Monothiol glutaredoxin Grx5 interacts with Fe–S scaffold proteins Isa1 and Isa2 and supports Fe–S assembly and DNA integrity in mitochondria of fission yeast

Biochemical and Biophysical Research Communications 392 (2010) 467–472

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Monothiol glutaredoxin Grx5 interacts with Fe–S scaffold proteins Isa1 and Isa2 and supports Fe–S assembly and DNA integrity in mitochondria of fission yeast Kyoung-Dong Kim, Woo-Hyun Chung, Hyo-Jin Kim, Kyung-Chang Lee, Jung-Hye Roe * Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742, South Korea

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Article history: Received 5 January 2010 Available online 18 January 2010 Keywords: Fe–S cluster assembly Glutaredoxin Monothiol Mitochondria Fission yeast

a b s t r a c t Mitochondrial monothiol glutaredoxins that bind Fe–S cluster are known to participate in Fe–S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Dgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe–S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1+ gene encoding a putative A-type Fe–S scaffold, in addition to mas5+ and hsc1+ genes encoding putative chaperones for Fe–S assembly process. Examination of other scaffold and chaperone genes revealed that isa2+, but not isu1+ and ssc1+, complemented the growth phenotype of Dgrx5 mutant as isa1+ did, partly through restoration of Fe–S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe–S assembly process through interaction with A-type Fe–S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe–S assembly. Ó 2010 Elsevier Inc. All rights reserved.

Fe–S clusters assembled in mitochondria are provided to apoproteins of various cell compartments. A current model based largely on extensive studies on Schizosaccharomyces cerevisiae proposes that Fe–S clusters are assembled on a scaffold composed of Isu1/Isu2 proteins (U-type scaffold), using cysteine desulfurase complex (Nfs1/Isd11) as a sulfur donor, Yfh1 (yeast frataxin homologue) as an iron donor, and Yah1 (ferredoxin) and its cognate reductase Arh1 system as electron donors [1,2]. The assembled Fe–S clusters are then proposed to be transferred to the target apo-proteins in mitochondria through the combined action of Grx5 and chaperone proteins such as Ssq1 (Hsp70 family chaperone), its cognate J-type co-chaperone Jac1, and the nucleotide exchange factor Mge1 [3–6]. In addition to these components, Isa1 and Isa2, which are homologues of bacterial A-type scaffold protein IscA, have been implicated to specifically assist Fe–S transfer to a limited group of targets such as aconitase-like proteins (Aco1 and Lys4), and SAM-dependent proteins biotin synthase (Bio2) and lipoic acid synthase (Lip5) in mitochondria [7,8].

Role of mitochondrial monothiol Grx5 in Fe–S assembly has been initially demonstrated in S. cerevisiae, where the growth defect of grx5 mutant was suppressed by multi-copy ISA2 and SSQ1 genes [9]. Two hybrid analysis hinted interaction of Grx5 with Isa1, but not with Isa2 or other Fe–S cluster assembly (ISC) components [10]. In order to investigate the function of mitochondrial monothiol glutaredoxin, we employed fission yeast Schizosaccharomyces pombe as a model system. Since the growth of fission yeast relies heavily on mitochondria as evidenced by the absence of petit (mitochondria-defective) mutants, some aspects of mitochondrial functions could be more sensitively studied in fission yeast. S. pombe possesses five Grxs, among which two (dithiol Grx2 and monothiol Grx5) reside in mitochondria [11,12]. We examined the phenotype of Dgrx5 mutant, and screened for multi-copy suppressors of growth-defective phenotype of the mutant. The partners of Grx5 through genetic and physical interactions in vivo were proposed, and evidences for expanded role of Grx5 were presented. Materials and methods

* Corresponding author. Address: School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, South Korea. Fax: +82 2 888 4911. E-mail address: [email protected] (J.-H. Roe). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.01.051

Yeast strains and culture media. Schizosaccharomyces pombe strains used in this study are ED665 (h ade6-M210 leu1-32 ura4D18), Dgrx2 (grx2::ura4 in ED665), Dgrx5 (grx5::kanR in ED665),

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and Dgrx2grx5 (grx2::ura4 grx5::kanR in ED665) [11,12]. Growth and maintenance of all the strains were done as described previously [13]. Cells were grown in YES (0.5% yeast extract, 3% dextrose) or Edinburgh minimal medium (EMM) with appropriate supplements. For respiratory growth, modified YES with glycerol (0.5% yeast extract, 0.1% dextrose, 3% glycerol, and 250 mg supplements/L) was used. Construction of the S. pombe genomic library and screening of multi-copy suppressors. Genomic DNAs from strain 972 (ED665) were prepared as described previously [13]. Following partial digestion with BamHI, DNA fragments from 5 to 6 kb in size were obtained from gel slices by electroelution. These were ligated with BamHIcut Splac551 vector and amplified in Escherichia coli. The Dgrx5 cells were transformed with the library, which covers >99% of the whole genome, and the transformants that grew on EMM plates were isolated. Total DNA was prepared from the transformants and was introduced into E. coli cells by electroporation, followed by preparation of plasmids that contain the multi-copy suppressor gene. The nucleotide sequence was determined, and the results were analyzed by using the S. pombe BLAST server at the Sanger Institute (http://www.sanger.ac.uk). Construction of recombinant plasmids. Construction of Splac551based recombinant plasmids were done as described previously for Splac551-grx5+ [12]. The isa1+, isa2+, isu1+, and ssc1+ genes were amplified with specific primer pairs that cover the whole coding region and upstream promoter, cut with PstI and XmaI, and ligated with PstI/XmaI-cut Splac551 vector. To employ bimolecular fluorescence complementation (BiFC) assay in S. pombe, N- or C-terminal fragments (VN or VC) of Venus, a variant of yellow fluorescent protein [14], were subcloned into pREP42 or pREP41 to fuse the Venus domains to the C-terminal end of the genes to be cloned. For VN tagging, the 540 bp PCR product was obtained from pFA6a-VN plasmid of S. cerevisiae [15], using primer pairs 50 -AACA GATCCATCGCCACTCGAGTG-30 (forward) and 50 -CGCTTATTTAGAA GTGCCATGGCC-30 (reverse). The DNA was digested with XhoI and NcoI, and replaced the EGFP gene in pREP42-EGFP [12] to generate pREP42-VN. For tagging VC, the 290 bp PCR product from FA6a-VC [15] generated by primer pairs 50 -AACCGCCCGGGGTGCA AAATCCCG-30 (forward) and 50 -CGCTTATTTAGAAGTGCCATGGCC30 (reverse) was digested with XmaI and NcoI, and replaced the EGFP gene in pREP41-EGFP [12] to generate pREP41-VC. PCRamplified isa1+ and isa2+ genes (NdeI/XhoI fragments) that contain the entire coding region from the start codon were then cloned into REP42-VN. PCR-amplified grx5+ gene (NdeI/XhoI fragment) was cloned into REP41-VC. Enzyme activity assays and measurement of intracellular iron. Cell extracts were prepared from cells grown in YES to early exponential phase or OD600 of 1.0 as described previously [16]. Activities of Fe–S enzymes in mitochondria (aconitase and succinate dehydrogenase) and cytoplasm (sulfite reductase) were measured as described previously [17,18]. To determine the amount of total intracellular iron, approximately 300 mg of dried cells was analyzed by ICP-atomic emission spectrometer (AES) (ICPS-1000IV; SHIMADZU) at the National Center for Inter-University Research Facilities at SNU. All the measurements in this study were carried out for at least three independent sample preparations. Real-time qPCR analysis. Aliquots of about 10-ng total DNA were added to each well in a 96-well plate containing Syber Green master mix (Applied Biosystems, CA). Triplicate PCRs for gene-specific primer pairs of 6 mitochondrial and 6 nuclear DNA regions were carried out according to manufacturer’s instruction, using a quantitative real-time PCR machine (ABI PRISMÒSequence Detection System, Applied Biosystems) with analysis software SDS2.2 (Applied Biosystems). DNA-specific primer pairs were designated as

described previously [19] in the regions of SPCC622.09 (htb1+), SPAC3F10.16C (hsr1+), SPAC1006.08 (etd1+), SPAC16A10.04 (rho4+), SPAC11E3.13C, SPAPB1E7.04c, and mitochondrial DNA (M1–M6, Fig. 4). Fluorescence microscopy. Wild-type (ED665) cells co-transformed with pREP41-grx5+-VC and either pREP42-isa1+-VN or pREP42-isa2+-VN were examined by fluorescence microscopy. For negative controls, cells co-transformed with a single species of fusion construct and a parental vector (pREP41-grx5+-VC and pREP42-VN, pREP42-isa1+-VN and pREP41-VC, or pREP42-isa2+VN and pREP41-VC) were examined. Cells grown in EMM to the exponential phase (OD595  1.0) were incubated in EMM for 20 min with 100 nM MitoTracker Red (M-7513, Molecular Probes), followed by rinsing three times with 10 mM potassium phosphate buffer. Fluorescence images from and MitoTracker were captured by Zeiss Axiovert 200 M microscope. Results The Dgrx5 mutant does not grow on glycerol as a carbon source and shows Cys auxotrophy In order to examine the role of mitochondrial glutaredoxins in S. pombe, null mutants of grx2+ or grx5+ encoding dithiol or monothiol glutaredoxins [11,12], respectively, were plated on rich (YES) media with either glucose or glycerol as carbon sources, or on minimal (EMM) media. The Dgrx5 mutant was unable to grow on glycerol or on minimal medium, whereas Dgrx2 mutant grew as well as the wild type (Fig. 1A). This indicates that Grx5 is critically required for mitochondrial and biosynthetic functions in S. pombe. The growth defect on EMM was partially relieved by adding cysteine (Fig. 1B), but not by lysine or glutamate (data not shown). This is unexpected since it has been reported in S. cerevisiae that the synthesis of lysine and glutamate depends on Fe–S enzymes [20,21]. We observed that additional provision of lysine and/or glutamate in addition to cysteine did not further restore the growth (data not shown), indicating that cysteine biosynthetic pathway is the primary bottleneck of growth in S. pombe that lacks Grx5. Sulfite reductase, which is an Fe–S enzyme in cysteine biosynthetic pathway, is a likely candidate that is affected in the grx5 mutant. The Dgrx5 mutant showed inactivation of several Fe–S enzymes and iron accumulation We then examined whether Grx5 of S. pombe is involved in Fe–S assembly in mitochondria as reported in other organisms. Activities of two mitochondrial (aconitase and succinate dehydrogenase) and one cytosolic Fe–S enzymes (sulfite reductase) in exponentially growing cells were measured, in addition to non-Fe–S enzyme (malate dehydrogenase) as a control. In Dgrx5 as well as in Dgrx2grx5 double mutants, the level of all three Fe–S enzymes was greatly reduced (Fig. 1C). The level of malate dehydrogenase decreased only slightly in all mutants. In Dgrx2 mutant, Fe–S enzymes did not change significantly, although a slight increase in aconitase and succinate dehydrogenase was observed. Therefore, monothiol Grx5 is critically required to maintain Fe–S enzymes in both mitochondria and the cytosol. Since a defect in Fe–S assembly system disturbs iron balance in the cell, we examined whether the grx5 mutation causes iron accumulation as observed in other mutants defective in Fe–S assembly [22–24]. Using ICP-AES, we found that the Dgrx5 mutant is elevated in total iron concentration by about 1.7-fold than the wild type, reflecting disregulation of iron homeostasis. The Dgrx2grx5 double mutant showed about 2.3-fold higher level (Fig. 1D).

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Fig. 1. Effect of mitochondrial glutaredoxin mutations on the growth and the activity of Fe–S enzymes. (A) The wild type, grx2, grx5, and grx2grx5 mutant cells grown to the exponential phase in YES medium were serially diluted and incubated on YES media containing glucose or glycerol as carbon sources, or on EMM media. (B) The wild type and grx5 mutant cells were incubated on YES or EMM supplemented with cysteine. Plates were incubated at 30 °C for 4 days. (C) Enzyme activities in cell extracts prepared from wild type (WT), grx2, grx5, and grx2grx5 mutant cells grown to exponential phase in YES medium (OD595 = 1.0–2.0). Activities of Fe–S enzymes such as aconitase (ACO), succinate dehydrogenase (SDH), and sulfite reductase (SR), and non-Fe–S enzyme malate dehydrogenase (MDH) were measured as described in the text. (D) The amount of total iron in each cell sample was measured by ICP-AES, and presented as nmol per mg dry cell weight. Values from three independent experiments were presented with standard deviations.

Isolation of Isa1, Mas5, and Hsc1 as multi-copy suppressors of growth defects of Dgrx5 As an initial trial to find epistatic or complementary components in the physiological pathway that involves Grx5, we screened for multi-copy suppressors of growth-defective phenotype of Dgrx5 on EMM plates. From Splac551-based DNA library of ED665 genome, we isolated 17 different clones that enabled Dgrx5 to grow on EMM plates. Among these, three clones persistently restored growth through subsequent re-introductions into Dgrx5 cells either on EMM plates or in liquid minimal media (Fig. 2A). These clones contained genes for isa1+, hsc1+, and mas5+ (SPBC1734.11), encoding a putative A-type Fe–S scaffold protein homologous to bacterial SufA/IscA, a chaperone of Hsp70 family [25], and a yeast homologue of DnaJ, respectively. Mitochondrial chaperones such as Ssq1 (Hsp70 family), Jac1 (DnaJ family), and Mge1 (GrpE family) have been previously reported to interact with Grx5 in S. cerevisiae to deliver Fe–S cluster from the U-type scaffold protein Isu1/2 to target apo-proteins [5]. Analogously, it is conceivable that the hsc1+ and mas5+ gene products in S. pombe may function as mitochondrial chaperones, and are involved in Fe–S assembly. Immunoblot analysis of Hsc1 and Mas5 proteins revealed that these proteins exist in both cytosolic and organellar fractions (data not shown). We examined whether these three multi-copy suppressors recovered the activities of Fe–S enzymes in Dgrx5. Cells grown in complex media (YES) to mid-exponential phase (OD595  1.0) were prepared to measure enzyme activities (Fig. 2B). Compared with the vector control (vc), Isa1 overproduc-

tion restored the level of aconitase, sulfite reductase, and succinate dehydrogenase significantly, approaching more than 70% of the level grx5+ complemented. The mas5+ and hsc1+ genes elevated only the aconitase activity, with no effect on succinate dehydrogenase and sulfite reductase. This observation coincides with the more complete restoration of growth in minimal media by isa1+ compared with the effect of hsc1+ and mas5+ genes (Fig. 2A). These results support a proposal that Isa1 functions in a more general way, whereas the role of Hsc1 and Mas5 is more limited to specific target proteins, in compensating Grx5 function. Both Isa1 and Isa2, but not Isu1 and Ssc1, suppress Dgrx5 growth phenotype Since isa1+, hsc1+, and mas5+ genes suppressed growth defect of Dgrx5, we examined whether other genes of related sequence similarity function in similar ways. Among candidates, we examined isa2+ whose gene product is 47% similar in amino acid sequence to Isa1 and is also localized in the mitochondria, ssc1+ whose gene product is the closest homologue of mitochondrial chaperone Ssq1p of S. cerevisiae, and isu1+ which encodes a putative U-type Fe–S scaffold protein of IscU/Nfu family. The results in Fig. 2C show that Isa2 suppresses the growth defect of Dgrx5 just as well as Isa1 does. On the other hand, both Ssc1 and Isu1 did not restore the growth of the mutant. The growth defect on rich media containing glycerol as a carbon source was also complemented by Isa2 (Fig. 2D). Therefore, since either Isa1 or Isa2 was effective to suppress the growth defect of Dgrx5, it can be postulated that each

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Fig. 2. Effects of multi-copy suppressors on the growth and Fe–S enzyme activities of grx5 mutant. (A) The growth of grx5 mutant cells transformed with Splac551-grx5+ (positive control), Splac551 (vc, negative control), Splac551-isa1+, Splac551-hsc1+, and Splac551-mas5+ was monitored in minimal liquid media. (B) Enzyme activities in each cell culture were measured as described in Fig. 1C from three independent experiments. (C) The grx5 mutant cells transformed with Splac551-grx5+ (positive control), Splac551 (negative control), Splac551-isa1+, Splac551-isa2+, Splac551-isu1+, and Splac551-ssc1+ were grown on EMM plates for 4 days at 30 °C. (D) The grx5 mutant cells transformed with Splac551-grx5+ (positive control), Splac551 vector (, negative control), Splac551-isa1+, or Splac551-isa2+ were spotted on YES plates containing glucose or glycerol as carbon sources.

of them may interact with Grx5 in Fe–S assembly pathway. Among mitochondrial chaperone homologues, Ssc1 does not collaborate with Grx5 in Fe–S assembly, whereas Hsc1 and Mas5 work together with Grx5, in ways more specific to target Fe–S proteins than Isa1 or Isa2 does. This contrasts with an observation in S. cerevisiae, where the growth defect of Dgrx5 is suppressed by SSQ1, the Ssc1 orthologue in S. cerevisiae [9]. Mitochondrial DNA is damaged in Dgrx5 Among multi-copy suppressors that partially restored growth of Dgrx5 mutant on minimal media, several clones were found to contain mitochondrial (mt) DNA fragments. When these recombinant plasmids were isolated and re-transformed into Dgrx5, they no longer restored the growth of the mutant. The cloned mt-DNA fragments from the initial suppressor screen were distributed throughout the mitochondrial genome as illustrated in Fig. 3A. Since these clones have been recovered from the initial screen, we suspected that the mitochondrial DNA may not be intact in Dgrx5 mutant. We therefore estimated the content of mitochondrial DNA in the mutant in comparison with the wild type by quantitative real-time PCR [19]. Six pairs of primers that can amplify different regions (M1–M6) of mt-DNA as indicated in Fig. 3A were used to detect the amount of template mt-DNA. We found that with all the pairs of primers the Dgrx5 mutant contained about half the amount of mt-DNA compared with the wild type (Fig. 3B). Therefore, lack of Grx5 causes damage in mt-DNA, which can be partially complemented by introducing undamaged mt-DNA fragments. Since the legion could be different among individual Dgrx5

mutant, the complementing effect may not have been reproduced upon re-introduction to different cells. Direct interaction of Grx5 with Isa1 and Isa2 We examined whether Grx5 directly interacts with Isa1 and Isa2 in vivo by bimolecular fluorescence complementation (BiFC) assay [26]. Using this tagging system, we constructed Grx5 fused with the C-terminal half of Venus (Grx5-VC) and Isa proteins fused with the N-terminal half of Venus (Isa1-VN and Isa2-VN) as described previously [15]. No Venus fluorescence was detected in cells transformed with only a single species of fusion constructs, Grx5-VC, Isa1-VN, or Isa2-VN. However, bright fluorescence was clearly detected in cells co-transformed with Grx5-VC and one of the two Isa-VN fusions (Fig. 4). The fluorescence shapes matched nicely with the images from MitoTracker, a mitochondria-specific dye, confirming that Grx5 interacts with Isa1 or Isa2 in mitochondria. We also examined the interaction between Grx5-VC and either Hsc1-VN or Mas5-VN. However, no significant fluorescences were detected (data not shown). Discussion In this study, we found that Grx5 is required for the activities of not only mitochondrial Fe–S enzymes but also a cytosolic one (sulfite reductase). This suggests that Grx5 serves a central role in providing Fe–S clusters that are used not only in mitochondria, but also to be transported out of mitochondria in fission yeast and probably in other eukaryotes.

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Fig. 3. The amount of mitochondrial DNA in grx5 mutant. (A) The position of cloned mitochondrial DNA fragments initially recovered from eight multi-copy suppressor clones. The innermost arcs indicate the region cloned in the eight multi-copy suppressor clones. Six short bars (M1–M6) indicate the region amplified with mitochondriaspecific DNA probes by real-time PCR. (B) The amount of mitochondrial DNA in the grx5 mutant relative to the wild type. In order to assess the copy number of mitochondrial DNA, six pairs of mt-DNA-specific probes were designed to perform real-time qPCR. As a control, the amount of nuclear DNA was assessed in parallel using six pairs of nuclear DNA-specific probes. For nuclear DNA, both the wild type and grx5 mutant gave nearly equal values for all six pairs of probes (N1–N6, 100%). More than three RT-qPCR measurements were done for each set of primers.

Fig. 4. Interaction of Grx5 with Isa proteins monitored by bimolecular fluorescence complementation (BiFC) in vivo. ED665 cells co-transformed with pREP41-grx5+-VC (Grx5-VC) and either pREP42-isa1+-VN (Isa1-VN) or pREP42-isa2+-VN (Isa2-VN) were examined by fluorescence microscopy. Negative controls examined in parallel were those cells co-transformed with pREP41-grx5+-VC and pREP42-VN (Grx5-VC and VN), pREP42-isa1+-VN and pREP41-VC (Isa1-VN and VC), or pREP42-isa2+-VN and pREP41-VC (Isa2-VN and VC). MitoTracker Red dye was added to visualize mitochondria. Fluorescence images were visualized by Axiovert 200 fluorescent microscope (Carl Zeiss).

Chaperones such as Hsc1 (Hsp70 family) and Mas5 (DnaJ family) may cooperate with Grx5 to support Fe–S transfer to target apo-proteins. However, among three Fe–S enzymes we examined, their contribution was limited to only aconitase. In S. cerevisiae, the closest sequence homologue of Hsc1 (Ssb1p and Ssb2p) are known to function in cytoplasmic protein synthesis [27], whereas Mas5 in S. cerevisiae is known to function in mitochondrial protein import [28]. On the contrary, Ssq1 (Hsp70 chaperone) suppresses the growth defect of Dgrx5 in S. cerevisiae [9], whereas its closest homologue (Ssc1) in S. pombe does not. Therefore, the kind of chaperone proteins that interact with Grx5 as well as the spectrum of their target apo-proteins may differ among different organisms. In S. pombe, each single mutant of Disa1 or Disa2 is not viable even on rich media (http://pombe.bioneer.co.kr), suggesting a critical role for each paralogue, unlike in S. cerevisiae where even the double mutation is not lethal [29]. Interaction of Grx5 with either of Isa1 and Isa2 was demonstrated through both genetic interaction (multi-copy suppression of grx5 mutant phenotype) and phys-

ical interaction in situ (bimolecular fluorescence complementation). Isa proteins suppressed defects in cysteine synthesis, and aerobic respiration. In S. cerevisiae, the growth phenotype of grx5 mutant is complemented by overproducing Isa1, but not Isa2, even though two hybrid analysis suggests interaction between Grx5 and Isa2 [9,10]. Our results clearly demonstrate that Grx5 interacts with A-type Fe–S scaffold proteins Isa1 and Isa2, and that these two proteins most likely serve a more central role in Fe–S assembly process rather than serving a limited role to activate a small number of mitochondrial Fe–S enzymes. The observation that the amount of mitochondrial DNA in grx5 mutant is about half the level of the wild type is intriguing. How the loss of Grx5 caused this reduction in mt-DNA is not clear. In S. cerevisiae, aconitase has been reported as an essential component of nucleoid complex for mitochondrial DNA maintenance [30], and failure to incorporate Fe–S into aconitase in Isa-depleted cells has been linked with the loss of mt-DNA [1]. On the basis of these observations in budding yeast, we can hypothesize that the

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reduction of mt-DNA in grx5 mutant could be due to reduction in functional aconitase in S. pombe. However, since mitochondrial nucleoid complex has not yet been determined in fission yeast, this hypothesis needs be tested in the future. Another possibility includes the involvement of DNA-damaging ROS that can arise from solvent-exposed iron generated from defective Fe–S assembly process in grx5 mutant. The increased iron content and sensitivity of grx5 mutant to various oxidants [12] support this possibility. However, a defect in nucleoid structure and an increase in DNA damage by ROS need not be exclusive each other. Further study is in need to understand the relationship between Fe–S assembly and mtDNA maintenance. We expect that the common and distinct features observed in fission and budding yeasts will continue to reveal universal and unique aspects of critical life processes. Acknowledgments This work was supported by a National Research Laboratory (NRL) Grant (0427-20090005) from the Ministry of Education, Science and Technology to J.H. Roe. K.D. Kim was supported by the second stage of BK21 program for Biological Sciences at SNU.

[11]

[12]

[13] [14]

[15]

[16]

[17]

[18] [19]

[20]

References [1] R. Lill, U. Muhlenhoff, Maturation of iron–sulfur proteins in eukaryotes: mechanisms, connected processes, and diseases, Annu. Rev. Biochem. 77 (2008) 669–700. [2] R. Lill, Function and biogenesis of iron–sulphur proteins, Nature 460 (2009) 831–838. [3] U. Muhlenhoff, J. Gerber, N. Richhardt, R. Lill, Components involved in assembly and dislocation of iron–sulfur clusters on the scaffold protein Isu1p, EMBO J. 22 (2003) 4815–4825. [4] E.A. Craig, J. Marszalek, A specialized mitochondrial molecular chaperone system: a role in formation of Fe/S centers, Cell. Mol. Life Sci. 59 (2002) 1658– 1665. [5] R. Lill, U. Muhlenhoff, Iron–sulfur-protein biogenesis in eukaryotes, Trends Biochem. Sci. 30 (2005) 133–141. [6] R. Lill, U. Muhlenhoff, Iron–sulfur protein biogenesis in eukaryotes: components and mechanisms, Annu. Rev. Cell Dev. Biol. 22 (2006) 457–486. [7] D.C. Johnson, D.R. Dean, A.D. Smith, M.K. Johnson, Structure, function, and formation of biological iron–sulfur clusters, Annu. Rev. Biochem. 74 (2005) 247–281. [8] U. Muhlenhoff, M.J. Gerl, B. Flauger, H.M. Pirner, S. Balser, N. Richhardt, R. Lill, J. Stolz, The ISC [corrected] proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae, Eukaryot. Cell 6 (2007) 495–504. [9] M.T. Rodriguez-Manzaneque, J. Tamarit, G. Belli, J. Ros, E. Herrero, Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes, Mol. Biol. Cell 13 (2002) 1109–1121. [10] F. Vilella, R. Alves, M.T. Rodriguez-Manzaneque, G. Belli, S. Swaminathan, P. Sunnerhagen, E. Herrero, Evolution and cellular function of monothiol

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28] [29] [30]

glutaredoxins: involvement in iron–sulphur cluster assembly, Comp. Funct. Genomics 5 (2004) 328–341. W.H. Chung, K.D. Kim, Y.J. Cho, J.H. Roe, Differential expression and role of two dithiol glutaredoxins Grx1 and Grx2 in Schizosaccharomyces pombe, Biochem. Biophys. Res. Commun. 321 (2004) 922–929. W.H. Chung, K.D. Kim, J.H. Roe, Localization and function of three monothiol glutaredoxins in Schizosaccharomyces pombe, Biochem. Biophys. Res. Commun. 330 (2005) 604–610. S. Moreno, A. Klar, P. Nurse, Molecular genetic analysis of fission yeast Schizosaccharomyces pombe, Methods Enzymol. 194 (1991) 795–823. T. Nagai, K. Ibata, E.S. Park, M. Kubota, K. Mikoshiba, A. Miyawaki, A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications, Nat. Biotechnol. 20 (2002) 87–90. M.K. Sung, W.K. Huh, Bimolecular fluorescence complementation analysis system for in vivo detection of protein–protein interaction in Saccharomyces cerevisiae, Yeast 24 (2007) 767–775. J. Lee, I.W. Dawes, J.H. Roe, Adaptive response of Schizosaccharomyces pombe to hydrogen peroxide and menadione, Microbiology 141 (Pt. 12) (1995) 3127– 3132. E.C. Chang, D.J. Kosman, O2-dependent methionine auxotrophy in Cu, Zn superoxide dismutase-deficient mutants of Saccharomyces cerevisiae, J. Bacteriol. 172 (1990) 6. A. Hausladen, I. Fridovich, Measuring nitric oxide and superoxide: rate constants for aconitase reactivity, Methods Enzymol. 269 (1996) 37–41. Z. Chu, J. Li, M. Eshaghi, R.K. Karuturi, K. Lin, J. Liu, Adaptive expression responses in the Pol-gamma null strain of S. pombe depleted of mitochondrial genome, BMC Genomics 8 (2007) 323. S.P. Gangloff, D. Marguet, G.J. Lauquin, Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate, Mol. Cell. Biol. 10 (1990) 3551–3561. J.K. Bhattacharjee, Alpha-aminoadipate pathway for the biosynthesis of lysine in lower eukaryotes, Crit. Rev. Microbiol. 12 (1985) 131–151. Y. Yamaguchi-Iwai, R. Stearman, A. Dancis, R.D. Klausner, Iron-regulated DNA binding by the AFT1 protein controls the iron regulon in yeast, EMBO J. 15 (1996) 3377–3384. G. Belli, M.M. Molina, J. Garcia-Martinez, J.E. Perez-Ortin, E. Herrero, Saccharomyces cerevisiae glutaredoxin 5-deficient cells subjected to continuous oxidizing conditions are affected in the expression of specific sets of genes, J. Biol. Chem. 279 (2004) 12386–12395. A. Hausmann, B. Samans, R. Lill, U. Muhlenhoff, Cellular and mitochondrial remodeling upon defects in iron–sulfur protein biogenesis, J. Biol. Chem. 283 (2008) 8318–8330. K. Oishi, R. Sugiura, H. Shuntoh, T. Kuno, Cloning and characterization of hsc1+, a heat shock cognate gene of the fission yeast Schizosaccharomyces pombe, Gene 181 (1996) 45–49. C.D. Hu, Y. Chinenov, T.K. Kerppola, Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation, Mol. Cell 9 (2002) 789–798. R.J. Nelson, M.F. Heschl, E.A. Craig, Isolation and characterization of extragenic suppressors of mutations in the SSA hsp70 genes of Saccharomyces cerevisiae, Genetics 131 (1992) 277–285. D.P. Atencio, M.P. Yaffe, MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import, Mol. Cell. Biol. 12 (1992) 283–291. L.T. Jensen, V.C. Culotta, Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis, Mol. Cell. Biol. 20 (2000) 3918–3927. X.J. Chen, X. Wang, B.A. Kaufman, R.A. Butow, Aconitase couples metabolic regulation to mitochondrial DNA maintenance, Science 307 (2005) 714–717.