics of MET3, MET16,and MET25 expression was identical in cells that expressed the Met4-Cbf1 chimera as compared with wild-type controls (Fig. 5A). Likewise,repression kinetics in response to methionine was similar in theMET4andmet4::MET4-CBF1 strains (Fig. 5B). Chromatin immunoprecipita-tionwas then used to assess the abundance of theMet4-Cbf1 chimera atindividual promoters. Cross-linked chromatin was prepared frommet4::MET4-CBF1 cells grown inminimal Bmediumbefore and 40minafter the addition of 1 mMmethionine and the Met4-Cbf1 chimera wascaptured with polyclonal Met4 antisera (25). Addition of methioninecaused a 5- to 15-fold decrease inMet4-Cbf1 chimera occupancy at theMET3, MET16, and MET25 gene promoters, in accord with theobserved methionine-mediated repression of these genes (Fig. 5C).To further test whether repression ofMET gene expression was due

to the destabilization of the Met4-Cbf1 chimera, we determined thestability of an HA epitope-tagged version of Met4-Cbf1, also expressedfrom the chromosomal locus. The abundance of HA3Met4-Cbf1 wasrapidly diminished upon addition of methionine (Fig. 5D). That theHA3Met4 and HA3Met4-Cbf1 proteins were converted to similar multi-ple low electrophoretic mobility species and were degraded with iden-tical kinetics argues that theMet4-Cbf1 chimera was effectively ubiqui-tylated by the SCFMet30 ligase. These results demonstrate thatMET generepression can occur independently of the means by which Met4 isrecruited to promoterDNAand that theMet4-Cbf1 chimera is an effec-tive substrate for the SCFMet30 ligase.

Methionine-dependentControl of Chromosome Segregation—In addi-tion to its role in methionine biosynthesis, the Cbf1 protein binds to thecentromere and participates in kinetochore complexes. Thus, in addi-tion to methionine auxotrophy, the cbf1� null mutation causes a 9-foldincrease in the rate of mitotic chromosome loss (21, 22, 34). As ourMet4-Cbf1 chimera contains the 210–351 carboxyl-terminal fragmentof Cbf1 that is sufficient to bind to the CDE1 element and to conferaccurate chromosome segregation (34), we investigated the functional-ity of theMet4-Cbf1 chimera in centromere function. For this purpose,we examined the loss rate of a CEN-based plasmid in wild-type, cbf1�,and cbf1� cells that expressed the Met4-Cbf1 chimera from the endog-enousMET4 promoter. TheMet4-Cbf1 fusion protein fully suppressedthe defects in plasmid segregation that are observed in the cbf1� nullmutant such that in the absence of high extracellular methionine, theloss rate of the CEN plasmid in themet4::MET4-CBF1,cbf1� strain wasindistinguishable fromwild type (Table 2). Chromatin immunoprecipi-tation confirmed that the Met4-Cbf1 chimera was bound to chromo-somal CDE1 elements (data not shown). To assess whether the Met4-

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FIGURE 3. Met4 degradation is proteasomedependent. A, stabilization of a GFP-Met4 fusionprotein in the presence of methionine and MG132.GFP-Met4 was expressed from the GAL1 promoter(strain CYS151, ura3::pGAL1-GFP-MET4::URA3,pdr1�, pdr3�) and then repressed by addition ofglucose in the presence of 1 mM L-methionine andin the absence or presence of 50, 100, or 200 �M

MG132. The GFP-Met4 fluorescent signal wasrecorded and quantified at the indicated timesusing a BD Biosciences FacScaliburTM flow cytom-eter. B, cells were grown as in panel A, and the sta-bility of GFP-Met4 was analyzed by fluorescencemicroscopy. C, Met4 that lacks lysine 163 isnot degraded upon methionine exposure. TheGFP-Met4�12 (strain C313, met4::TRP1,ura3::pGAL1-GFP-MET4�12::URA3) fusion proteinwas expressed in the cells from the GAL1 promoterand then repressed by the addition of glucose,either in the absence of methionine (�Met) or inthe presence of 1 mM L-methionine (�Met). Fluo-rescence images were acquired at the indicatedtimes. D, stabilization of the HA3Met4�12 in thepresence of 1 mM L-methionine. C323 (met4::TRP1,ura3::pGAL1-HA3MET4�12::URA3) cells were treatedas in panel B. At the indicated times, proteins wereextracted using the trichloroacetic acid procedureand immunoblotted with anti-HA antibody.

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Cbf1 chimera was capable of responding to methionine in thecontext of CEN DNA, we measured CEN plasmid stability in themet4::MET4-CBF1,cbf1� strain in the presence of high levels ofmethionine. Addition of high extracellular methionine dramaticallyincreased the rate of plasmid loss up to that observed in cbf1� nullmutant cells (Table 2). This genetic readout for Met4-Cbf1 functionparallels themethionine-induced elimination ofMet4-Cbf1 and accom-panyingMET gene repression and thus provides additional in vivo evi-dence that Met4 is regulated by proteolytic mechanism in minimalmedium.

DISCUSSION

The central role of sulfur metabolism in protein and nucleic acidbiosynthesis and in overall redox status of the cellmandates an exquisitemanagement system in which Met4 is a critical nexus. Not surprisinglythen, theMet4 system is subject to multiple layers of regulation, includ-ing combinatorial transcriptional complexes that operate differentiallyat various MET gene promoters, auto-regulation through Met4-dependent transcription of MET30, and both degradation-dependentand -independent control by SCFMet30. Here, we have delineated theintracellular signal for MET gene regulation and Met4 degradation,unequivocally demonstrated that Met4 is degraded in vivo under mini-mal medium conditions, and shown that Met4 can be modulated bySCFMet30 independently of local protein and DNA contexts.

Met4 Degradation Is Triggered by the Conversion of AdoMet intoCysteine—The ubiquitin-dependent degradation of Met4 was previ-ously thought to be triggered by an increase in the intracellular AdoMetpool, as derived from the rapid conversion of extracellular methionineinto AdoMet by the S-adenosylmethionine synthases encoded by theSAM1 and SAM2 genes. Thus, in cells that lack Sam1 and Sam2, methi-onine fails to repress MET genes, whereas high levels of extracellularAdoMet efficiently repress MET genes (35). Consistent with this view,the SAM1 and SAM2 genes were originally identified as eth2 and eth10

mutations that impaired the negative regulation of sulfur amino acidbiosynthesis (36). In addition, single point mutations within MET30allow cells to grow in the presence of a high amount of S- adenosyl-methionine, a toxic analogue of AdoMet (37). We have extended thisscheme to demonstrate that Met4 degradation actually depends on thesubsequent conversion of AdoMet into cysteine. Thus, when expressedfrom the endogenous MET4 promoter, both GFP-Met4 and Ha3Met4proteins are degraded when str4� cells are exposed to extracellular cys-teine, but not to either AdoMet or methionine. The requirement forStr4 inMET gene repression by methionine and AdoMet, but not cys-teine, has been noted previously (38). The here-reported cellular cys-teine-triggered destabilization of both GFP-Met4 and Ha3Met4expressed from the endogenousMET4 promoter exactly correlateswiththe results of Hansen et al. (38) demonstrating that cysteine does con-stitute the proximal sulfur-containing signal that mediates the repres-sion of MET genes in cells grown in minimal medium and exposed tohigh extracellular methionine.Cysteine serves as a precursor for the synthesis of glutathione and is

involved in the synthesis of different vitamins, such as thiamin andbiotin (39). Our results have shown that the inactivation of the GSH1gene that codes the first enzyme of glutathione synthesis does notimpair Met4 degradation as triggered by cysteine, indicating that gluta-thione or its derivatives are not involved in Met4 regulation, as previ-ously suggested (40). That cysteine is the intracellular signal that con-trols the fate of Met4 concurs with the prominent role of Met4 both inthe response to cadmium (41, 42) and in glutathione biosynthesis, whichgenerates compensating reducing equivalents in cells exposed to severeoxidative stress (43–45). In contrast to methionine and AdoMet, cys-teine and glutathione are both thiol compounds and thus directlyresponsive to global changes in oxidation. Intracellular cysteine is thusan ideal intermediary to control the Met4 regulon in response to thedifferent cues of sulfur amino acid metabolism and oxidative stressdefense.

FIGURE 4. The Met4-Cbf1 chimera activates methionine biosynthesis. A, the Met4-Cbf1 chimera relieves the methionine auxotrophy of single and double met4�,cbf1� mutantcells. Serial dilutions of CC849 – 8A (met4�), CC857–2A (cbf1�), and CY110 –11B (met4�,cbf1�) cells transformed by a plasmid expressing Met4 or Met4-Cbf1 chimera from the GAL1promoter were plated on medium containing 2% glucose or 2% galactose as carbon source in the absence or presence of 1mM L-methionine. B, the Met4-Cbf1 chimera fullycomplements growth rate. CC857-2A (cbf1�) cells transformed by a plasmid expressing Met4-Cbf1 chimera from GAL1 promoter were grown in minimal medium containing either2% glucose in the absence (black squares) or presence (black circles) of 1 mM L-methionine, or 2% galactose in the absence of methionine (grey triangles). C, the Met4-Cbf1 chimerastimulates sulfate reduction. W303–1A (wild-type) and CY172–3D (met4::MET4-CBF1) cells were grown in B medium containing 0.2 mM homocysteine as sulfur source. After filtration,cells were transferred in B medium containing radioactive sulfate. After 5 min, the cells were centrifuged, washed, and boiled. Increasing amounts of boiled extracts were analyzedby thin layer chromatography. Left and right control lanes were loaded with radioactive sulfate and methionine � glutathione, respectively.

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Met4 Degradation Requires Nuclear Localization and ProteasomeFunction—AlthoughMet4 is known to be a nuclear protein (23, 25), thesequence requirements for Met4 nuclear localization have not beendetermined. Our results suggest that Met4 contains two NLS regionsthat appear to function independently of each other. Importantly,nuclear localization of Met4 is required for its rapid elimination inresponse to methionine, consistent with the nuclear localization ofMet30 (23, 32). Nuclear localization of the SCFCdc4 complex is requiredfor degradation of one of its nuclear substrates, the cyclin-dependentkinase inhibitor Far1 (46). Finally, nuclear, but not cytoplasmic, elimi-nation of the GFP-Met4�7 derivative in response to methionine andstabilization of GFP-Met4 by proteasome inhibitors demonstrates that

Met4 is degraded in the nucleus in a conventional proteasome-depend-ent manner.

Context-independent Degradation of Met4—TheMET gene networkis controlled by several distinct transcription factors that operate inconjunction with Met4, namely Cbf1, Met28, Met31, and Met32 (16).Depending on theMet4 target gene, different complexes are formed: theMet4-Cbf1-Met28 complex assembles on the TCACGTG elementpresent upstream of the MET16 gene, whereas the Met4-Met28-Met31/32 complexes bind to the core motif AAACTGTG presentupstream of theMET3 andMET28 genes (19, 20). In the latter case, thebinding of Cbf1 in the vicinity of the AAACTGTG sequences enhancesthe affinity of Met4-Met28-Met31/32 complexes (20). Recently, Cbf1

FIGURE 5. Methionine triggers degradation ofthe Met4-Cbf1 chimera. A, MET genes areinduced with normal kinetics in a Met4-Cbf1strain. CD233 (wild-type) and CY172–3D(met4::MET4-CBF1) cells were grown in B mediumcontaining a repressive amount of L-methionine toearly log phase, transferred to a B medium withoutsulfur compound, and total RNA was extracted atthe indicated times and then analyzed by North-ern blot. B, MET genes are repressed with normalkinetics in a Met4-Cbf1 strain. MET gene expres-sion was analyzed by Northern blot in CD233(wild-type), CY172–3D (met4::MET4-CBF1), andCY172– 4C (met4::MET4-CBF1, cbf1�) before andafter 1 mM L-methionine addition. C, methioninecauses disappearance of Met4-Cbf1 at the METgene promoters. CY202– 4B (met4::HA3MET4-CBF1,cbf1�) cells were grown in B medium and exposedto 1 mM L-methionine. After cross-linking andimmunoprecipitation with Met4 antibodies, totalDNA was analyzed by quantitative PCR withprimer pairs specific to the indicated promoters.The IME2 open reading frame was used as a con-trol. D, the Met4-Cbf1 chimera is degraded uponmethionine exposure. CD233 (met4::HA3MET4) andCY202–1B (met4::HA3MET4 -CBF1) cells were grownin the absence of methionine (�Met) or in pres-ence of 1 mM methionine (�Met). At the indicatedtime, proteins were extracted using the trichloro-acetic acid procedure and immunoblotted withanti-HA antibody.

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has been shown to interact with the chromatin-remodeling ATPaseIsw1 at MET promoters and to be required for normal nucleosomepositioning in promoter-proximal regions that contain TCACGTGmotifs (47, 48). An important issue in the regulation of transcription bythe ubiquitin system is the role of discrete contexts, both in terms ofchromatin structure and protein interactions. We used a fully func-tional Met4-Cbf1 fusion protein to probe such effects. The kinetics ofMET gene repression in response tomethionine correlate preciselywithpromoter occupancy and abundance of the Met4-Cbf1 chimera. Theseresults support the view that Cbf1 functions atMET gene promoters torecruitMet4 and that the syntheticMet4-Cbf1 derivative is regulated bya proteolysis in minimal medium. These experiments would appear torule out the possibility that SCFMet30-dependent ubiquitylation ofMet4repressesMET gene transcription through specific dissociation of Cbf1frompromoter DNA (24). A second important conclusion that could bederived from the properties of the Met4-Cbf1 chimera is that Met4 canbe targeted by Met30 at entirely heterologous regions, as suggested bythe methionine-dependent instability of CEN plasmid DNA in strainsbearing Met4-Cbf1 as their only source of Cbf1 activity. Notably, cen-tromeric DNA is transcriptionally inert, suggesting that chromatin con-formation does not limit the access of SCFMet30 toMet4. In addition, themethionine-dependent plasmid instability conferred by the Met4-Cbf1fusion is consistent with the ubiquitin-mediated degradation ofMet4 inminimal medium.

Mechanism of SCFMet30 Regulation—An outstanding issue raised bythese studies is the mechanism whereby intracellular cysteine levelsdictate the activity of SCFMet30 towardMet4. The role of cysteine in thisprocess can in part be considered from the perspective of the evolutionof the ubiquitin activation pathway. Structural studies suggest thateukaryotic ubiquitin and prokaryotic sulfur carrier ThiS (involved inthiamine biosynthesis) evolved from a common ancestor (49, 50). Thefirst steps of ubiquitin activation are similar to the first steps of thiazolesynthesis: ThiS is activated as a carboxyl-terminal acyladenylate andthen converted to a thiocarboxylate by cysteine, precisely analogous tothe activation of ubiquitin by E1 enzyme (51). It is perhaps noteworthythat apart from mutations in MET30 and core SCF subunits, no othermutations that impair SCFMet30-mediated regulation ofMet4 have beenidentified. Thus, only a few protein components might be involved inthe cysteine-dependent regulation of SCFMet30 and/or Met4. As onepossibility, we suggest that cysteine may directly control the activity ofSCFMet30 toward Met4 through a thiol-based mechanism that actseither on SCFMet30 and/or on Met4. Among different hypotheses, aninteresting one is that cysteine may stabilize the Met4-SCFMet30 inter-

action by binding directly to the Met30 receptor subunit. It is worthnoting that such amechanism has been recently evidenced for the planthormone auxin, which triggers the degradation of the Aux/IAA tran-scriptional repressors. Auxin was indeed demonstrated to be a ligand ofthe SCFTir1 ubiquitin ligase, and the binding of auxin to the receptorsubunit Tir1 was shown to induce Aux/IAA degradation by promotingtheAux/IAA-SCFTir1 interaction (52). The known versatility of the thiolgroup in allosteric control of enzyme action (53)would certainly accom-modate a mechanism in which cysteine directly regulates the activity ofa specific ubiquitin ligase.

Acknowledgments—We thankP. Zarzov and J. DeRoyer for help in flow cytom-etry experiments.

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TABLE 2Plasmid stabilityThe stability of a centromeric plasmid (CEN6) expressing the HIS3 gene was measured in wild-type, cbf1�, and the double mutantmet4::MET4-CBF1,cbf1� strains. Thestrains were grown in synthetic medium without histidine and diluted in a medium containing histidine and either a repressive or non-repressive concentration ofL-methionine. At the indicated time the cells were plated on medium either containing or lacking histidine and either a repressive or non-repressive concentration ofL-methionine.

Strains Growthconditions

Cell numberT 0 T 1 T 2 T 3 T 4

W303–1A � PRS313 � His 348 352 388 277 378� His 307 291 257 247 277% 12 18 11 11 13

cbf1� � PRS313 � His 322 417 195 150 186� His 119 145 27 19 13% 63 65 86 87 93

met4::MET4-CBF1 �His 223 337 394 313 258cbf1� � PRS313 �His 195 296 338 298 227

% 13 12 14 5 12met4::MET4-CBF1 �His 259 286 376 326cbf1� � PRS313 1 mM L-Met �His 181 137 221 137 122

% 30 52 62 58 61

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Dominique ThomasAlexandra Menant, Peggy Baudouin-Cornu, Caroline Peyraud, Mike Tyers and

FactorDeterminants of the Ubiquitin-mediated Degradation of the Met4 Transcription

doi: 10.1074/jbc.M600037200 originally published online February 23, 20062006, 281:11744-11754.J. Biol. Chem. 

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