`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`THE JOURNAL OF BlOLOGlCAL CHEMISTRY VOL. 281, NO. 26, pp. 17661-77669, June 33, 2006
`© 2006 by The Ane'ican Society for Biochemistry and Molecular Biology, inc.
`Printed in the U SA.
`
`Role of Glutaredoxin-3 and GIutaredoxin-4 in the Iron
`Regulation of the Aft1 Transcriptional Activator
`in Saccharomyces cerevisiae*
`Received for publication, March 7, 2006, and in revised form,April 24, 2006 Published, JBC Papers in Press, April 28, 2006, DOl l0.lO74/jbc.l\/i602165200
`
`Luis Ojedai, Greg Kelleri, Ulrich Muhlenhoff§, Julian C. Rutherfordl, Roland Lill§, and Dennis R. Winge“
`From the *University of Utah Health Sciences Center, Departments of Medicine and Biochemistry, Salt Lake City, Utah 84132
`and §Institut fur Zytobiologie und Zytopathologie Philipps-Universitat Marburg, Robert-Koch-Strasse 6, 35033 Marburg, Germany
`
`The transcription factors Aft1 and Aft2 from Saccharomyces cer—
`evisiae regulate the expression of genes involved in iron homeosta—
`sis. These factors induce the expression of iron regulon genes in
`iron—deficient yeast but are inactivated in iron-replete cells. Iron
`inhibition of Aft1/Aft2 was previously shown to be dependent on
`mitochondrial components required for cytosolic iron sulfur pro-
`tein biogenesis. We presently show that the nuclear monothiol glu—
`taredoxins er3 and er4 are critical for iron inhibition of Aft1 in
`yeast cells. Cells lacking both glutaredoxins show constitutive
`expression of iron regulon genes. Overexpression of er4 attenu—
`ates wild type Aft1 activity. The thioredoxin-like domain in er3
`and er4 is dispensable in mediating iron inhibition o Aft1 activity,
`whereas the conserved cysteine that is part of the co served CGFS
`motif in monothiol glutaredoxins is essential for this f nction. er3
`and er4 interact with Aft1 as shown by two-hybrid interactions
`and co—immunoprecipitation assays. The interaction between glu-
`taredoxins and Aft1 is not modulated by the iron status of cells but
`is dependent on the conserved glutaredoxin domain Cys residue.
`Thus, er3 and er4 are novel components required for Aft1 iron
`regulation that most likely occurs in the nucleus.
`
`Iron, an indispensable nutrient in cell physiology, is used in iron—
`sulfur clusters, hemes, and diiron-oxo metal centers in enzymes. Sac—
`charomyces cerevisme, a model organism in metal metabolism, main-
`tains iron homeostasis largely through the regulation of iron uptake and
`storage. In this yeast, survival under low iron conditions is ensured
`through the utilization of the iron—responsive transcriptional activa»
`tors Aft1 and Aft2 (1—3). These factors are activated in iron—deficient
`cells and induce the expression of more than 20 genes that are
`referred to as the iron regulon (4—7). This regulon includes genes
`whose products function in ionic iron acquisition, iron siderophore
`uptake, and vacuolar iron utilization. Activated Aft1 also induces the
`expression of CTH2 that encodes an RNA—binding protein. Cth2
`mediates the degradation of transcripts of some iron-requiring
`enzymes to conserve iron in the cell (8).
`Aft1 is localized to the nucleus under low iron conditions and to
`
`cytoplasm under iron—sufficient conditions (9). Under iron—sufficient
`conditions, Aft1 remains inactive due to its cytoplasmic localization
`where it is unable to drive transcription (9). Aft1 has two nuclear local—
`
`
`* This work was supported by National Institutes of Health Grant CA61286 (to D. R. W.)
`and grants of the Sonderforschungsbereich 593, Deutsche Forschungsgemeinschaft
`{Gottfried Wilhelm Leibniz program), Fonds der chemischen lndustrie, and the Euro-
`pean Commission (to R. L.). The costs of publication of this article were defrayed in
`part by 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.
`1Towhom correspondence should be addressed. Tel.: 801585-5 1 03; Fax: 801 -585»5469;
`E-mail: dennis.winge@hsc.utah.edu.
`
`ization sequences and a nuclear export sequence (NES),2 which map to
`its N—terminal DNA binding domain (9, 10). Mutations of two leucines
`within the NES result in retention of Aft1 within the nucleus and con—
`
`stitutive transcriptional activity regardless of iron levels. In addition,
`Aft1 contains a functionally important conserved 291CXC293 sequence
`motif adjacent to the DNA binding domain and 190 residues down—
`stream of the NES. Cys to Phe substitutions at either Cys within this
`motif in Aft1 result in constitutive transcriptional activation in iron—
`replete cells (9, 11). As expected, the constitutively active C2911: Aft1
`variant (Aft1—1“?) is retained within the nucleus. Thus, iron—regulation
`of Aft1 is dependent on its cycling between the nuclear and the cyto—
`plasmic compartments.
`The mechanism by which Aft1 and Aft2 sense cellular iron levels has
`been a topic ofinterest and intense research. Clues on the mechanism of
`iron sensing came from the observation that cells defective for Fe—S
`cluster biogenesis within the mitochondrial matrix exhibited constitu—
`tive expression of the iron regulon (12). Since disruption of Fe—S cluster
`biogenesis results in mitochondrial iron accumulation, it was initially
`thought that Aft1 was constitutive by virtue of depletion of cytosolic
`iron (12). It was later shown that disruption ofFe—S cluster biogenesis by
`diminution in the levels of the cysteine desulfurase (Nfsl) or the frataxin
`homologue (thI) did not decrease cytosolic iron (13). This is an indi»
`cation that Aft1 becomes constitutive due to impairment of a signal
`created by the mitochondrial Fe-S biosynthetic machinery and not to an
`indirect effect of alteration in iron compartmentalization.
`In S. cerevisiae mitochondria are required for maturation of Fe—S
`proteins both inside and outside of the organelle (14). For synthesis of
`cytosolic and nuclear Fe—S proteins, mitochondria export a still
`unknown compound via the mitochondrial inner membrane trans—
`porter Atml (15). Other components of this export machinery are the
`mitochondrial intermembrane space sulfhydryl oxidase Ervl as well as
`glutathione (15, 16). Depletion of glutathione activates Aft1 (ll, 17).
`After export to the cytosol, the cytosolic Fe—S protein assembly machin»
`ery (CIA) matures Fe—S Clusters and inserts them into target proteins
`(14). The CIA machinery includes the proteins Narl, Cfdl. Nbp35, and
`Cia1(18—20).
`Iron sensing by Aft1 and Aft2 requires proper mitochondrial Fe—S
`cluster biosynthesis as well as a functional export to the cytoplasm.
`However, it does not require the CIA machinery (ll), demonstrating
`that iron sensing by Aft1/Aft2 is not linked to the maturation of cyto—
`solic 4FE-4S clusters.
`
`Since the CIA complex is not required to mediate iron inhibition of
`Aft1 function, we predicted that other proteins may be involved in sens—
`ing the iron inhibitory signal extruded by Atml. One attractive candi—
`
`2The abbreviations used are: NES, nuclear export sequence; CM, complete synthetic
`medium; TAP, tandem affinity purification; CIA, cytosolic Fe-S, protein assembly; BPS,
`bathophenanthroline sulfonate.
`
`JOURNAL OF BIOLOGICAL CHEM/STRY 17661
`JUNE 30, 2006~VOLUME 281 -NUMBER 26
`ks
`
`BUTAMAX 1007
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`

`

`Glutaredoxins Modulate Aft1 Function
`
`TABLE 1
`
`Strains used in the present study
`
`Description
`Strain
`BY4742 Mata, hisBAl, leuZAO, lysZAO, uraSAO
`Wild type
`BY4-742 Mata, hisSAI, leuZAO, lysZAO, uraSAO, grx322KanMX
`AgrxS
`BY4742 Mata, his3A1, leuZAO, lysZAO, ura3A0, grx4zzKanMX
`Agrx4
`BY4-742 Mata, hisSAI, leu2A0, lysZAO, uraSAO, grx3::LEU2 grx4zzKanMX
`Agrx3,Agrx4
`BY4742 Mata, his3A1, leu2A0, lysZAO, ura3AO, glrl::Kar1MX
`Aglrl
`BY4742 Mata, hi53A1, leuZAO, lysZAO, uraBAO, aftlz:KanMX
`Aaftl
`CY4 Mat a ura3—52, leu2-3, 112 trp1—1 ade2-1, hi53—11 can1»100
`Wild type
`CY4 Mat a ura3-52, leu2—3, 112 trp1-1 ade2—1, hisB-II can1—100 trr1::H1$3
`Atrrl
`Mata, ura3-52, 1163-13200, ade2-101, 13152—801, leu2-3,112, trpI—901 tyri—SOJ gal4—A512gal80—AS38, adeS::hisG
`YM4271
`
`
`Aaft](YM4271)
`Mata, uraB—SZ, hisB—AZOO, aa'e2—101, 13252-801, leu2—3,112, trp1-901 tyrI—SOZ gal4—A512gal80—A538, adeS::hisG, aftlzzHISB
`
`date protein was the nuclear glutaredoxin—3 (er3), which was reported
`to interact with Aft1 in a global yeast two—hybrid interaction study (21).
`Glutaredoxins are glutathione-dependent thiol—disulfide oxidoreducta—
`ses that function in maintaining the cellular redox homeostasis. S. cer—
`evisiae has two dithiol glutaredoxins (erI and er2) and three mono—
`thiol glutaredoxins (er3, er4, and er5) (22—24). The monothiol
`glutaredoxins are believed to reduce mixed disulfides formed between a
`protein and glutathione in a process known as deglutathionylation. In
`contrast, dithiol glutaredoxins can participate in deglutathionylation as
`well as in the direct reduction of disulfides (25). erS, the most studied
`monothiol glutaredoxin, is localized to the mitochondrial matrix, where
`it participates in the maturation of Fe—S clusters (24). er3 and er4 are
`predominantly localized to the nucleus (26). These proteins can substi—
`tute for er5 when overexpressed and targeted to the mitochondrial
`matrix (23); no information on their natural function has been reported.
`In addition to the reported interaction between er3 and Aft1, iron
`inhibition of Aft1 requires glutathione (11). Based on these clues, we
`evaluated the role of er3 and er4 in the iron inhibition of Aft1 and
`
`show presently that iron sensing is dependent on the presence of the
`redundant er3 and er4 proteins.
`
`MATERIALS AND METHODS
`
`Yeast Strains and Culture Conditions-The yeast strains used in this
`study are listed in Table 1. BY4742, Agrx3, Agrx4, and Aglrl strains were
`obtained from Research Genetics. A PCR»created LEU2 cassette was
`
`integrated by homologous recombination at the GRX3 locus in a Agrx4
`cell to create the Agrx3Agrx4 strain. The Atrrl and YM4~271 strains were
`previously described (27). A PCR»created H183 cassette was integrated
`by homologous recombination at the AFT] locus in a YM4271 strain to
`create the Aaftl strain used in yeast two-hybrid experiments. Cells were
`grown at 30 ”C either in YPD medium, containing yeast extract, tryp—
`tone, and dextrose, or in complete synthetic medium (CM) or incom-
`plete synthetic medium lacking, for example, uracil (CM—Ura) or
`leucine (CM—Leu). For several experiments, the growth medium was
`supplemented with 0.1 mM bathophenanthroline sulfonate (BPS) as a
`ferrous iron chelator to lower the availability of iron or supplemented
`with 0.1 mM FeCl2. Doxycycline was added to the medium at a 5 ug/ml
`final concentration for the indicated periods of time to modulate
`expression from the tetO7 promoter (28). All cells were harvested during
`log phase.
`Plasmids—All plasmid constructs were confirmed by DNA sequenc—
`ing. Full—length wild type GRX3 and GRX4 coding sequences as well as
`their mutant forms (GRX3 C2115 and GRX4 C1715) were tagged at the
`3’-end with one Myc epitope. In addition, full—length wild type GRX4 as
`well as its mutant forms (GRX4 C1715 and GRX4 GPm) were also
`tagged at the 3’—end with l-Iis6 epitopes. All of these constructs were
`Cloned in the YCp pCM189 and YEp pCM19O plasmids under the con—
`trol of the doxycycline—regulated tetO7 promoter (28). In the previous
`
`cloning procedures, GRX3 constructs were cloned between NotI/PstI
`sites, and GRX4 constructs were Cloned at BamHI/Pstl sites.
`
`For the two—hybrid experiments, the plasmid pBG4D~1, which con—
`tains the ADI-11 promoter and the GAL4 (codons 1—147) DNA binding
`domain, was used. The GRX3 and GRX4 coding sequences were ampli»
`tied and ligated into pBG4D-1, resulting in GRX3 and GRX4 3’—ends
`being fused in frame with the GAL4 DNA binding domain. The VP16
`activation domain fused to the CYCJ terminator (5' to 3' orientation)
`was amplified and ligated into pRS416. The AFT] promoter and open
`reading frame was PCR—amplified with SpeI/BglII sites and ligated into
`cut pRS416 VP16-CYC1 plasmid. resulting in APT] being fused in frame
`with VP16. The resulting AFT] VPJ6 was used as a template for PCR
`mutagenesis
`fC291F and C293F. The previous AFT] constructs are
`under the con rol of the AFT] promoter.
`AFT] was AP—tagged at its C terminus by homologous recombina-
`tion in its chr mosomal locus (29), The genomic AFTJ—TAP was later
`used as a template in a PCR where AFTJ—TAP was amplified, cut, and
`ligated into pCM190, where it is under the control of the tetO7 pro-
`moter. Wild type AFT1,AFT1-l"p, and AFT] L99A were subcloned in
`plasmid pRS416 under the control ofits own promoter.
`The C—terminal 375 bp of GRX4 (including the glutaredoxin domain
`but excluding the thioredoxin domain) as well as the C—terminal 381 bp
`of GRXS (excluding the mitochondrial target sequence) was PCR—am-
`plified and ligated into pCM190, where they were under the control of
`the tetO7 promoter.
`S] Nuclease Assays—RNA was extracted from cells grown to midlog
`phase using the hot acid phenol method. and S1 analysis was performed
`as previously described (30). For each reaction, 12 ug of total RNA were
`hybridized to a 32F end—labeled DNA oligonucleotide probe before
`digestion with $1 nuclease and separation on an 8% polyacrylamide, 8 M
`urea polyacrylamide gel. Dried gels were imaged using a Bio—Rad FX
`phosphor imager and quantified using Quantity One software prior to
`autoradiography.
`DNA Microarray Analysis—RNA was extracted from wild type
`BY4742 and Agrx3Agrx4 cells grown in YPD medium supplemented
`with 200 MM FeClz. Total RNA was isolated using the hot acid phenol
`method. mRNA was isolated from total RNA by using the Poly(A) Tract
`mRNA isolation system IV kit from Promega following the manufactur—
`er’s instructions. Fabrication of DNA microarray, synthesis of fluores-
`cence-labeled CDNA, hybridization of the microarrays, and subsequent
`scanning were performed in the Huntsman Cancer Institute Microarray
`Core Facility at the University of Utah.
`B—Galactosidase Assays—Cells were grown to midexponential phase
`(A600 0.5) in CM—Ura~Leu—I—Iis—Trp, 2% glucose either with supple—
`mented iron or in the presence of BPS. B—Galactosidase activity was
`measured in permeabilized cells as previously described (31) and is
`expressed in Miller units that are calculated as follows (A420 X 1000)/
`(min >< ml of culture used X absorbance of the culture at 600 nm).
`
`17662 JOURNAL OF B/OLOG/CAL CHEM/STRY
`VOLUME 281- NUMBER 26'JUNE 30, 2006 ,
`
`
`

`

`Glutaredoxins Modulate Aft1 Function
`
`Agrx3
`
`Agrx4 Agrx3Agrx4
`
`
`
`BPS Fe
`
`
`BPS Fe
`BPS‘Fe
`
`
`
`
`BPs Fe
`
`
`
`FET3
`
`CMD1
`
`B. WT
`
`Ang3
`
`Ang4 Agrx3Ang4
`
`
`
` FI T3
`
`
`
`
`(Fe
`BPS“
`BPS Fe
`BPS Fe '
`FIGURE 1. Aft1 is partially activated in the absence of er3 or er4 but fully acti-
`vated in cells lacking both glutaredoxins. Wild type, Agrxj, Agrx4, and Ang3Agrx4
`cells were grown in YPD medium in the presence of either 100 uM BPS or 200 uM FeCI2
`prior to $1 nuclease analyses of FET3 (A) and FIT3 (3) mRNA levels. CMD1 encoding cal-
`modulin was used as the loading control.
`
`CMD1
`
`
`
` Immunoprecipitation and Immunodetection—Cellular lysates for
`
`immunoprecipitation analysis were prepared by glass beading in 50 mM
`Tris—Cl, pH 7.5, 150 mM sodium chloride, 0.1% Nonidet P—40, 0.05%
`sodium deoxycholate, and a protease inhibitor mixture. The superna»
`tant was incubated with a rabbit polyclonal anti—Myc antibody for 1 h at
`4 ”C. Protein A—agarose was added and incubated overnight at 4 “C. The
`protein A—agarose was collected by centrifugation, washed three times,
`and boiled in SDS sample buffer. The immunoprecipitated protein was
`resolved by SDS—10% polyacrylamide gel electrophoresis and trans—
`ferred to nitrocellulose. Membranes were blocked and probed with
`either PAP peroxidase anti»peroxidase (for TAP detection) or rabbit
`polyclonal anti»Myc. Detection was performed by enhanced chemilu—
`minescence after incubation with a horseradish peroxidaseconjugated
`secondary antibody.
`In addition, an aliquot of the supernatant was used for immunode—
`tection analysis by immunoblotting, using PAP (Sigma), rabbit poly—
`clonal anti-Myc (Santa Cruz Biotechnology, Inc., Santa Cruz. CA),
`mouse monoclonal anti-ng1 (Molecular Probes), and mouse mono—
`clonal anti—His (Novagen).
`Cellular lysates were prepared for immunoblotting by glass beading
`using 10% trichloroacetic acid in Tris acetate buffer, pH 8. Proteins were
`resolved by SDS—polyacrylamide gel electrophoresis and transferred to
`nitrocellulose, The membranes were probed with antibodies previously
`described and detected using chemiluminescence (ECL; Pierce).
`Labeling ofyeast cells with radioactive iron (55Pe) and the determina—
`tion of iron incorporation into Fe—S proteins by immunoprecipitation
`and liquid scintillation counting were carried out as previously
`described (15).
`Miscellaneous Procedures—The following published methods were
`used. The sulfite reductase assay was performed as previously described
`(11). For aconitase activity assays, cells were lysed by bead beating, and
`aconitase activity was determined by coupled reaction of aconitase (EC
`4.2.1.3) and isocitrate dehydrogenase (EC 11.1.42) (32). Mutagenesis
`was performed by either PCR mutagenesis or by introducing the muta—
`tion in the primer followed by homologous recombination (33). Yeast
`transformation was performed using standard lithium acetate protocol
`(34).
`
`RESULTS
`
`To evaluate the role of er3 and er4 in the iron inhibition of Aft1,
`
`we quantified the expression oftwo iron regulon genes, PET3 and FITS,
`in cells lacking either er3 or er4 or in cells lacking both molecules
`(Fig. 1, A and B). Gene expression was assessed by quantifying mRNA
`levels using the SI nuclease protection assay. Whereas expression of
`PET3 and PITS was inhibited in iron—supplemented wild type cells,
`expression of PETS, but not PITB, was elevated 3.5— and 25—fold in
`iron—supplemented AgrxS or Agrx4 cells, respectively. relative to wild
`type cells. The absence of er3 or er4 did not affect the full induction
`of PET3 observed when the iron bioavailability is limited in cells treated
`with the iron chelator bathophenanthroline sulfonate (BPS).
`Cells lacking both er3 and er4 exhibited constitutive expression of
`both PETS and PIT3. To verify that the major iron regulon genes were
`expressed in Agrx3Agrx4 cells, DNA microarray analysis was performed
`comparing wild type and AgrxSAgrx4 cells cultured in YPD medium
`supplemented with iron (Table 2). The same genes induced by the con—
`stitutively active Aft1—1uP were highly expressed in Agrxé’ Agrx4 cells,
`although the observed induction ratios varied. Such variation in the
`induction ratios of iron regulon genes is observed under other condi—
`tions that activate Aft1 (4—7). The high expression of the Aft2 target
`gene MRS4 (7, 35) suggested that both Aft1 and Aft2 are constitutively
`
`TABLE 2
`
`Genes induced in Agrx3Agrx4 cells compared with
`Aft1-1"p-containing cells
`Microarray analysis was conducted on Agrx3Agrx4 cells compared with wild type
`(WT) cells cultured in YPD containing 0.2 mM FeClJ. RNA was extracted from these
`cells, and poly(A) RNA was recovered. The mean from two duplicate experiments is
`shown. The -fold induction data are compared with transcript profile data ofAPTI-
`1”” cells published previously. Only a subset of the iron regulon genes are shown to
`document that the iron regulon genes are induced in Agrx3Agrx4 cells. In the dupli»
`cate experiments with Agrx3Agrx4 cells, the variation in the -fold induction was
`within 10%.
`
`Gene
`
`Aft1 — 1up versus
`Agrx3Agrx4 versus
`WT (mean n = 3)
`WT (mean n = 2)
`fold induction
`
`9
`38
`F]T]
`43
`19
`PIT3
`30
`1 2
`F1T2
`5
`11
`CTHZ
`25
`8
`PETB
`7
`8
`51T1
`7
`5.5
`ENE]
`1.3
`4.7
`ISUZ
`1.7
`4.3
`CADI
`2.5
`4.1
`ARN]
`1.7
`3.8
`MRS4
`
`
`COT] 2.2 3.7
`
`active in the Agrx3Agrx4 strain. These data confirm that the iron regu—
`lon is induced in the absence ofer3 and er4. The induced expression
`of iron regulon genes observed in Agrx3Agrx4 cells cultured in iron—
`replete medium was due to the absence of er3 and er4, since trans—
`formation of the double null cells with GRX4 under the TET promoter
`restored inhibition of PET3 expression (Fig. 2). As expected, the addi»
`tion of doxycycline to repress GRX4 expression yielded elevated PETS
`expression.
`er3 and er4 appear to be redundant molecules. Agrx4 cells trans—
`formed with a low copy plasmid containing either GRX3 or GRX4
`restored full iron inhibition of PET3 transcription (Fig. 3A). er3 and
`er4 are members of the monothiol glutaredoxin family and as such
`possess a single functional cysteinyl residue within a CGFS sequence
`motif (36). We tested whether the single conserved cysteine residue
`within the CGFS motif in each protein is essential for iron inhibition of
`Aft1 activity. Transformation of Agrx4 cells with a YCp plasmid-borne
`GRX4 restored iron inhibition of PET3 expression, whereas transfor-
`mants containing a mutant GRX4 allele encoding a C1715 substitution
`failed to inhibit PET3 expression (Fig. 3A). In the same way, mutant
`er3 containing a C2115 substitution failed to mediate iron inhibition
`
`JOURNAL OF BlOLOG/CAL CHEMISTRY 17663
`JUNE 30, 2006'VOLUME 281 -NUMBER 26
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`

`

`Glutaredoxins Modulate Aft1 Function
`
`FE T3
`
`CMD 1
`
`-
`
`+
`
`Doxycyc/ine
`FIGURE 2. The iron regulon induction phenotype seen in Agrx3Agrx4 cells is
`reversed by introducing a wild type GRX4 gene. Agrx3Agrx4 cells transformed with a
`low copy plasmid containing GRX4 were grown in CM—Ura, 2% glucose for 19 h in the
`presence or absence of doxycycline (5 pug/ml). Si nuclease analysis was used to assess
`the expression of FET3 mRNA. CMD] encoding calmodulin was used as the loading
`control.
`
`of PET3 expression in AgrxS cells (Fig. BB). Although the mutant er3
`and er4 proteins were inactive, they were stably expressed (data not
`shown). Thus, the putative functional cysteinyl residue in each glutare—
`doxin is important to mediate iron inhibition of Aftl.
`Since depletion of er3 and er4 resulted in constitutive Aftl activ-
`
`ity, the effect of overexpression of GRX4 on Aftl function in wild type
`cells was evaluated. Wild type cells cultured in iron-limited SC medium
`showed partial FET3 expression that could be completely inhibited by
`the addition of iron salts to the culture medium. The overexpression of
`GRX4 in cells cultured in this iron-limited medium markedly attenu—
`ated FET3 expression (Fig. 4, A and B, two lanes on the left). The inhib—
`itory effect of GRX4 overexpression was also seen when Aftl was fully
`activated in BPS—supplemented, iron—deficient cells (data not shown). In
`addition, the C1715 substitution in er4 partially abrogated the ability
`of overexpressed er4 to attenuate FET3 expression in wild type cells
`(Fig. 4A).
`Aftl becomes constitutively active when the 291CXC293 motif or the
`NES motif is mutated (1, 9). To address whether overexpression of er4
`can attenuate the function of constitutively active variants of Aftl, the
`TET—GRX4—containing high copy vector was transformed into Aafll
`cells containing either an AFT] allele encoding the C291F variant (Aftl-
`lup) or the L99A NES variant. Overexpression of er4 inhibited wild
`type Aftl activity and resulted in a partial, reproducible attenuation of
`Aftl (L99A) (Pig. 4, C and D) but no significant attenuation of the
`activity of the Aftl—lUP constitutive mutant (Fig. 4E).
`Monothiol glutaredoxins are believed to function in the deglutathi0~
`nylation of target proteins (37). In the deglutathionylation reaction,
`monothiol glutaredoxins are predicted to be transiently glutathionyl—
`ated themselves (36). We evaluated whether the conserved residues that
`
`form the glutathione—binding pocket in other monothiol glutaredoxins
`are important for er4»mediated iron inhibition of Aftl activity. We
`tested a double mutant of er4 in which the conserved 209\)(/P210 was
`converted to 209DAZIO. The 209DA210 mutant er4, designated er4
`GPm for “glutathione pocket mutant,” was unable to mediate iron inhi—
`bition of FET3 expression in Agrx4 cells (Fig. 3C). The er4 GPm
`mutant did not affect the full induction of FET3 by iron deprivation and
`was shown to be equally stable to the wild type protein by immunoblot
`ting (data not shown). Thus, glutathione binding may be important for
`er4 activity.
`
`A
`
`
`
`V
`
`er3 er4 er4
`c1713
`
`V GtX3 Crx3
`02113
`
`FET3
`
`cum
`
`C
`
`v
`
`er4
`
`er4 GPm
`
`N A: C FET3
`
`
`
`BP
`
`BP
`BPS
`Fé
`3
`Fe
`Fe
`8
`FIGURE 3. The conserved cysteine in the glutaredoxin domain of er4 as well as the
`putative glutathione pocket residues tryptophan and proline are important for the
`control of Mn activity. A, Ang4 cells transformed with a low copy plasmid alone (V) or
`with the same plasmid containing either the wild type GRX3 gene (er3), the wild type
`GRX4 gene (er4), or the GRX4 C7775 mutant gene (er4 C1775). B, .3ng3 cells trans-
`formed with a low copy plasmid alone or with the same plasmid containing either the
`wild type GRX3 gene or the GRX3 C27 75 mutant gene (er3 C2175). Cells from A and B
`were grown in CMiLeu, 2% glucose plus 50 um FeCl2 prior to $1 nuclease analyses of
`FET3 and CMDl mRNA levels. C, Agrx4 cells were transformed with a low copy plasmid
`alone or with the same plasmid containing either the wild type GRX4 gene or the GRX4
`glutathione pocket mutant (er4 GPm). These cells were grown in CM~ Leu, 2% glucose
`supplemented with either 100 MM BPS or 50 uM FeCI2 prior to Si nuclease analyses of
`FET3 and CMD? mRNA levels.
`
`er3 and er4 differ from the mitochondrial monothiol glutare—
`doxin er5 in that they contain an N—terminal thioredoxin domain in
`addition to the C—terminal glutaredoxin domain (26). The thioredoxin
`domain of er3 is believed to be responsible for the predominant
`nuclear localization of this glutaredoxin (26). To test whether the thi—
`oredoxin domain is important for iron inhibition of Aftl, we engineered
`a er4 truncate lacking the N—terminal thioredoxin domain. FET3
`expression was iron—inhibited in Agrx3Agrx4 cells harboring a high copy
`plasmid containing the er4 truncate, designated er4T (Fig. 5A).
`However, when the er4T was expressed in a low copy plasmid, it had
`no effect (data not shown). A green fluorescent protein fusion of the
`er4 truncate, expressed in a low copy plasmid, was found to have a
`diffused localization throughout the cell (data not shown), suggesting
`that insufficient protein existed within the nucleus to mediate iron inhi-
`bition of Aftl activity.
`Cells lacking er3 and er4 exhibit a growth defect in synthetic
`culture medium (Fig. SB) but are less impaired in rich YPD medium
`(data not shown). The thioredoxin domain is nonessential for normal
`
`cell growth, because expression of er4 or er4T restored wild type
`growth (Fig. SB).
`.
`er5 functions in the mitochondrial Fe—S biogenesis pathway (24).
`er3 and er4 were shown to substitute for er5 in mitochondrial
`
`function when overexpressed and targeted to the mitochondrial matrix
`(23). Suppression of the AgrxS phenotypes requires both the thiore-
`doxin and glutaredoxin domains and the essential glutaredoxin domain
`Cys residue (23). To address whether the mitochondrial er5 can com-
`
`plement AgprAgrx4 cells, a truncated GRXS construct was engineered
`that lacked the N~terminal mitochondrial target sequence (designated
`er5 C). Expression of the erS truncate restored wild type growth of
`Ag'prAgrxéi cells (Fig. SB), yet FET3 expression was constitutive (Fig.
`5A), suggesting that the growth defect is unrelated to constitutive Aftl
`
`17664 JOURNAL OF BlOLOG/CAL CHEMISTRY
`
`VOLUME 281 °NUMBER 26'JUNE 30,2006 .
`
`

`

`Glutaredoxins Modulate Aft1 Function
`
`B A
`e: 10°
`.5
`80
`‘8
`a.
`9
`60
`E
`40
`i2
`LLI
`LL
`
`20
`0
`
`
`
`V
`
`H'
`er4
`
`2:);8
`
`FET3
`
`D A
`\ é
`g c 160
`g -%
`9 8 120
`E ‘5.
`(3’; §
`”i: E
`LL E 40
`0
`
`80
`
`CMD1
`
`0
`
`
`
`
`V er4
`V er4
`Aft1
`Aft1 L99A
`
`
`..44..4,WW___—————ug__.__.
`
`FIGURE 4. Overexpression of er4 decreases
`the activity of wild type and NES mutant
`Aft1. A, wild type cells transformed with a high
`copy plasmid alone(V) orwith the same plasmid
`containing either the wild type GRX4 gene or
`the GRX4 C1715 mutant gene were grown in
`CM—Ura, 2% glucose containing the BlOiOi
`low iron nitrogen base. Si nuclease analysis was
`used to assess the expression of FET3 mRNA.
`CMDi encoding calmodulin was used as the load—
`ing control. B, the FET3 mRNA levels in A were
`quantified and normalized to CMDi mRNA levels.
`C,Aafticellsweretransformed withtwo plasmids,
`a low copy plasmid containing either a wild type
`AFTi (Afti)ora NES mutant AFT] (Aft1 L99A)gene
`and a high copy plasmid alone or containing the
`wild type GRX4 gene. These cells were grown in
`CM~Ura—Leu,2%glucose containingthe BlOiOi
`low iron nitrogen base. Si nuclease analysis was
`used to assess the expression of FET3 mRNA. D,
`FET3 mRNA levels in C were quantified and nor-
`malized to CMDi mRNA levels. E, cells containing
`Afti»iup eitherinthe presence or absence ofGRX4
`were grown in CM—Ura—Leu, 2% glucose, and
`FET3 expression was assessed by Si nuclease anal-
`ysis.
`in three independent experiments done in
`duplicate, the mean FET3 expression in er4-over-
`expressing Afti—i “9 cells was 85% of that in con—
`trol Aft1-i“p cells.
`
`A
`
`FET3
`
`CMD1
`
`
`
`,,__
`,_
`
`‘
`‘
`V
`er‘l er4
`C1713
`
`C Aft1
`
`Aft1 L99A
`
`_
`'
`
`.
`;
`
`
`
`
`
`
`v er4
`
`E
`
`Aft1-1 up
`
`
`
`FET3
`
`CMD1
`
`
`
`A 9
`
`. FET3
`
`9
`
`. . . CMD1
`
`er4
`
`er4 T er5 C
`
`B
`
`1/1
`
`1/10
`
`1/100
`
`
`
`FIGURE 5. The thioredoxin domain of er4 is not necessary for complementation of
`either growth or the control of Aft1 activity in a Agrx3Agrx4 strain. Agrx3Agrx4 cells
`weretransformed with a low copy plasmid containing the wild type GRX4 gene (60(4) orwith
`a high copy plasmid either alone (V) or containing the GRX4 gene lacking the thioredoxin
`domain (Gnr4 T) or the GXR5 gene lacking the mitochondrial target sequence (er5 C).A, cells
`were grown in CM—Ura, 2% glucose prior to Si nuclease analyses of FEB and CMDI mRNA
`levels. B, cells from A were plated on CMeUra, 2% glucose plates. The double null cells have
`a long lag phase in growth and this manifests in the low growth seen in 8.
`
`activity. Since the er5 truncate was not stably expressed in the yeast
`cytoplasm (data not shown), we cannot be certain whether the lack of
`iron inhibition of PET3 expression was a result of low protein levels or
`inactivity in that function.
`The previous results are consistent with er3 and er4- having a
`direct role in the iron inhibition of Aft1 activity. The original motivation
`to consider er3 was its reported interaction with Aft1 in a global two—
`hybrid study. A two—hybrid assay system for detecting protein—protein
`interactions was set up to confirm the binding interaction between er3
`(or er4) and Aft1. The Gal4~ DNA binding domain was fused to er3
`and er4. generating er3/Gal4- and er4/Gal4 fusion proteins. The
`transactivation domain from the herpes simplex VP16 was fused to the
`C terminus ofAftl. Cells harboring the two fusion proteins and a GALI/
`lacZ reporter fusion were assayed for B—galactosidase activity. The com—
`bination of either glutaredoxin fusion with Aft1/VP16 resulted in ele—
`vated B—galactosidase activity (Fig. 6A). Mutation of the critical Cys in
`either glutaredoxin abrogated lacZ expression consistent with a loss of
`interaction. The mutant er3/Gal4 and er4/Gal4 fusion proteins
`were shown to be equally abundant as the wild type fusion proteins by
`immunoblotting (data not shown). In addition, the interaction of er3
`and er4 with Aft1 was markedly diminished when the constitutively
`active Aft1 C291F,C293F mutant variant was used (data not shown).
`The observed interaction between the two glutaredoxins and Aft1 was
`not altered by changes in the cellular iron status (Fig. 6B).
`To confirm the observed interaction between the glutaredoxins and
`Aft1, constructs were engineered in which AFT] was TAP—tagged and
`GRX4 was either Myc-tagged or poly-His~tagged. Cells harboring vec—
`
`JUNE 30, 2006°VOLUME281-NUMBER 26
`
`5‘
`
`jOURNAL OF BlOLOG/CAL CHEMISTRY 17665
`
`

`

`Glutaredoxins Modulate Aft1 Function
`
`A
`
`1 20 —
`
`Aft1
`er4
`
`C171S
`
` V
`
`V
`
`Af‘t1
`
`Aft1 V er3 V
`
`Aft1
`
`er3 er4 er3 er4 C2118 er4 er3
`C171S C211s
`
`1 00 '-
`
`80 —
`
`A
`3‘ c")
`E E
`*5 (D
`(U \— 60 __
`N E
`o
`_
`8
`_J o\
`v
`
`40
`
`20 _
`
`_
`
`0
`
`B
`
`C00
`
`O) O
`
`is0
`
`
`
`LacZactIVIty(Arbitraryunits)
`
`
`
`Fe
`
`BPS
`
`Fe
`
`BPS
`
`Aft1 + er3
`
`Aft1 + er4
`
`FlGURE 6. Yeast two-hybrid analyses shows
`.
`.
`.
`that Aft1 interaction with er3 or er4
`requires the conserved glutaredoxrn domaln
`cysteine and is independent of iron levels.
`Aaftl cells were transformed with a high copy
`plasmid containing the GALl/lacZ reporter, a
`low copy plasmid either empty (V) or containing
`the AFT] VP16 construct, and a low copy pias-
`mid with either GRX3 or GRX4 in frame with the
`GAL4 DNA binding domain. Interaction be-
`tween Aftl with the glutaredoxins results in
`increased IacZ expression.A, cells were grown in
`CMAUraALeu —Trp—His, 2% glucose supple»
`mented with 100 [LM FeCl2 prior to [ad activity
`assays. Aft1 interaction with er3 or er4 depends
`on the conserved glut