PERSPECTIVE

Mitochondrial iron trafficking and the integration of iron between the and

Des R. Richardsona,1, Darius J. R. Lanea, Erika M. Beckera, Michael L.-H. Huanga, Megan Whitnalla, Yohan Suryo Rahmantoa, Alex D. Sheftelb, and Prem Ponkac,d,1 aIron Metabolism and Chelation Program, Discipline of Pathology, University of Sydney, NSW 2006, Australia; bInstitut für Zytobiologie, Philipps-Universität Marburg, Marburg 35037, Germany; cLady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada H3T 1E2; and dDepartments of Physiology and Medicine, McGill University, Montreal, QC, Canada H3A 2T5

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 30, 2010 (received for review March 15, 2010)

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)-containing involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial path- ways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreich’s ataxia) has established that communication exists between iron metabolism in the mitochondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron- processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron- processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism.

iron sulfur cluster | heme | Friedreich’s ataxia | frataxin | sideroblastic anemia

he mitochondrion is mostly ap- a biological catalyst. Consequently, iron is plays a role in organizing the actin cyto- preciated for its role in energy a crucial element required for growth. skeleton and is up-regulated by iron- transduction. However, it is less However, the very chemical properties of depletion through the iron regulatory T – well known that this organelle iron that allow this versatility also create (IRP) iron-responsive element can be considered a focal point when it a paradoxical situation, making acquisition (IRE) interaction (see below) (6). MRCKα comes to the metabolism of the most by the organism very difficult. Indeed, at colocalizes with Tf-TfR1 complexes fol- common transition metal in cells, namely pH 7.4 and physiological tension, lowing their internalization and it has been iron (1). It is the reversible oxidation states the relatively soluble iron(II) is readily shown that attenuation of MRCKα ex- of iron that enable the mitochondrion to oxidized to iron(III), which upon hydrolysis pression causes a significant decrease in catalyze electron transport via heme- and forms insoluble ferric hydroxides. As a Tf-mediated iron uptake (7). Additionally, iron sulfur cluster (ISC)-containing pro- result of this virtual insolubility and po- it is known that Sec15l1, which is involved teins and use this process in energy trans- tential toxicity because of redox activity, in the mammalian exocyst complex (8), duction. Considering this alone, it is no iron must be constantly chaperoned. In plays a role in iron uptake from Tf via its wonder that the mitochondrion plays such fact, specialized molecules for the acqui- role in exocytosis (9, 10). In fact, Sec15l1 is a critical role in iron metabolism. In fact, sition, transport, and storage of iron in linked to the Tf cycle through its inter- the mitochondrion is the sole site of heme a soluble, nontoxic form have evolved to action with Rab11 (a GTPase involved in synthesis and a major generator of ISCs, meet the organism’s iron requirements. vesicular trafficking) and a mutation in both of which are present in mitochondria Over the last 15 years there has been a Sec15l1 leads to the anemia found in he- and cytosol (2). Although the molecular wide variety of unique molecules discov- moglobin-deficit mice (9, 10). pathways involved in the generation of ered that play a role in iron metabolism, Within the endosome, the affinity of Tf heme and ISCs are well known, only more and the most relevant of these to this re- for iron is decreased by the low pH gen- recently have some of the molecular view are listed in Table S1. erated through the activity of a proton players responsible for mitochondrial iron Because of its redox properties, iron can pump (11, 12). Importantly, the TfR1 fa- transport been identified. Clearly, these catalyze the production of reactive oxygen cilitates liberation of iron from Tf in the molecular circuits are vital for the supply species (ROS) that can be highly toxic pH range attained by the endosome (pH of the metal ion that is needed for gen- (3). Therefore, under normal physiological 5–5.5) (13). In vitro, iron release from erating the final biologically important conditions, iron is specifically transported Tf requires a “trap,” such as pyrophos- end-products, namely heme and ISCs. In in the blood by diferric transferrin (Tf) phate (13), but a physiological chelator contrast to the mitochondrion, at the (4, 5). All tissues acquire iron by the serving this role has not been identified. In whole-cell level the molecular pathways binding of Tf to the transferrin receptor 1 erythroid cells, once iron(III) is released and regulation of iron uptake and storage (TfR1), with this complex then being in- have been well characterized. Hence, these ternalized by receptor-mediated endocy- fi fl A Author contributions: D.R.R., D.J.R.L., E.M.B., M.L.-H.H., M.W., will be rst brie y described to provide tosis (4, 5) (Fig. 1 ). Recent studies have Y.S.R., A.D.S., and P.P. wrote the paper. basic background on the field of iron me- demonstrated that the internalization of The authors declare no conflict of interest. tabolism (3, 4) before examining what is the Tf-containing endosome via the cyto- This article is a PNAS Direct Submission. known regarding the mitochondrion. skeleton is under control of intracellular 1To whom correspondence may be addressed. E-mail: prem. iron levels (6, 7). In part, this uptake [email protected] or [email protected]. Cellular Iron Metabolism and Transport mechanism is mediated by myotonic dys- This article contains supporting information online at Iron is an essential metal for the organism trophy kinase-related Cdc42-binding ki- www.pnas.org/lookup/suppl/doi:10.1073/pnas.0912925107/-/ because of its unparalleled versatility as nase alpha (MRCKα) (6). This molecule DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0912925107 PNAS | June 15, 2010 | vol. 107 | no. 24 | 10775–10782 Downloaded by guest on September 29, 2021 from Tf in the endosome, it is thought to IRP-1 performs two functions: (i) regu- Furthermore, the inhibitor of heme syn- be reduced to iron(II) by a ferrireductase lating iron homeostasis via binding IREs thesis, succinylacetone, led to a reduction in the endosomal membrane known as the and (ii) having cytosolic aconitase activity in this low Mr iron, suggesting it was heme six-transmembrane epithelial antigen of when containing an [4Fe-4S] cluster (21). or a heme-containing molecule (30). These the prostate 3 (14, 15) (Fig. 1A). After this IRP-2 is thought to be the principal RNA- studies, coupled with the findings in pre- step, iron(II) is then transported across binding protein in vivo and is regulated by vious investigations by others, led to a hy- the endosomal membrane by the divalent iron-dependent proteasomal degradation pothesis that iron is always transported, at metal transporter-1 (DMT1) (16) and (22–24). Although the IRP-IRE mecha- least in erythroid cells, bound within hy- forms, as generally believed, the cytosolic nism plays crucial roles in the regulation of drophobic pockets of proteins that act as low Mr labile or chelatable iron pool (17). iron metabolism, complete understanding intermediates (or chaperones) and prevent This pool of iron is thought to supply the of the homeostatic mechanisms involved cytotoxic redox chemistry (30). metal for storage in the cytosolic protein in iron metabolism in relation to commu- As yet, such iron chaperone molecules ferritin and for metabolic needs, including nication between the cytosol and mito- remain elusive, although Vyoral et al. (31) iron uptake by the mitochondrion for chondrion have yet to be deciphered, and identified a high Mr intermediate that ap- heme and ISC synthesis. are considered in the following sections. peared to donate its iron to ferritin after Iron can also be released from the cell incubation of K562 cells with Tf. More by the transporter, ferroportin1 (18) (Fig. Intracellular Iron Transport and Communication recently, a protein known as poly (rC)- 1A). Ferroportin1 expression can be reg- with the Mitochondrion. Once iron is trans- binding protein 1 has been identified that ulated by the hormone of iron metabo- ported out of the endosome via DMT1, it donates iron to ferritin and may play an lism, hepcidin. Hepcidin is a key regulator enters the chelatable or labile iron pool (Fig. important role in this process (32). Al- of systemic iron metabolism (18) and is 1A) that traditionally was thought to consist though work identifying chaperones that transported in the blood bound to α2- of low Mr complexes (e.g., iron-citrate) (17, transport iron remain preliminary, it is macroglobulin (18). Hepcidin secretion by 25, 26). The only strong evidence that such notable that for copper, which is the sec- the liver is stimulated by high iron levels a pool exists comes from studies with che- ond most-abundant transition metal ion in and also inflammatory cytokines, such as lators that mobilize iron from cells (27, 28). mammalian tissues, much evidence of interleukin-6 (19). However, it is just as likely that these com- chaperone-mediated transport has been pounds remove iron from organelles and described (33, 34). Like iron, copper is Regulation of Cellular Iron Homeostasis proteins as it is that they chelate iron from also cytotoxic because of its redox activity, fi The chelatable iron pool is thought to genuine cytosolic low Mr complexes (29). and is never found free at signi cant con- control the activity of IRPs-1 and -2 Studies using reticulocytes, which are centrations, but is always bound to trans- (Fig. 1A). The IRPs are RNA-binding highly active in terms of iron uptake, porters, chaperone molecules, and target – proteins that bind to IREs in the 3′- and demonstrate that these cells contain very proteins (33 36). In fact, copper uptake fl 5′-untranslated regions in mRNAs of little iron as low Mr complexes (30). In fact, and ef ux involves chaperones as well as – molecules playing crucial roles in the up- the only low Mr iron present had kinetics organelle interactions (33 36). take, utilization, export, and storage of of iron uptake consistent with an end- Considering the arguments above, it has fi iron (e.g., TfR1, ferritin, etc.) (20). product rather than an intermediate (30). been shown that highly ef cient iron tar- geting to the mitochondrion is evident in erythroid cells where ferrochelatase inserts iron(II) into protoporphyrin IX [PPIX (11)]. Because Tf-bound iron is efficiently used for heme synthesis (11, 30) and no low Mr cytoplasmic iron-transport inter- mediate has been found in reticulocytes, an intimate direct transfer of iron from Tf to the mitochondrion was proposed to occur (37, 38). This idea has developed in more recent years and has led to the “kiss and run” hypothesis (11) (Fig. 1B). This model suggests that a direct transfer of iron from the Tf-containing endosome to the mitochondrion occurs, by-passing the cytosol (11, 28, 30, 39). The precise mo- lecular details involved in a possible con- tact between the endosome and mito- chondrion remain unknown. However, molecular motors, docking complexes, myosin Vb (40), and molecules involved in regulating the cytoskeleton, namely MRCKα (5), and also vesicular docking [e.g., Sec15l1 (9, 10)], point to mechanisms that may facilitate kiss and run (29).

Communication Between the Cytosol and Mitochondrion: Regulation of Iron Uptake. If a direct system of iron transfer exists be- tween the endosome and mitochondrion, Fig. 1. Schematics of cellular iron uptake. (A) The process or iron uptake and utilization. (B) The “kiss regulation of iron metabolism by the cell and run” hypothesis (see text). mustbecoordinatedwithironuptakeand

10776 | www.pnas.org/cgi/doi/10.1073/pnas.0912925107 Richardson et al. Downloaded by guest on September 29, 2021 transport to the mitochondrion. Such ho- tated by a better understanding of the meostatic mechanisms are not fully un- mechanisms of mitochondrial iron import derstood, but may work in concert with the and release. IRP-IRE mechanism to regulate iron ho- Considering the regulation of whole-cell meostasis. Evidence that regulatory pro- iron metabolism via molecular defects cesses exist that couple cytosolic and in the mitochondrion described above, it is mitochondrial iron metabolism can be de- intriguing to consider the possibility that duced from studies in vitro and in vivo. For similar mechanisms of communication example, when heme synthesis is inhibited in exist for other organelles that play key roles the mitochondrion using succinylacetone, in iron metabolism. For example, the ly- iron continues to enter the organelle (30, 41, sosome is involved in ferritin degradation, 42). This finding could be interpreted in two recycling of stored iron, and autophagy of ways. First, it may indicate little communi- other organelles, including the iron-rich cation and coupling between the cytosolic mitochondrion (55). The rupture of a and mitochondrial iron-metabolism ma- small number of lysosomes is an early chineries, as iron continues to be trans- upstream event in many cases of apopto- ported into the organelle irrespective of the sis, particularly oxidative stress-induced lack of heme synthesis. Second, it could apoptosis; necrosis results from a major suggest that iron continues to enter the mi- lysosomal rupture. Consequently, it has tochondrion because of a failure to generate been suggested that regulation of the ly- heme, which leads to a signal-to-import iron sosomal content of redox-active iron ap- in an effort to rescue heme synthesis. Fig. 2. Model of alteration in iron uptake in pears essential for cell survival (56). The latter concept suggests a coupling Friedreich’s ataxia. Frataxin-deficiency results in between the cytosolic- and mitochondrial increased mitochondrial-targeted iron uptake and Mitochondrial Iron Import iron-processing pathways and is supported cytosolic iron-deficiency (see text). Although DMT1 is responsible for the by several lines of evidence. For example, export of iron(II) from the endosome, the Tf and Tf-dependent iron uptake are in- itinerary of the metal from the cytosol to creased when heme synthesis is inhibited tured cells in vitro, have also demon- the inner mitochondrial membrane is using reticulocytes in vitro (41, 42), and strated that there is a cytosolic iron-deficit not well understood. However, it is known this is accomplished by an increase in in fibroblasts and lymphoblasts from that the inner membrane of the mito- TfR1 cycling rate (42). This response oc- Friedreich’s ataxia (FA) patients, as shown chondrion contains proteins capable of curs despite on-going mitochondrial iron- by increased IRP1/2-RNA-binding activ- transporting iron into the mitochondrial loading because of the inhibition of heme ity (49). Finally, overexpression of mito- matrix. Foury and Roganti suggested a role synthesis. Hence, the lack of heme gener- chondrial ferritin (Ftmt) in the mitochon- for the eukaryotic, mitochondrial solute ation appears to result in a compensatory drion leads to mitochondrial iron-loading carriers, Mrs3 and Mrs4, in mediating increase in Tf-bound iron uptake. Other and cytosolic iron-deprivation (50). Hence, mitochondrial iron metabolism in yeast evidence comes from studies in vivo using there is evidence that alterations in mito- (57). A further study demonstrated that the muscle creatine kinase conditional chondrial iron homeostasis lead to changes these proteins are essential for yeast frataxin knockout mouse (43), where con- in cellular iron metabolism, suggesting growth under iron-limiting conditions, ditional frataxin-deletion in cardiomyocytes communication between compartments suggesting that, at least in this organism, leads to depressed ISC and heme synthesis, and a modulating influence of the mi- an additional iron transport mechanism is mitochondrial iron-loading, TfR1 up-regu- tochondrion. present (58). A recent investigation of the lation, and thus increased iron uptake from The discussion above demonstrates that function of Mrs3 and -4 in small mito- Tf (44, 45) (Fig. 2). cytosolic iron metabolism is regulated, at chondrial particles showed these proteins Under these latter conditions, in the least in part, by the mitochondrial demand transport iron(II) along a concentration absence of frataxin, the defect in ISC and for iron that is critical for heme and ISC gradient (59). heme synthesis appears to lead to a “res- synthesis. This finding appears logical, Studies with the zebrafish mutant with cue response,” where iron uptake is in- because the mitochondrion is a focal point profound anemia, frascati, led to the dis- creased and targeted away from the of iron processing. Such compensatory covery of the Mrs3 and -4 homolog, and ferritin toward the mito- alterations in iron trafficking, demon- termed mitoferrin-1 (SLC25A37) and chondrion. This response leads to a rela- strating communication between the cy- mitoferrin-2 (SLC25A28) (60). These ho- tive cytosolic iron-deficiency and mito- tosol and mitochondrion, are also found mologous proteins are important for mi- chondrial iron-loading (44, 45). In the in other conditions where mitochondrial tochondrial iron uptake in erythroid and latter case, it was hypothesized that a sig- iron-processing is disturbed (51). For ex- nonerythroid cells, respectively (60, 61) nal (potentially caused by decreased ISC ample, similar mitochondrial iron-loading (Fig. 1A). Mitoferrin-1 is localized on the or heme levels) was sent from the mito- occurs in patients with a splice mutation inner mitochondrial membrane and func- chondrion to the cytoplasm to up-regulate in the ISC , Iscu (52), or tions as an essential importer of iron for iron uptake to compensate for the de- with a deficiency in -5 that is mitochondrial heme and ISC in erythro- pressed ISC and heme synthesis resulting involved in ISC/heme synthesis (53, 54). blasts and is necessary for erythropoiesis from frataxin-deficiency (45). Although Moreover, in a patient with a glutaredoxin- (60). Mitoferrin-1 is highly expressed in increased directional targeting of iron to 5deficiency, this problem led to decreased erythroid cells and in low levels in other the mitochondrion occurred, leading to ferritin and increased TfR1 expression tissues, whereas mitoferrin-2 is ubiqui- iron-loading in this organelle, it does not (54), which was a similar metabolic phe- tously expressed (61). The half-life of lead to an effective reversal of the mito- notype to that found in frataxin knockout mitoferrin-1 (but not mitoferrin-2) in- chondrial defect (44, 45), because frataxin, mice (44, 45). Further studies are necessary creases in developing erythroid cells and which plays a crucial role in ISC and heme to define at the molecular level how this this may be part of a regulatory mecha- synthesis, is deleted in cardiomyocytes communication occurs between the cytosol nism aiding mitochondrial iron uptake (43, 46–48). Similarly, Li et al., using cul- and the mitochondrion, and may be facili- (61). Recently, Abcb10, a mitochondrial

Richardson et al. PNAS | June 15, 2010 | vol. 107 | no. 24 | 10777 Downloaded by guest on September 29, 2021 inner-membrane ATP-binding cassette by an atypical intronless (66). How- and leads to sequential assembly leading transporter, was found to physically inter- ever, its role is unclear, particularly con- to [2Fe-2S] units that then form a [4Fe-4S] act with mouse Mfrn1, and thereby en- sidering its tissue distribution pattern. cluster (75). In yeast cells, these reactions hance the stability of the protein and Indeed, Ftmt was found at the highest lev- are accomplished by Isu1 and -2 (76), increase mitochondrial iron-import (62). els in the testes and the erythroblasts of but in humans, the function of IscU is Interestingly, Abcb10 has been suggested sideroblastic anemia patients (67, 68). Mi- performed via the splicing of IscU mRNA to play some role in heme synthesis and tochondrial ferritin has also been detected to lead to two transcripts which generate can be rapidly induced by the transcription in the , brain, spinal cord, kidney, and a cytosolic (Iscu1) or mitochondrial (Is- factor, GATA-1, which plays a role in pancreas (67). Unlike cytosolic ferritin, cu2) isoform. The maturation of extra- erythroid differentiation (63). Ftmt is not highly expressed in the liver and mitochondrial ISC proteins requires the It is still unknown how iron is trans- spleen, suggesting that it plays a distinct mitochondrial ISC assembly system (77). ported across the outer mitochondrial role. These findings led to the hypothesis The mitochondrion contributes a yet-to-be membrane and clearly other transporters that Ftmt plays a role in protection from discovered compound necessary for the may yet be identified. Considering this, iron-dependent oxidative damage (67). biogenesis of ISCs outside of this com- a large-scale computational screen identi- The role of Ftmt in iron metabolism was partment (i.e., in the cytosol or other or- fied three potential transporters that may examined by employing a stably transfected ganelles) (70). be involved in mitochondrial iron metab- cell line that hyper-expresses Ftmt (50). The mechanism involved in iron delivery olism, namely SLC25A39, SLC22A4, and These studies showed that overexpression to Iscu1 is not clear, but it has been sug- TMEM14C (64). In fact, targeted knock- of Ftmt resulted in increased IRP-1/2- gested to involve frataxin as an iron donor down of these in zebrafish resulted RNA-binding activity, decreased cytosolic (2). Moreover, frataxin has been shown to in profound anemia. Furthermore, silenc- and mitochondrial aconitase activity (sug- play a critical role in the early stages of ing of Slc25a39 in murine erythroleukemia gesting decreased ISC synthesis), de- ISC genesis (78), and this is discussed in cells impaired iron incorporation into creased cytoplasmic ferritin, increased detail below. In yeast, Isu1 only provides PPIX to form heme (64). TfR1 expression, decreased heme synthe- a cluster for de novo cluster production It is also notable that a mutation in the sis, decreased frataxin expression, and in- from which an HSP70-type chaperone transmembrane mitochondrial protein, creased iron-loading of Ftmt (50). Hence, system transfers the new clusters to apo- sideroflexin1, is responsible for the skeletal Ftmt overexpression leads to partitioning proteins (2). Yet another molecule, abnormalities and hematological phenotype of iron away from heme and ISC synthesis ABCB7, appears to mediate cytosolic ISC in the flexed-tail mouse model, namely in the mitochondrion. This effect not only biogenesis (79). The maturation of cyto- hypochromic, microcytic anemia, and side- alters mitochondrial iron metabolism, but solic ISCs is inhibited by mutations in rotic granules in erythrocytes (65). Because also whole-cell iron metabolism (50), lead- ABCB7 and this causes the disease, X- of its predicted five-transmembrane do- ing to a cytosolic iron-deficiency that re- linked sideroblastic anemia with cerebellar mains, this molecule has been suggested duces the proliferation of neoplastic cells ataxia (XLSA/A). This condition is char- to be a transporter essential for mitochon- hyper-expressing Ftmt in vivo (69). As al- acterized by loss of cytosolic ISC proteins, drial iron metabolism. Indeed, it has been ready discussed, very similar alterations of defects in heme metabolism, and in- speculated to transport molecules into or out also occur after ablation of creased mitochondrial iron levels (79). of the mitochondrion for iron utilization or frataxin expression (44, 45) and indicate Heme biosynthesis. The third major mito- heme synthesis (65). Sideroflexin may not communication between the cytosol and chondrial metabolic pathway that utilizes necessarily transport iron and could mediate mitochondrion. iron is that of heme synthesis that is ex- the uptake of other metabolites essential for Iron-sulfur cluster biosynthesis. Being a major clusive to this organelle (1, 11). Heme is heme synthesis (e.g., pyridoxine) that is site of ISC assembly, the mitochondrion synthesized by a pathway composed of necessary for the formation of pyridoxal plays a pivotal role in the biosynthesis of eight sequential reactions in the mito- phosphate, a coenzyme required for the first ISC proteins (1, 2). ISCs consist of iron chondrion and cytoplasm (1, 11). The first step in heme production (65). and sulfide anions (S2-), which assemble to and last three steps in the heme biosyn- form [2Fe-2S] or [4Fe-4S] clusters (2). thesis pathway take place in the mito- Mitochondrial Iron Metabolism These clusters form cofactors in proteins chondrion. The first in the path- Three Major Pathways: Heme Synthesis, ISC that perform vital functions, such as elec- way, 5-aminoleuvulinate synthase (ALAS), Synthesis, and Storage. Once iron is trans- tron transport, redox reactions, metabolic catalyzes the condensation of glycine and ported into the mitochondrion it can then catalysis, and other such functions (70). succinyl CoA to form 5-aminolevulinate be used for heme synthesis, ISC synthesis, In eukaryotic cells, more than 20 mole- (1, 11). There are two genes for ALAS, the or stored in Ftmt. It is essential that mi- cules facilitate maturation of ISC proteins ubiquitously expressed ALAS1 and the tochondrial iron is maintained in a safe in the mitochondria, cytosol, and nucleus erythroid-specific ALAS2, which is under form to prevent oxidative damage, as mi- (2). Functional defects in ISC proteins the regulation of iron via the IRP system tochondria are a major source of cytotoxic or components involved in their biogenesis (1). In nonerythroid cells, the rate of ROS (3). Hence, it is likely that as in the lead to human diseases (71, 72). Bio- heme synthesis is dependent on the rate cytosol, iron is carefully transported within synthesis of ISCs and their insertion into of 5-aminolevulinate synthesis via ALAS1 the mitochondrion in a form distal to the apo-proteins requires both the mitochon- (11). In contrast, in erythroid cells, the rate- aqueous environment, deep in the hydro- drial and cytosolic machinery. The first limiting step may be the delivery of Tf- phobic pockets of communicating proteins molecule identified in the ISC machinery iron (11). that form iron transport pathways. Al- was the enzyme NifS from Azobacter vin- The subsequent four steps of heme though the molecular nature of these cir- landii, which is a that biosynthesis take place in the cytosol, fol- cuits remains unclear, the pathways that participates in ISC assembly as a sulfur do- lowing which coproporphyrinogen III use iron are well known and are discussed nor (73). This enzyme is highly conserved is decarboxylated and then oxidized in below. and in humans is known as Nfs1 (74). the mitochondrial intermembrane Mitochondrial iron storage: mitochondrial ferritin. ISCs are assembled on scaffold proteins space to produce PPIX (1, 11). Cop- Like a typical ferritin, Ftmt shows ferrox- and then transferred to apo-proteins. In roporphyrinogen may be transported into idase activity and binds iron (66). In contrast Escherichia coli, this scaffold protein is mitochondria by either peripheral-type to cytoplasmic ferritin, Ftmt is encoded known as ISC assembly protein U (IscU) benzodiazepine receptors (80) or

10778 | www.pnas.org/cgi/doi/10.1073/pnas.0912925107 Richardson et al. Downloaded by guest on September 29, 2021 potentially ABCB6 (81). The seventh step, tions are discussed in detail in the follow- which is catalyzed by protoporphyrinogen ing sections. IX oxidase generates PPIX. In the final reaction of the pathway, iron(II) is inserted Friedreich’s Ataxia and the Metabolic Role of into PPIX by the ISC protein, ferrochela- Frataxin. FA is a rare disease leading to tase, in the mitochondrial matrix (11). severe neuro- and cardio-degeneration and is caused by a deficiency of frataxin (85). Mitochondrial Iron Export. Apart from The decrease in frataxin expression is importing iron, the mitochondrion synthe- caused by an intronic GAA-repeat ex- sizes heme and ISCs that subsequently are pansion in -1 of FRDA. Frataxin is transported out to the cytosol. These export a vital protein that is highly expressed in processes are important to understand, as tissues rich in mitochondria (e.g., heart decreased release of iron as heme or ISCs and neurons) (86), with deletion leading to —or their precursors—can contribute to lethality (87). mitochondrial iron-loading, as found in the The suggested functions for frataxin all frataxin knockout mouse (45). A candidate relate to iron metabolism (Fig. 3) and in- iron exporter has been identified, namely clude ISC and heme biogenesis, as well mammalian mitochondrial ABC protein 3 as iron storage. Frataxin was linked to ISC (MTABC3; also known as ABCB6) (82). proteins by the observation that there was Fig. 3. Schematic of the possible functions of This protein can rescue mitochondrial iron- adeficiency in ISC cluster proteins in frataxin. (A) Frataxin may perform a mitochondrial loading, respiratory dysfunction, and mito- knock-out mice, FA patients, and yeast iron-storage function similar to ferritin. (B) Fra- atm1 fi – taxin may function as an iron chaperone by binding chondrial DNA damage in -de cient (43, 46 48). Frataxin has also been impli- iron and then delivering it. (C) The frataxin or- yeast cells (82). Of relevance, the human cated in iron scavenging (88), regulating tholog, CyaY may function as an “iron-sensing”- ortholog of atm1 is ABCB7, which can oxidative stress (89), and as an iron chap- negative regulator that inhibits the rate of ISC as- complement atm1 deficiencies in yeast erone (90). To date, the cumulative evi- sembly under conditions of high mitochondrial cells, enabling the maturation of ISC-con- dence suggests frataxin is involved in the iron and low ISC apo-acceptor availabilities. (D) taining proteins in the cytosol (79). It has maintenance of iron homeostasis (44, 45). Frataxin may also function as a “metabolic switch” been suggested that ABCB7 transports Frataxin is an inner mitochondrial mem- that allows the mitochondrion to favor heme or ISCs to the cytoplasm (79), although this brane and mitochondrial-matrix protein ISC/heme syntheses, depending on frataxin and has not been directly shown. (85). However, as there does not appear to protoporphyrin IX levels (91, 99) (see text). It is unknown how heme is exported be any structural feature that would an- from the mitochondrion. However, its low chor frataxin to the mitochondrial mem- Spontaneously oligomerized recombi- solubility and highly toxic nature suggest an brane, it is possible that membrane- nant human frataxin appears capable of efficient heme-carrier must be involved. associated frataxin is bound to other pro- binding and storing iron (97, 98). However, Considering this, heme-binding protein 1 teins in a macromolecular complex con- recombinant human frataxin monomers, has been identified as a candidate for this taining ferrochelatase (91). unlike those of Yfh1, cannot be induced to carrier (83). The expression of this mole- Recent insight into frataxin function has oligomerize in vitro by iron (98). More- cule is ubiquitous, but it is also increased come from studies of frataxin orthologs over, the spontaneous oligomerization of during erythroid differentiation and high in yeast (i.e., Yfh1) and bacteria (i.e., human frataxin appears to be dependent levels are found in the liver (83). Heme- CyaY) that share a high degree of sequence on heterologous overexpression (98). Such binding protein 1 binds one heme per identity to the human protein (92). A considerations argue against an in vivo mole of protein (83) and, although it could consensus is that the functions of frataxin iron-storage function for human frataxin. be a candidate for heme transport, direct share a requirement for iron-binding (92– The observation that the manipulation evidence for this is lacking. 94). Although there are several propo- of mitochondrial iron levels does not affect sals, the exact function of frataxin remains frataxin expression levels is not consistent Diseases of Mitochondrial Iron controversial and is discussed in the with a significant role in mitochondrial Metabolism following sections. iron storage (99). Theoretical support for Mechanisms of iron transport across mi- Frataxin and iron storage. Frataxin has been the generality of the “iron-storage” hy- tochondrial membranes have evolved to proposed to function as a mitochondrial pothesis was also lessened upon the dis- supply the necessary iron to mitochondria iron storage protein (95) (Fig. 3A). The covery of Ftmt in higher organisms (66, and also maintain the balance of cytosolic frataxin ortholog, Yfh1, was reported to be 100). The existence of Ftmt appears to iron (1). Mitochondrial iron levels must capable of self-assembling into oligomers make redundant any significant iron- be tightly controlled as iron induces ROS, and then into higher-order multimers in storage role for human frataxin. However, which can damage the organelle (84). the presence of increasing iron(II) (96). yeast cells do not express (100), The importance of these tight controls is The iron-dependent oligomerization of and it is possible that Yfh1 may possess highlighted by the fact that alterations in Yfh1 is associated with the development an additional iron-storage capacity not mitochondrial iron homeostasis have of activity that appears to exist shared by frataxin orthologs in higher pathological consequences (1, 70). In organisms. only in the oligomeric/multimeric struc- recent years, cellular and animal models of Frataxin as an iron chaperone. Perhaps the ture (92). This finding was proposed to mitochondrial iron dysfunction have pro- most promising emerging role for frataxin vided valuable information in identifying allow Yfh1 oligomers/multimers to store is as an iron chaperone (Fig. 3B) for ISC and new proteins to elucidate the pathways iron in a mineralized and redox-inactive heme biosyntheses. Frataxin has been ob- involved in mitochondrial iron homeosta- iron(III) state (92, 97). However, because served to interact with, and presumably sis. Interesting examples of mitochondrial the iron-dependent oligomerization of donate iron to, iron-dependent proteins diseases that have provided important Yfh1 only occurs in the absence of Ca2+ and involved in ISC and heme biosyntheses (90, insight into the processes of mitochondrial Mg2+, which are typically abundant in the 91). For example, Yfh1 appears capable of iron metabolism are Friedreich’s ataxia mitochondrion (92, 93, 96), this diminishes interacting with the central ISC assembly and sideroblastic anemia. These condi- support for the iron-storage hypothesis. complex comprising the scaffold protein,

Richardson et al. PNAS | June 15, 2010 | vol. 107 | no. 24 | 10779 Downloaded by guest on September 29, 2021 Isu, and the cysteine desulfurase, Nfs1, in esis is supported by the observation that without a heme synthesis defect in ery- a manner enhanced by iron(II) (90, 101). the immediate precursor for heme syn- throblasts. Recently, it has been suggested that thesis, PPIX, down-regulates frataxin ex- It can be proposed that the combination frataxin interacts with Isu1 via a highly pression (99). Hence, increased PPIX of several factors plays a role in the path- conserved tryptophan residue (W131a) in levels, which indicate a requirement for ogenesis of mitochondrial iron accumula- its conserved β-sheet region (102). Anal- heme synthesis, lead to decreased frataxin tion in sideroblastic anemias associated ogously, human frataxin demonstrates expression and a diversion of iron from with a heme synthesis defect: (i) iron is iron-enhanced interactions with Isu and other mitochondrial pathways (i.e., ISC specifically targeted toward erythroid mi- ferrochelatase (91, 103). Importantly, the synthesis or iron storage) to heme bio- tochondria; (ii) iron cannot be used for interaction of frataxin with either Isu or genesis (99). heme synthesis because of the lack of ferrochelatase appears to increase the rate This latter hypothesis is supported by the PPIX; (iii) there is a lack of heme, the of ISC synthesis (90) or the ferrochelatase- observation that an increase in frataxin negative regulator of iron uptake from Tf; catalyzed insertion of iron(II) into PPIX levels relative to ferrochelatase (i.e., above and (iv) iron can leave erythroid mito- (91), respectively. Such observations sug- a molar ratio of 1:1 frataxin:ferrochelatase chondria only after being inserted into gest frataxin may function as an iron- dimer) results in decreased rates of PPIX. A key example of this scenario is X- donor to Isu and ferrochelatase. Emerging heme synthesis in vitro (91). It has also linked sideroblastic anemia, which is data indicate that the nature of frataxin’s been observed that iron-bound human caused by mutations in erythroid-specific facilitatory interactions with the ISC frataxin has a higher putative binding af- ALAS2 (104). As already discussed, a dis- and heme synthesis machinery may be finity for ferrochelatase (17 nM) than tinct form of X-linked sideroblastic ane- more complex than just simple iron- Isu (480 nM) (91). These observations mia is XLSA/A. This condition is caused donation. As discussed in the next sec- provide a basic mechanism that supports by mutations in ABCB7 (79), whose tion, a possible primary function of fra- the hypothesis that frataxin may allow product is thought to transfer an ISC ’ taxin s interactions may be to additionally metabolic switching between ISC and precursor from mitochondria to the cyto- negatively regulate the respective bio- heme synthesis pathways depending on sol. In XLSA/A, as is the case in ALAS2- synthetic interactions. expression levels relative to those of Isu associated sideroblastic anemia, decreased Frataxin as an iron-sensing negative-regulator. and ferrochelatase (91, 99). levels of heme are likely to contribute to A recent study with CyaY suggests that ’ the pathogenesis of ring sideroblast for- “ ” A frataxin metabolon? Frataxin sputative the protein may act as an iron-sensor ability to modulate iron-dependent bio- mation. In refractory anemia with ring (Fig. 3C) that negatively regulates the rate sideroblasts (RARS), there is no evidence chemical reactions through protein- ’ of ISC biosynthesis under conditions of protein interactions is suggestive of the for a defect in PPIX formation in patients high iron and low ISC apo-acceptor possibility that it may form one or more erythroblasts. In some patients with availabilities (89). If we extend this model metabolons, or protein complexes, with RARS, acquired mutations in subunits to eukaryotic systems, a deficiency in fra- of cytochrome oxidase encoded by mito- proteins involved in ISC and heme bio- taxin expression is presumably deleteri- chondrial DNA have been described syntheses (94). The conserved tryptophan ous because the rate of ISC biosynthesis (105). It has been proposed that this defect residue-131 in frataxin, which is respon- may exceed the availability of ISC apo- could lead to impaired iron reduction sible for its interaction with Isu1, suggests acceptors, resulting in the overproduction that is needed for heme and ISC synthesis, of ISCs that are unstable in an unbound the association is crucial for its function, and without this, mitochondrial iron de- form (89). Essentially, this model suggests which is underlined by the fact that mu- posits occur. that over and above functioning as an iron tating this residue results in a loss of mi- It can also be hypothesized that ery- donor in ISC biosynthesis, frataxin may tochondrial aconitase activity (102). This throid progenitors of patients with RARS, exert “kinetic control” over the rate of hypothesis suggests that the loss of the characterized by genomic instability and ISC biosynthesis, depending on the rela- interaction with Isu1 results in an im- premature apoptosis, exhibit anomalous tive availabilities of iron and ISC apo- pairment of ISC synthesis and supports induction of mitochondrial ferritin that acceptors. This possible iron-sensing role the notion of a functional protein com- would lead to a shift of iron into their is consistent with the relatively low (i.e., plex involving frataxin. In general, me- mitochondria (50, 69). This would prevent micromolar) affinities of iron-binding by tabolons are dynamic protein complexes the use of iron for hemoglobin synthesis bacterial, yeast and human frataxin or- that greatly enhance the efficiency of and cause a ring-sideroblast phenotype. In thologs (92). The applicability of this metabolic reactions through processes, fact, any metabolic abnormality that model remains to be examined in mam- such as substrate channeling. On the basis markedly affects the synthesis of ISCs or mals. It also needs to be assessed whether of the possibility that frataxin may act heme is likely to result in mitochondrial any such kinetic control is elicited by fra- as an iron-donor, it might be expected iron-loading. Actually, it has recently been taxin’s interaction with ferrochelatase that frataxin could be part of a mitochon- shown that mutations in the putative gly- during heme biosynthesis. drial metabolon consisting of ISC as- cine transporter, SLC25A38, lead to Frataxin as an expression-regulated “metabolic sembly components, such as Iscu, and a rare form of sideroblastic anemia (106). switch.” An extension of the notion of fra- heme biosynthesis , such as fer- This iron-loading probably occurs be- taxin as a negative regulator to frataxin’s rochelatase (Fig. 3). cause ALAS catalyzes the reaction of functional role in heme biosynthesis may glycine with succinyl CoA to generate 5- help to explain the decline in frataxin Sideroblastic Anemias. The characteristic aminolevulinate. Without glycine, PPIX levels during erythroid differentiation (99) feature of all sideroblastic anemias is the synthesis would be inhibited which pre- (Fig. 3D). That is, because frataxin ex- ring sideroblast. This is a pathological vents heme generation. Defects in other pression is markedly decreased during erythroid precursor containing excessive metabolic pathways, which affect mito- Friend cell hemoglobinization (99), it deposits of nonheme iron in mitochondria chondrial iron metabolism, can also lead is possible that frataxin may be down- with peri-nuclear distribution responsi- to sideroblastic anemia. An example of regulated during erythroid differentiation ble for the ring appearance. With consid- this would be patients with mutations in to allow higher rates of heme synthesis, erable simplification, and from the point of pseudouridine synthase-1, which is in- potentially at the expense of decreased view of pathogenesis, sideroblastic ane- volved in the processing of mitochondrial levels of ISC synthesis (99). This hypoth- mias can be divided into those with or tRNAs (107).

10780 | www.pnas.org/cgi/doi/10.1073/pnas.0912925107 Richardson et al. Downloaded by guest on September 29, 2021 Summary strong evidence to suggest that the mito- ACKNOWLEDGMENTS. D.R.R.’s laboratory Although the mitochondrion is a focal chondrion can modulate the cellular iron was supported by grants from the National Health point for iron utilization in heme and ISC uptake machinery to satisfy its demand. and Medical Research Council of Australia, the US Muscular Dystrophy Association (MDA), synthesis, there has been little realization Considering this evidence, it is likely that ’ signaling pathways exist that allow com- MDA New South Wales, Friedreich s Ataxia Re- that the mitochondrion can play an im- search Alliance (FARA) Australia, and FARA USA. portant role in orchestrating whole-cell munication between the mitochondrion P.P and A.D.S. were supported by grants from iron metabolism. Indeed, analysis of dis- and cytoplasm, enabling the mitochon- the Canadian Institutes of Health Research. ease states has enabled understanding of drial iron processing machinery to be Y.S.R. was a grateful recipient of a Cancer Institute the role of the mitochondrion in regu- supplied with this metal to allow heme and New South Wales Early Career Development lating cellular iron metabolism. There is ISC synthesis. Fellowship.

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