Mitochondrial iron-sulfur protein biogenesis and human disease.

Biochimie 100 (2014) 61e77

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Review

Mitochondrial ironesulfur protein biogenesis and human disease Oliver Stehling a, Claudia Wilbrecht a, Roland Lill a, b, c, * a

Institut für Zytobiologie, Philipps-Universität Marburg, Robert-Koch-Str. 6, 35032 Marburg, Germany Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str. 10, 35043 Marburg, Germany c LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans-Meerwein-Str., 35043 Marburg, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 November 2013 Accepted 13 January 2014 Available online 23 January 2014

Work during the past 14 years has shown that mitochondria are the primary site for the biosynthesis of ironesulfur (Fe/S) clusters. In fact, it is this process that renders mitochondria essential for viability of virtually all eukaryotes, because they participate in the synthesis of the Fe/S clusters of key nuclear and cytosolic proteins such as DNA polymerases, DNA helicases, and ABCE1 (Rli1), an ATPase involved in protein synthesis. As a consequence, mitochondrial function is crucial for nuclear DNA synthesis and repair, ribosomal protein synthesis, and numerous other extra-mitochondrial pathways including nucleotide metabolism and cellular iron regulation. Within mitochondria, the synthesis of Fe/S clusters and their insertion into apoproteins is assisted by 17 proteins forming the ISC (ironesulfur cluster) assembly machinery. Biogenesis of mitochondrial Fe/S proteins can be dissected into three main steps: First, a Fe/S cluster is generated de novo on a scaffold protein. Second, the Fe/S cluster is dislocated from the scaffold and transiently bound to transfer proteins. Third, the latter components, together with specific ISC targeting factors insert the Fe/S cluster into client apoproteins. Disturbances of the first two steps impair the maturation of extra-mitochondrial Fe/S proteins and affect cellular and systemic iron homeostasis. In line with the essential function of mitochondria, genetic mutations in a number of ISC genes lead to severe neurological, hematological and metabolic diseases, often with a fatal outcome in early childhood. In this review we briefly summarize our current functional knowledge on the ISC assembly machinery, and we present a comprehensive overview of the various Fe/S protein assembly diseases. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Ironesulfur cluster Mitochondrial ISC system Iron regulation Genome integrity

1. Introduction Mitochondria are eukaryotic organelles not only serving as cellular energy suppliers but also hosting a multitude of important metabolic processes. As the cell’s major consumers of iron they are involved in the formation of heme and the biosynthesis of ironesulfur (Fe/S) clusters which are present in all kingdoms of life. In eukaryotes proteins carrying Fe/S clusters are located in mitochondria, cytosol, nucleus, and plastids. The essential character of Fe/S clusters was first identified in the context of oxidoreductive processes including electron transfer in the respiratory chain complexes I to III or in photosystem I of photosynthetic organisms. Some Fe/S proteins like aconitase are required for enzymatic catalysis whereas others play a role in the regulation of gene expression. For example, human iron regulatory protein 1 (IRP1) gets activated as a mRNA binding protein in * Corresponding author. Institut für Zytobiologie und Zytopathologie, PhilippsUniversität Marburg, Robert-Koch Str. 6, 35032 Marburg, Germany. Tel.: þ49 6421 286 6449; fax: þ49 6421 286 6414. E-mail address: [email protected] (R. Lill). 0300-9084/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2014.01.010

the absence of its Fe/S cofactor, thereby modulating the biosynthesis of proteins involved in iron metabolism [1]. Other proteins including the bacterial FNR (fumarate and nitrate reduction) regulatory proteins or the protein IscR carry oxygen-sensitive Fe/ S clusters thus operating as O2 sensors [2e4]. Recently, proteins involved in DNA replication and repair have been shown to harbor Fe/S clusters that stabilize the protein structure. These proteins include the eukaryotic replicative DNA polymerases (Pol a, Pol ε, Pol d and Pol z) and the DNA helicases involved in DNA damage repair such as XPD and FancJ [5,6]. Functional impairment of the latter proteins causes Xeroderma pigmentosum and Fanconi anemia, respectively. The most abundant Fe/S clusters in eukaryotes are the rhomboid [2Fee2S] and the cubane [4Fee4S] clusters, yet more complex forms harboring other heavy metal ions have been characterized, predominantly in bacterial species [7,8]. Fe/S clusters are typically bound to proteins via cysteine residues of the polypeptide chain, but other amino acid residues including histidine, arginine, and serine are also used. A well-known example is the [2Fee2S] Rieske cluster of respiratory complex III in which one of the two Fe ions is coordinated by two histidine residues.

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O. Stehling et al. / Biochimie 100 (2014) 61e77

Formation and insertion of Fe/S clusters in a cell does not occur spontaneously but rather requires complex machineries which are highly conserved from yeast to man. In this review, we will first provide a brief summary of our current knowledge of the components and mechanisms involved in the maturation of mitochondrial Fe/S proteins. Second, we discuss various human ‘Fe/S diseases’ caused by mutations in genes coding for proteins required for mitochondrial Fe/S protein maturation. These diseases both underline the crucial character of this mitochondrial biosynthetic process and deepened our functional understanding of the Fe/S protein assembly process. 2. A primer on mitochondrial Fe/S protein assembly The assembly system for mitochondrial Fe/S proteins was inherited from bacteria during an endosymbiotic event [9]. The function of this so-called Fe/S cluster (ISC) assembly machinery has been worked out for mitochondria most explicitly in the model system Saccharomyces cerevisiae [10e13]. Hence, we will refer to the yeast names when introducing ISC assembly factors in this review, but keep the human nomenclature in the subsequent disease part of this review (see Table 1). The chain of events leading to the assembly of Fe/S proteins can be dissected into three consecutive major steps (Fig. 1). First, a Fe/S cluster is assembled de novo in a transient fashion on the heterodimeric scaffold protein Isu1 (and its yeast paralog Isu2 which is due to gene duplication). The sulfur required for this process is provided by conversion of cysteine to alanine by the desulfurase complex Nfs1eIsd11. Additionally, the sulfur has to be reduced to sulfide in order to be combined with ferrous iron (Fe2þ) to a [2Fee2S] cluster on Isu1. Ferrous iron is imported into the mitochondrion by carrier proteins using the proton motive force as an energy source for transport. In a second step, the Fe/S cluster is released from the scaffold, an event that is mediated by a dedicated chaperone system. The [2Fee2S] cluster then transiently binds to the monothiol glutaredoxin Grx5 from which it can be directly handed over to [2Fee2S] target proteins. In

Table 1 Inventory of mitochondrial ISC assembly factors. Assembly step

Yeast name

Human name

(Putative) function

Step I: Core ISC assembly factors involved in de novo Fe/S cluster synthesis

Isu1/Isu2a Nfs1 Isd11 Yfh1

ISCU NFS1 ISD11 Frataxin (FXN)

Yah1

Jac1 Mge1 Grx5 Isa1

Ferredoxin 2 (FDX2) Ferredoxin reductase (FDXR) Mortalin (GRP75, HSPA9) HSCB GRPEL1/2 GLRX5 ISCA1

Scaffold protein Sulfur donor Stabilizer of Nfs1 Iron donor? Regulator of Nfs1? Electron transport

Isa2

ISCA2

Iba57

IBA57

Nfu1

NFU1

Aim1

BOLA3

Ind1b

IND1

Arh1 Step II: ISC factors involved in cluster transfer

Step III: Late-acting ISC targeting factors

a b

Ssq1a, Ssc1

Electron transport Fe/S cluster transfer Fe/S cluster transfer Nucleotide exchange Fe/S cluster transfer [4Fee4S] cluster assembly [4Fee4S] cluster assembly [4Fee4S] cluster assembly Dedicated ISC targeting factor Dedicated ISC targeting factor? Maturation of respiratory complex I

Present only in S. cerevisiae and few other yeasts. Not present in S. cerevisiae but in other fungi containing respiratory complex I.

a third step, the Isa and Iba57 proteins help to convert the [2Fee2S] into [4Fee4S] clusters which then are inserted into apoproteins by various ISC targeting factors like Nfu1 and Ind1. The ISC assembly machinery is not only required for the maturation of mitochondrial but also of cytosolic and nuclear Fe/S proteins. Mitochondria are known to export a sulfur containing compound necessary for Fe/S protein biogenesis within the cytosol and the nucleus (Fig. 1). The core component of the so-called ISC export machinery is the ABC transporter Atm1 of the mitochondrial inner membrane [14,15]. The export process is further supported by the intermembrane space sulfhydryl oxidase Erv1 and the tripeptide glutathione (GSH) [16,17]. In the cytosol the sulfur is used by the cytosolic Fe/S protein assembly (CIA) machinery to mature cytosolic and nuclear Fe/S proteins. Comprehensive reviews of the CIA machinery have been published recently [18,19]. In the following, we will discuss the individual steps of mitochondrial Fe/ S protein assembly in more detail. 3. Mitochondrial Fe/S protein assembly is a three-step process 3.1. Step 1: the de novo synthesis of a [2Fee2S] cluster on the scaffold protein Isu1 To date six different components have been identified to contribute to the formation of a transiently bound [2Fee2S] cluster on the scaffold protein Isu1 (Fig. 2) [14,20,21]. Central to this process is the cysteine desulfurase complex Nfs1-Isd11 which converts cysteine to alanine. During this reaction sulfur is released from cysteine and transiently bound as a persulfide group on a conserved cysteine residue on the desulfurase [22,23]. The function of Nfs1 in vivo depends on the eukaryote-specific, LYRM family protein Isd11, forming a hetero-oligomer with Nfs1 [24e27]. Isd11 seems to be a stabilizing partner of Nfs1 and may not directly participate in the desulfurase reaction, as it is not essential for desulfurase activity but for Fe/S cluster synthesis. The persulfide sulfur may then be transferred from Nfs1 to Isu1, likely involving the formation of a persulfide group on one of the three cysteine residues of the scaffold. For subsequent cluster assembly the sulfur needs to be reduced from sulfane sulfur (S ) to sulfide (S2). The electrons required for the reduction process are probably provided by an electron transport chain including NAD(P)H, the ferredoxin reductase Arh1, and the ferredoxin Yah1 [21,28,29]. Since Yah1 itself is an [2Fee2S] protein, it is likely that its Fe/S cluster gets reduced in an intermediate step prior to transmission of the electrons to the persulfide group on Isu1. This scenario is supported by the observation that Yah1 exclusively binds to the scaffold in its reduced state (Fig. 2; Webert et al., unpublished data). In humans, the Yah1 orthologue ferredoxin 2 (FDX2) and ferredoxin reductase FdxR have been shown to support Fe/S protein biogenesis [30,31]. Assembly of the Fe/S cluster on Isu1 requires ferrous iron which is imported into mitochondria in a membrane potential-dependent manner via the Mrs3/4 mitochondrial solute carriers [32e34] (Fig. 1). In vertebrates the Mrs3/4 homologs mitoferrin 1 and 2 are expressed in a tissue-specific way. Whereas mitoferrin 2 is expressed ubiquitously, mitoferrin 1 is essential for mitochondrial iron import in developing erythroid cells [35,36]. In yeast, the mitochondrial pyrimidine nucleotide transporter Rim2 supports the co-import of pyrimidine and iron into the matrix, yet under normal physiological conditions this does not significantly contribute to iron supply, as pyrimidine nucleotide transport usually occurs with magnesium or other metals [37]. The mechanism by which the imported iron gets access to the scaffold complex is still under debate. A protein presumably playing a role in iron delivery is Yfh1 (frataxin in humans; Table 1). One suggestion is that the protein directly acts as an iron-donor for the scaffold


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Fig. 1. An overview of the three steps of mitochondrial Fe/S protein biogenesis. In the first step, a [2Fee2S] cluster is assembled on the Isu1 scaffold protein. This process requires ferrous iron which is imported into mitochondria via the mitochondrial carriers Mrs3/4 and possibly other carriers, a mechanism driven by the proton motive force (pmf). The functions of the cysteine desulfurase complex Nfs1eIsd11, the [2Fee2S] ferredoxin Yah1, the ferredoxin reductase Arh1, and frataxin are explained in detail in Fig. 2. In the second step, the Fe/S cluster is released from Isu1 involving a dedicated chaperone system and the glutaredoxin Grx5 which together with glutathione (G) binds a [2Fee2S] cluster (see Fig. 3). The ISC components of steps 1 and 2 constitute the core ISC assembly machinery and are sufficient for the assembly of mitochondrial [2Fee2S] proteins. Moreover, these components are involved in the biogenesis of extra-mitochondrial Fe/S proteins via the ISC export machinery. Its central component is the ABC transporter Atm1 which exports an unknown sulfur-containing moiety (XeS) towards the cytosolic Fe/S protein assembly (CIA) machinery for maturation of cytosolic and nuclear Fe/S proteins (dotted arrow). In the third step, dedicated ISC targeting factors including Isa1eIsa2 and Iba57 allow for the formation of [4Fee4S] clusters and their target-specific insertion into mitochondrial apoproteins (see Fig. 4).

protein Isu1, a possibility which is consistent with the finding that Yfh1 tightly interacts with the Nfs1eIsd11eIsu1 complex [38,39]. Recently, Yfh1 was shown to significantly stimulate the desulfurase activity of Nfs1 which raises the alternative possibility that Yfh1 acts as an allosteric activator of the cysteine desulfurase [40,41]. Both models are not mutually exclusive. 3.2. Step 2: a dedicated chaperone system and the glutaredoxin Grx5 mediate the release of the [2Fee2S] cluster from the scaffold After formation of the transient [2Fee2S] cluster on Isu1 it has to be released from the scaffold and further be transferred to target

proteins. In yeast, dissociation of the Fe/S cluster from Isu1 requires a dedicated chaperone system comprising the Hsp70-type protein Ssq1, the co-chaperone Jac1, and the nucleotide exchange factor Mge1 [42]. The general mechanism of these chaperones in Fe/S cluster release is reminiscent of the action of Hsp70 chaperones in protein folding [43,44]. Yeast has developed a dedicated Hsp70/cochaperone system for the cluster transfer process apart from the mitochondrial import-folding Hsp70 system. In contrast, mammals and many other eukaryotes rely on the action of a single, multifunctional mitochondrial Hsp70 protein which performs import, folding, and Fe/S cluster insertion of proteins [45]. The Hsp70 reaction cycle in the process of Fe/S cluster release from Isu1 is

Fig. 2. De novo [2Fee2S] cluster formation on the scaffold protein Isu1. The desulfurase complex Nfs1eIsd11 converts cysteine to alanine and releases sulfur which is transiently bound in form of a persulfide group on a conserved cysteine residue on Nfs1. After putative transfer to a cysteine residue on the scaffold protein Isu1 the sulfane sulfur is reduced to sulfide to allow for Fe/S cluster formation. Sulfur reduction is presumably mediated by the electron transport chain comprised of NAD(P)H, Arh1, and Yah1. Yah1 binds to Isu1 only in its reduced (red) but not in its oxidized (ox) state. The delivery of iron to Isu1 might involve frataxin (Yfh1) which is needed for maximal desulfurase activity of Nfs1.

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initiated by binding of Jac1 to the scaffold (Fig. 3, step 1), a step facilitating the scaffold to interact at its conserved LPPVK loop with ATP-laden Ssq1 (Fig. 3, step 2) [46,47]. The attachment of both Jac1 and Isu1 triggers the ATPase activity of Ssq1 causing a conformational change which further strengthens the binding to Isu1 [48,49] (Fig. 3, step 3). ATP hydrolysis is accompanied by the dissociation of Jac1 from the complex and by a conformational change in Isu1 resulting in a less stable binding of the Fe/S cluster [50,51]. Fe/S cluster release from Isu1 also involves the monothiol glutaredoxin Grx5 [21]. Dimerization of Grx5 allows the coordination of a bridging [2Fee2S] cluster via the active-site cysteine residue and non-covalently bound GSH [52e54] (Fig. 1). Recently, Grx5 was found to bind to Ssq1 at a site which is in close proximity to but not overlapping with the peptide binding region of Ssq1 where Isu1 is attached to the chaperone (Fig. 3, step 4) [55]. Even though Grx5 is able to interact with both the ATP- and the ADP-form of Ssq1, binding to the ADP conformation of Ssq1 is preferred. The vicinity of holo-Isu1 and apo-Grx5 may facilitate Fe/S cluster transfer from the scaffold to the glutaredoxin (Fig. 3, step 5). At the end of the chaperone cycle the nucleotide exchange factor Mge1 binds to Ssq1 and drives the exchange of ADP for ATP (Fig. 3, step 6) [48]. This results in the release of all components from Ssq1 including [2Fee 2S]-laden Grx5 which supports maturation of all cellular Fe/S proteins, either directly or via the participation of additional ISC (see 3.3.; Fig. 3, step 7) and CIA factors [55]. Apo-Isu1 and the chaperones are then ready for utilization in another cycle. In contrast to the chaperones and other core ISC assembly factors the deletion of Grx5 in yeast is not lethal [56,57]. This observation indicates that the Grx5-mediated cluster transfer is possibly not essential and can be bypassed by a direct dislocation of the Isu1-bound [2Fee2S] cluster to ISC targeting factors and/or target apoproteins [55] (Fig. 4). According to the current view Grx5 plays a role as a transient Fe/S cluster carrier which passes the cluster on to other ISC targeting factors and/or target apoproteins (Fig. 1). However, based on available data it cannot be excluded that Grx5

plays another, possibly enzymatic role in facilitating Fe/S cluster release from Isu1 and/or transfer to downstream proteins. 3.3. Step 3: specific ISC targeting factors assist the assembly of [4Fee4S] proteins After its release from Isu1 the Fe/S cluster may be directly used for the assembly of [2Fee2S] proteins (Fig. 1). In contrast, maturation of [4Fee4S] proteins requires numerous additional ISC factors (Fig. 4). Conversion of the [2Fee2S] to a [4Fee4S] cluster is mediated by the A-type ISC proteins Isa1 and Isa2, and by the folatebinding protein Iba57 [58e60]. The three proteins physically interact with each other suggesting that they function in the same biochemical reaction by forming a complex [58]. As they are not required for Fe/S cluster assembly on Isu1, they are not part of the core ISC assembly machinery [59]. Notably, both Isa1 and Isa2 have been shown to interact with Grx5 supporting a role of this glutaredoxin in linking the early and late phases of mitochondrial Fe/S protein assembly [57,61]. The precise function of the Isa proteins and of Iba57 in the late phase of Fe/S protein assembly is unknown to date. Several studies with human, yeast and bacterial proteins have demonstrated that Isa-like proteins bind iron as a cofactor which might be used for the conversion of the primordial [2Fee2S] into the definite [4Fee4S] cluster [59,62]. Since in yeast iron binding to the Isa proteins is not dependent on Nfs1 or Isu1, the Isa-bound iron is not part of a Fe/S cluster. This is in contrast to studies with bacterial and human proteins that have been shown to bind a [2Fee2S] cluster [63e66]. Together, these results may imply that Isa-type proteins may bind either iron or [2Fee2S] clusters. How the Isa proteins use iron or the rhombic [2Fee2S] cluster to assist the assembly of a cubic [4Fee4S] cluster remains to be determined. Apart from the general ISC targeting proteins Isa1, Isa2, and Iba57 also other, more specialized targeting factors have evolved to assist the maturation of dedicated subsets of [4Fee4S] proteins.

Fig. 3. Chaperone- and Grx5-assisted Fe/S cluster dissociation from Isu1. Holo-Isu1 with its transient [2Fee2S] cluster is recruited by the co-chaperone Jac1 and guided to the ATPbound form of the Hsp70 chaperone Ssq1 (step 1). Ssq1 binds to the conserved LPPVK loop of Isu1 (step 2) thus triggering the ATPase activity of Ssq1 which in turn leads to tight binding of Isu1 and to the release of Jac1 (step 3). ATP hydrolysis is believed to induce a conformational change on Isu1 to weaken Fe/S cluster binding to Isu1. The monothiol glutaredoxin Grx5 binds to Ssq1 in close vicinity to Isu1 (step 4) thereby enabling Fe/S cluster transfer from the scaffold to Grx5 (step 5). Next, ADP is exchanged for ATP by the exchange factor Mge1 triggering a conformational change of the peptide binding domain of Ssq1 from the closed to an open state. Subsequently, the Ssq1eIsu1 complex disassembles, allowing the components to resume with a new cycle (steps 6 and 7). The Fe/S cluster on holo-Grx5 is required for maturation of all cellular Fe/S proteins, comprising the mitochondrial [4Fee4S] and [2Fee2S] proteins and the cytosolic-nuclear Fe/S proteins.


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Fig. 4. Dedicated maturation of mitochondrial [4Fee4S] proteins by specific ISC targeting factors. Maturation of mitochondrial [4Fee4S] proteins requires the Grx5-bound (or Isu1bound) [2Fee2S] clusters and the Isa1eIsa2eIba57 complex. Iba57 may bind folate, and the two Isa proteins bind iron. The precise mechanism of [4Fee4S] cluster generation is unclear. Dedicated ISC targeting factors are involved in the insertion of the Fe/S cluster into specific [4Fee4S] proteins. Nfu1, which like Isu1, may transiently coordinate a [4Fee4S] cluster, and Aim1 participate in the maturation of respiratory complex I, SDH (complex II), and lipoic acid synthase, and are thus exhibiting similar substrate specificity. The highly specific P-loop NTPase Ind1 also binds a transferrable [4Fee4S] cluster and is specifically required for Fe/S cluster incorporation into complex I (c.f. Fig. 5). G, Glutathione.

Nfu1 is an ISC assembly component which had initially been thought to serve as an alternative scaffold acting in parallel to Isu1, as the human protein was found to transiently bind a [4Fee4S] cluster [67]. Only recently the protein could be characterized as a late ISC targeting factor due to the identification and detailed examination of patients with impaired expression or function of human NFU1 [68,69]. Based on the biochemical phenotype of these patients it turned out that NFU1 is specifically required for the proper assembly of only a subset of [4Fee4S] proteins including subunits of respiratory complexes I and II, as well as the radical SAM protein lipoic acid synthase (LIAS). The latter enzyme converts octanoic acid into lipoic acid and is needed for the maturation of the four mitochondrial lipoic acid-dependent enzymes pyruvate dehydrogenase (PDH), a-ketoglutarate dehydrogenase (a-KGDH), branched-chain ketoacid dehydrogenase (BCKDH) and the H protein of the glycine cleavage system (Fig. 4). In contrast, [2Fee2S] proteins or mitochondrial aconitase are not dependent on yeast or human Nfu1. Additional studies in yeast showed that the [4Fee4S] cluster on Nfu1 requires the function of the earlier-acting ISC components including Isu1, supporting the function of Nfu1 in late stages of the biogenesis [69]. Another dedicated ISC targeting factor probably acting along with NFU1 is BOLA3, the human homolog of yeast Aim1 [68]. BOLA3 was identified as an ISC assembly factor since patients harboring a frame-shift mutation in the BOLA3 gene presented with a biochemical phenotype similar to NFU1-deficient individuals. BOLA3 deficiency results in the impaired maturation of respiratory complexes I and II as well as of LIAS (Fig. 4). The BOLA family of proteins contains two other members: BOLA1, like BOLA3, is located within mitochondria, while BOLA2 and its yeast counterpart Fra2 are located in the cytosol where the latter plays a role in iron uptake regulation [70,71]. The molecular function of the BOLA proteins, particularly that of BOLA3, and their potential cooperation with the other late-acting ISC factors remains to be resolved. An even more specific ISC targeting factor is the mitochondrial P-loop NTPase Ind1. It has been identified in the yeast Yarrowia lipolytica and in human cells, and is closely related in sequence to two cytosolic P-loop NTPases, Cfd1 and Nbp35, which act as a heteromeric scaffold complex in cytosolic Fe/S cluster assembly [72,73]. Like these two CIA factors also Ind1 is able to transiently bind a [4Fee4S] cluster. Studies in various organisms including Y. lipolytica, humans, and plants could clearly link Ind1 to the

maturation of [4Fee4S] cluster-containing subunits of respiratory chain complex I whereas other tested mitochondrial [4Fee4S] proteins are matured independently of Ind1 [72,73]. The yeast S. cerevisiae does not harbor a typical respiratory chain complex I, and this is probably the reason why it also lacks an Ind1 homolog. However, a function of Ind1 in the maturation of mitochondrial Fe/ S proteins other than complex I subunits cannot completely be excluded to date. In order to directly test of whether Ind1 was involved in the maturation of the [4Fee4S] protein lipoic acid synthase, an RNAi-depletion system for human IND1 in HeLa cells was used [73]. Immunoblotting revealed that low levels of Ind1 had no detectable effect on the lipoic acid content of the E2 subunits of PDH and a-KGDH, suggesting that Ind1 is not required for the maturation of LIAS (Fig. 5A). In contrast, a severe decrease was observed in the steady-state protein levels of two subunits of the Fe/S cluster-containing soluble arm of complex I (NDUFS3, NDUFA13) while subunit NDUFA9 and subunits of other respiratory complexes were not affected (Fig. 5B). These results extend previous studies showing the specificity of human IND1 [73]. The RNAi approach was validated by ectopic expression of a silently mutated, RNAi-resistant IND1 version which fully complemented the loss of endogenous IND1 (Fig. 5). The fact that the function of LIAS is independent of IND1 supports the view that IND1 is a dedicated ISC targeting factor specifically required for the maturation of respiratory chain complex I (Fig. 4). The situation may be more complex in plant mitochondria where IND1 has been suggested to be required for an unknown step of protein translation [74]. 4. Human diseases associated with defects in Fe/S protein biogenesis The ISC assembly and export systems are highly conserved from yeast to humans, and their proper function is of vital importance for all eukaryotic organisms inspected so far. In vertebrates, particularly in mammals, the loss of individual ISC components is lethal during early embryonic development [75e78]. Thus, it is not surprising that mutations in ISC genes may cause severe disorders in humans. To date, alterations in ten of these genes are known to be relevant for ironesulfur protein assembly diseases (Fig. 6). Many of them are fatal, sometimes already in early childhood. Mutations have been found in the genes FXN (frataxin, homologue of yeast

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Fig. 5. The human P-loop NTPase IND1 is specific for maturation of complex I. A, HeLa cells were transiently transfected with a plasmid encoding a short hairpin RNA directed against human IND1 mRNA (shIND1, lane 2). In order to complement IND1 deficiency, a plasmid was co-transfected encoding a silently mutated (sm) IND1-EGFP fusion protein whose mRNA is resistant to RNAi treatment (lane 3). As a control, respective backbone plasmids (pEGFP-N1, pSuperior) were transfected as indicated (lane 1). Transfections were performed three times at a three day interval. After nine days of depletion, cells were harvested, lysed, and subjected to SDS-PAGE. Immunoblotting was performed with anti-IND1 serum and anti-tubulin (loading control) monoclonal antibodies. Note the efficient depletion of endogenous IND1 in lanes 2 and 3. B, Cell extracts from A were blotted and the indicated respiratory complex subunits were immunostained. The lipoic acid-content of the E2 components of the pyruvate dehydrogenase complex (PDH-E2) and the a-ketoglutarate dehydrogenase complex (a-KGDH-E2) was determined using a lipoate-specific antiserum [69].

Yfh1), ISCU (Isu1), FDX2 (one of the two homologues of yeast Yah1), LYRM4 (Isd11), GLRX5 (Grx5), IBA57, NFU1, BOLA3 (Aim1), IND1, and ABCB7 (Atm1) (Table 1). In addition, aberrant mRNA splicing has been identified for mitoferrin 1 (encoded by MFRN1, a homologue of yeast Mrs3/4), but its impact on the Fe/S protein assembly process requires further investigation. Discovery and characterization of the ISC assembly and export systems in model systems paved the way for the identification of relevant mutations and associated metabolic changes in Fe/S protein assembly diseases affecting humans. For example, the critical functions of ISCU, GLRX5, and IND1 in Fe/S cluster and protein assembly had been already studied before respective patients were identified [21,57,72]. Many consequences resulting from depletion of ISC assembly or export components in experimental systems like yeast, zebrafish, and human cell culture are recapitulated in patients, thus providing strong support for the proposed mechanisms of the Fe/S protein assembly process. Vice versa, discovery of disease-causing mutations in genes like NFU1 and BOLA3 provided important clues to the function of the respective gene products [68,69]. Depending on the identity of the ISC component and the site of mutation within the respective gene, the clinical presentation and biochemical characteristics differ substantially between affected individuals. In case of the ISCU gene an intronic point mutation favors tissue-specific mRNA mis-splicing, causes low levels of ISCU protein, and results in a myopathy with exercise intolerance [79,80]. Intronic expansions within the frataxin gene interfere with transcription in multiple tissues causing severe neurological dysfunctions [81]. At variance, point mutations affecting the ABCB7 gene elicit a pronounced hematopoietic phenotype [82,83], while mutations within the IND1 gene primarily affect cellular respiration [72,84]. Despite this phenotypical heterogeneity, a valid criterion for the classification of ‘ironesulfur diseases’ might be the presence or absence of mitochondrial iron accumulation. Mitochondria are important sites of both heme synthesis and Fe/S cluster assembly, thus processing a major amount of cellular iron [85]. Improper activity of one of the two iron-consuming pathways is sufficient to disturb the entire cellular iron metabolism and to produce an ironstarvation phenotype. Consistent with experimental findings in model organisms, only mutations in human genes of the core ISC assembly or export systems give rise to mitochondrial iron accumulation [20,57,86,87], while mutations in genes of the ISC targeting factors do not [59,60,72]. The mechanistic basis why

mitochondria accumulate iron remains elusive so far, but in humans (and other animals) the process involves the two cytosolic iron regulatory proteins IRP1 and IRP2 which are responsible for the posttranslational regulation of cellular iron metabolism [for review see, e.g., [88]]. IRP1 is a Fe/S protein with aconitase activity, yet in its apoform it can bind to iron-responsive elements (IREs) of mRNAs encoding proteins involved in iron trafficking (transferrin receptor (TfR) and ferroportin), storage (ferritin), or utilization (aconitase and ALAS2), thereby regulating the translation efficiency or mRNA stability. The equilibrium between apo- and holo-IRP1 is shifted by Fe/S cluster assembly and thus depends on both the mitochondrial ISC systems and the CIA machinery [85]. Vice versa, the efficiency of mitochondria to generate Fe/S clusters is influenced by cellular iron availability. Any shortage of iron will lead to its increased uptake and supply to the mitochondrial matrix. In contrast to IRP1, IRP2 does not carry a Fe/S cluster and binds constitutively to IREs. Regulation of IRP2 is dependent on iron-dependent ubiquitinylation and proteasomal degradation involving the iron-sensing E3 ubiquitin ligase subunit FBXL5. Since all core ISC assembly and export components determining mitochondrial iron accumulation are also required for proper function of the CIA system [89,19], a discrimination of mitochondrial Fe/S protein assembly diseases according to the mitochondrial iron status immediately allows the evaluation of whether the maturation of cytosolic-nuclear Fe/S proteins is affected or not. Furthermore, other disease characteristics like the occurrence of ring sideroblasts (see Section 4.1.5.), the metabolic pathways affected, and the defective Fe/S protein may provide important insights into the course of events in mitochondrial Fe/S protein assembly and may offer valuable information for the clinician. In the following we will summarize available information about currently known mitochondrial Fe/S protein assembly diseases (Table 2) and discuss how their characteristic phenotypes might relate to disturbances in the ISC assembly and export process. 4.1. Fe/S diseases associated with mitochondrial iron accumulation 4.1.1. Friedreich’s ataxia (FRDA) The prototypical mitochondrial Fe/S disease is Friedreich’s ataxia (FRDA), being the most common hereditary ataxia within the Caucasian population with a prevalence of 1:20,000 to 1:125,000. The disease is characterized as a neurodegenerative condition with


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Fig. 6. Diseases linked to mitochondrial Fe/S protein biogenesis. The model depicts the three steps of mitochondrial Fe/S protein assembly described in Figs. 1e4 using the human ISC protein names. Yellow boxes highlight ISC proteins whose genes are mutated in human Fe/S diseases. They can be classified into two groups: ISC proteins that are associated with mitochondrial iron accumulation are indicated by red crosses, whereas green asterisks highlight ISC proteins associated with diseases that do not affect the iron metabolism. In addition, the model depicts a mitochondrial iron import disorder associated with a mutation in the inner membrane carrier mitoferrin 1 (MFRN1) in erythroid cells (gray box).

spinocerebellar and sensory atrophy resulting in progressive ataxia, which is sometimes accompanied by deafness and blindness. Nonneurological manifestations frequently include fatal hypertrophic cardiomyopathy, muscle weakness, and diabetes mellitus (reviewed in Ref. [90]). The condition is reminiscent of respiratory chain disorders and was already known when a positional cloning approach identified disease-causing alterations in a ubiquitously expressed nuclear gene termed frataxin [81]. In most FRDA cases GAA-triplet repeat expansions of variable length within the first intron of the FXN gene interfere with transcription and result in a severe decrease of frataxin protein levels by 70%e95% [91]. About 4% of the FRDA patients are compound heterozygotes for a GAA expansion on one FXN allele and an inactivating missense mutation on the other one. The carrier frequency of FXN mutations is estimated 1:60 to 1:110 [90]. Even heterozygote individuals harboring GAA expansions on only one of the FXN alleles exhibit lowered frataxin levels, yet they are clinically not affected [92]. Patients with

inactivating missense mutations in both frataxin alleles are not known, in line with mouse studies showing that a complete loss of frataxin function is embryonically lethal [75]. A pronounced genotypeephenotype correlation in FRDA is probably contributing to the broad spectrum of disease traits [90]. The length of the shortest GAA expansion determines the magnitude of frataxin expression and hence onset and severity of the condition, in line with a crucial function of the protein in the early phase of mitochondrial Fe/S protein assembly. The proposed function of frataxin as an allosteric switch leads to a decrease in the activity of the cysteine desulfurase complex [40], and in turn to impaired maturation of many mitochondrial and cytosolic-nuclear Fe/S proteins, including respiratory complexes I to III and aconitases of both compartments [93e96] (Fig. 6). Infrequently, low activities of PDH and a-KGDH complexes were detected in FRDA patients [97e100], indicating impaired maturation of the lipoic acid-forming Fe/S enzyme LIAS (Fig. 6). In some patient tissues, citrate synthase activity and

Table 2 Diseases linked to defects in mitochondrial Fe/S protein assembly. Disease 1) Fe/S diseases associated with mitochondrial iron accumulation 1.1.) Friedreich’s ataxia (FRDA) 1.2.) Hereditary Myopathy with Lactic acidosis (HML) 1.3.) Mitochondrial muscle myopathy with deficiency of ferredoxin 2 1.4.) Combined oxidative phosphorylation defect with ISD11 deficiency 1.5.) Sideroblastic anemias 1.5.1.) Inherited sideroblastic anemias 1.5.1.1.) Sideroblastic anemia with deficiency of glutaredoxin 5 1.5.1.2.) X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A) 1.5.2.) Acquired sideroblastic anemias 1.5.2.1.) Refractory anemia with ring sideroblasts (RARS) 1.5.2.2.) Refractory anemia with ring sideroblasts and isodicentric (X)(q13) chromosome 2) Fe/S diseases without mitochondrial iron accumulation 2.1.) Multiple mitochondrial dysfunction syndromes 2.1.1.) Juvenile encephalomyopathy with deficiency of IBA57 (MMDS3) 2.1.2.) Multiple mitochondrial dysfunction syndrome with functional NFU1 deficiency (MMDS1) 2.1.3.) Multiple mitochondrial dysfunction syndrome with functional BOLA3 deficiency (MMDS2) 2.2.) Mitochondrial encephalomyopathy with deficiency of IND1 3) Variant erythropoietic protoporphyria with abnormal expression of mitoferrin 1

Affected gene (protein)

Affected process

FXN (Frataxin) ISCU FDX1L (ferredoxin 2) LYRM4 (ISD11)

Core Core Core Core

GLRX5 (Glutaredoxin 5) ABCB7

Cluster transfer ISC export

ABCB7 ? ABCB7 ?

ISC export ISC export

IBA57 NFU1 BOLA3 NUBPL (IND1) SLC25A37 (Mitoferrin 1, MFRN1)

[4Fee4S] assembly [4Fee4S] assembly [4Fee4S] assembly Complex I assembly Mitochondrial iron import

ISC ISC ISC ISC

assembly assembly assembly assembly

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mitochondrial DNA content are decreased [95], suggesting that mitochondria are lost due to damage as observed in a mouse model for FRDA [101]. Expression analyses indicated that frataxin levels are particularly high in tissues and organs with high energy demands like brain, heart, and skeletal muscle, suggesting that limited mitochondrial energy supply due to improper assembly of the respiratory complexes may be a major determinant in the development of FRDA [81,102]. In line with the requirement of the core ISC assembly system for the maturation of extra-mitochondrial Fe/S proteins, gene expression studies and RNAi-based approaches suggest that cytosolicnuclear Fe/S proteins are also affected in FRDA [103,104]. Defects in both the ISC assembly and CIA systems have been shown to impair Fe/S enzymes assisting nuclear DNA replication and repair, hence interfering with DNA metabolism [105e107]. A clear association of FRDA with malignancy is not evident [108], but analysis of peripheral blood samples revealed an increased number of mitochondrial and nuclear DNA lesions [109]. Alterations in DNA damage response pathways, in nucleotide excision repair, in RNA metabolism, and in protein biosynthesis might be linked to impaired maturation of extra-mitochondrial Fe/S proteins XPD, FANCJ, PRI2, and RLI1, respectively. The best studied cytosolic Fe/S protein in FRDA is IRP1 due to its impact on cellular iron homeostasis [94,96]. Cytosolic aconitase activity of IRP1 is decreased in FRDA, while its IRE-binding activity is elevated, driving TfR expression and attenuating ferritin synthesis [103,104]. As a consequence, IRP2 is stabilized and exerts additional effects on cellular iron metabolism. Eventually, deterioration of iron homeostasis results in mitochondrial iron accumulation [95,110,111]. Mitochondrial iron together with the compromised respiratory chain complexes possibly drives the formation of reactive oxygen species (ROS) which in turn are supposed to attack Fe/S centers, thus fueling a vicious cycle of deteriorated Fe/S protein activity and aggravating the disease [112,113]. Hence, impaired assembly of mitochondrial and extra-mitochondrial Fe/S proteins in multiple tissues with effects on manifold metabolic pathways makes FRDA a true systemic disease. 4.1.2. Hereditary Myopathy with Lactic acidosis (HML) Hereditary Myopathy with Lactic acidosis (HML) is a rare and recessively inherited disease characterized by early onset muscle weakness, exercise intolerance, and lactic acidosis, in line with a defect in oxidative phosphorylation [114e116] (Table 2). Myoglobinuria due to rhabdomyolysis is a frequent secondary consequence of physical strain and may cause fatal renal failure. The disease was known and a defect in the biosynthesis of Fe/S clusters already suspected [117] when two independent approaches identified a single base transition in intron 5 of the ISCU gene being the major cause of the disease [79,80] (Fig. 6). The mutation is prevailing mainly in individuals of northern Swedish descent and can be tracked back to a founder haplotype [116]. It favors aberrant splicing of the primary mRNA transcript preferentially in skeletal muscle cells, resulting in the retention of an additional exon and the introduction of a premature stop codon. As a consequence, levels of both the mature mRNA [118] and the truncated ISCU protein are profoundly decreased, impairing the activity of the ISC assembly system particularly in striated muscle. Only few HML patients have been identified as compound heterozygotes for the intronic transition and a missense mutation within the coding region of the ISCU gene [118]. The resulting amino acid substitution does not destabilize the protein but rather abrogates its activity in all tissues. Affected individuals present with a more severe clinical phenotype including hypertrophic cardiomyopathy, in line with the indirect function of ISCU in cellular energy metabolism. Accordingly, mutations lead to impaired maturation of mitochondrial and extra-

mitochondrial Fe/S enzymes, and mitochondrial iron accumulation. Due to the biochemical phenotype of the affected individuals and their provenance HML is also known as ‘Swedish Type Myopathy with Exercise Intolerance’, ‘Myopathy with Deficiency of Succinate Dehydrogenase and Aconitase’, and ‘IroneSulfur Cluster Deficiency Myopathy’. Though the disease phenotype is restricted to skeletal muscle and not evident in other tissues like blood vessels or visceral smooth muscle [114,117e119] mis-splicing and lowered ISCU levels are observed also in other cell types [77,120]. Heterozygous carriers of the intronic mutation do not develop any conspicuous condition [118,121]. In fact, aberrantly spliced ISCU mRNA has also been found in healthy individuals, but only to a minor degree and without any deteriorating effect [77,79,120]. The tissue-specific mis-splicing of the ISCU mRNA probably involves the nuclear factors IGF2BP1, RBM39, and PTBP1 [122], and appears to be dependent on the differentiation level of myoblasts as the muscle-specific transcription factor MyoD interferes with normal processing of ISCU mRNA [121]. Accordingly, in regenerating and thus less differentiated muscles of patients recovering from episodes of rhabdomyolysis, ISCU protein levels were comparably high and hallmarks of HML were barely detectable [123]. ISCU is a critical constituent of the mitochondrial core ISC assembly complex [39,124] (Fig. 6). Its deficiency in HML not only restricts the availability of assembly platforms for the formation of Fe/S clusters but also impairs the steady-state protein level of the desulfurase complex NFS1-ISD11 [121]. In contrast, levels of frataxin are increased suggesting a counterbalancing cellular response in order to force Fe/S cluster assembly. Because ISCU is not sufficiently available in the disease state, respiratory chain complexes I to III are not properly assembled and their protein levels and activities are considerably decreased [117,119,120]. In addition, respiratory complex IV (cytochrome c oxidase; COX) is affected [118], probably due to impaired maturation of ferrochelatase [125]. The limited respiratory chain activity is reflected by poor oxygen consumption in muscle tissue and associated with low ATP generation [115,119], probably a major determinant of the myopathy. Moreover, aconitase is compromised [117,119], and already before the identification of the ISCU mutations defects in the LIAS-dependent enzymes PDH and a-KGDH have been implicated in the disease [126] (see also Section 4.2.1., Multiple Mitochondrial Dysfunction Syndromes). As a consequence, mitochondrial energy metabolism is already restricted at the level of the citric acid cycle [119]. The assembly of extra-mitochondrial Fe/S enzymes in HML is only poorly examined, but an impaired maturation of IRP1 has been observed [79,121,127], suggesting that cytosolic-nuclear Fe/S protein assembly is also affected in the disease. Moreover, there is some indication that protein levels of IRP2 are altered [121,127] which like in FRDA (see Section 4.1.1.) together with IRP1 might contribute to another hallmark of HML, namely the accumulation of iron within mitochondria [77,118,119]. Microarray analysis of HML patient muscle biopsies revealed an elevated expression of the mitochondrial iron importer mitoferrin-2 (MFRN2) [127], pointing to a mechanistic link between impaired mitochondrial Fe/S cluster assembly and increased mitochondrial iron content. 4.1.3. Mitochondrial muscle myopathy with deficiency of ferredoxin 2 A second type of isolated muscle myopathy caused by impaired mitochondrial Fe/S cluster assembly has been linked to deficiency of FDX2 (Fig. 6). Only one affected individual has been identified so far, suffering from progressive muscle weakness, exercise-induced rhabdomyolysis, and myoglobinuria [128] similar to HML patients (see Section 4.1.2.). Exome sequencing combined with homozygosity mapping suggested the autosomal recessive inheritance of a


deleterious mutation in the start codon of the FDX1L gene although a Kozak-sequence-embedded alternative AUG is located downstream in close proximity. Despite the tissue-specific phenotype FDX2 was undetectable in both muscle and fibroblasts. Since the disease condition manifested only during early adulthood, one might suspect the accumulation of secondary defects particularly in skeletal muscle [129]. In line with the critical role of FDX2 as a core ISC assembly component the function of mitochondria obtained from patient skeletal muscle was severely impaired [128]. Particularly, activities of mitochondrial aconitase and of respiratory chain complexes I to III were substantially decreased. FDX2 deficiency not only affected Fe/S protein subunits but destabilized entire complexes as demonstrated by low levels of complex II subunit A (SDHA). COX activity was diminished in comparison to activities of both respiratory chain complex V (FoF1 ATP synthase) and citrate synthase, suggesting deprived heme formation as a sign of improper ferrochelatase maturation and activity similar to HML (see Section 4.1.2.). Elevated concentrations of lactate, ketobodies, citric acid cycle metabolites, and 3-methyl glutaconic acid indicated low activities of the lipoic acid-dependent enzymes PDH, a-KGDH, and BCKDH, pointing to an insufficient assembly of the [4Fe/4S] enzyme LIAS (see Section 4.2.1.). Whether extra-mitochondrial Fe/S enzymes are affected in patient skeletal muscle or other tissues has not been determined yet, but cell culture studies demonstrated a crucial function of FDX2 also for the maturation of cytosolic Fe/S proteins, particularly of IRP1 [30]. Accordingly, experimental depletion of FDX2 resulted in profound changes in iron homeostasis. In patient tissues mitochondrial iron deposits were not detectable [128], but ISD11 deficiency (see Section 4.1.4.) demonstrates that disturbances in cellular and systemic iron metabolism are sometimes only mild, and hence can also not be excluded in FDX2 myopathy. 4.1.4. Combined oxidative phosphorylation defect with ISD11 deficiency Exome sequencing in tissue material of two individuals with combined respiratory chain deficiency identified a homozygous missense mutation in LYRM4, the gene encoding ISD11, resulting in the substitution of a highly conserved arginine at position 68 of the polypeptide chain by leucine [129]. As a consequence, ISD11 protein was detectable neither in skeletal muscle, nor in liver and fibroblasts. Expression studies in yeast and Escherichia coli demonstrated that the mutant ISD11 protein does not support the desulfurase activity of NFS1. According to the function of ISD11 as a core ISC assembly component (Fig. 6), steady-state-protein levels of mitochondrial Fe/S enzymes aconitase and ferrochelatase were decreased in patients. Particularly the lack of ferrochelatase was consistent with the low activity of the heme-containing respiratory chain complex IV in some tissue samples. As a consequence of impaired mitochondrial respiration patients developed neonatal lactic acidosis in cerebrospinal fluid and blood plasma, and suffered from respiratory distress and hypotony. One of the infants presented with even more severe neurologic and metabolic symptoms including epileptiform changes, hepatomegaly, and steatosis. Notably, proper lipid metabolism is dependent on the function of the [4Fee4S] protein ETFDH (electron transfer flavoprotein:ubiquinone oxidoreductase) participating in fatty acid oxidation [131], providing a possible mechanistic link to the observed phenotype. In addition, the infant showed elevated blood pyruvate levels and developed ketosis, suggesting impaired branched-chain keto acid dehydrogenase (BCKDH) and PDH activity due to hampered LIAS assembly (see Section 4.2.1., Multiple Mitochondrial Dysfunction Syndromes). Mitochondria of this individual contained only few cristae membranes, indicative of a poor

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supramolecular organization of respiratory chain complexes [132]. Besides defective mitochondrial Fe/S protein assembly, the cytosolic-nuclear compartment was also affected in both patients as indicated by low steady-state protein levels of cytosolic aconitase, providing a mechanistic link to the mild iron overload observed in the more severely affected infant. Surprisingly, one of the patients recovered from the neonatal crisis and developed well [130]. Though ISD11 was still not detectable in adulthood, steady-state levels of several mitochondrial Fe/S proteins improved considerably. Particularly, fibroblasts did not show any conspicuous biochemical phenotype. Expression analysis in mouse and men revealed that ISD11 mRNA levels in brain rise during ontogenesis and reach their maximum only in adulthood [133], suggesting that mutations in the ISD11 gene might be deleterious predominantly in neonates. However, in adult humans, attenuated expression of ISD11 has been linked to the development of mental disorders such as schizophrenia [133]. This condition is frequently associated with both low respiratory chain activities and mild neurologic signs including impaired motor coordination and cognitive defects. Intriguingly, recent studies suspect a general causative role of mitochondrial dysfunctions in the development of mental disorders [134,135], in line with the critical task of mitochondria in neuronal function. 4.1.5. Sideroblastic anemias Sideroblastic anemias (SAs) are a heterogeneous group of inherited or acquired disorders with the common trait of disturbed mitochondrial iron metabolism. Erythrocytes of affected individuals are hypochromic due to ineffective heme formation, and mitochondria of bone marrow erythrocyte precursors are characterized by crystalline iron deposits which are usually encased by mitochondrial ferritin [reviewed in Refs. [136,137]]. The perinuclear localization of iron-laden mitochondria gives rise to a ring-shaped appearance, and erythroblasts are thus called ring sideroblasts. So far, two rather rare forms of congenital SAs have been identified as mitochondrial Fe/S protein assembly diseases. One is caused by a genetic alteration impairing the expression of the ISC assembly core component GLRX5 [78], although some mutations in the GLRX5 gene have been identified which do not elicit an SA phenotype [138]. The second form of congenital SA associated with impaired Fe/S protein assembly is caused by a mutation in the gene for the export component ABCB7 [83] (Fig. 6). ABCB7 additionally has been implicated in the development of two types of acquired sideroblastic anemias associated with myelodysplastic syndromes (MDS), called ‘refractory anemia with ring sideroblasts’ [139] and ‘refractory anemia with ring sideroblasts and isodicentric (X)(q13) chromosome’ [140]. The exact mechanism by which disturbed heme metabolism produces sideroblastic anemias has not been resolved yet, but mitochondrial iron mishandling is likely involved. It has been suggested that the lowering of protoporphyrin creates a situation in which mitochondrial iron cannot be sequestered, thus giving rise to mitochondrial iron overload [136]. Distorted mitochondrial Fe/S cluster assembly and export may cause such an imbalance in multiple ways [137]. First, ferrochelatase, the [2Fee2S] enzyme catalyzing the final step in heme formation, i.e. insertion of iron into protoporphyrin IX, is not active without its cofactor. Second, the mRNA of the erythroid-specific 5-aminolevulinate synthase 2 (ALAS2), the initial and rate-limiting enzyme of heme biosynthesis, carries an iron-responsive element (IRE) at its 50 -end. Improperly matured IRP1 and IRP2 may not only drive TfR-dependent cellular iron uptake but also block ALAS2 expression, thereby limiting the availability of protoporphyrin IX. A similar connection has been hypothesized for the classical form of X-linked sideroblastic anemia which is caused by mutations in the X-chromosomal ALAS2 gene,

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resulting in functional ALAS2 deficiency [137]. However, a simple elevation of iron delivery alone is not sufficient to explain the sideroblastic phenotype. Although FRDA and HML lead to mitochondrial iron accumulation, and although at least in FRDA the two proteins IRP1 and IRP2 probably interfere with ALAS2 expression, sideroblastic anemia does not develop. Moreover, profound cellular iron overload is also present in both hereditary and transfusional hemochromatosis but neither disorder results in mitochondrial iron accumulation [136] or in the formation of ring sideroblasts. It has been proposed that the unique combination of tissue-specific demands for some distinct ISC assembly or export components together with a particular set of Fe/S target proteins might give rise to the sideroblastic phenotype [141] but the molecular basis for this phenomenon remains elusive. 4.1.5.1. Inherited sideroblastic anemias 4.1.5.1.1. Sideroblastic anemia with deficiency of glutaredoxin 5. So far, four individuals have been identified suffering from mutations in the gene of GLRX5 with one of them developing the typical characteristics of SA in late adulthood [78,138]. Secondary to the profound iron overload this patient developed type II bronze diabetes, hepatosplenomegalie, and cirrhosis. A recessively inherited mutation in the last codon of exon 1 is predicted to interfere with the correct splicing of the primary GLRX5 transcript, resulting in severely decreased mRNA and protein levels in both hematopoietic and non-hematopoietic cells [78,141]. Expression studies in healthy mice revealed an extremely high level of GLRX5 in erythropoietic tissues [141,142], providing an explanation for the tissue restriction of the disease, although other cell types are also affected. For example, fibroblasts showed a decrease in both activity and protein content of respiratory chain complex I. Moreover, diminished activities of both mitochondrial and cytosolic aconitase as well as an increase in IRP1’s IRE binding activity were detected in patient fibroblasts and in cells of the hematopoietic lineage [78,141], suggesting that GLRX5 deficiency causes a generalized assembly defect of mitochondrial and extra-mitochondrial Fe/S proteins (Fig. 6). Deregulation of cellular iron metabolism is indicated by elevated levels of IRP2 and TfR, and by diminished amounts of ferritin heavy chain protein. At the transcriptional level, expression of the iron import carrier DMT1 and of the ferric reductase DCYTB was increased, in line with an increase in TfR-mediated iron uptake [141]. Additionally, mRNA levels of heme oxygenase 1, ferritin light chain, and ferroportin 1 were increased, possibly in response to iron-mediated oxidative stress. In cells of the erythroid compartment mitochondrial ferritin was detectable, in line with the presence of ring sideroblasts [78]. Moreover, the GLRX5-associated Fe/S protein assembly defect hampered the maturation of ferrochelatase as indicated by its low protein levels, while the increase in the IREbinding activity of IRP1 and IRP2 are in line with a decline in the amount of ALAS2 [141]. Both mechanisms interfere with heme synthesis, thus contributing to the hematopoietic phenotype. In contrast to the patient suffering from SA, three other individuals harbor GLRX5 mutations which give rise to a completely different phenotype. Instead of developing SA they present with a variant form of nonketotic hyperglycinemia characterized by a delayed childhood onset of spastic paraplegia, optic atrophy, spinal lesions, and encephalopathy [138,143,144]. Single-nucleotide polymorphism analysis and sequencing revealed a recessively inherited deletion of a base pair triplet in exon 1 of GLRX5, resulting in the deletion of the surface-exposed residue Lys51. One individual is compound heterozygous with a deletion in one allele and a frame shift mutation in the other, resulting in a premature stop codon. Immunoblotting demonstrated that the triplet deletion does not interfere with GLRX5 protein levels in fibroblasts, but biochemical analyses uncovered a severe defect in the formation of lipoic acid,

pointing to improper LIAS activity. None of the E2 subunits of PDH and a-KGDH contained the cofactor, and enzyme activities of both PDH and the glycine cleavage system (GCS) were strongly decreased. As a consequence, glycine accumulates in blood and brain and probably induces the neurodegenerative phenotype due to its neurotoxic effects [145]. Whether conversely the low GLRX5 protein levels in the SA patient also impact on LIAS activity has not been determined, but neurological symptoms were absent [78] indicating at least basal activities of the lipoate-containing enzymes. Surprisingly, the three patients lacking Lys51 of GLRX5 do not show any other typical signs of Fe/S diseases except for the LIAS defect. Neither respiratory chain complexes, nor mitochondrial and cytosolic aconitases are affected [138], indicating reasonable mitochondrial and cytosolic-nuclear Fe/S cluster biosynthesis. Accordingly, the iron metabolism was normal in the three patients. The isolated lipoic acid defect suggests that GLRX5 might particularly contribute to the function of LIAS by a yet disregarded mechanism which critically involves the surface-exposed Lys51 residue. 4.1.5.1.2. X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). Another rare congenital sideroblastic anemia is caused by missense mutations in the gene of the mitochondrial export component ABCB7 (Fig. 6), entailing defective Fe/S protein assembly predominantly in the cytosolic-nuclear compartment [82,83]. Disease-causing mutations in ABCB7 are frequently located close to or in transmembrane domains, suggesting an interference with proper protein folding and/or membrane insertion [82,83,146,147]. The X-chromosomal location of the ABCB7 gene [148,149] is eponymous for the recessively inherited disease and accounts for the predominant manifestation of XLSA/A in males [150]. In contrast to GLRX5 deficiency, patients develop only mild hypochromic microcytic anemia and their tissue iron status is usually inconspicuous [82,151]. Rather, individuals present with a severe early-onset spinocerebellar syndrome characterized by cerebellar atrophy and motor impairment, pointing to the secondary acquisition of respiratory chain defects. Heterozygous females may develop a hematologic phenotype including few ring sideroblasts in bone marrow but remain otherwise asymptomatic [146,151]. The tissue distribution of ABCB7 is broad [148] with particular high expression in hematopoietic tissue and peripheral nervous system [152], as well as in skeletal muscle, heart, and pancreas [83,149], indicating that these tissues are most severely affected by functional impairment of the transporter. Studies in mice [76,153] and tissue culture cells [87] demonstrated that ABCB7 is essential in all tissues but particularly required for hematopoiesis, in line with its tissue distribution. Functional deficiency of the transporter results in mitochondrial damage, but does not lead to a general inactivation of mitochondrial Fe/S enzymes [14]. Ferrochelatase and mitochondrial aconitase maintain their activities [76,87,147] but respiration is impaired [76], probably as a secondary consequence of mitochondrial iron accumulation. Severe defects were also observed in the maturation of the extra-mitochondrial Fe/S proteins xanthine oxidase and cytosolic aconitase [76,87,154], demonstrating the critical function of ABCB7 in the biogenesis of cytosolic Fe/S proteins. Elevated levels of apo-IRP1 and of IRP2 are consistent with increased TfR expression [76] and indicate deterioration of cellular iron metabolism. Hence, XLSA/A is primarily associated with Fe/S protein defects in the cytosolic-nuclear compartment. 4.1.5.2. Acquired sideroblastic anemias. Acquired SAs are much more common than the hereditary forms and represent specific traits of the myelodysplastic syndrome (MDS) [136,137]. MDS comprises a heterogeneous group of hematopoietic malignancies arising from somatic mutations in pluripotent hematopoietic stem cells.


Growing evidence suggests that at least some types of acquired SAs are associated with impaired expression or ablation of ABCB7, but in contrast to the congenital sideroblastic anemia XLSA/A, they are restricted to the hematopoietic compartment. 4.1.5.2.1. Refractory anemia with ring sideroblasts (RARS). Refractory anemia with ring sideroblasts (RARS) is an isolated anemia of clonal origin characterized by erythroid hyperplasia and ineffective erythropoiesis [137]. Consistent with deteriorated heme formation the ratio of protoporphyrin IX to heme is elevated, and gene expression profiling of patient erythroblasts revealed abnormally increased ALAS2 mRNA levels [155] which point to a counterbalancing transcriptional response. In contrast, expression of ABCB7 was decreased, providing a mechanistic link to XLSA/A. Moreover, the amount of ring sideroblasts in the hematopoietic system of RARS patients was found to be inversely related to the level of ABCB7 mRNA [139]. A causative connection was proposed when ABCB7 was experimentally overproduced in RARS patient cells [156]. At least erythroid growth improved, and cells turned down the expression of mitochondrial ferritin. Vice versa, depletion of ABCB7 in normal erythroblasts elicited a RARS-related phenotype. Apparently, ABCB7 deficiency is a major determinant in the development of SA and might trigger a similar chain of events as in XLSA/A. The genetic basis for the low expression of ABCB7 in RARS requires further examination, but mutations in the splicing factor SF3B1, a core component of the RNA splicing machinery, have been proposed to be involved. Conspicuously, SF3B1 haploinsufficiency leads to the formation of ring sideroblasts [157], and experimental silencing of SF3B1 in K562 cells reduces ABCB7 expression [156]. However, mutations in the SF3B1 gene have not always been found to interfere with ABCB7 expression in RARS [157] suggesting that other factors contribute to the disease-associated changes in ABCB7 levels. 4.1.5.2.2. Refractory anemia with ring sideroblasts and isodicentric (X)(q13) chromosome. A rare type of RARS is associated with complete ablation of the ABCB7 gene due to formation of an isodicentric (X)(q13) chromosome (idic (X)(q13)) [140]. Generation of such a chromosome is assumed to occur during mitosis and probably involves DNA double strand breaks within two sister chromatids at site of the q13 band, followed by incorrect DNA rejoining proximal to the centromere [158]. Consequently, the idic (X)(q13) harbors two centromeres and two p-arms while a major part of the q-arm including the ABCB7 locus is lost. Notably, the XLSA-associated ALAS2 gene resides at the p-arm [159] and is thus not deleted by the ablation. The idic (X)(q13) abnormality is restricted to women, usually in advanced age, and always present as a single copy [140]. In males an idic (X)(q13) likely causes cell death because the sole copy of the X chromosome is lost. Sideroblast formation is observed only in some cases of idic (X)(q13) neoplasia, probably since always only a single copy of ABCB7 is deleted in affected females [158,160]. It has been suggested that the RARS phenotype might particularly develop when the deletion affects the active and not the inactive X-chromosome [140]. Genetic analyses support this view [161], thus emphasizing the critical importance of the export component ABCB7 for cellular and mitochondrial iron homeostasis. 4.2. Fe/S diseases without mitochondrial iron accumulation Defects in genes of the mitochondrial ISC assembly components acting downstream of GLRX5 result in severe mitochondrial (metabolic) dysfunctions but hardly impair cytosolic-nuclear Fe/S protein assembly, indicating that they do not affect the ISC export process (Fig. 6). Particularly, such defects are not associated with disturbed iron metabolism and mitochondrial iron accumulation, thus emphasizing the importance of a dynamic Fe/S cluster-based

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crosstalk between the mitochondrial and cytosolic-nuclear compartment in order to maintain proper mitochondrial and cellular iron homeostasis. Nevertheless, defects in late-acting ISC assembly components cause serious and fatal conditions (see Section 4.2.1.). However, mutations in the NFU1 and BOLA3 genes also shed light on the function of the encoded proteins, and the identification of disease-causing mutations in the genes encoding IBA57 or IND1 provided in vivo evidence for their late function in the ISC assembly process, as indicated by earlier studies in yeast and human cell culture models [59,60,72,73]. 4.2.1. Multiple mitochondrial dysfunction syndromes Mutations in the genes IBA57, NFU1, and BOLA3 may severely impair diverse mitochondrial metabolic pathways and interfere with energy production [68,69,162]. Hence, these diseases have been designated as multiple mitochondrial dysfunction syndromes (MMDS) which may be fatal at perinatal stages. Affected individuals present with hypotonia, respiratory insufficiency, encephalopathy, brain malformations, and neurological regression. At the biochemical level, patients exhibit metabolic acidosis, hyperglycinemia, and severely increased concentrations of lactate and ketoacids. Enzyme activities of respiratory complexes I, II, and IV, and of the lipoic aciddependent enzymes PDH, a-KGDH, and GCS are diminished. Closer inspection of cellular Fe/S proteins in patient material or tissue culture models of IBA57 and NFU1 mutations revealed that a subset of mitochondrial [4Fee4S] proteins including respiratory complexes I and II are impaired in their assembly, while both maturation and function of [2Fee2S] proteins (e.g., of Rieske Fe/S protein of complex III) are normal [69,162]. Likewise, cytosolic-nuclear Fe/S proteins are unchanged in MMDS. Thus, the disease phenotypes are consistent with mechanistic models derived earlier from yeast and HeLa cell culture studies that assigned a late function of IBA57 and NFU1 in mitochondrial Fe/S protein assembly (Fig. 6). Some of the affected [4Fee4S] target proteins are involved in lipid metabolism, including ETFDH and LIAS. Particularly, the radical SAM enzyme LIAS appears to play a key role in the development of MMDS. Insufficient assembly and hence enzyme activity decreases the lipoic acid content of the E2 subunits of PDH and a-KGDH [68,69,162]. This in turn impacts on the citric acid cycle and consequently on energy metabolism [163]. Moreover, lipoic acid is required for the function of BCKDH and of the H protein of the GCS (H-GCS) [69]. Compromised activities of these enzymes are evident in the patients because they present with metabolic acidosis and hyperglycinemia, which may be causative of toxic encephalopathy [162]. A frequent, yet puzzling observation in IBA57, NFU1, and BOLA3 patients as well as in experimental tissue culture systems is the lowered activity of COX [60,68,162]. This respiratory complex does not contain a Fe/S cofactor and hence should be normal. Conspicuously, this phenotype is reproduced in respective yeast ISC mutants and upon RNAi-mediated depletion of these proteins in HeLa cells [58,59,69]. To date, the molecular reason for the COX defect is unclear. Destabilization of COX due to the disappearance of respiratory chain supercomplexes as a consequence of general Fe/S cluster assembly defects seems unlikely, since isolated complex I deficiencies hardly affect the function of COX [73]. In zebrafish, ablation of IBA57 suggested a link to heme formation [164], but hemoglobin and hematocrit levels in the IBA57 patients were within the normal range [162]. One might suspect that a yet illdefined pathway crucial for the biosynthesis of COX might involve a mitochondrial [4Fee4S] protein, but so far such a possibility has not been substantiated. 4.2.1.1. Juvenile encephalomyopathy with deficiency of IBA57 (MMDS3). Among the three forms of MMDS the most severe

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phenotype was caused by a homozygous missense mutation in the IBA57 gene, resulting in a Gln314Pro substitution in a nonconserved region of the primary translation product [162]. The alteration was identified in two siblings who died shortly after birth and had completely lost the protein. Biochemical analysis of the mutation in a HeLa tissue culture model revealed that the proline substitution only partially affected the activity of IBA57, yet caused a profound proteolytic sensitivity and degradation of the protein. Though the precise molecular function of IBA57 is not solved yet, its loss in both patients and human tissue culture resulted in impaired assembly of all mitochondrial [4Fee4S] proteins inspected so far, underlining the vital importance of IBA57 for the generation of [4Fee4S] clusters in mitochondria (Fig. 6). 4.2.1.2. Multiple mitochondrial dysfunction syndrome with functional NFU1 deficiency (MMDS1). NFU1 is a ubiquitously expressed ISC assembly component which transiently binds a [4Fee4S] cluster [67]. Mutations in NFU1 cause the first MMDS identified [68,69]. One group of MMDS1 patients harbors a homozygous mutation in the donor splice site of exon 6, resulting in improper mRNA processing, generation of a premature stop codon, and degradation of both mRNA and the truncated polypeptide [68]. Another group of patients carries a Gly208Cys missense mutation close to the Fe/S cluster binding motif, but NFU1 protein levels remain unchanged [69]. In both patient cohorts maturation of a subset of mitochondrial [4Fee4S] proteins is impaired, yet in contrast to the IBA57 patients mitochondrial aconitase is unaffected. This indicates that NFU1 shows higher target specificity distinguishing its function from that of IBA57 which acts more upstream of NFU1 and is responsible for maturation of virtually all mitochondrial [4Fee4S] proteins (Fig. 6). For the severity of the disease phenotype these biochemical differences seem to be of minor relevance, as the complete loss of NFU1 due to mRNA mis-splicing was fatal shortly after birth [165], similar to IBA57 deficiency in MMDS3 (see 4.2.1.1.). In contrast, the Gly208Cys exchange in NFU1 causes a less severe phenotype, and the patients deceased later [69]. Subsequent studies in yeast revealed that the cysteine residue added by the Gly208Cys substitution resulted in a more stably bound Fe/S cluster on NFU1, suggesting that Fe/S cluster transfer from mutated NFU1 to respective target proteins might have been severely impaired [69]. The two types of mutations in NFU1 affected different patient tissues in a diverse fashion. While lipoylation of PDH-E2 and aKGDH-E2 was severely hampered in muscle and liver, the respective cofactors were present in kidney and brain [166], suggesting a sufficient activity and hence maturation of LIAS. Likewise, COX activity was not affected in the Gly208Cys NFU1 patients [69], while the strong depletion of NFU1 in case of the splice site mutation resulted in a decreased COX activity [68,165]. Obviously, the effects on COX activity depend on the severity of the mutations in the lateacting ISC assembly components. Analysis of the Gly208Cys NFU1 mutation in yeast also helped to uncover the chain of events during the late phase of mitochondrial Fe/S protein maturation [69]. Cluster assembly on mutant yeast Nfu1 was found to be dependent on the core ISC assembly components including Isu1, but was independent of Isa1eIsa2. In contrast, the function of both yeast Isu1 and human ISCU did not require Nfu1, indicating that Nfu1 does not act as an alternative scaffold upstream or in parallel to Isu1 (Figs. 1 and 6). Rather, Nfu1 acts downstream of both Isu and Isa proteins, presumably fulfilling a dedicated function in Fe/S cluster insertion into target apoproteins. Moreover, complementation and expression studies in cell lines of patients and of healthy controls did not support the idea of a previously discussed extra-mitochondrial function of NFU1 [68]. Only the mitochondrial NFU1 isoform but not the putative cytosolic

version complemented the NFU1 deficiency phenotype. In line with these results, immunofluorescence analyses in cells of healthy donors suggested that NFU1 is located within mitochondria. Accordingly, NFU1 levels are particularly high in tissues with high mitochondrial content [166], supporting a dedicated role of the protein within these organelles. 4.2.1.3. Multiple mitochondrial dysfunction syndrome with functional BOLA3 deficiency (MMDS2). Three different types of missense mutations in the BOLA3 gene have been identified so far, affecting at least six individuals and resulting in clinical and biochemical phenotypes that closely resemble the condition caused by NFU1 deficiency [68,138,165,167]. In line with the impaired mitochondrial function most of the BOLA3 patients developed cardiomyopathy, optic atrophy, and neurodegeneration accompanied by epileptic seizures [168]. The least severe biochemical phenotype was caused by a c.136C > T point mutation in exon 2 of the BOLA3 gene producing a premature p.R46* stop codon [138], mainly affecting respiratory chain complex II (succinate dehydrogenase, SDH) and LIAS, as indicated by low PDH and GCS activities. As a consequence, lactate and glycine levels were elevated, probably exerting neurotoxic effects (see Section 4.1.5.1.1.). One individual was homozygote for a single c.123dupA base pair duplication within the BOLA3 gene, leading to a frame shift and a premature stop codon (p.Glu42Argfs*13) without affecting mRNA levels [68]. Two other individuals harbored a homozygous mutation located on exon 3 of the BOLA3 gene, resulting in an Ile67Asn substitution in a highly conserved region of the protein and impairing COX and citric acid cycle activity, as indicated by the high amounts of its metabolites in urine [167]. Both the base pair duplication and the Ile67Asn substitution defects could be complemented in patient fibroblast lines by ectopic expression of the longer of two annotated BOLA3 isoforms, underlining the critical function of BOLA3 in the assembly of a subset of mitochondrial [4Fee4S] proteins. The similar presentation of MMDS1 and MMDS2 individuals points to a function of BOLA3 in concert with NFU1 (Fig. 6), but this hypothesis requires biochemical testing. 4.2.2. Mitochondrial encephalomyopathy with deficiency of IND1 The mitochondrial P-loop NTPase IND1 (also termed NUBPL) performs a dedicated function in the maturation of respiratory chain complex I (Figs. 5 and 6). So far, eight individuals were identified suffering from functional impairment of this assembly factor [169,170]. They present with typical characteristics of an isolated, early-onset complex I deficiency and develop a mitochondrial encephalomyopathy with ataxia and ragged-red fibers, frequently accompanied by seizures and nystagmus [84,169e171]. Particularly, atrophy of brain white matter exhibits a unique magnetic resonance imaging pattern, possibly characteristic for impaired IND1 function and thus helpful as diagnostic criteria [172]. At the biochemical level impaired maturation of complex I is associated with a decrease in glucose turnover in brain and with lactate enrichment in cerebrospinal fluid and plasma [84,169,170]. Other respiratory chain complexes and the lipoate-dependent enzyme PDH are not affected, neither in a tissue culture model of IND1 deficiency ([73] and above) nor in fibroblast lines and muscle biopsies of IND1-deficient individuals. This is in line with the dedicated function of the protein (c.f. Fig. 5). All known IND1-deficient patients carry at least one IND1 allele with two mutations: a functionally silent Gly56Arg substitution at a highly conserved position, and a c.815-27T > C branching site mutation resulting not only in the formation of the wild-type but also of two additional splicing products [169,171]. One of the two aberrant mRNAs is instable and immediately degraded by nonsense-mediated decay. Processing of the other by exon-


skipping results in an instable IND1 isoform which is nonfunctional due to a truncated and altered C-terminus [171,173]. Surprisingly, this mutated allele is present in more than 1% of European haplotypes, and even heterozygote carriers show markedly decreased levels of IND1 wild-type mRNA and protein [169,171]. However, residual IND1 protein is apparently sufficient to allow for proper complex I assembly and respiratory function, indicating that the amount of IND1 is usually not rate-limiting for maturation of complex I. Only in case of homozygosity the amounts of IND1 are too low to support the assembly process [169]. Most of the IND1 patients are compound heterozygotes for the c.815-27T > C branching site mutation and one of several yet ill-defined null mutations, again resulting in low IND1 protein levels. Residual amounts of IND1 do not correlate with the severity of the patient phenotypes, in line with observations in other complex I disorders. The high frequency of the c.815-27T > C branching site mutation raises the possibility that it contributes to other complex I diseases of yet unknown genesis. Tissue culture studies indicated that IND1 deficiency particularly affects the assembly of the peripheral arm of complex I, while the membrane-embedded part is formed [73]. Accordingly, assembly intermediates of the peripheral arm have not been detected in patient material [169]. In an early phase of the assembly pathway two Fe/S cluster-coordinating subunits, NDUFS7 and NDUFS8, are required [174]. When these two Fe/S subunits are missing, formation of the peripheral arm is abrogated suggesting that at least NDUFS7 and NDUFS8 are dedicated targets of IND1 [169]. 4.3. Variant erythropoietic protoporphyria with abnormal expression of mitoferrin 1 Both Fe/S protein assembly and heme formation are dependent on proper mitochondrial iron import which in erythroblasts is primarily mediated by the solute carrier mitoferrin 1 (MFRN1, SLC25A37) [35] (Fig. 6). Hence, MFRN1 is not a classical mitochondrial ISC assembly component, yet critical for Fe/S cluster formation. Absence of the Fe/S enzyme ferrochelatase impairs heme biosynthesis and causes erythropoietic porphyria (EPP), a disease characterized by accumulation of protoporphyrin IX leading to photosensitivity of the skin and liver damage [175]. A variant form of EPP has been linked to aberrant splicing of MFRN1 mRNA, though affected individuals harbor defects also in genes of ferrochelatase and ALAS2 [176]. Activation of a cryptic donor splice site in intron 2 of the MFRN1 gene results in the insertion of an additional nucleotide sequence containing a premature stop codon. Transcript analysis revealed that some mis-splicing of MFRN1 mRNA is a common phenomenon, but in patients with variant EPP up to 50% of the primary transcripts are improperly processed. The majority of these abnormal transcripts are degraded by nonsensemediated decay, and the remaining portion encodes a truncated protein which is neither translocated to mitochondria nor functional. Ferrochelatase activity is decreased in patients with variant EPP, suggesting that iron import into mitochondria is severely hampered and limiting for Fe/S protein assembly. Both low ferrochelatase activity and insufficient iron supply for heme synthesis possibly contribute to variant EPP. Mitochondrial iron import and Fe/S protein assembly in other tissues are dependent on MFRN2, and thus are not affected, consistent with the dedicated function of MFRN1 in erythropoietic cells. 5. Conclusions Mitochondria integrate two major iron-consuming metabolic pathways, Fe/S cofactor assembly and heme formation, and consequently fulfill a key role in regulating cellular iron metabolism

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[85]. Diseases characterized by mitochondrial iron accumulation indicate that defects in core ISC components and in the export component Atm1 lead to severe alterations in iron homeostasis and mis-localization of the metal to mitochondria, yet the molecular basis of these alterations have not been elucidated hitherto. Biogenesis of cellular Fe/S proteins requires multiple complex and interdependent assembly systems inside and outside mitochondria, indicating the critical cellular function of Fe/S cofactors [89]. For example, Fe/S proteins contribute to mitochondrial energy production and fatty acid catabolism, maintain nuclear genome integrity due to their involvement in RNA and DNA metabolism, are involved in cytosolic protein translation, and directly participate in cellular iron homeostasis. The fact that mitochondria are primary sites of the assembly of cellular Fe/S clusters provides a clue why these organelles are essential for eukaryotic life [177]. Hence, it is not surprising that defects in the mitochondrial ISC assembly and export systems give rise to severe and often even fatal disorders. The clinical picture usually includes defects in mitochondrial energy production and cellular metabolism, and can now be addressed in more detail based on the largely improved understanding of the ISC assembly processes. Vice versa, analysis particularly of the biochemical phenotypes in ISC assembly diseases will help to improve our knowledge on the function of the ISC assembly system. Future goals include the better characterization of individual biogenesis components and their dynamic interaction during Fe/S protein assembly. Only little is known about the regulation of the ISC assembly systems, especially with respect to transcriptional control. Elucidation of the mechanisms underlying ISC regulation could be valuable for the development of new therapeutic approaches targeting disorders caused by defective mitochondrial Fe/S protein assembly. Acknowledgments We thank the members of our group for discussion. RL acknowledges generous support from Deutsche Forschungsgemeinschaft (SFB 593, SFB 987, and GRK 1216), MaxPlanck Gesellschaft, von Behring-Röntgen Stiftung, and LOEWE program of state of Hesse. References [1] K. Volz, The functional duality of iron regulatory protein 1, Curr. Opin. Struct. Biol. 18 (2008) 106e111. [2] J.L. Giel, A.D. Nesbit, E.L. Mettert, A.S. Fleischhacker, B.T. Wanta, P.J. Kiley, Regulation of ironesulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fee2S]-IscR in Escherichia coli, Mol. Microbiol. 87 (2012) 478e492. [3] P.J. Kiley, H. Beinert, Oxygen sensing by the global regulator, FNR: the role of the ironesulfur cluster, FEMS Microbiol. Rev. 22 (1998) 341e352. [4] B. Zhang, J.C. Crack, S. Subramanian, J. Green, A.J. Thomson, N.E. Le Brun, M.K. Johnson, Reversible cycling between cysteine persulfide-ligated [2Fee 2S] and cysteine-ligated [4Fee4S] clusters in the FNR regulatory protein, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 15734e15739. [5] D.J. Netz, C.M. Stith, M. Stumpfig, G. Kopf, D. Vogel, H.M. Genau, J.L. Stodola, R. Lill, P.M. Burgers, A.J. Pierik, Eukaryotic DNA polymerases require an irone sulfur cluster for the formation of active complexes, Nat. Chem. Biol. 8 (2012) 125e132. [6] J. Rudolf, V. Makrantoni, W.J. Ingledew, M.J. Stark, M.F. White, The DNA repair helicases XPD and FancJ have essential ironesulfur domains, Mol. Cell. 23 (2006) 801e808. [7] Y. Hu, M.W. Ribbe, Nitrogenase assembly, Biochim. Biophys. Acta 1827 (2013) 1112e1122. [8] J.W. Peters, J.B. Broderick, Emerging paradigms for complex ironesulfur cofactor assembly and insertion, Annu. Rev. Biochem. 81 (2012) 429e450. [9] M. Müller, W. Martin, The genome of Rickettsia prowazekii and some thoughts on the origin of mitochondria and hydrogenosomes, BioEssays 21 (1999) 377e381. [10] R. Lill, Function and biogenesis ironesulphur proteins, Nature 460 (2009) 831e838. [11] R. Lill, B. Hoffmann, S. Molik, A.J. Pierik, N. Rietzschel, O. Stehling, M.A. Uzarska, H. Webert, C. Wilbrecht, U. Mühlenhoff, The role of

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MYC and mitochondrial biogenesis.

Apolipoprotein a1 increases mitochondrial biogenesis through AMP-activated protein kinase.

GCN5-like protein 1 (GCN5L1) controls mitochondrial content through coordinated regulation of mitochondrial biogenesis and mitophagy.

The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis.

Mitochondrial biogenesis during fungal spore germination: products of mitochondrial protein synthesis in vivo.

Maintaining ancient organelles: mitochondrial biogenesis and maturation.

Oxidative protein biogenesis and redox regulation in the mitochondrial intermembrane space.

Optimizing intramuscular adaptations to aerobic exercise: effects of carbohydrate restriction and protein supplementation on mitochondrial biogenesis.

The MIA pathway: a key regulator of mitochondrial oxidative protein folding and biogenesis.

Mitochondrial biogenesis in plants during seed germination.

Biogenesis of mitochondrial porin: the import pathway.

Therapeutic effects of baicalein on rotenone-induced Parkinson's disease through protecting mitochondrial function and biogenesis.

Biogenesis of mitochondrial proteins in HeLa cells.

Mitochondrial DNA copy numbers in pyramidal neurons are decreased and mitochondrial biogenesis transcriptome signaling is disrupted in Alzheimer's disease hippocampi.

Differentiation of Human Neural Stem Cells into Motor Neurons Stimulates Mitochondrial Biogenesis and Decreases Glycolytic Flux.

Mitochondrial biogenesis in the acutely injured kidney.

Biogenesis of mitochondrial c-type cytochromes.

Mitochondrial disease genes COA6, COX6B and SCO2 have overlapping roles in COX2 biogenesis.

Regular postexercise cooling enhances mitochondrial biogenesis through AMPK and p38 MAPK in human skeletal muscle.

Human mitochondrial DNA replication machinery and disease.

Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1.

Biogenesis of mitochondrial outer membrane proteins, problems and diseases.