Maturation of cytosolic and nuclear iron–sulfur proteins Daili J.A. Netz1, Judita Mascarenhas1, Oliver Stehling1, Antonio J. Pierik1*, and Roland Lill1,2,3 1

Institut fu¨r Zytobiologie, Philipps-Universita¨t Marburg, Robert-Koch-Strasse 6, 35032 Marburg, Germany Max-Planck-Institut fu¨r Terrestrische Mikrobiologie, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany 3 ¨ konomischer Exzellenz) Zentrum fu¨r Synthetische Mikrobiologie LOEWE (Landes-Offensive zur Entwicklung Wissenschaftlich-O (SynMikro), Hans-Meerwein-Strasse, 35043 Marburg, Germany 2

Eukaryotic cells contain numerous cytosolic and nuclear iron–sulfur (Fe/S) proteins that perform key functions in metabolic catalysis, iron regulation, protein translation, DNA synthesis, and DNA repair. Synthesis of Fe/S clusters and their insertion into apoproteins are essential for viability and are conserved in eukaryotes. The process is catalyzed in two major steps by the CIA (cytosolic iron– sulfur protein assembly) machinery encompassing nine known proteins. First, a [4Fe–4S] cluster is assembled on a scaffold complex. This step requires a sulfur-containing compound from mitochondria and reducing equivalents from an electron transfer chain. Second, the Fe/S cluster is transferred from the scaffold to specific apoproteins by the CIA targeting complex. This review summarizes our molecular knowledge on CIA protein function during the assembly process. Overview of Fe/S clusters Fe/S clusters are primordial protein cofactors ubiquitously found in all organisms [1]. The ability of these clusters to transfer electrons is used in enzymes participating in nitrogen fixation, photosynthesis and mitochondrial or bacterial respiration [2]. Fe/S proteins also play key roles in a multitude of non-redox processes [3]. For instance, the [4Fe–4S] cluster of aconitase functions as a Lewis-acid catalyst, and the clusters in biotin and lipoate synthases are engaged in radical-mediated catalysis and possibly serve as sulfur donors for the synthesis of these cofactors [4,5]. The instability of Fe/S clusters, especially in response to varying oxygen and iron levels, allows Fe/S proteins such as the bacterial transcription factors FNR and IscR or eukaryotic IRP1 (iron-regulatory protein 1) to sense ambient oxygen and iron concentrations for maintaining cellular homeostasis [6–8]. For some Fe/S proteins Corresponding author: Lill, R. ([email protected]). Keywords: Iron–sulfur cluster; mitochondrial ISC system; DNA polymerases; DNA helicase; iron regulation; genome integrity. * New address: Faculty of Chemistry-Biochemistry, Technical University Kaiserslautern, 67663 Kaiserslautern, Germany. 0962-8924/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved.

the molecular function of their cofactor(s) in the assisted processes remains enigmatic. For instance, the essential Fe/S protein Rli1 (human ABCE1) supports various steps of translation [9,10], whereas numerous Fe/S proteins are involved in the maintenance of genome integrity, including DNA polymerases, primases, helicases, and various DNA repair enzymes [11,12]. Other Fe/S proteins are involved in various steps of tRNA modification but the role of their Fe/S cluster is also unclear [13]. Fe/S proteins can be reconstituted in vitro under anaerobic conditions in the presence of ferrous iron and sulfide ions. However, living organisms use complex proteinaceous machineries to synthesize the clusters and assemble them into apoproteins. Eukaryotes contain the ISC (iron– sulfur cluster) assembly apparatus in mitochondria, the CIA system in the cytosol, and the SUF (mobilization of sulfur) machinery in chloroplasts to facilitate the assembly of Fe/S proteins in various cellular compartments [14]. The ISC and SUF proteins were inherited during evolution from similar components of the bacterial ancestors of mitochondria and plastids, respectively [2,4,15]. Numerous new CIA proteins have been identified recently and much progress has been made in understanding their molecular function. Hence, is it worthwhile to review the CIA pathway in general and describe its intimate connections to other cellular processes. Detailed reviews on the mitochondrial ISC assembly and export systems have been published elsewhere [8,13,16–20]. Cytosolic and nuclear Fe/S proteins are involved in basic cellular processes such as the biosynthesis of amino acids and nucleotides, protein translation, tRNA base modification, DNA synthesis and repair, and telomere length regulation (Table 1). The list of known cytosolic-nuclear Fe/S proteins is still growing. Their maturation not only requires the CIA machinery but also parts of the mitochondrial ISC assembly machinery. In particular, the mitochondrial cysteine desulfurase complex Nfs1–Isd11 and the scaffold protein Isu1 were shown to be essential for extramitochondrial Fe/S protein biogenesis [21–23]. These and other ISC proteins synthesize a sulfur-containing component (X–S in Figure 1), which provides the sulfur for cytosolic and nuclear Fe/S clusters. The export of this unknown compound is mediated by the mitochondrial inner membrane ATP-binding cassette (ABC) transporter Trends in Cell Biology, May 2014, Vol. 24, No. 5



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Table 1. Inventory of cytosolic and nuclear Fe/S proteins in eukaryotesa Fe/S protein (human) Yeast homolog DNA maintenance Pri2 PRIM2 Pol1 POLA Pol2 POLE1 Pol3 POLD1 Rev3 REV3L absent FANCJ Ntg2 NTHL1 Rad3 XPD absent MUTYH absent RTEL1 Chl1 CHLR1 Dna2 DNA2 Amino acid and nucleotide metabolism Leu1 Absent Ecm17 Absent Glt1 Absent Absent AOX1 Absent DPYD Absent GPAT Absent XDH Ribosome function and tRNA modification Rli1 ABCE1 Elp3 ELP3 Tyw1 TYW1 Absent CDKAL1 Absent CDKRAP1 Other processes Grx3–Grx4 GRX3 (PICOT) Absent IRP1 Absent RSAD1 (Viperin) Absent mitoNEET (CISD1) MINER1 (NAF1, CISD2) MINER2

Absent Absent

Fe/S cluster type

Proposed main function

[4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S] [4Fe–4S]

Primase, synthesis of RNA primers for DNA replication Catalytic subunit of polymerase a, DNA replication Catalytic subunit of polymerase e, DNA replication Catalytic subunit of polymerase d, DNA replication Catalytic subunit of polymerase z, DNA repair Helicase, DNA repair DNA glycosylase, DNA repair Helicase, nucleotide excision repair DNA glycosylase, DNA repair Helicase, telomere stability, anti-recombinase Helicase, chromosome segregation Helicase/nuclease, DNA repair Okazaki fragment processing,

[4Fe–4S] [4Fe–4S] [4Fe–4S] 2 [2Fe–2S] 4 [4Fe–4S] [4Fe–4S] 2 [2Fe–2S]

Isopropylmalate isomerase (leucine biosynthesis) Sulfite reductase, required for biosynthesis of methionine Glutamate synthase Aldehyde oxidase (catabolism of xenobiotics) Dihydropyrimidine dehydrogenase (pyrimidine catabolism) Phosphoribosyl pyrophosphate amidotransferase (purine biosynthesis) Xanthine dehydrogenase/oxidase

2 [4Fe–4S] 2 [4Fe–4S] 2 [4Fe–4S] [4Fe–4S] [4Fe–4S]

Ribosome biogenesis, translation initiation and termination Elongator protein 3 (tRNA wobble base modification), SAM tRNA wybutosine biosynthesis, SAM tRNA modification tRNA modification

[2Fe–2S] [4Fe–4S] [4Fe–4S] [2Fe–2S]

Intracellular iron homeostasis Intracellular iron regulation Antiviral activity, SAM Insulin sensitivity, unknown (located in mitochondrial outer membrane facing cytosol) Ca2+ metabolism, unknown Ca2+ metabolism, unknown

[2Fe–2S] [2Fe–2S]


Known Fe/S proteins of the cytosol and nucleus are provided for human and yeast cells. Alternative names are provided in parentheses. Note that four additional Fe/S proteins are members of the CIA machinery (see Table 2). SAM, S-adenosyl-methionine as an additional cofactor.

Atm1 [21,24,25]. In addition, the export process requires the sulfhydryl oxidase Erv1 of the intermembrane space and glutathione (GSH). Currently, nine CIA factors have been identified (Figures 1 and 2; Table 2). Previous work, mainly performed in yeast and human cells, has shown a high degree of conservation for both the components and mechanisms of biogenesis [26]. The overall reaction can be dissected into two major steps. First, a transiently bound [4Fe–4S] cluster is assembled on the scaffold complex formed by the two P-loop NTPases Cfd1 and Nbp35 [27–29]. This synthesis reaction further requires the electron transfer chain from NADPH to the diflavin reductase Tah18 and the Fe/S protein Dre2 [30,31]. In the second step, the transiently bound [4Fe–4S] cluster of Cfd1–Nbp35 is transferred to apoproteins, which involves the function of the iron-hydrogenase-like protein Nar1 [32] and the ‘CIA targeting complex’ comprised of the b-propeller protein Cia1, the HEAT repeat protein Mms19, and the small acidic protein Cia2 (Figure 1) [33–37]. Biogenesis also requires the function of the monothiol glutaredoxins Grx3–Grx4, which transiently bind a bridging [2Fe–2S] cluster (Figure 1; 304

Table 1) [38,39]. Because Grx3 and Grx4 are assumed to be generally involved in iron homeostasis, they are not considered as CIA proteins. We introduce here the individual CIA components and the molecular mechanism by which they assemble target Fe/S proteins, then highlight differences between the yeast and human CIA systems, and finally discuss the importance of both the mitochondrial and cytosolic biogenesis machineries for maintaining nuclear genome integrity. Assembly of [4Fe–4S] clusters on the Cfd1–Nbp35 scaffold complex Cfd1 and Nbp35 belong to the Mrp/MinD subfamily of P-loop NTPases [40]. They form a heterotetrameric protein complex which binds two different pairs of [4Fe–4S] clusters [27–29,41]. One type is coordinated by a ferredoxinlike CX13CX2CX5C motif at the N-terminus of Nbp35 (Figure 3). Intriguingly, maturation of this cluster depends on the electron transfer chain NADPH–Tah18–Dre2 (see below) [31]. Another [4Fe–4S] cluster binds to conserved C-terminal CX2C motifs in both Cfd1 and Nbp35, bridging these two proteins. Both Cfd1 and Nbp35 can individually


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Fe/S scaffold B


CIA targeng complex


N Nbp 35 Cfd1





1 Nbp35 N Cfd


Cytosolic Fe/S proteins


Dre2 FAD


2 e–

Nuclear Fe/S proteins








Electron transfer chain


Nucleus ISC assembly machinery


Cytosol TRENDS in Cell Biology

Figure 1. Model for the maturation of cytosolic and nuclear Fe/S proteins in yeast. The assembly process can be dissected into two different steps. First, bridging [4Fe–4S] clusters (B) are assembled on the Cfd1–Nbp35 heterotetrameric scaffold complex. This reaction requires a sulfur source (X–S) generated by the mitochondrial ISC assembly machinery and exported by the mitochondrial ABC transporter Atm1. Generation of the functionally essential N-terminal Fe/S cluster (N) of Nbp35 depends on the flavoprotein Tah18 and the Fe/S protein Dre2, which serve as an NADPH-dependent electron (e) transfer chain. Second, the bridging Fe/S clusters are released from Cfd1– Nbp35 and transferred to apoproteins (Apo), a reaction mediated by the Fe/S protein Nar1 and the CIA targeting complex Cia1–Cia2–Mms19. The latter three proteins interact with target (apo)proteins, and assure specific Fe/S cluster insertion. Biogenesis further requires, at an unknown step, the cytosolic multidomain monothiol glutaredoxins Grx3–Grx4, which bind a glutathione-coordinated, bridging [2Fe–2S] cluster. The thick arrows mark the flow of the Fe/S clusters. Abbreviations: CIA, cytosolic iron–sulfur protein assembly; ISC, iron–sulfur cluster.

bind Fe/S clusters in vivo and in vitro, demonstrating that the homodimers are able to coordinate the bridging [4Fe–4S] cluster [41]. However, under physiological conditions the bridging [4Fe–4S] cluster may be primarily bound by the Cfd1–Nbp35 heterotetrameric complex [42]. Several lines of in vitro evidence support the scaffold function of the Cfd1–Nbp35 complex: The bridging [4Fe– 4S] clusters (i) are bound in a labile fashion, (ii) can be efficiently transferred to apoproteins in a stoichiometric fashion, and (iii) are transferred several orders of magnitude faster to target proteins than Fe/S clusters are assembled by chemical reconstitution [29]. Recent in vivo studies corroborate the scaffold function. Pulse-chase experiments with 55Fe-labeled yeast cells show the differential lability of the two types of Fe/S clusters on Cfd1– Nbp35 [42]. Moreover, the Cfd1–Nbp35-bound clusters are much more labile than those associated with canonical target proteins. Although Cfd1 and Nbp35 belong to the large family of P-loop NTPases, no significant ATPase or GTPase binding or hydrolysis activity has been detected for the purified yeast proteins. Nevertheless, the Walker A nucleotide-binding motif of these proteins is essential for their in vivo function in Fe/S protein assembly because mutations in this motif are lethal in yeast, abolish Fe/S cluster incorporation into Cfd1 and Nbp35 and, consequently, disrupt the CIA pathway [41]. Strikingly, when wild type copies of Cfd1 or Nbp35 were present in addition to the mutated versions, Fe/S clusters could be assembled on the mutant proteins at almost wild type efficiency. These results indicate that nucleotide function is needed for Fe/S cluster loading onto Cfd1–Nbp35 but this function can be supplied in trans.

Human and murine NBP35 form a tight complex with CFD1, as indicated by co immunoprecipitation and yeast two-hybrid analysis (Table 2) [43,44]. Depletion of human NBP35 by RNA interference in HeLa cells resulted in a severe defect in the assembly of cytosolic and nuclear Fe/S proteins without an apparent effect on mitochondrial Fe/S protein maturation [44]. Cytosolic aconitase is among the affected target proteins. In the absence of its [4Fe–4S] cluster it functions as ‘iron regulatory protein 1’ (IRP1) and binds to stem–loop structures of specific mRNAs of proteins involved in iron uptake and distribution [7]. Consequently, Nbp35 depletion severely affects cellular iron homeostasis by decreasing cellular H-ferritin and increasing transferrin receptor levels [44]. Overall, this leads to higher transferrin–iron uptake, indicating that the efficiency of human cells to generate cytosolic and nuclear Fe/ S proteins is utilized for intracellular iron sensing. These characteristics clearly distinguish human NBP35 (and other CIA proteins) from its yeast counterpart which plays no detectable role in cellular iron regulation. Interestingly, murine NBP35 and CFD1 are associated with KIFC5A, a microtubule-binding, minus-end-directed motor protein involved in spindle assembly [43]. Depletion of KIFC5A, NBP35, and CFD1 by siRNA results in a cytokinesis defect, as seen by supernumerary centrosome formation with persistence in interphase. Recently, NBP35 and CFD1 were suggested to be part of both centrioles and basal bodies by interacting with members of the CCT/TRiC molecular chaperone complex [45]. These observations raise the question of whether NBP35 and CFD1 might fulfill moonlighting functions in the regulation of centriole and basal body duplication, or whether these effects are 305


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Iron regulaon IRP2



Mito. ISC Early CIA



IOP1 Holo



Cytosolic Fe/S proteins



CIA targeng complex


Nuclear Fe/S proteins TRENDS in Cell Biology

Figure 2. The dual role of the late-acting, human cytosolic iron–sulfur protein assembly (CIA) components in Fe/S protein maturation and iron regulation. The components and mechanisms of the human CIA machinery are similar to those defined in yeast (see Figure 1). As a major difference, humans possess two isoforms of CIA2, which both bind to CIA1. CIA2B is the functional ortholog of yeast Cia2 and is involved in the biogenesis of canonical cytosolic and nuclear Fe/S proteins. By contrast, CIA2A is specifically involved in the Fe/S cluster maturation of iron regulatory protein 1 (IRP1). This protein, together with the related IRP2, which does not assemble a Fe/S cluster, regulates intracellular iron homeostasis in humans by a post-translational mechanism. CIA2A binds to and stabilizes IRP2, thus introducing a second layer of regulation by the CIA machinery on cellular iron metabolism. Abbreviation: Mito., mitochondrial.

secondary to impairment of the assembly of (unknown) Fe/ S proteins involved in these processes. Photosynthetic eukaryotes contain only Nbp35, as demonstrated by the presence of the N-terminal Nbp35-like motif [46]. In the Viridiplantae the C-terminal CX2C motif is substituted by a conserved stretch of 35 amino acids with a divergent CX17CX8CX5C pattern, possibly involved in binding the bridging Fe/S cluster. The Arabidopsis thaliana Nbp35 protein is mainly localized in the cytosol and binds two [4Fe–4S] clusters per monomer, as does yeast Nbp35 [46]. The Tah18–Dre2 electron transfer system for cytosolic Fe/S protein biogenesis DRE2, encoding an Fe/S protein, was identified by its synthetic lethality with the two mitochondrial iron importers MRS3–MRS4 [30]. Functional analysis showed that Dre2 is part of the CIA machinery. Dre2 is composed of a Nterminal S-adenosylmethionine (SAM) methyltransferaselike domain [47,48] connected by a flexible linker to the Cterminal domain (CTD), which encompasses two pairs of four conserved cysteine residues: motif I, with the pattern CXnCX2CXC; and motif II, with the invariant pattern CX2CX7CX2C (Figure 3). Purified yeast Dre2 is almost devoid of bound Fe/S clusters [30,31,48,49]. After chemical reconstitution, Dre2 can bind up to six Fe and S ions per monomer. Electron paramagnetic resonance (EPR) spectroscopy identified a mixture of [2Fe–2S] and [4Fe–4S] clusters [30,31]. In vivo studies and spectroscopic investigation of site-directed cysteine mutants of Dre2 showed a 306

labile [2Fe–2S]2+ cluster bound to motif I and a [4Fe–4S]2+ cluster attached to motif II (Netz et al., unpublished). The latter cluster readily breaks down to a [2Fe–2S]2+ cluster upon aerobic purification. Dre2 physically interacts with Tah18, an essential protein containing FMN-, NAD(P)(H)- and FAD-binding domains [50] (Figure 3). Enzyme activity measurements and 55Fe incorporation in Tah18-depleted yeast cells identified this proteins as a CIA factor [31]. EPR studies using different mixtures of NADPH, Tah18, and holo-Dre2 demonstrated that electrons are transferred from NADPH via the FMN and FAD centers of Tah18 to the [2Fe–2S]2+ cluster of Dre2 [31]. One possible function of the electron transfer chain may be the maturation of Dre2. This hypothesis seems unlikely, however, because Tah18 depletion did not significantly decrease the amount of Fe/S clusters bound to Dre2. Furthermore, Fe/S cluster assembly on Dre2 was not affected by depletion of any other CIA protein but required the mitochondrial ISC system. These findings imply that Dre2 and Tah18 function at an early step in the CIA pathway (Figure 1). Both Dre2 and Tah18 are required for the incorporation of the stable [4Fe–4S] cluster into Nbp35, and hence for the function of the Cfd1– Nbp35 scaffold complex. The cytosolic Tah18–Dre2 complex may be considered as a formal counterpart of the mitochondrial electron transfer chain of the [2Fe–2S] ferredoxin, Yah1, which receives its electrons from the NADH-reducible flavoprotein Arh1. Both chains share a common biophysical principle: an obligate two-electron donor (NADPH) transfers


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Table 2. Components of the CIA machinerya Human CIA proteins CFD1 (NUBP2)

Yeast CIA proteins Cfd1

Important functional groups Bridging [4Fe–4S] cluster with Nbp35



CIAPIN1 (Anamorsin) NDOR1 IOP1 (NARFL)

Dre2 Tah18 Nar1

Bridging [4Fe–4S] cluster with Cfd1; N-terminal [4Fe–4S] cluster [2Fe–2S] and [4Fe–4S] clusters FAD, FMN 2 [4Fe–4S] clusters


Cia1 Cia2 Mms19 (Met18)

Hyper-reactive Cys Hyper-reactive Cys? -

Proposed main function Scaffold complex, assembly, and transient binding of [4Fe–4S] cluster Scaffold complex, assembly, and transient binding of [4Fe–4S] cluster Electron acceptor from NADPH–Tah18 Electron transfer from NADPH to Dre2 CIA adaptor protein, mediates contact between early and late parts of CIA machinery Docking site of the CIA targeting complex Insertion of Fe/S clusters into target apoproteins Insertion of Fe/S clusters into IRP1 Insertion of Fe/S clusters into target apoproteins


Known yeast and human CIA proteins are listed. Note that in this review we mainly use the yeast nomenclature (bold face). Alternative names are provided in parentheses.

reducing equivalents to a flavoprotein (Tah18 or Arh1), which is able to hand over the electrons to a one-electron acceptor (the [2Fe–2S] cluster of Dre2 or Yah1). An apparent difference between the two systems is that the diflavin (FMN and FAD) protein Tah18 has a preference for NADPH, whereas Arh1 is a monoflavin enzyme (FAD) with NADH specificity. In addition to Fe/S protein biogenesis, the reducing equivalents of the NADH-Arh1-Yah1 chain are used for at least two other processes: the hydroxylation of heme O to heme A [51] and the biosynthesis of coenzyme Q6 [52]. Likewise, Dre2 function is not confined to Fe/S cluster biogenesis. Depletion of yeast Dre2 impairs the activity of the binuclear iron center-containing enzyme ribonucleotide reductase (RNR), which catalyzes the formation of deoxyribonucleotides for DNA synthesis [53]. The requirement of Dre2 (and possibly Tah18) links Fe/ S cluster biogenesis to DNA synthesis, two essential biological processes in eukaryotic cells. The human Tah18 and Dre2 homologs, NDOR1 and CIAPIN1 (Table 2), form a stable complex [31] but their potential role in human Fe/S protein biogenesis has not been addressed. Individual human Dre2 and Tah18 homologs and a combination of both homologs of Arabidopsis can rescue cell growth and Fe/S cluster assembly of yeast cells depleted for Dre2 and Tah18 [30,31,54]. Thus, the Tah18– Dre2 electron transfer chain appears to be evolutionarily conserved. Some studies link CIAPIN1 function to cancer, but whether the protein plays a direct role in cancer development requires further examination (reviewed in [55]). The [FeFe]-hydrogenase-like Nar1 links early and late steps of biogenesis Nar1 is a Fe/S protein and shows sequence homology to bacterial [FeFe] hydrogenases [56,57]. All eukaryotic Nar1 homologs possess an N-terminal ferredoxin-like domain with four cysteine residues, corresponding to the ligands of the medial [4Fe–4S] cluster of [FeFe] hydrogenases, and a C-terminal domain which is similar to the active site of [FeFe] hydrogenases (Figure 3) [32]. The absence of hydrogenase activity and biogenesis genes in yeast suggests that, instead of a [4Fe–4S] cluster linked to the Fe2(CO)3(CN)2 active-site cluster, Nar1 only retains the [4Fe–4S] cluster. Cell biological and biochemical studies identified two magnetically coupled [4Fe–4S] clusters in yeast and plant Nar1, but the recombinant proteins

contained substoichiometric amounts of Fe/S clusters, and they may not correspond to the native versions [32,58,59]. The assembly of the C-terminal Fe/S cluster of yeast Nar1 depends on the N-terminal cluster, showing that the two Fe/S clusters act cooperatively [58]. Homology modeling and stability studies suggest that the C-terminal cluster is buried in the protein and that the more labile N-terminal cluster is surface-exposed. Depletion of Nar1 in yeast strongly impairs the maturation of all cytosolic and nuclear Fe/S proteins studied, whereas mitochondrial Fe/S proteins are unaffected [32]. Similar results have recently been obtained for the human Nar1 homolog IOP1 (iron-only hydrogenase-like protein; Table 2) and Chlamydomonas Nar1 [60–62]. Nar1 physically interacts with Nbp35 in vivo [32], and shows a genetic interaction with Cfd1 because the growth defect of Cfd1depleted yeast cells is exacerbated by overproduction of Nar1 (or Nbp35) [28]. Further, yeast Nar1 interacts with the CIA targeting complex Cia1, Cia2, and Mms19 [33,37,63]. Likewise, human IOP1 associates with CIA1, CIA2A, CIA2B, and MMS19 (see below), but not with target Fe/S proteins [35–37,64,65], suggesting that Nar1 and IOP1 function at the interface between early (Cfd1– Nbp35, Tah18–Dre2) and late (Cia1, Cia2, and Mms19) steps of CIA function (Figures 1 and 2). Functional studies in yeast have strongly supported this view. Depletion of all four early-acting CIA factors impairs Fe/S cluster incorporation into Nar1 in vivo [29,31,32]. Conversely, depletion of Nar1 has no influence on the Fe/S cluster assembly on Cfd1, Nbp35, or Dre2. By contrast, none of the late-acting CIA factors are required for Fe/S cluster assembly on Nar1 [33,35] (Mascarenhas et al., unpublished). Nar1 therefore acts as an adapter between early and late phases of the CIA pathway, but its precise molecular role remains to be elucidated. IOP1 knockout mice show embryonic lethality before embryonic (E) day E10.5, and have decreased IRP1 aconitase activity, providing evidence that IOP1 is essential for viability in mammals [62]. Humans possess a second Nar1related protein termed IOP2 (or NARF [66]). Only depletion of human IOP1 (but not of IOP2) by siRNA technology diminishes the activities of the cytosolic Fe/S proteins IRP1 and xanthine oxidase and causes alterations in cellular iron metabolism [61]. IOP2 function remains unclear and is possibly not related to the CIA machinery because it is 307



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Cfd1 293

Key: Fe/S cluster binding cysteine


Electron transfer



Reacve cysteine

Methyltransferaselike domain

Walker A






WD40 repeats



HEAT repeats Nar1

Targeng complex


[2Fe–2S] cluster [4Fe–4S] cluster

Cia1 330


DUF59 231

Mms19 1032 TRENDS in Cell Biology

Figure 3. Domain structure and functional modules of the yeast cytosolic iron–sulfur protein assembly (CIA) components. The cartoons depict the structures of the various CIA proteins. The numbers indicate the length of each CIA protein. The functional units and features are described in the box.

found exclusively in the nucleus and binds to the nuclear protein prelamin A [66]. Nar1 has been suggested to sense oxygen and oxidative stress, as indicated by complex formation of human IOP1 with PHD2, an iron-containing protein that functions as an oxygen-sensing component of the HIF1a system [67]. In human cells and in A. thaliana [68], IOP1 represses HIF1a expression under normoxia. In Caenorhabditis elegans and yeast, defects in Nar1 result in hypersensitivity to oxidative stress [69]. Currently, it is not clear whether Nar1– IOP1 have a separate regulatory role under oxidative stress or whether these effects are indirect consequences of a general defect in CIA function. Cia1, the docking site of the CIA targeting complex In fission yeast the P-loop NTPase Cfd1 forms a fusion protein with the WD40-repeat protein Cia1, suggesting that the latter may be a CIA component. This expectation was verified in budding yeast and recently in human cells [33,37]. Cia1 is required for virtually all tested target Fe/S proteins, but its function is dispensable for the maturation of the Fe/S cluster-containing CIA proteins (Figures 1 and 2) [31,33,37]. These results are consistent with a late requirement of Cia1 in the CIA pathway. The Cia1 crystal ˚ resolution, shows a b-propeller structure, solved at 1.7 A structure with seven pseudo-symmetrically orientated blades around a central axis (Figure 3) [70]. This doughnut-shaped geometry is highly conserved among the large family of WD40 proteins, which serve as docking sites in a multitude of cellular networks and mediate molecular recognition of partner proteins mainly through the top and side surfaces [71,72]. The function of Cia1 as a general docking site has been supported by co immunoprecipitation and proteomics 308

experiments which revealed an interaction of Cia1 with Nar1–IOP1 and the other members of the CIA targeting complex (CIA2B and MMS19) [33,37]. Moreover, human CIA1, together with CIA2B and MMS19, binds to the majority of cytosolic and nuclear Fe/S proteins [35–37], suggesting that the CIA targeting complex specifically delivers the Fe/S clusters to the apoproteins (see also below). It is not yet clear to which regions of Cia1 the individual protein partners bind. In particular, the conserved regions of Cia1 may function as binding sites and have been mapped to the top, bottom, and side faces and to the central channel of the Cia1 doughnut structure [70]. Notably, Arg127 at the top of yeast Cia1, is important for protein–protein interaction because mutation to Glu affects growth and CIA machinery function. Future structure–function studies will need to define where Cia1 recognizes its CIA partners and possibly Fe/S target proteins to serve as a docking site within the CIA targeting complex. The function of Mms19 as a CIA factor resolves a longlasting mystery Mms19, known as Met18 in yeast, contains several HEAT repeats which are enriched at its C-terminus, forming a predicted armadillo structure (Figure 3; Table 2). In contrast to all other known CIA proteins, Mms19 is not essential for viability in yeast. However, MMS19 deletion in both yeast and mammalian cells is associated with a multitude of phenotypes that for many years created a puzzle as to what molecular function(s) might explain all these different observations. For instance, yeast cells lacking MMS19 show both methionine auxotrophy and increased sensitivity to DNA damage (by, e.g., UV irradiation or the DNA damaging agent methyl methanesulfonate, MMS) [73]. Yeast and human Mms19 were

Review suggested to be accessory proteins of the transcription factor IIH (TFIIH) complex, which is required for transcription and nucleotide excision repair [74]. Yeast MMS19 was identified in a screen for factors required for telomere length regulation [75]. Mms19-deficient yeast cells contain low levels of the Fe/S cluster-containing DNA helicase Rad3, which is related to human XPD (xeroderma pigmentosum group D protein; Table 1). Hence, a regulatory role of Mms19 in nucleotide excision repair was proposed [76]. Recently, human MMS19 was described as part of a TFIIHindependent complex composed of MMS19, CIA1, and CIA2B [77]. siRNA-mediated knockdown of MMS19 led to improper chromosome segregation and abnormal mitosis, with striking similarity to phenotypes seen in patient cells with xeroderma pigmentosum. Consequently, MMS19 and its partner proteins were implicated to function in chromosome segregation. The recent characterization of yeast and human Mms19 as a CIA component now explains the majority, if not all, of the Mms19-associated (pleiotropic) phenotypes [35,36]. Mms19 assists in the maturation of numerous Fe/S proteins involved in diverse cellular processes that are compromised in Mms19-mutant cells, thus eliciting a number of indirect phenotypes. The Fe/S maturation function of Mms19 has been directly documented for sulfite reductase involved in methionine synthesis, some DNA helicases, glycosylases, and polymerases involved in DNA synthesis and repair, and the helicase RTEL1 involved in telomere length regulation. Thus, the primary function of Mms19, as part of the CIA targeting complex, is the delivery of Fe/S clusters to various target Fe/S proteins, including some involved in genome maintenance (Figures 1 and 2) [35,36]. Cia2, the CIA targeting factor with a hyper-reactive cysteine residue Cia2 is a small acidic protein containing a C-terminal domain of unknown function 59 (DUF59; Figure 3). Yeast Cia2 and its human ortholog CIA2B were recently shown to function as part of the CIA targeting complex in Fe/S protein maturation (Figures 1 and 2; Table 2) [37] (Mascarenhas et al., unpublished). As mentioned above, various yeast high-throughput screens and proteomic studies in human cells identified Cia2–CIA2B not only as part of the CIA targeting complex but also in contact with many Fe/S target proteins. Mutation of the Cia2 homolog AE7 in Arabidopsis led to lower activity of cytosolic aconitase and nuclear glycosylase, both being [4Fe–4S] proteins [63]. Hence, the function of Cia2 in cytosolic-nuclear Fe/ S protein biogenesis seems to be conserved in eukaryotes. Structural information has been obtained for three bacterial Cia2 homologs whose function is unknown [78]. In addition, a nuclear magnetic resonance (NMR) structure and two different X-ray structures of human CIA2A (see below) have been reported [79] (Table 2). So far, the structural information did not provide any decisive insights into the molecular function of Cia2. A hyperreactive cysteine identified in both yeast Cia2 (Cys161) and human CIA2B (Cys93) appears to perform a crucial role in Cia2 function [34]. Mutation of the surface-exposed cysteine residue is lethal in yeast and abolishes the activity of the Fe/S protein Leu1. Elucidation of the molecular role

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of this reactive cysteine residue might clarify not only the role of Cia2 but also that of the entire CIA targeting complex in Fe/S protein maturation. Differential targeting functions of the two human CIA2A and CIA2B isoforms Previous studies have demonstrated that both the components and mechanisms of the CIA pathway are highly conserved from yeast to man (Table 2). However, as a characteristic difference to yeast, human cells encode a second CIA2 homolog, termed CIA2A, which also binds to CIA1 (Figure 2). Strikingly, depletion of CIA2A does not elicit any defects in canonical cytosolic and nuclear Fe/S proteins [37]. Instead, CIA2A is exclusively required for the maturation of IRP1 to cytosolic aconitase. Consequently, depletion of CIA2A (but not of CIA2B or MMS19) increases the IRE binding activity of IRP1. As a result, ferritin levels are diminished and the expression of the transferrin receptor is increased. Together, this indicates that CIA2A, together with the early-acting CIA factors and the ISC machinery, is crucial for IRP1 maturation, thus separating the regulation of cellular iron homeostasis from general Fe/S protein maturation (Figure 2). An additional important effect of CIA2A on cellular iron regulation is mediated via its binding to IRP2 [37], which does not contain a Fe/S cluster, but is regulated by ironand oxygen-dependent degradation by the proteasome [80]. This reaction is mediated by the E3 ubiquitin ligase FBXL5 that senses iron and oxygen levels via its hemerythrin domain. The binding of CIA2A to IRP2 stabilizes the latter and introduces a second, unexpected level of iron regulation by the CIA machinery. Overall, the CIA2A branch of the CIA machinery, through the regulation of both IRP1 and IRP2, affects cellular iron homeostasis in multiple ways. Another level of Fe/S protein target specificity has been found for the other members of the human CIA targeting complex, CIA1 and MMS19 [35–37]. For instance, MMS19 is less important for glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) maturation than the other CIA proteins, and CIA2B is not crucial for DNA polymerase D1 (POLD1) assembly [37]. It therefore appears that the individual CIA targeting complex constituents specifically deliver the Fe/S clusters to dedicated client apoproteins. These functional observations are corroborated by systematic proteomic studies which show differential binding of CIA1, CIA2B, and MMS19 to the collection of cytosolic and nuclear Fe/S proteins [35– 37,64,65]. How this target binding specificity is achieved remains to be elucidated. The essential role of the CIA and mitochondrial ISC systems in genome integrity Virtually all CIA factors (and many of the mitochondrial ISC components) are essential for cell viability. It is now clear that this is explained by several cytosolic and nuclear Fe/S proteins with central functions in protein translation, DNA replication and damage repair, transcription, and chromosome segregation (Table 1; Box 1) [35–37]. Notably, the essentiality of the mitochondrial ISC system is not due to the presence of numerous Fe/S clusters in the respiratory 309

Review Box 1. An unexpectedly large number of Fe/S proteins involved in DNA metabolism Until recently DNA glycosylases were the only Fe/S proteins known to be involved in DNA metabolism. This picture substantially changed in the past few years when hitherto unrecognized Fe/S clusters were found in key proteins of this process. These proteins include DNA helicases, nucleases, primase, and polymerases (see Table 1 in main text). The Fe/S clusters in these proteins are usually essential but their precise molecular function remains elusive. Better understanding of the role of these Fe/S clusters is crucial because many of these Fe/S enzymes are linked to severe diseases. Exchange of cysteine residues in the Fe/S helicase XPD (Rad3 in yeast) results in failure to couple ATP hydrolysis to DNA translocation, loss of helicase activity, and destabilization of the tertiary structure (reviewed in [85]). XPD is a core component of TFIIH, which catalyzes the melting of duplex DNA at a promoter site in transcription or at a site of DNA damage in transcriptionally coupled nucleotide excision repair. In humans there are three additional XPD-related Fe/S cluster-containing helicases [12]. FANCJ directionally unwinds the DNA duplex and has a key role in homologous recombination and double-strand break repair [85]. Genetically, mutations of the Fe/S domain in XPD and FANCJ are linked to the diseases xeroderma pigmentosum and Fanconi anemia, respectively. The Fe/S helicase RTEL1 unravels hidden DNA 30 -ends during fork progression in replication or in DNA repair [86]. Depletion, deletion, or mutation of CHLR1 causes chromosome loss, as observed in patients with the Warsaw chromosome breakage syndrome [87]. The helicase-nuclease fusion protein DNA2, containing a [4Fe–4S] cluster in its nuclease domain, is essential for Okazaki fragment processing in replication and in DNA break resection during repair [88]. Although the precise function of the Fe/S cluster in DNA2 is unknown, mutations of the Fe/S cluster-coordinating cysteine residues in the prokaryotic homolog impair the ability to bind broken DNA [89]. The primase subunit Pri2 contains a [4Fe–4S] cluster which is essential for replication [90]. Finally, all three replicative DNA polymerases and the major translesion polymerase Rev3 bind a [4Fe– 4S] cluster by their C-terminal domains (see Table 1 in main text) [11]. The cluster is required for interaction with accessory subunits of the polymerase complexes, and is hence needed for the processivity at the replication fork. In all these cases it remains an important open question whether the role of the Fe/S cluster in these proteins extends beyond the stabilization of protein structure.

chain. Many eukaryotes (including baker’s yeast) can live anaerobically without making use of respiration or do not contain respiratory complexes such as mitosomes [81], thus refuting the possibility that oxidative phosphorylation is the essential task of the ISC machinery. Rather, the requirement of the mitochondrial ISC system in the biogenesis of a multitude of cytosolic and nuclear Fe/S proteins involved in, for example, protein translation, DNA replication, and DNA repair, explains its essentiality [36,37,82,83]. The fact that these basic processes of life depend on Fe/S clusters may be related to the suggested Fe/S origin of life [84]. This theory proposed that the first biochemical reactions were catalyzed on Fe/S mineral surfaces. In any case, the maintenance of Fe/S cluster chemistry in many central enzymes of modern DNA, RNA, and protein metabolism reinforces the importance of these ancient cofactors for life. Concluding remarks The past decade has seen the discovery and initial functional characterization of the CIA machinery. In the near future more components of this machinery will be discovered and the molecular mechanism will be better defined. 310

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Box 2. Outstanding questions Progress on our understanding of Fe/S protein assembly in the cytosol and nucleus has rapidly increased in the past decade by studies mainly performed in yeast and human cells. During the next decade several important questions need to be answered by genetic, cell biological, biochemical, and ultrastructural studies.  Which sulfur-containing compound is exported by Atm1 from mitochondria?  Are there any additional components of the CIA machinery?  What is the precise function and site of requirement of the glutaredoxins Grx3–Grx4 in the CIA pathway?  What is the biochemical role of the electron transfer from Tah18– Dre2 to the Cfd1–Nbp35 complex?  Do the human Cfd1, Dre2, and Tah18 homologs fulfill the same function as their yeast CIA counterparts?  What is the mechanism of the synthesis of the bridging [4Fe–4S] cluster on the scaffold complex Cfd1–Nbp35, and what is the role of the N-terminal Fe/S cluster of Nbp35 in this reaction?  What is the role of ATP or GTP (hydrolysis) for Fe/S cluster synthesis on Cfd1–Nbp35? Are the nucleotides required for Fe/S cluster transfer?  What is the specific role of the two Fe/S clusters of Nar1?  How does Cia1 bind to its partner proteins?  Are chaperones and/or dedicated factors involved in stabilizing specific cytosolic and nuclear Fe/S targets before Fe/S cluster insertion?  What is the role of the hyper-reactive cysteine residue in Cia2? Does it transiently bind a Fe/S cluster or activate cysteine residues of the Fe/S apoproteins?  What is the specific functional contribution of the Fe/S clusters in different proteins required for DNA metabolism?  Are there any diseases connected to the CIA proteins?  How are mitochondrial function, Fe/S protein biogenesis, and DNA metabolism interconnected in aging and aging-related diseases?

The challenge of the next decade will be to answer key questions centered on a better structural and functional understanding of the individual CIA components and of the overall process (Box 2). It seems likely that future research will discover diseases caused by dysfunction of CIA components. It will be informative, also for medical reasons, to compare the phenotypes of these diseases to those linked to Fe/S target proteins such as FANCJ or XPD. For sure, there is an exciting decade of Crucial Investigations Ahead of us. Acknowledgments We thank all other members of our group for discussion. R.L. acknowledges generous support from Deutsche Forschungsgemeinschaft (SFB 593, SFB 987, and GRK 1216), Max-Planck Gesellschaft, von Behring-Ro¨ntgen Stiftung, LOEWE program of state Hessen, Feldberg Foundation, and Fonds der chemischen Industrie.

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Maturation of cytosolic and nuclear iron-sulfur proteins.

Eukaryotic cells contain numerous cytosolic and nuclear iron-sulfur (Fe/S) proteins that perform key functions in metabolic catalysis, iron regulation...
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