Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System*

Background: The mitochondrial sulfhydryl oxidase Erv1 is implicated in cytosolic iron-sulfur protein maturation and iron regulation. Results: An erv1 yeast strain used in previous iron metabolism studies is glutathione-deficient, and erv1/mia40 mutants with sufficient glutathione do not exhibit iron-related defects. Conclusion: Iron homeostasis is independent of Erv1/Mia40 function. Significance: The unexplained role of Erv1 in yeast iron metabolism is resolved. The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/ Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.
Erv1 is an FAD-dependent sulfhydryl oxidase localized to the mitochondrial intermembrane space (IMS). 3 Erv1 has an essen-tial, well established role in importing small, cysteine-containing proteins into the IMS via a disulfide relay with the import receptor Mia40 (1,2). Substrate proteins for this pathway enter the IMS in an unfolded, reduced state, where Mia40 catalyzes oxidation of cysteine pairs in the imported proteins, facilitating their folding and retention in the IMS (3,4). Erv1 catalyzes reoxidation of Mia40 and shuttles the electrons to the respiratory chain (5,6). The substrate proteins of the Mia40-Erv1 pathway play critical roles in mitochondrial protein import, assembly of respiratory chain components, and removal of reactive oxygen species. As such, defects in Mia40 or Erv1 lead to depletion of these IMS proteins causing a variety of phenotypes, including respiratory deficiency, loss of mitochondrial DNA, and aberrant mitochondrial morphology. The importance of this pathway is highlighted by the fact that yeast deletion mutants for ERV1 and MIA40 are inviable (6).
In addition to participating in IMS protein import, Erv1 was suggested to have a role in exporting a sulfur-containing compound from the mitochondrion to the cytosol required for maturation of cytosolic Fe-S cluster proteins and regulation of iron homeostasis (7,8). The mitochondrion houses the iron-sulfur cluster (ISC) pathway for Fe-S cluster assembly, which is required not only for the biogenesis of Fe-S containing proteins in the mitochondria, but also for cytosolic and nuclear Fe-S proteins (8). The maturation of most Fe-S proteins in the cytosol and nucleus relies on the CIA (cytosolic iron-sulfur protein assembly) machinery (9). The connection between the ISC and CIA assembly pathways is proposed to occur via export of a sulfur-containing compound from the mitochondrial matrix to the cytosol that is used to build and/or insert Fe-S clusters into cytosolic proteins. This compound is a product of the ISC pathway and is exported by the ATP binding cassette transporter Atm1 (10). One of the substrates for Atm1 was recently identified as glutathione polysulfide (GS-S-SG), suggesting that the thiol-containing tripeptide glutathione (GSH) helps transport an activated persulfide (S 0 ) from the mitochondria to the cyto-sol for Fe-S cluster assembly (11). This finding is supported by the recent crystal structures of yeast Atm1 and its bacterial homologue with bound GSH and/or GSSG (12,13). The ISC pathway, Atm1, and GSH also impact on activity of the ironresponsive transcriptional regulators Aft1 and Aft2 (14 -16). In the proposed regulation model, the substrate exported by Atm1 is used to assemble Fe-S clusters on the cytosolic glutaredoxins Grx3 and Grx4, which use GSH to coordinate the Fe-S clusters and help deliver them to Aft1 and Aft2. In turn, Fe-S cluster binding by Aft1 and Aft2 inhibits their ability to bind to and activate iron uptake and mobilization genes (17,18).
The putative role of Erv1 in this regulation pathway and in the maturation of cytosolic Fe-S cluster proteins stems from a single report in which a temperature-sensitive erv1 mutant strain (erv1-1) displayed Fe-S related phenotypes similar to strains depleted of GSH or Atm1 (7, 19 -21). These phenotypes included diminished iron incorporation into two cytosolic Fe-S proteins, Leu1 and Rli1, and accumulation of mitochondrial iron caused by dysregulation of iron homeostasis. Based on these results, it was suggested that the IMS-localized protein Erv1 functions together with Atm1 and GSH to export the mitochondrial ISC-derived substrate required for cytosolic Fe-S cluster assembly and iron sensing (8).
Because the specific role of Erv1 in cytosolic Fe-S cluster biogenesis and iron metabolism was unclear, we analyzed Fe-S protein activity in a number of erv1 mutant strains. Furthermore, we extended our studies to include mia40 mutants to determine whether the role of Erv1 in these iron pathways is linked to its disulfide relay function with Mia40. Surprisingly, we discovered that the erv1-1 strain originally tested for Fe-S cluster defects by Lange and co-workers (7) was the only strain to exhibit defects in the cytosolic Fe-S enzymes isopropylmalate isomerase and sulfite reductase. Mitochondrial and cytosolic Fe-S enzyme activities in the other erv1 and mia40 mutants tested were similar to or higher than the WT control. We found that the erv1-1 strain had dramatically depleted GSH levels in both the mitochondria and cytosol, whereas GSH levels were normal in other erv1 and mia40 mutants. The cause of GSH deficiency in the erv1-1 strain was a previously undetected mutation in the GSH1 gene encoding glutamate cysteine ligase, which catalyzes the first step in GSH biosynthesis. Addition of GSH to the growth media rescued both the sulfite reductase activity and iron regulation. These results demonstrate that the defects in cytosolic Fe-S enzymes and iron homeostasis in erv1-1 are due to GSH depletion. Taken together, these results indicate that neither Erv1 nor Mia40 play significant roles in cytosolic Fe-S cluster assembly and iron homeostasis.

Materials and Methods
Yeast Strains, Media, and Growth Conditions-Saccharomyces cerevisiae strains used in this study are listed in Table 1. Temperature-sensitive strains were grown overnight at 24°C on synthetic complete (SC) medium supplemented with 2% glucose and the appropriate amino acids. One set was then diluted, shifted to 37°C, and grown for 5 more hours, whereas the other set was diluted and maintained at 24°C. GAL-regulated strains were maintained at 30°C on SC medium in the presence of 2% raffinose ϩ 0.5% galactose to induce expression or grown in 2% raffinose for at least 64 h to repress expression. For plasmid shuffling, an erv1 deletion strain with wild-type Erv1 expressed on a URA3 plasmid (Spore 2A) was transformed with a TRP1 plasmid carrying Erv1(F124S) (pYX232-erv1(F124S) described below). Transformants were selected on SC-Trp plates and shuffling of the URA3-marked plasmid expressing WT Erv1 was carried out with 5-fluoroorotic acid. Yeast transformations were performed by the lithium acetate procedure. All assays were performed on strains at mid-log phase.
Construction of Plasmids-The 2 TRP1 plasmid pYX232-erv1(F124S) was generated by site-directed mutagenesis of pYX232-ERV1 (22)) using the QuikChange II Mutagenesis kit (Agilent). The CEN LEU2 plasmid p415-ADH-GSH1 was constructed by insertion of the GSH1 open reading frame in the XbaI and XhoI sites of p415-ADH (23). A triple tandem HA epitope was incorporated at the C terminus of Gsh1 at an engineered NotI site. All plasmid inserts were verified by DNA sequencing.
Subcellular Fractionation-Yeast cells were grown aerobically to mid-log phase in selective SC medium with 2% glucose. Mitochondrial and post-mitochondrial supernatant (PMS) fractions were obtained as previously described by converting cells to spheroplasts followed by gentle lysis by Dounce homogenization and differential centrifugation (24). Incubation with DTT was omitted from the spheroplasting step to avoid perturbation of the intracellular thiol redox state. Protein concentrations in extracts were determined using the Bradford method (Bio-Rad) with bovine serum albumin as the calibration standard.
Glutathione Assay-Total glutathione (GSH ϩ GSSG) in PMS and mitochondrial extracts was measured by the 5,5Јdithiobis(nitrobenzoic acid)-GSSG reductase recycling assay as described previously (25) or the GSH/GSSG-Glo TM Assay following the manufacturer's protocol with slight modifications (Promega). For whole cell GSH measurements, 1-4 ϫ 10 5 cells (or 0.5-1 ϫ 10 7 for erv1-1 strains) were harvested by centrifugation, resuspended in lysis buffer supplied by the GSH/GSSG-Glo TM Assay kit, and lysed via mechanical disruption with glass beads. After addition of luciferin generation and detection reagents, stable luciferin luminescent signals were detected using the Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek). The results are expressed as nanomoles of GSH per 10 7 cells (assuming 1 A 600 ϭ 2 ϫ 10 7 cells).
Non-reducing SDS-PAGE-Non-reducing SDS-PAGE was performed as previously described (26). Briefly, cells were grown in SC media to mid-log phase and then acid quenched with TCA (Sigma) (15% (w/v) final concentration) at 4°C for 20 min. Five A 600 units of cells were harvested by centrifugation and resuspended in 1 ml of 10% TCA. Following glass bead lysis, lysed cells were transferred to a new tube and pelleted by centrifugation. The pellet was resuspended in 500 l of 1ϫ nonreducing SDS sample buffer containing 40 mM N-ethylmaleimide (Sigma). Following a 10-min incubation at room temperature, proteins were separated on 16% Tris glycine gels (Invitrogen). Reduced and oxidized forms of Mia40 were analyzed by quantitative immunoblot using an Odyssey Infrared Imaging System (LI-COR).
Immunoblotting Techniques-Yeast extracts subjected to gel electrophoresis were analyzed by Western blotting using anti-Erv1 or anti-Mia40 antibodies (both kind gifts of J. Riemer) or anti-Gsh1 antibodies using a secondary anti-rabbit IgG (IRDye, LI-COR, Lincoln, NE). Recombinant yeast Gsh1 was produced in Escherichia coli as described previously (27) and the purified enzyme was used to produce a rabbit anti-Gsh1 antibody (Covance). Human IRP1 antibody was purchased from Santa Cruz Biotechnology. Cytosolic and mitochondrial fractions were monitored by anti-phosphoglycerate kinase (PGK1) and anti-porin antibodies (Invitrogen), respectively, using a secondary anti-mouse IgG (IRDye, LI-COR, Lincoln, NE). Western blots were visualized and quantified using an Odyssey Infrared Imaging System (LI-COR).
GSH1 Gene Sequencing-Genomic DNA for JRY-675 and erv1-1 strains was isolated using the Promega Wizard Genomic DNA Purification Kit and the GSH1 gene was amplified from Ϫ720 to ϩ2380 by PCR using the primers listed in Table 2. The purified DNA fragments were subjected to Sanger sequencing at the Selah Clinical Genomics Center at Innovista (Columbia, SC).
RNA Isolation and Quantitative Real-time PCR-Total RNA was isolated from yeast strains JRY-675 and erv1-1 with the PureLink RNA Mini kit (Invitrogen) following the manufacturer's protocol. Yeast RNA was reverse transcribed into cDNA with the SuperScript III First-strand Synthesis System (Invitrogen) following the manufacturer's recommendations. Using the primers shown in Table 2, quantitative RT-PCR was carried out on an IQ ICycler (Bio-Rad) with the RT 2 SYBR Green Fast Mastermix (Qiagen). The relative quantification of GSH1 expression was calculated by the 2 Ϫ⌬⌬C T method (28) using the ACT1 gene (actin) as a control.
Cytosolic Fe-S cluster Enzyme Activity Assays-For Leu1 (isopropylmalate isomerase) assays, temperature-sensitive strains and corresponding parent strains were grown in SC-glucose media at 24°C until A 600 1-1.5. The A 600 was adjusted to 1.0 and cells were incubated at 37°C for 5 h with or without 1 mM GSH. Yeast cells were harvested, washed, and broken with glass beads in TNETG buffer (20 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 150 mM NaCl, 10% (v/v) glycerol, 0.5% (w/v) Triton X-100). Cell debris was removed by centrifugation. Leu1 enzyme activity was measured as the conversion of 2-isopropylmaleate to 2-isopropylmalate at 235 nm. As an indirect assay for sulfite reductase, yeast cells were plated on bismuth sulfite (Bi 2 (SO 3 ) 3 ) plates and incubated at 30°C for 48 h. Reduction of sulfite (SO 3 2Ϫ ) to sulfide (S 2Ϫ ) results in formation of bismuth sulfide (Bi 2 S 3 ) as a brown precipitate. 1 mM GSH was included in the plates where indicated.
In-gel Aconitase Activity Assay-Cytosolic and mitochondrial aconitase activities were monitored using an in-gel activity assay described previously (29). We note that these assays were performed in cells with a complete leucine biosynthetic pathway because the LEU2 IRP1 plasmid was selected for in media lacking leucine. Thus, aconitase activity should be unaffected by increased expression of Leu1 as occurs in leu2⌬ strains, which subsequently impacts Fe-S delivery to aconitase (30,31). Yeast cells expressing human cytosolic aconitase IRP1 from pRS425-IRP1 (YEp-LEU2) were grown to mid-log phase in selective SC media, harvested, and washed with sterile water. The pellets were then resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 50 mM NaCl, 2.5% Triton X-100, 0.5 mM PMSF, 1 mM DTT, 2 mM citrate, protease inhibitor mixture, 200 units/ml catalase) and subjected to glass bead lysis. Extracts were centrifuged and the supernatants were collected for assaying. Protein concentration of the extracts was determined by the Bradford method (Bio-Rad). A chilled 8% Tris borate-citrate polyacrylamide gel was pre-electrophoresed in Tris glycine-citrate running buffer at 140 V for 40 min. Subsequently, 100 g of protein was loaded to the gel and electrophoresed at 140 V for 3.5 h on ice. Gels were incubated at 37°C for 30 min in the dark with aconitase activity assay stain (100 mM Tris-HCl, pH 8.0, 1 mM NADP ϩ , 2.5 mM cis-aconitate, 5 mM MgCl 2 , 1.2 mM methylthiazolyldiphenyl-tetrazolium bromide, 0.3 mM phenazine methosulfate, and 5 units/ml isocitrate dehydrogenase) and scanned.

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␤-Galactosidase Assays-All strains tested were transformed with the FET3-LacZ reporter construct pFC-W (YEp-URA3) that contains the FET3 iron-response element in a minimal promoter (32) or the FIT3-LacZ reporter construct pFIT3-LacZ (YEp-URA3) that contains the FIT3 promoter (33). Temperature-sensitive erv1, mia40, and corresponding parent strains were grown in selective SC glucose media at 24°C to an A 600 of 1 and then divided into 3-ml aliquots for induction. The cultures were incubated at 24 and 37°C with either 50 M FeCl 3 (high iron), 100 M bathophenanthroline disulfonate (low iron), or no addition (normal iron) for 5 h. GAL-ERV1 and the parent W303A strain were grown for 64 h at 30°C to an A 600 of 1 in inducing (SC-raffinose/galactose) or repressing (SC-raffinose) media and similarly divided into high, low, and normal iron aliquots and grown for an additional 4 h at 30°C. Cells were then harvested and assayed for ␤-galactosidase activity as previously described (34).
Intracellular Iron Analysis-Mitochondrial and cytosolic iron content was measured using atomic absorption spectroscopy. Yeast cells were grown to mid-log phase in SC glucose media for temperature-sensitive strains, or SC galactose/raffinose for W303A, GAL-ERV1, and GAL-ATM1 strains, and mitochondria and PMS fractions were prepared as described above. Extracts were diluted in Milli-Q water and iron analysis of fractions was performed on a PerkinElmer PinAAcle 900T graphite furnace atomic absorption spectrometer using the manufacturer's recommended conditions.
Data Analysis-For all assays and quantifications, averages and standard deviations were calculated from at least three independent experiments.

Results
The erv1-1 Strain but Not Other erv1 Mutants Exhibits Cytosolic Fe-S Protein Defects-To better understand the connection between Erv1 function and cytosolic Fe-S cluster biogenesis, we obtained several different erv1 mutants. The temperature-sensitive erv1 strain (pet492-6A), named erv1-1, was first described by Lisowsky (19,35), and shown to exhibit defects in IMS protein import (4) and maturation of cytosolic Fe-S clusters (7). This strain harbors an Erv1 F124S mutation located at the dimer interface (36) and therefore likely impairs Erv1 dimerization, which is essential for catalytic function (22). The erv1-2 and erv1-5 strains each contain single point mutations in the ERV1 gene creating N166D and C159S substitutions, respectively (37). Asn-166 and Cys-159 are both located near the FAD binding site, with Cys-159 forming a structural disulfide with Cys-176 (36). As such, the C159S Erv1 mutant exhibits decreased FAD binding in vitro (38), whereas both erv1-2 and erv1-5 are impaired in import of IMS proteins (3,37,39). We confirmed the Erv1 functional defect by assessing the in vivo redox state of the import receptor Mia40 at the permissive (24°C) and restrictive (37°C) temperatures. As expected, the redox-active cysteine pair in Mia40 is ϳ88% oxidized in both the WT and erv1-1 strains at 24°C, but is only ϳ33% oxidized in erv1-1 at 37°C (Fig. 1, left). The defect in Mia40 oxidation is similar to that found in the erv1-2 and erv1-5 strains (Fig. 1, right).
Consistent with the report by Lange et al. (7), the erv1-1 strain exhibited a 40% decrease in the activity of isopropylmalate isomerase (Leu1) compared with the genetically matched WT strain ( Fig. 2A). This decrease, however, was not as severe as measured for the temperature-sensitive nbp35-1 strain (ϳ90% activity loss), which is known to be defective in cytosolic Fe-S cluster protein maturation (40,41). Leu1 protein levels were similar for erv1-1, nbp35-1, and their respective wild-types (Fig. 2B), thus differences in protein synthesis did not cause the Fe-S enzyme defects in erv1-1 or nbp35-1. To test whether the Fe-S defect in the erv1-1 strain is linked to the Erv1-Mia40 mitochondrial import system, we measured Leu1 activity in other erv1 and mia40 temperature-sensitive strains. Leu1 activity was actually increased in the erv1-2 and erv1-5 strains compared with the WT control ( Fig. 2A). Similarly, Leu1 activity measured for mia40-3 was comparable with WT. We further measured sulfite reductase activity, another cytosolic [4Fe-4S]-dependent enzyme, using a bismuth sulfite plate assay in which the sulfide product reacts with bismuth to give a brown color. As shown in Fig. 2C (left panel), the erv1-1 strain exhibited a marked defect in sulfite reductase activity. We note that sulfite reductase activity could not be assessed for the erv1-2 and erv1-5 alleles using this assay, because the strains were unable to grow on bismuth plates for unknown reasons. Because GSH and Erv1 are proposed to function together in cytosolic Fe-S cluster assembly, we tested whether GSH addition influenced Fe-S activity in the erv1-1 mutant. Interestingly, addition of 1 mM GSH to the growth media rescued the sulfite reductase defect in this strain (Fig. 2C, right panel).
As a complementary approach to examine the role of Erv1 in Fe-S cluster assembly, we expressed human IRP1 in the yeast cytosol, which acts as a cytosolic Fe-S assembly reporter (42)(43)(44). The Fe-S-dependent aconitase activities of IRP1 and the native yeast mitochondrial aconitase (Aco1) were then assessed in parallel via an in-gel activity assay (29). Both aconitase activ- ities were very low in the WT(1) and erv1-1 strains (Fig. 2D). However, addition of 1 mM GSH to the growth medium increased cytosolic aconitase activity in the erv1-1 mutant at both 24 (permissive) and 37°C (restrictive temperature) with little effect on the WT control. In addition, we tested aconitase activity in the erv1-2, erv1-5, mia40-3, and mia40-4 mutants in the YPH499 background (Fig. 2E). Although these assays display some variability from one data set to another, none of these mutants showed any clear difference in cytosolic or mitochondrial aconitase activity when compared with WT controls at either 24 or 37°C. Furthermore, addition of GSH did not influence the observed aconitase activities in these strains (data not shown). Taken together, these data suggest that Fe-S cluster defects are unique to the erv1-1 mutant and argue against a major role for either Erv1 or Mia40 in the maturation of cytosolic Fe-S cluster proteins.  (2)) and isogenic erv1 and mia40 mutant strains expressing human IRP1 (pRS425-IRP1). C, WT control strain without human IRP1 expression plasmid. Cells were grown in SC glucose media with or without the addition of 1 mM GSH as indicated. Mitochondrial (Aco1) and cytosolic aconitase (IRP1) run separately in native gels. Bottom, the same protein extracts were separated by reducing SDS-PAGE and immunoblotted against human IRP1 and yeast Pgk1 (loading control).  NOVEMBER 13, 2015 • VOLUME 290 • NUMBER 46

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Cytosolic and Mitochondrial GSH Pools Are Depleted in erv1-1 but Not in Other erv1 or mia40 Mutant Strains-Because GSH addition increased Fe-S enzyme activity in the erv1-1 strain, we measured endogenous GSH levels in this mutant. Surprisingly, the erv1-1 strain exhibited strongly decreased levels of GSH at both 24 and 37°C (Fig. 3A, left), as compared with the WT control. We further fractionated yeast cells into mitochondrial and cytosolic extracts to determine whether GSH deficiency impacted these specific compartments differently. We measured a ϳ30-fold decrease in cytosolic GSH levels in the erv1-1 strain compared with WT at 24°C that further decreased to ϳ120-fold at 37°C (Fig. 3B, left). In the mitochondria, the differences were less extreme because the GSH levels in erv1-1 were ϳ7-fold lower that WT at 24°C and ϳ80-fold lower at 37°C (Fig. 3C, left).
To test whether the GSH depletion phenotype discovered in the erv1-1 strain is directly related to the Erv1-Mia40 import system or unique to that specific mutant, we measured total GSH levels in the other erv1 and mia40 temperature-sensitive strains we obtained (3,37,39). Total GSH levels in whole cell, mitochondrial, and cytosolic extracts for these erv1 and mia40 strains were similar to the WT control (Fig. 3, A-C, right panels). To investigate whether the GSH depletion phenotype is related to changes in Erv1 protein levels, we performed Erv1 immunoblot analysis for these strains. Erv1 expression levels in mitochondrial fractions consistently decreased at 37°C for all erv1 and mia40 mutants tested, including erv1-1 (Fig. 3D). This result is expected given that Erv1 itself is a substrate of the Erv1-Mia40 import pathway, and thus its import into the IMS is impaired with disruption of either Erv1 or Mia40 function (45). However, the data indicate that alterations in Erv1 protein levels do not correlate with GSH deficiency in these temperaturesensitive mutants. We next questioned whether the specific Erv1 mutation harbored by the erv1-1 strain is the cause of the GSH defect. The erv1-1 mutation (F124S) was recreated in the W303A strain background by plasmid shuffling. This erv1(F124S) strain exhibited a defect in Mia40 oxidation at the restrictive temperature confirming Erv1 dysfunction (Fig. 4, A  and B, left); however, total GSH levels were similar to WT at both temperatures (Fig. 4C, left). From these results we conclude that the Erv1 F124S mutation is not the cause of GSH deficiency in the erv1-1 strain.
To further understand how changes in Erv1 expression influence GSH levels independent of temperature effects, we measured total GSH levels in a GAL-regulated ERV1 strain that allows Erv1 overexpression or depletion depending on the presence or absence of galactose in the growth media (Fig. 4A,  right). Increased expression of Erv1 led to a more oxidized in vivo redox state for Mia40, whereas decreased expression led to a more reduced state (Fig. 4, A and B, right), as previously reported (6). Interestingly, both overexpression and depletion of Erv1 resulted in a 2-3-fold increase in total GSH levels compared with the WT control (Fig. 4C, right), rather than a severe decrease as measured for the erv1-1 strain. Taken together, these results demonstrate that GSH deficiency is unique to the erv1-1 strain, unrelated to Erv1 protein levels, and not a general phenotype of Erv1 or Mia40 dysfunction. erv1-1 Has Markedly Decreased Gsh1 Protein Levels Due to a Mutation in GSH1-To uncover the origin of the GSH deficiency in erv1-1, we tested for defects in the GSH biosynthetic pathway in this strain. The tripeptide GSH (␥-glutamylcysteinyl glycine) is synthesized in two steps via glutamate cysteine ligase (Gsh1) and GSH synthase (Gsh2). Gsh1 forms the linkage between glutamate and cysteine, whereas Gsh2 catalyzes the addition of glycine. Addition of cysteine, glycine, or glutamate to the growth media did not rescue the GSH deficiency in the erv1-1 strain (data not shown), suggesting that the GSH defect is not due to a lack of these GSH precursors. We next tested for changes in expression of the first biosynthetic enzyme, glutamate cysteine ligase (Gsh1). Interestingly, the erv1-1 strain has strongly decreased Gsh1 protein levels compared with WT controls and other erv1 and mia40 mutants at both 24 and 37°C (Fig. 5, A and B). However, GSH1 RNA levels were consistently higher in the erv1-1 strain (Fig. 5C) compared with WT, ruling out a transcriptional or post-transcriptional defect. Therefore, we sequenced the GSH1 gene in the erv1-1 strain to determine whether the decrease in Gsh1 levels is due to a mutation in the GSH1 open reading frame. A G to A mutation was discovered at ϩ839 in the coding sequence, changing an Arg at position 280 to His (Fig. 5D). Arg-280 is well conserved across eukaryotic Gsh1 protein sequences (Fig. 5D). It forms a salt bridge with Asp-403 (Fig. 5E) as well as backbone hydrogen bonds with Leu-369 and Gly-370. Disruption of these interactions may impact the placement of Tyr-362 (in a loop region), which helps coordinate the ␣-carboxylate of the glutamate substrate (27). Thus, the R280H mutation likely affects both the enzymatic activity and the protein stability of glutamate cysteine ligase (Fig. 5E). Expressing WT GSH1 on a plasmid or adding GSH to the culture medium rescues the GSH deficiency phenotype (Fig. 6, A and B). Taken together, these results demonstrate that GSH deficiency in erv1-1 is caused by a previously undetected mutation in the gene encoding glutamate cysteine ligase (GSH1).
Defects in Iron Regulation in erv1-1 Are Rescued by GSH-In addition to exhibiting cytosolic Fe-S cluster deficiency, mutations in erv1 have also been implicated in dysregulation of iron homeostasis leading to mitochondrial iron accumulation (46,   NOVEMBER 13, 2015 • VOLUME 290 • NUMBER 46 47). To determine whether iron regulation is dysfunctional in erv1 and mia40 mutants, we measured the expression of two iron regulon genes, FET3 and FIT3, under varying iron growth conditions. FET3 encodes a multicopper oxidase involved in high affinity iron uptake, whereas FIT3 encodes a cell wall mannoprotein involved in siderophore uptake (48,49). Under ironlimiting conditions, Aft1/2 induces the expression of FET3 and FIT3, whereas under normal and high iron conditions their expression is deactivated. Using FET3-LacZ and FIT3-LacZ reporters, we measured ␤-galactosidase activity in extracts from cells grown in excess, normal, or limiting iron conditions. Both the FET3 and FIT3 reporters show misregulation of iron homeostasis in the erv1-1 mutant (Fig. 7, A and B) because the genes are highly expressed regardless of iron growth conditions. Therefore, we tested whether addition of GSH to the growth media rescued iron misregulation in this mutant. With both reporters, we saw higher overall expression compared with WT, but the pattern of expression showed functional regulation with GSH addition (lower expression when iron is normal or in excess, higher expression when iron is limiting). In addition, the other temperature-sensitive erv1 (erv1-2, erv1-5) and mia40 (mia40-3, mia40-4) mutants that have normal GSH levels exhibited iron regulation that was similar to the WT control under both permissive and restrictive growth temperatures (Fig. 8A). Addition of 1 mM GSH to these mutants has no effect on gene expression (data not shown). To show that these effects were not dependent on growth temperature, we also tested expression of the reporter genes in the GAL-ERV1 strain. Unlike the erv1-1 strain, FET3 expression was decreased in iron-deficient media with both up-and down-regulation of ERV1 in the presence or absence of GSH. However, expression of these genes in normal and excess iron was similar to the isogenic wild-type control (Fig. 8B). A similar pattern was observed for FIT3 expression in this strain (data not shown). Taken as a whole, these results indicate that dysfunctional iron regulation in the erv1-1 mutant is primarily due to low levels of GSH, rather than specific defects in the Erv1 protein. Furthermore, the assays shown in Fig. 8A similarly rule out a significant role for Mia40 in iron regulation.

Yeast Iron Metabolism is Independent of Erv1/Mia40 Function
Mitochondrial Iron Accumulation in erv1-1 Is Rescued by GSH-Previous results demonstrate that deletion or depletion of the ISC Fe-S cluster assembly or export machinery, including the glutathione-persulfide exporter Atm1, causes accumulation of iron in mitochondria (21, 46, 50 -52). This effect is attributed to constitutive activation of the iron regulon leading to intracellular iron accumulation. To determine whether iron levels are affected by erv1 dysfunction, iron levels in mitochondrial and cytosolic extracts were measured in several erv1 mutants grown in SC media (normal iron levels) using atomic absorption spectroscopy (Fig. 9). Cytosolic and mitochondrial iron levels are elevated in the erv1-1 mutant at the restrictive temperature (37°C) by ϳ2.5 and ϳ7-8 times, respectively, rel-  ative to temperature-matched WT controls (Fig. 9, A and B, left  panels). These results are consistent with up-regulation of Aft1/2-activated genes in this strain as shown in Fig. 7. Addition of 1 mM GSH rescued this high iron phenotype (Fig. 9, A and B, right panels) as observed for the iron misregulation phenotype. We further measured mitochondrial and cytosolic iron levels in an erv1(F124S) mutant in the W303A background because a previous report suggested that mitochondrial iron is elevated in this strain (47). However, the temperature-sensitive erv1(F124S) mutant did not exhibit substantial changes in mitochondrial or cytosolic iron levels at 37°C, with only 2-fold higher cytosolic iron and wild-type levels of mitochondrial iron (Fig. 9, C and D, left panels). Finally, we measured subcellular iron levels in a GAL-ERV1 strain and detected a similar trend. When Erv1 is depleted, cytosolic iron is slightly elevated (ϳ2 times WT levels), whereas mitochondrial iron remains similar to WT. As a positive control, we measured iron levels in a GAL-ATM1 strain that was shown to accumulate mitochondrial iron when ATM1 expression is turned off (21,51,53). Similar to previous results, mitochondrial iron levels were found to be 7 times higher than WT when Atm1 is depleted, whereas cytosolic levels remained unchanged (Fig. 9, C and D, right panels). Taken together, these data reaffirm that only the erv1-1 mutant harboring a GSH1 mutation exhibits significant defects in iron homeostasis that are rescued with exogenous GSH, and furthermore, rule out an essential role for Erv1 in iron regulation.

Discussion
The specific role of Erv1 in cytosolic Fe-S cluster maturation and iron signaling has been a long-standing mystery ever since Fe-S cluster defects were reported for a temperature-sensitive (ts) erv1 strain in 2001 (7). This report is widely cited, yet no subsequent study in yeast has revisited these results to provide a molecular explanation for these observations. To address this issue, we obtained the original erv1 ts strain (erv1-1) as well as additional temperature-sensitive and GAL-regulated erv1 mutants in a variety of strain backgrounds. We measured the in vivo Mia40 redox state in each of these strains and confirmed the defect in Erv1 sulfhydryl oxidase activity at the restrictive temperature or upon Erv1 depletion. However, only the erv1-1 strain exhibited decreased activity for the cytosolic Fe-S enzymes isopropylmalate isomerase and sulfite reductase. Because some of these defects were rescued with GSH addition to the growth media, we measured GSH levels in the erv1 and mia40 mutants and discovered that the erv1-1 strain has dramatically lowered GSH levels compared with the other WT and mutant strains. By sequencing the GSH1 gene in erv1-1, an R280H mutation in the glutamate cysteine ligase coding sequence was identified. Because Erv1 was also reported to impact iron regulation in addition to cytosolic Fe-S cluster maturation (8), we extended these studies to examine the effect of Erv1 and Mia40 dysfunction or depletion on iron regulation. Similar to the Fe-S enzyme activity defects, iron misregulation leading to mitochondrial iron accumulation was only detected in the GSH-depleted erv1-1 strain. Addition of GSH to the growth media rescued the iron-dependent phenotypes in erv1-1, suggesting that GSH deficiency is the underlying cause rather than non-functional Erv1.
In light of the new information provided by our study, we re-evaluated the results from the original 2001 erv1 article (7). In that publication, the authors reported that 55 Fe incorporation into cytosolic Fe-S proteins Leu1 and Rli1 in erv1-1 is ϳ10 -30% lower than WT at 24°C, whereas ϳ90% lower at 37°C. Given that GSH is also critical for maturation of cytosolic Fe-S cluster proteins (20), we suggest that these differences in iron loading may be due to changes in GSH levels in erv1-1 at 24 versus 37°C. We found that whole cell GSH levels in erv1-1 are 80ϫ lower than WT at 24°C but 400ϫ lower at 37°C (Fig. 3A). Thus, the intracellular GSH concentration in erv1-1 at 24°C  NOVEMBER 13, 2015 • VOLUME 290 • NUMBER 46 may provide sufficient GSH to sustain cytosolic Fe-S cluster assembly. However, at 37°C, the GSH concentration likely drops below a critical threshold due to increased instability of the R280H Gsh1 mutant (Fig. 5, A and B), leading to a defect in maturation of cytosolic Fe-S proteins. This explanation is supported by numerous studies showing that GSH must be severely depleted (levels Ͼ100 times below WT) to impact cytosolic Fe-S cluster biogenesis and iron regulation (14,20), reinforcing the proposal that GSH is required in only trace amounts to function in these pathways (16).

Yeast Iron Metabolism is Independent of Erv1/Mia40 Function
The 2001 Lange (7) article also reported that expression of Erv1 or its human homologue ALR (augmenter of liver regeneration) on a plasmid restored 55 Fe incorporation into Leu1. We note that Erv1 and ALR were both expressed from a high copy yeast episomal plasmid (LEU2 selection for Erv1 and URA3 selection for ALR) under the control of a constitutive promoter in that study. Leu1 Fe-S incorporation in these assays is complicated by using LEU2 as a selectable marker for some strains and not others, since expression of LEU2 is shown to impact Leu1 expression levels and Fe-S loading (30,31). Furthermore, Erv1 and ALR were likely overexpressed under those conditions. Interestingly, we note that constitutive overexpression of Erv1 in the GAL-ERV1 strain (Fig. 4A) leads to a 2.8-fold increase in GSH levels (Fig. 4C). Therefore it is possible that increased Erv1/ALR expression in the previous study may have boosted the GSH concentration above the threshold required for sustain Fe-S cluster protein maturation. Pinpointing the mechanism by which increased Erv1 expression impacts GSH levels is beyond the scope of this study but will be investigated in the future.
We also investigated the possibility that Erv1 may play an indirect role in the maturation of cytosolic Fe-S proteins and iron homeostasis. Erv1 has a clear, undisputed function in facilitating import of nuclear-encoded proteins into the mitochondrial intermembrane space. Substrates of the Erv1-Mia40 import system include assembly factors and components of the respiratory chain, chaperones that facilitate protein import into mitochondrial compartments and membranes, and chaperones for antioxidant enzymes (54). Thus, it is not surprising that Erv1 dysfunction has pleotropic effects on mitochondrial biogenesis, function, and morphology. Because cytosolic Fe-S cluster biogenesis is dependent on the mitochondrial ISC pathway and Atm1 function, Erv1 dysfunction may indirectly impact synthesis and assembly of the Atm1-exported substrate (GS-S-SG) required for maturation of cytosolic Fe-S proteins and regulation of iron homeostasis. However, under our growth and assay conditions using multiple erv1 mutants we did not detect major effects of Erv1 dysfunction on cytosolic Fe-S protein activity nor iron regulation, leading us to conclude that Erv1 does not have a direct or indirect role in these pathways. We note that our conclusions are reinforced by a recent study in human cells, demonstrating that depletion of ALR (the human Erv1 homologue) does not impact the maturation of cytosolic Fe-S proteins assembled via the CIA pathway (55).
Our results also demonstrate that Mia40, the functional partner for Erv1, has little impact on Fe-S cluster assembly and iron regulation. It was important to address this issue because previous studies have demonstrated that recombinant human and yeast Mia40 expressed in E. coli can be purified aerobically with a bound [2Fe-2S] cluster (56,57). Furthermore, 55 Fe incorporation experiments demonstrated that yeast Mia40 binds iron in vivo via the redox active CXC motif (57). Although the physiological function of the Fe-S cluster in Mia40 is unknown, the authors of that report speculated that Fe-S binding to Mia40 could be involved in regulating the thiol oxidoreductase activity of Mia40 or Fe-S-Mia40 may have a role in ISC biogenesis (57). Our results argue against the latter possibility because mia40 mutants display normal Leu1 and cytosolic and mitochondrial aconitase activities and normal expression of Fe-S-dependent, Aft1/2-regulated genes.