Cell Metabolism
`
`Article
`
`Cytosolic Monothiol Glutaredoxins Function in
`Intracellular Iron Sensing and Trafficking via
`Their Bound Iron-Sulfur Cluster
`
`Ulrich Mu¨ hlenhoff,1 Sabine Molik,1 Jose´ R. Godoy,1 Marta A. Uzarska,1 Nadine Richter,1 Andreas Seubert,2 Yan Zhang,3
`JoAnne Stubbe,3 Fabien Pierrel,4 Enrique Herrero,5 Christopher Horst Lillig,1 and Roland Lill1,*
`1Institut fu¨ r Zytobiologie und Zytopathologie
`2Fachbereich Chemie, Philipps-Universita¨ t Marburg
`35032 Marburg, Germany
`3Chemistry Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
`4Laboratoire de Chimie et Biologie des Me´ taux, CEA Grenoble, 38054 Grenoble Cedex 9, France
`5Departament de Cie` ncies Me` diques Ba` siques, IRB Lleida, Universitat de Lleida, Lleida 25198, Spain
`*Correspondence: lill@staff.uni-marburg.de
`DOI 10.1016/j.cmet.2010.08.001
`
`SUMMARY
`
`Iron is an essential nutrient for cells. It is unknown
`how iron, after
`its import
`into the cytosol,
`is
`specifically delivered to iron-dependent processes
`in various cellular compartments. Here, we identify
`an essential function of the conserved cytosolic
`monothiol glutaredoxins Grx3 and Grx4 in intracel-
`lular iron trafficking and sensing. Depletion of Grx3/4
`specifically impaired all
`iron-requiring reactions in
`the cytosol, mitochondria, and nucleus, including
`the synthesis of Fe/S clusters, heme, and di-iron
`centers. These defects were caused by impairment
`of iron insertion into proteins and iron transfer to mito-
`chondria,
`indicating that intracellular iron is not
`bioavailable, despite highly elevated cytosolic levels.
`The crucial task of Grx3/4 is mediated by a bridging,
`glutathione-containing Fe/S center that functions
`both as an iron sensor and in intracellular iron
`delivery. Collectively, our study uncovers an impor-
`tant role of monothiol glutaredoxins in cellular iron
`metabolism, with a surprising connection to cellular
`redox and sulfur metabolisms.
`
`INTRODUCTION
`
`Iron is essential for virtually all organisms, because it functions as
`a cofactor in central cellular processes such as respiration, DNA
`synthesis and repair, ribosome biogenesis, and metabolism.
`Research over the past decade has uncovered sophisticated
`systems facilitating the specific transport of iron across the
`plasma and various intracellular membranes (Hentze et al.,
`2004; Kaplan and Kaplan, 2009; Philpott and Protchenko, 2008;
`Vergara and Thiele, 2008). Despite its central metabolic function,
`little is known about the passage of iron through the eukaryotic
`cytosol to become incorporated into proteins and transported
`into various subcellular compartments. A soluble, low-molec-
`ular-mass form of iron was described in the 1970s, but ever since
`
`the discovery of this ‘‘labile iron pool,’’ its physiological impor-
`tance and composition have been under debate (Crichton and
`Charloteaux-Wauters, 1987; Richardson and Ponka, 1997).
`Presumably, iron may also be bound to dedicated proteins
`ensuring specific delivery and insertion into iron-requiring sites.
`A metallo-chaperone function has been worked out for insertion
`of copper and nickel into respective metal-containing enzymes
`(Finney and O’Halloran, 2003; Lyons and Eide, 2007), but proteins
`performing a general role in iron trafficking or insertion are
`unknown. An iron donor function has been suggested for frataxin
`in mitochondrial Fe/S cluster biosynthesis (Bencze et al., 2006;
`Lill, 2009). In humans, the poly (rC) binding protein 1 (PCBP1)
`was shown to specifically deliver bound iron to ferritin, the major
`iron storage protein in higher eukaryotes (Shi et al., 2008). The
`apparently specific role of the PCBP1 iron chaperone and the
`fact that both ferritin and PCBP1 are not universally conserved
`leave open the possibility that other proteins with a general
`importance for iron trafficking exist. Clearly, the mode of specific
`iron delivery within the eukaryotic cytosol remains one of the
`fundamental unresolved problems of iron homeostasis.
`Because iron is not only essential but also toxic at higher
`levels, cells have developed sophisticated systems for ensuring
`a tightly regulated iron homeostasis (Hentze et al., 2004; Kaplan
`and Kaplan, 2009). In mammals this process is executed by
`iron-regulatory proteins in a posttranscriptional fashion, and
`the yeast Saccharomyces cerevisiae uses the iron-sensing
`transcription factors Aft1 and Aft2. Under iron deprivation,
`Aft1-Aft2 activate transcription of genes of the iron regulon
`encoding cell-surface iron transporters and proteins involved in
`intracellular iron utilization (Kaplan and Kaplan, 2009; Philpott
`and Protchenko, 2008). Sensing of intracellular iron by Aft1
`also requires the regulatory proteins Fra1-Fra2 and the cyto-
`solic-nuclear monothiol glutaredoxins Grx3 and Grx4, which
`are essential for the nuclear export of Aft1 in response to iron
`sufficiency (Kumanovics et al., 2008; Ojeda et al., 2006; Pujol-
`Carrion et al., 2006). The regulatory role of Grx3/4 is functionally
`conserved in fungi
`that utilize iron-regulated transcription
`systems unrelated to those from S. cerevisiae (Haas et al.,
`2008; Kaplan and Kaplan, 2009; Mercier and Labbe, 2009).
`Grx3/4 belong to the large thioredoxin (Trx) fold family and are
`composed of an N-terminal Trx and a C-terminal monothiol
`
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`

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`Role of Cytosolic Glutaredoxin in Iron Trafficking
`
`Cell Metabolism
`
`glutaredoxin (Grx) domain (Herrero and de la Torre-Ruiz, 2007;
`Lillig et al., 2008). Although the Grx3/4 subfamily of multidomain
`monothiol glutaredoxins is conserved in eukaryotes, no universal
`function has been assigned to this family so far. In contrast to
`most members of the Grx protein family that catalyze dithiol-di-
`sulfide redox reactions, monothiol Grx proteins rarely possess
`oxidoreductase activity. Instead, after in vitro reconstitution or
`upon overexpression in Escherichia coli, they are capable of
`binding a bridging [2Fe-2S] cluster utilizing the active-site
`cysteine residue of the Grx domain and glutathione (GSH) as
`ligands (Li et al., 2009; Picciocchi et al., 2007). The existence
`of this unusual Fe/S center under physiological conditions,
`however, has not been demonstrated, and its functional role
`has remained unclear.
`Here, we have used yeast as a model to define an essential
`role of Grx3/4 in intracellular iron trafficking. Depletion of Grx3/
`4 led to functional
`impairment of virtually all
`iron-dependent
`processes,
`including heme biosynthesis, mitochondrial and
`cytosolic Fe/S protein biogenesis, and the formation of di-iron
`centers in mitochondria and the cytosol, eventually leading to
`the loss of cell viability. We provide evidence for the in vivo
`binding of a bridging Fe/S cluster to Grx3/4 and we assign
`a crucial physiological function to this cofactor both in cytosolic
`iron trafficking and as an iron sensor. Thus, the conserved cyto-
`solic monothiol glutaredoxins use their bound Fe/S cofactor for
`a general role in intracellular iron trafficking.
`
`RESULTS
`
`Deficiency in Grx3/4 Is Associated with Defects
`in Iron-Dependent Enzymes
`To facilitate the functional analysis of Grx3/4, we constructed
`a regulatable yeast strain (Gal-GRX4; strain background W303-
`1A; see Table S1 available online) in which GRX3 was deleted
`and GRX4 was expressed under the control of the glucose-
`repressible GAL-L promoter. Upon Grx4 depletion, Gal-GRX4
`cells failed to grow on both fermentable and nonfermentable
`carbon sources (Figure 1A; see below). Likewise, double deletion
`of GRX3-GRX4 was lethal
`in the W303 strain background,
`distinguishing these cells from strain BY4742 grx3/4D, which
`shows only severely retarded growth (Figure 1A) (Ojeda et al.,
`2006). The strong effect of Grx3/4 deficiency on cell viability is
`not explained by their role in iron regulation, since Aft1 is not
`essential under iron-replete conditions (Kaplan and Kaplan,
`2009). These data and the general conservation of Grx3/4 in
`eukaryotes suggest
`that
`these proteins perform a so far
`unknown, important function.
`Gal-GRX4 cells were used to investigate the immediate conse-
`quences of Grx3/4 deficiency. Gal-GRX4 cells were cultivated in
`minimal medium supplemented with glucose and iron chloride to
`gradually deplete Grx4 (Figures 1B and 1C). A strong activation
`of the Aft1-dependent FET3 gene was observed using a FET3
`promoter-GFP fusion as a reporter (Figure 1B) (Ojeda et al.,
`2006). Surprisingly, the activities of the mitochondrial Fe/S
`protein aconitase and cytosolic catalase, a heme-containing
`protein, drastically decreased, despite the presumed sufficient
`cellular iron supply. These effects resemble those upon deple-
`tion of Ssq1, a component the iron-sulfur cluster (ISC) assembly
`machinery, even though the changes occurred later as a result of
`
`slower depletion of Ssq1 (strain Gal-SSQ1). Grx3/4 deficiency
`was associated with a severe activity loss of
`respiratory
`complexes II (succinate dehydrogenase) and IV (cytochrome
`oxidase) but was fully complemented by expression of GRX4
`from a plasmid (Figure 1D). Likewise, low activities of both
`aconitase and respiratory complexes III and IV were observed
`in BY4742 grx3/4D cells (Figure S1A), consistent with our earlier
`observation of an impaired 55Fe/S cluster incorporation into
`aconitase (Ojeda et al., 2006). Immunostaining of cell extracts
`from Grx4-depleted Gal-GRX4 cells and BY4742 grx3/4D cells
`further revealed changes in the levels of several iron-containing
`proteins, including the aconitase-type Fe/S proteins Aco1 and
`Leu1, ferrochelatase Hem15, and the core mitochondrial ISC
`assembly protein Isu1 (Figure 1C). These changes of protein
`levels correlate with those of the transcriptome of both iron-
`deprived and ISC machinery–compromised cells (Hausmann
`et al., 2008; Shakoury-Elizeh et al., 2004). In contrast, other
`iron-dependent proteins, such as succinate dehydrogenase
`subunit 2 (Sdh2) and the ubiquinone biosynthesis enzyme
`Coq7, were hardly changed and behaved similarly to the noniron
`proteins mitochondrial cytochrome oxidase subunit 4 (Cox4) and
`porin (Por1), cytosolic Hsp70, and ribosomal subunit Rps3.
`Together, these findings indicate that Grx3/4-deficient cells
`develop severe defects in several mitochondrial and cytosolic
`iron-dependent proteins, despite the induction of the Aft1-
`dependent iron uptake system. Notably, these global
`iron-
`related defects are not detected upon the constitutive activation
`of Aft1 (Hausmann et al., 2008; Ihrig et al., 2010), suggesting that
`these consequences of Grx3/4 deficiency occur independently
`of Aft1 and a deregulated iron homeostasis.
`
`Deficiency in Grx3/4 Impairs the De Novo Synthesis
`of Cellular Fe/S Clusters and Heme
`We asked whether the decreased Fe/S protein activities in Grx3/
`4-depleted cells might be explained by an impaired de novo
`synthesis of their Fe/S clusters and addressed this problem by
`using an established 55Fe radiolabeling and immunoprecipitation
`assay (Molik et al., 2007). First, the essential cytosolic Fe/S
`proteins Rli1, Dre2, and Nar1 were analyzed by expressing these
`proteins from a high-copy vector in wild-type and Gal-GRX4
`cells. Fe/S cluster insertion into all three Fe/S protein targets
`was decreased 4–10-fold upon Grx4 depletion (Figure 2A). The
`amount of Dre2 in Gal-GRX4 cells was comparable to that in
`wild-type cells,
`indicating a specific Fe/S cluster assembly
`defect (Figure 2A, inset). In the case of Rli1 and Nar1, protein
`levels were diminished, likely suggesting that the apoforms of
`these Fe/S proteins were degraded. Similar apoprotein insta-
`bility is frequently observed upon strong defects in Fe/S protein
`biogenesis (Balk et al., 2004). Analysis of 55Fe incorporation into
`the mitochondrial Fe/S proteins Bio2 (biotin synthase) and Ilv3
`(dihydroxyacid dehydratase) and the essential mitochondrial
`ISC scaffold protein Isu1 revealed an up to 4-fold lower 55Fe
`incorporation upon Grx4 depletion (Figures 2B and 2C). Protein
`levels of Bio2, Ilv3, and Isu1 did not change upon depletion of
`Grx4 (Figures 2B and 2C,
`insets). These findings indicate
`a general impairment in the de novo assembly of Fe/S proteins
`upon depletion of Grx3/4.
`In principle, the defect of Fe/S protein maturation upon Grx3/4
`depletion could be explained by a primary mitochondrial Fe/S
`
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`Cell Metabolism
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`Role of Cytosolic Glutaredoxin in Iron Trafficking
`
`Figure 1. Deficiency in Grx3/4 Is Associated with Defects in Iron-Dependent Enzymes
`(A) Wild-type (WT), Gal-GRX4, grx3D (strain background W303-1A), and grx3/4D (strain background BY4742) were grown in SD medium for 40 hr. Ten-fold serial
`
`C.
`dilutions were spotted onto YPEG plates and incubated for 2 days at 30
`(B and C) WT, Gal-GRX4, and Gal-SSQ1 strains harboring plasmid pFET3-GFP were grown in SD minimal medium. At the indicated times, FET3 promoter activ-
`ities were determined by measuring the GFP-specific fluorescence emission of cells (B), and cell extracts were assayed for aconitase and catalase activities, or
`were analyzed for the indicated proteins by immunostaining (C).
`(D) Enzyme activities of respiratory complexes II (SDH) and IV (COX) were determined relative to malate dehydrogenase (MDH) in mitochondria isolated from
`Gal-GRX4 cells cultivated in rich glucose medium for 40 hr and 64 hr, and from Gal-GRX4 cells expressing GRX4 from vector pCM189 (+Grx4). Error bars indicate
`the SEM (n R 4).
`
`protein assembly defect, because mitochondria are involved in
`generation of all cellular Fe/S proteins (Lill and Mu¨ hlenhoff,
`2008). However, we note that the observed effects were less
`severe in mitochondria compared to the cytosol. To directly
`test the functionality of the mitochondrial ISC assembly ma-
`chinery, we made use of an anaerobic in vitro system analyzing
`the capacity of mitochondrial detergent extracts to support Fe/S
`cluster insertion into the apoform of purified Yah1, a [2Fe-2S]
`
`ferredoxin (Molik et al., 2007). Apo-Yah1 was incubated with
`mitochondrial lysates and 55Fe. The radiolabeled Yah1 holopro-
`tein was bound to Q-sepharose, and the amount of radioactivity
`associated with holo-Yah1 was quantified by scintillation count-
`ing. Remarkably, extracts derived from Grx3/4-depleted mito-
`chondria were even more competent in synthesizing the Fe/S
`cluster on Yah1 than Grx4-complemented Gal-GRX4 cells
`(Figure 2D). This finding strongly suggests that the mitochondrial
`
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`

`

`Role of Cytosolic Glutaredoxin in Iron Trafficking
`
`Cell Metabolism
`
`Figure 2. Deficiency in Grx3/4 Impairs the De Novo Synthesis of Cellular Fe/S Clusters and Heme
`(A–C) Wild-type (WT) and Grx4-depleted Gal-GRX4 cells overproducing the cytosolic Fe/S proteins Rli1-HA, Dre2 or Nar1 (A), mitochondrial Bio2, Ilv3-Myc (B), or
`Isu1 (C) were radiolabeled with 10 mCi 55Fe for 2 hr. The Fe/S proteins were immunoprecipitated, and bound 55Fe was quantified by scintillation counting. Protein
`levels were assessed by immunostaining. Porin (Por1) served as a loading control. Gal-GRX4 cells were depleted for Grx4 by growth in SD medium for 40 hr and
`64 hr (in case of Isu1).
`(D) Purified apo-Yah1 was incubated under anaerobic conditions in the presence of 55Fe and cysteine either alone (-) or with detergent extracts of mitochondria
`isolated from 40 hr or 64 hr depleted Gal-GRX4 cells or Gal-GRX4 cells overproducing Grx4 (+Grx4). 55Fe/S cluster reconstitution on re-isolated Yah1 was quan-
`tified by scintillation counting.
`(E) WT and Gal-GRX4 cells (40 hr depletion) harboring either vector pCM189 (-) or pCM189-GRX4 (+Grx4) were radiolabeled with 55Fe. 55Fe-heme was extracted
`with butyl-acetate and quantified by scintillation counting. Error bars indicate the SEM (n R 4).
`
`in Grx3/4-depleted cells,
`ISC assembly system is functional
`rendering it likely that the decreased Fe/S cluster incorporation
`into apoproteins is explained by impaired iron supply.
`Consistent with this idea, 55Fe insertion into heme was 5.5-fold
`lower
`in Grx4-depleted Gal-GRX4 cells, compared with
`wild-type cells or Gal-GRX4 cells complemented by GRX4
`(Figure 2E). This diminished heme synthesis activity may explain
`the loss of function of heme-dependent enzymes, such as cata-
`lase and cytochrome oxidase, upon depletion of Grx3/4 (see
`above). In summary, Grx3/4-depleted cells are strongly impaired
`in both cellular Fe/S protein maturation and heme biosynthesis.
`Such defects are not observed in Aft1-activated cells (Haus-
`mann et al., 2008; Ihrig et al., 2010).
`
`Deficiency in Grx3/4 Leads to Impairment of Di-Iron
`Enzymes Despite Cytosolic Iron Overload
`The strong decrease of cellular Fe/S clusters and heme in Grx3/
`4-deficient cells is somewhat paradoxical, because these cells
`are expected to accumulate iron as the result of a constitutively
`activated cellular iron uptake system (Ojeda et al., 2006). To
`verify this idea, we measured the cellular iron content by ICP-MS
`analysis of wild-type and Grx4-depleted Gal-GRX4 cells grown
`in minimal medium supplemented with 100 mM FeCl3. Total
`cellular
`iron increased 6-fold upon depletion of Grx4
`
`(Figure 3A). The level of chelatable iron increased similarly and
`was mainly present as Fe2+ (Figure S2). Cellular levels of other
`metals, with the exception of Zn (3-fold higher), were hardly
`changed. In contrast, mitochondrial iron levels were up to 2.3-
`fold lower in Grx4-depleted Gal-GRX4 cells compared with
`wild-type levels (Figure 3B). Mitochondria from BY4742 grx3/
`4D cells contained even 7.5-fold less iron. Mitochondrial Mn,
`Co, and Zn levels were hardly altered, but Cu changed in parallel
`to iron. The decrease in mitochondrial
`iron levels in Grx3/4-
`depleted cells is the more remarkable, because cells with mito-
`chondrial Fe/S protein assembly defects usually display strongly
`elevated mitochondrial iron levels (Lill and Mu¨ hlenhoff, 2008).
`The fact that this was not observed, despite high levels of total
`cellular iron, indicates a defective delivery of iron to mitochondria
`in Grx3/4-deficient cells.
`A reasonable explanation for these general defects in iron
`handling in the absence of Grx3/4 may be a sequestration of
`iron into the vacuole, the major iron storage compartment in
`fungi
`(Kaplan and Kaplan, 2009; Philpott and Protchenko,
`2008). To test this idea, we varied the amount of Ccc1, the major
`importer of divalent metals into the vacuole. Deletion of CCC1
`did not restore growth of Grx4-depleted Gal-GRX4 cells and
`did not increase the low enzyme activities of aconitase and
`catalase (Figure 3C,
`top panel, and Figure 3D). Similarly,
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`Cell Metabolism
`
`Role of Cytosolic Glutaredoxin in Iron Trafficking
`
`Figure 3. Deficiency in Grx3/4 Results in Cytosolic Iron and GSH Accumulation
`(A and B) The metal content of (A) wild-type (WT) and Gal-GRX4 cells (depleted for 64 hr) and (B) mitochondria isolated from the indicated strains was determined
`by ICP-MS.
`(C) The indicated strains lacking (ccc1D) or overproducing Ccc1 (Ccc1[) were cultivated in SD medium for 40 hr. Ten-fold serial dilutions were spotted onto SC
`C under aerobic (+O2) or anaerobic (O2) conditions.
`
`agar plates containing glycerol (Glyc) or glucose (Glc), and were cultivated at 30
`(D) WT, Gal-GRX4 and Gal-GRX4/ccc1D cells were grown in SD medium for 64 hr, and aconitase and catalase enzyme activities were determined.
`(E) GSH levels were determined in cell extracts from WT, BY4742 grx3/4D, Gal-GRX4 (depleted for 40 hr or 64 hr) and Gal-GRX4 cells expressing GRX4 from
`a plasmid (+Grx4).
`
`overproduction of Ccc1 failed to restore growth (Figure 3C,
`middle panel). These data suggest that the accumulated iron is
`not stored in the vacuole.
`Grx4-depleted Gal-GRX4 cells failed to grow under anaerobic
`conditions (Figure 3C, bottom panel). Thus, reactive oxygen
`species (due to increased iron levels) are not responsible for
`the lethal phenotype of Grx3/4-depleted cells. Moreover,
`oxidized glutathione levels (GSSG; measured under anaerobic
`conditions) were below the detection limit (not shown). Rather,
`reduced glutathione (GSH) was strongly elevated in Grx4-
`in BY4742 grx3/4D
`depleted Gal-GRX4 cells, but not
`(Figure 3E). Together, these results and the predominant pres-
`ence of iron in its ferrous form (Figure S2) indicate that reducing
`conditions prevail in Grx3/4-deficient cells.
`
`The experiments presented above showed a maturation
`defect in cellular Fe/S and heme proteins in Grx3/4-deficient
`cells despite a cytosolic iron accumulation. Because this
`indicated a defective delivery of iron, we asked whether other
`iron-dependent enzymes were affected by Grx3/4 deficiency.
`First, the iron status of ribonucleotide reductase (Rnr), a cytosolic
`diferric-tyrosyl radical enzyme essential for deoxyribonucleotide
`synthesis, was analyzed (Perlstein et al., 2005). Upon depletion
`of Grx4 in Gal-GRX4 cells, the protein levels of Rnr2 decreased
`slightly (Figure S3). Nevertheless, the specific activity of Rnr
`was 6-fold lower compared with wild-type cells (Figure 4A).
`This was likely due to inefficient metallation, because 55Fe inser-
`tion into Rnr in vivo (followed by immunoprecipitation of subunit
`Rnr2) was 5–7-fold decreased in Grx4-depleted cells (Figure 4B).
`
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`Role of Cytosolic Glutaredoxin in Iron Trafficking
`
`Cell Metabolism
`
`Figure 4. Deficiency in Grx3/4 Leads to Functional Impairment of Di-iIron Enzymes
`(A) Permeabilized wild-type (WT) and Grx4-depleted Gal-GRX4 cells were assayed for specific ribonucleotide reductase activity (see also Figure S3).
`(B) WT and Gal-GRX4 cells were grown in SD medium for 40 and 64 hr and were radiolabeled, and 55Fe binding to Rnr2 was analyzed by immunoprecipitation and
`scintillation counting. Protein levels of Rnr2 and Por1 were determined by immunoblotting (inset).
`(C) The substrate (demethoxyubiquinol DMQ6) and product (ubiquinone CoQ6) of mitochondrial mono-oxygenase Coq7 were analyzed by electrochemical
`detection coupled to HPLC separation of mitochondrial lipid extracts from WT and Grx4-depleted Gal-GRX4 cells. CoQ4 is a commercial standard.
`(D) Ratio of DMQ6 and CoQ6 levels was determined in mitochondria isolated from WT Gal-GRX4 cells (depleted for 40 hr or 64 h) containing either vector pCM189
`or pCM189-GRX4 (+Grx4).
`(E) WT and Grx4-depleted Gal-GRX4 cells overproducing Fe-only superoxide dismutase from E. coli (FeSod) were analyzed for superoxide dismutase in-gel
`activities and FeSod by immunoblotting.
`
`As a second di-iron protein, we analyzed the mitochondrial
`mono-oxygenase Coq7, which catalyzes the hydroxylation of
`demethoxyubiquinol (DMQ6), the penultimate reaction of ubiqui-
`none (CoQ6) biosynthesis (Tran et al., 2006). HPLC analysis of
`mitochondrial
`lipid extracts revealed diminished CoQ6 and
`increased DMQ6 levels upon Grx4 depletion (Figure 4C). This
`effect was reversed to wild-type ratios by expression of GRX4
`from a plasmid (Figure 4D). The simultaneous accumulation of
`the substrate (DMQ6) and decrease of the product (CoQ6) of
`the enzyme Coq7 indicates a diminished activity of this di-iron
`mono-oxygenase upon depletion of Grx3/4. Collectively, these
`
`data demonstrate that a Grx3/4 deficiency causes a severe
`defect in cellular di-iron enzymes.
`Are the observed defects in Grx3/4-depleted cells specific for
`iron-dependent proteins? This question was addressed by
`analyzing the activities and protein levels of several metal-
`dependent enzymes. The in-gel activities of the endogenous
`Cu/Zn- and Mn-dependent superoxide dismutases (Sod1 and
`Sod2,
`respectively)
`remained unchanged in Grx4-depleted
`Gal-GRX4 cells (Figure 4E). In marked contrast, both the level
`and activity of ectopically expressed iron-only superoxide
`dismutase (FeSod) from E. coli strongly declined. Furthermore,
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`Cell Metabolism
`
`Role of Cytosolic Glutaredoxin in Iron Trafficking
`
`the Zn-dependent alcohol dehydrogenase (ADH) was 3-fold
`more active upon Grx4 depletion (Figure S1B). This increased
`ADH activity is characteristic for a switch toward fermentative
`metabolism and is typically observed upon iron deprivation.
`The normal function of several metal-reliant enzymes indicates
`that the described defects in Grx3/4-depleted cells are specific
`for iron-related processes. We conclude that Grx3/4 perform
`an essential
`role in cellular
`iron trafficking,
`in addition to
`and independently of their nonessential function in iron uptake
`regulation.
`
`Grx3/4 Assemble a Bridging Fe/S Cluster Independently
`of the CIA Machinery
`For a function of monothiol glutaredoxins in cellular iron traf-
`ficking, iron binding may be a necessary prerequisite. Recent
`in vitro studies have shown that various glutaredoxins, including
`yeast Grx3/4, can bind a bridging, GSH-liganded [2Fe-2S]
`cluster (Bandyopadhyay et al., 2008; Berndt et al., 2007; Johans-
`son et al., 2007; Li et al., 2009). We asked whether this unusual
`Fe/S cofactor is of physiological relevance and can be observed
`in a native environment using the 55Fe radiolabelling assay.
`Significant amounts of 55Fe were coimmunoprecipitated with
`anti-Grx4-antibodies from cell extracts derived from wild-type
`cells (strain BY4742), whereas only background levels were
`observed in grx3/4D cells (Figure 5A). Similar amounts of 55Fe
`were coimmunoprecipitated from both grx3D and grx4D cells,
`indicating that the homologous Grx3 and Grx4 bind iron with
`similar efficiency and independently of each other. The amount
`of Grx4-bound 55Fe was unchanged both in wild-type cells of
`our standard strain W303-1A, and, remarkably, under anaerobic
`or oxidative stress conditions prevailing after addition of H2O2 or
`upon deletion of SOD1 (sod1D cells), indicating that iron binding
`to Grx3/4 was insensitive to oxidative stress (Figure 5B).
`To analyze whether the iron bound to Grx3/4 is part of an
`Fe/S cluster, we investigated the requirement of 55Fe binding
`for components of Fe/S protein biogenesis (Lill and Mu¨ hlenhoff,
`2008). We first analyzed the role of the cysteine desulfurase
`complex Nfs1-Isd11, which serves as the sulfur donor for
`Fe/S protein biogenesis. For regulated depletion of Nfs1 or
`Isd11, we used Gal-NFS1 or Gal-ISD11 cells, respectively.
`55Fe binding to Grx4 (Figure 5C) and to the cytosolic Fe/S
`protein Leu1 as a control (Figure S4) declined to background
`levels upon depletion of either Nfs1 or Isd11. The same result
`was seen in Gal-ISU1Disu2 cells depleted for the core ISC
`scaffold proteins Isu1/2. No recovery of iron binding to Grx3/
`4 was observed when a cytosolic-nuclear version of Nfs1
`(DN-Nfs1) was produced in Gal-NFS1 cells lacking mitochon-
`drial Nfs1 (Figure 5C). We found during these experiments
`that the Grx4 protein levels decreased upon Nfs1 depletion,
`whereas the Grx3 levels remained unchanged. Although the
`reason for this specific decrease is unknown, we note that
`the remaining Grx3 did not bind 55Fe above background levels,
`unlike in grx4D cells (Figure 5A). The dependence of
`iron
`binding to Grx3/4 on core components of the mitochondrial
`ISC assembly system demonstrates that Grx3/4 bind an Fe/S
`cluster in vivo.
`Does incorporation of the Fe/S cluster into Grx3/4 also require
`the cytosolic iron-sulfur protein assembly (CIA) system (Lill,
`2009)? Depletion of the essential CIA components Nbp35 and
`
`Dre2 in the respective GAL promoter-exchange mutants signifi-
`cantly increased rather than impaired 55Fe binding to Grx3/4
`(Figure 5D), whereas hardly any 55Fe binding was observed to
`Leu1 as a control (Figure S4). This surprising finding suggests
`that incorporation of the Fe/S cluster into Grx3/4, although
`requiring mitochondrial Nfs1-Isd11-Isu1, does not depend on
`the cytosolic CIA machinery.
`The noncanonical pathway used for Fe/S cluster assembly on
`Grx3/4 may be due to the special nature of this cofactor, with
`a conserved Cys and GSH serving as ligands of a [2Fe-2S]
`cluster bridged between two Grx monomers, as observed after
`in vitro reconstitution or after overexpression in E. coli (Li et al.,
`2009; Picciocchi et al., 2007). We therefore sought to obtain
`in vivo evidence for this configuration. First, we determined
`which ligands might coordinate the Fe/S cluster. The importance
`of the conserved active-site cysteine residues C171 in the
`C-terminal Grx domain and C37 in the N-terminal Trx domain
`(Figure 5E, top) for 55Fe binding was tested in wild-type cells
`expressing a plasmid-encoded Myc-tagged Grx4 (Grx4-Myc)
`in which these residues were mutated to serine (mutant proteins
`C171S and C37S) or alanine (C171A). Myc-tagged wild-type
`Grx4 was fully functional
`(see below) and was produced at
`similar levels as the mutant proteins (Figure 5E, bottom). Mutant
`proteins C171S and C171A did not bind 55Fe above background
`levels (Figure 5E, middle). In contrast, mutant protein C37S
`bound wild-type amounts of 55Fe, suggesting that iron is bound
`via C171. Second, the importance of GSH for 55Fe binding to
`Grx3/4 in vivo was tested using the GSH synthesis-deficient
`mutant gsh1D that can be depleted for GSH upon growth in
`media lacking GSH. Although significant amounts of 55Fe were
`bound to Grx4-Myc in the presence of exogenously added
`GSH, only background levels of 55Fe were found in GSH-
`deprived cells (Figure 5F). Together, these findings suggest
`that the active-site C171 of the Grx domain and GSH serve as
`ligands of the Grx3/4-bound Fe/S cluster.
`Finally, we tried to find in vivo evidence for the presence of
`a bridging Fe/S cluster on Grx3/4. A yeast strain was constructed
`that simultaneously expressed C-terminally HA- and Myc-
`tagged Grx4. Cell extracts were subjected to immunoprecipita-
`tion with anti-HA or anti-Myc immunobeads followed by
`immunoblotting. Immunoprecipitation with anti-HA antibodies
`led to coisolation of Grx4-HA and a smaller amount of Grx4-
`Myc (Figure 5G). The same result, yet with inversed intensities,
`was observed using anti-Myc beads, whereas no cross-reacting
`bands were visible in wild-type cells. In cells expressing a C171S
`mutant Grx4-Myc and wild-type Grx4-HA, coimmunoprecipita-
`tion was far less efficient but still detectable. These observations
`document the importance of residue C171 for efficient Grx4
`dimer formation and thus are consistent with the idea of
`a bridging Fe/S cluster between two Grx monomers. Collec-
`tively, our findings suggest that, under physiological conditions,
`Grx3/4 bind a bridging Fe/S cluster that is coordinated by the
`active-site cysteine and GSH. These results are consistent with
`the structure of glutaredoxins reconstituted in vitro.
`
`The Grx3/4-Bound Fe/S Cluster Is Important for Iron
`Metabolism
`Is the Grx3/4-bound Fe/S cluster important for the function of
`Grx3/4 in iron trafficking and is this cofactor also required for
`
`Cell Metabolism 12, 373–385, October 6, 2010 ª2010 Elsevier Inc. 379
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`Role of Cytosolic Glutaredoxin in Iron Trafficking
`
`Cell Metabolism
`
`380 Cell Metabolism 12, 373–385, October 6, 2010 ª2010 Elsevier Inc.
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`Cell Metabolism
`
`Role of Cytosolic Glutaredoxin in Iron Trafficking
`
`iron sensing? As shown above, the C171S and C171A Grx4-Myc
`mutant proteins have lost the ability to stably bind the Fe/S cluster
`(Figure 5E). Grx4-depleted Gal-GRX4 cells expressing these
`mutant proteins from the endogenous GRX4 promoter failed to
`grow, whereas wild-type and C37S Grx4-Myc that retained
`normal
`iron binding supported wild-type growth (Figure 6A,
`top). Aft1-dependent transcription of FET3 was fully activated,
`and virtually no aconitase and catalase activities were observed
`in Gal-GRX4 cells producing C171S and C171A Grx4-Myc
`instead of wild-type Grx4 (Figure 6B). In contrast, wild-type and
`C37S Grx4-Myc-expressing Gal-GRX4 cells showed wild-type
`signals in these assays. All Grx4-Myc proteins were present at
`similar levels, with the exception of C171A, which apparently
`was less stable (Figure 6A, bottom). These results demonstrate
`the crucial role of residue C171 and thus the Grx3/4-bound
`Fe/S cluster for both iron regulation and iron trafficking.
`Formally, it is possible that the active-site C171 performs its
`essential role in Grx4 via thiol-dependent redox chemistry
`(Herrero and de la Torre-Ruiz, 2007; Lillig et al., 2008) rather
`than by coordination of the Fe/S cluster. Such a function can
`be excluded from the observation that overexpression (from
`a Tet promoter) of the C171S (but not C171A) Grx4-Myc mutant
`protein in Grx4-depleted Gal-GRX4 cells restored wild-type
`growth (Figure 6C). We noted that Grx4 overexpression generally
`diminished FET3 induction (Figure 6D). The corresponding
`C171S cells still displayed strong FET3 activation and wild-
`type aconitase, and strongly increased catalase activities. The
`restored iron loading of these and