Cytosolic Iron-sulfur Protein Assembly Research Articles

Characterization of the SUF FeS cluster synthesis machinery in the amitochondriate eukaryote Monocercomonoides exilis

Monocercomonoides exilis is the first known amitochondriate eukaryote. Loss of mitochondria in M.exilis ocurred after the replacement of the essential mitochondrial iron-sulfur cluster (ISC) assembly machinery by a unique, bacteria-derived, cytosolic SUF system. It has been hypothesized that the MeSuf pathway, in cooperation with proteins of the cytosolic iron-sulfur protein assembly (CIA) system, is responsible for the biogenesis of FeS clusters in M.exilis, yet biochemical evidence is pending. Here, we address the M.exilis MeSuf system and show that SUF genes, individually or in tandem, support the loading of iron-sulfur (FeS) clusters into the reporter protein IscR in Escherichia coli. The Suf proteins MeSufB, MeSufC, and MeSufDSU interact invivo with one another and with Suf proteins of E.coli. Invitro, the M.exilis Suf proteins form large complexes of varying composition and hence may function as a dynamic biosynthetic system in the protist. The putative FeS cluster scaffold MeSufB-MeSufC (MeSufBC) forms multiple oligomeric complexes, some of which bind FeS clusters and form selectively only in the presence of adenosine nucleotides. The multi-domain fusion protein MeSufDSU binds a PLP cofactor and can form higher-order complexes with MeSufB and MeSufC. Our work demonstrates the biochemical property of M. exilis Suf proteins to act as a functional FeS cluster assembly system and provides insights into the molecular mechanism of this unique eukaryotic SUF system.

The critical role of the iron-sulfur cluster and CTC components in DOG-1/BRIP1 function in Caenorhabditis elegans.

FANCJ/BRIP1, initially identified as DOG-1(Deletions Of G-rich DNA) in Caenorhabditiselegans, plays a critical role in genome integrity by facilitating DNA interstrand cross-link repair and resolving G-quadruplex structures. Its function is tightly linked to a conserved [4Fe-4S] cluster-binding motif, mutations of which contribute to Fanconi anemia and various cancers. This study investigates the critical role of the iron-sulfur (Fe-S) cluster in DOG-1 and its relationship with the cytosolic iron-sulfur protein assembly targeting complex (CTC). We found that a DOG-1 mutant, expected to be defective in Fe-S cluster binding, is primarily localized in the cytoplasm, leading to heightened DNA damage sensitivity and G-rich DNA deletions. We further discovered that the deletion of mms-19, a nonessential CTC component, also resulted in DOG-1 sequestered in cytoplasm and increased DNA damage sensitivity. Additionally, we identified that CIAO-1 and CIAO-2B are vital for DOG-1's stability and repair functions but unlike MMS-19 have essential roles in C. elegans. These findings confirm the CTC and Fe-S cluster as key elements in regulating DOG-1, crucial for genome integrity. Additionally, this study advances our understanding of the CTC's role in Fe-S protein regulation and development in C. elegans, offering a model to study its impact on multicellular organism development.

CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders

PurposeThe functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. MethodsGenetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. ResultsMetabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. ConclusionA general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide.

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO- tripeptide is present at the C-terminus of more than a quarter of clients or their adaptors. When present, this targeting complex recognition (TCR) motif is necessary and sufficient for binding to the CTC in vitro and for directing Fe-S cluster delivery in vivo. Remarkably, fusion of this TCR signal enables engineering of cluster maturation on a nonnative protein via recruitment of the CIA machinery. Our study advances our understanding of Fe-S protein maturation and paves the way for bioengineering novel pathways containing Fe-S enzymes.

Metformin inhibits oral squamous cell carcinoma progression through regulating RNA alternative splicing

AimsOral squamous cell carcinoma (OSCC) is considered as the sixth most common cancer worldwide characterized by high invasiveness, high metastasis rate and high mortality. It is urgent to explore novel therapeutic strategies to overcome this feature. Metformin is currently a strong candidate anti-tumor drug in multiple cancers. However, whether metformin could inhibit cancer progression by regulating RNA alternative splicing remains largely unknown. Main methodsCell proliferation and growth ability of CAL-27 and UM-SCC6 were analyzed by CCK8 and colony formation assays. Cell migration was judged by wound healing assay. Mechanistically, RNA-seq was applied to systematically identify genes that are regulated by metformin. The expression of metformin-regulated genes was determined by real-time quantitative PCR (RT-qPCR). Metformin-regulated alternative splicing events were confirmed by RT-PCR. Key findingsWe demonstrated that metformin could significantly inhibit the proliferation and migration of oral squamous cell carcinoma cells. Mechanistically, in addition to transcriptional regulation, metformin induces a wide range of alternative splicing alteration, including genes involved in centrosome, cellular response to DNA damage stimulus, GTPase binding, histone modification, catalytic activity, regulation of cell cycle process and ATPase complex. Notably, metformin specifically modulates the splicing of NUBP2, a component of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA). Briefly, metformin favors the production of NUBP2-L, the long splicing isoform of NUBP2, thereby inhibiting cancer cell proliferation. SignificanceOur findings provide mechanistic insights of metformin on RNA alternative splicing regulation, thus to offer a potential novel route for metformin to inhibit cancer progression.

Isolation and characterization of IbAE7-like 1 gene encoding the cytosolic iron-sulfur cluster assembly pathway in response to skinning injury in sweetpotato

Abstract. Effendy J. 2022. Isolation and characterization of IbAE7-like 1 gene encoding the cytosolic iron-sulfur cluster assembly pathway in response to skinning injury in sweetpotato. Biodiversitas 23: 4223-4233. Skinning injury is a major constrain in postharvest loss in sweetpotato due to the loss of skin from the surface of the roots. ASYMMETRIC LEAVES1/2 ENHANCER7(AE7) is an important cytosolic iron-sulfur (Fe-S) protein assembly (CIA) pathway. A partial cDNAIbI51 encoding AE7-like 1 was isolated from a skinning injury cDNA library to evaluate its potential role in response to skinning injury. The open reading frameof IbAE7-like 1 consisted of 324 nucleotides and the deduced polypeptide sequences consistedof 108 amino acids with missing 46 amino acid residues at 5? end. The amino acid sequence ofIbAE7-like 1 shared 100% identity toAE7-like 1 previously identified in Ipomoea triloba and Ipomoea nil and 89.91-100% identity from other plants species. The findings indicated that AE7-like 1 was highly conserved in Ipomoea sp. In addition to similarity to AE7-like 1, IbAE7-like 1 also showed similarities to MIP18 (89.91-93.52%); DUF59 (91.67%); FAM96B (90.74%); and proteins with unknown function (90.74-91.67%). IbAE7-like 1 mRNA transcripts showed transient expression in response to skinning injury. The phylogenetic analysis classified IbAE7-like 1 into three main classes. This study revealed IbAE7-like 1 may play a key role in plant defense and wound repair to prevent DNA damage due to skinning injuryin storage roots of sweetpotato.

The human YAE1-ORAOV1 complex of the cytosolic iron-sulfur protein assembly machinery binds a [4Fe-4S] cluster

Iron-sulfur (Fe-S) clusters are among the most versatile cofactors in biology. Although Fe-S clusters formation can be achieved spontaneously in vitro with inorganic iron and sulfur sources, the in vivo behaviour is more complex and requires the so-called Fe-S biogenesis machineries. In the cytosol, the biogenesis of Fe-S proteins is assisted by the cytosolic Fe-S protein assembly machinery, which comprises at least thirteen known proteins, among which there are human ORAOV1 and YAE1. A hetero-complex formed by the two latter proteins facilitates Fe-S cluster insertion in the human ABC protein ABCE1 within a chain of binding events that are still not well understood. In the present work, ORAOV1 and the YAE1-ORAOV1 complex were produced and their structural and cluster binding properties spectroscopically investigated. It resulted that both ORAOV1 and the YAE1-ORAOV1 complex are characterized by well-structured, α -helical regions and by unstructured, flexible regions, and are both able to bind a [4Fe-4S]2+ cluster. Bioinformatics and site-directed mutagenesis studies indicated that the [4Fe-4S] cluster in ORAOV1 is bound by a conserved cluster binding motif, while YAE1, which does not have a metal-binding consensus motif, is not essential for the [4Fe-4S]2+ cluster binding in the YAE1-ORAOV1 hetero-complex. Overall, these results support a model that the YAE1-ORAOV1 complex might actively participate in the Fe-S cluster insertion into ABCE1 thanks to the [4Fe-4S]2+ cluster binding properties of ORAOV1.

CIAO3 protein forms a stable ternary complex with two key players of the human cytosolic iron-sulfur cluster assembly machinery.

The CIAO3 protein operates at a crossroad of the cytosolic iron-sulfur protein assembly (CIA) machinery. Although the functional role of CIAO3 has been recently characterized, a description of its interaction network is still not complete. Literature data suggested that CIAO3 interacts individually with CIA2A and CIAO1 protein, with the latter two interacting each other. However, no experimental data are available yet showing the formation of a possible ternary complex composed by CIAO3, CIAO1, and CIA2A. This work shows, for the first time, via size exclusion chromatography coupled with multiangle light scattering, UV-vis absorption and electron paramagnetic resonance (EPR) spectroscopies, the formation of a stable, [4Fe-4S]-bound, complex, composed by CIAO3 and the hetero-CIA2A-CIAO1 complex. Moreover, site-directed mutagenesis data suggested a structural role for the C-terminal [4Fe-4S] cluster of the CIAO3 protein. These findings can provide solid bases for further investigation of the molecular mechanisms involving these CIA machinery proteins.

Iron-Sulfur Cluster Biosynthesis in Algae with Complex Plastids.

Plastids surrounded by four membranes harbor a special compartment between the outer and inner plastid membrane pair, the so-called periplastidal compartment (PPC). This cellular structure is usually presumed to be the reduced cytoplasm of a eukaryotic phototrophic endosymbiont, which was integrated into a host cell and streamlined into a plastid with a complex membrane structure. Up to date, no mitochondrion or mitochondrion-related organelle has been identified in the PPC of any representative. However, two prominent groups, the cryptophytes and the chlorarachniophytes, still harbor a reduced cell nucleus of symbiont origin, the nucleomorph, in their PPCs. Generally, many cytoplasmic and nucleus-located eukaryotic proteins need an iron–sulfur cofactor for their functionality. Beside some exceptions, their synthesis is depending on a so-called iron–sulfur complex (ISC) assembly machinery located in the mitochondrion. This machinery provides the cytoplasm with a still unknown sulfur component, which is then converted into iron–sulfur clusters via a cytosolic iron–sulfur protein assembly (CIA) machinery. Here, we investigated if a CIA machinery is present in mitochondrion-lacking PPCs. By using bioinformatic screens and in vivo-localizations of candidate proteins, we show that the presence of a PPC-specific CIA machinery correlates with the presence of a nucleomorph. Phylogenetic analyses of PPC- and host specific CIA components additionally indicate a complex evolution of the CIA machineries in organisms having plastids surrounded by four membranes.

Fe-S cluster coordination of the chromokinesin KIF4A alters its subcellular localization during mitosis.

Fe-S clusters act as co-factors of proteins with diverse functions, for example, in DNA repair. Downregulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability through the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B (also known as FAM96B) and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, colocalize with components of the mitotic machinery. Downregulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon downregulation of the CIA targeting complex contributes to the mitotic defects.

Biochemical Reconstitution and Spectroscopic Analysis of Iron-Sulfur Proteins.

Iron-sulfur (Fe/S) proteins are involved in numerous key biological functions such as respiration, metabolic processes, protein translation, DNA synthesis, and DNA repair. The simplest types of Fe/S clusters include [2Fe-2S], [3Fe-4S], and [4Fe-4S] forms that sometimes are present in multiple copies. De novo assembly of Fe/S cofactors and their insertion into apoproteins in living cells requires complex proteinaceous machineries that are frequently highly conserved. In eukaryotes such as yeast and mammals, the mitochondrial iron-sulfur cluster assembly machinery and the cytosolic iron-sulfur protein assembly system consist of more than 30 components that cooperate in the generation of some 50 cellular Fe/S proteins. Both the mechanistic dissection of the intracellular Fe/S protein assembly pathways and the identification and characterization of Fe/S proteins rely on tool boxes of in vitro and in vivo methods. These cell biological, biochemical, and biophysical techniques help to determine the extent, stability, and type of bound Fe/S cluster. They also serve to distinguish bona fide Fe/S proteins from other metal-binding proteins containing similar cofactor coordination motifs. Here, we present a collection of in vitro methods that have proven useful for basic biochemical and biophysical characterization of Fe/S proteins. First, we describe the chemical assembly of [2Fe-2S] or [4Fe-4S] clusters on purified apoproteins. Then, we summarize a reconstitution system reproducing the de novo synthesis of a [2Fe-2S] cluster in mitochondria. Finally, we explain the use of UV-vis, CD, electron paramagnetic resonance, and Mössbauer spectroscopy for the routine characterization of Fe/S proteins.

Cellular requirements for iron–sulfur cluster insertion into the antiviral radical SAM protein viperin

Viperin (RSAD2) is an interferon-stimulated antiviral protein that belongs to the radical S-adenosylmethionine (SAM) enzyme family. Viperin's iron-sulfur (Fe/S) cluster is critical for its antiviral activity against many different viruses. CIA1 (CIAO1), an essential component of the cytosolic iron-sulfur protein assembly (CIA) machinery, is crucial for Fe/S cluster insertion into viperin and hence for viperin's antiviral activity. In the CIA pathway, CIA1 cooperates with CIA2A, CIA2B, and MMS19 targeting factors to form various complexes that mediate the dedicated maturation of specific Fe/S recipient proteins. To date, however, the mechanisms of how viperin acquires its radical SAM Fe/S cluster to gain antiviral activity are poorly understood. Using co-immunoprecipitation and 55Fe-radiolabeling experiments, we therefore studied the roles of CIA2A, CIA2B, and MMS19 for Fe/S cluster insertion. CIA2B and MMS19 physically interacted with the C terminus of viperin and used CIA1 as the primary viperin-interacting protein. In contrast, CIA2A bound to viperin's N terminus in a CIA1-, CIA2B-, and MMS19-independent fashion. Of note, the observed interaction of both CIA2 isoforms with a single Fe/S target protein is unprecedented in the CIA pathway. 55Fe-radiolabeling experiments with human cells depleted of CIA1, CIA2A, CIA2B, or MMS19 revealed that CIA1, but none of the other CIA factors, is predominantly required for 55Fe/S cluster incorporation into viperin. Collectively, viperin maturation represents a novel CIA pathway with a minimal requirement of the CIA-targeting factors and represents a new paradigm for the insertion of the Fe/S cofactor into a radical SAM protein.

Anamorsin/Ndor1 Complex Reduces [2Fe-2S]-MitoNEET via a Transient Protein-Protein Interaction.

Human mitoNEET is a homodimeric protein anchored to the outer mitochondrial membrane and has a C-terminal [2Fe-2S] binding domain located in the cytosol. Recently, human mitoNEET has been shown to be implicated in Fe/S cluster repair of cytosolic iron regulatory protein 1 (IRP1), a key regulator of cellular iron homeostasis in mammalian cells. The Fe/S cluster repair function of mitoNEET is based on an Fe/S redox switch mechanism: under normal cellular conditions, reduced [2Fe-2S]+-mitoNEET is present and is inactive as an Fe/S cluster transfer protein; under conditions of oxidative cellular stress, the clusters of mitoNEET become oxidized, and the formed [2Fe-2S]2+-mitoNEET species reacts promptly to initiate Fe/S cluster transfer to IRP1, recycling the cytosolic apo-IRP1 into holo-aconitase. Until now, no clear data have been available on which is the system that reduces the mitoNEET clusters back once oxidative stress is not present anymore. In the present work, we used UV-vis and NMR spectroscopies to investigate the electron transfer process between mitoNEET and the cytosolic electron-donor Ndor1/anamorsin complex, a component of the cytosolic iron-sulfur protein assembly (CIA) machinery. The [2Fe-2S] clusters of mitoNEET are reduced via the formation of a transient complex that brings the [2Fe-2S] clusters of mitoNEET close to the redox-active [2Fe-2S] cluster of anamorsin. Our data provide in vitro evidence of a possible direct link between the CIA machinery and the mitoNEET cluster transfer repair pathway. This link might contribute to recovery of CIA machinery efficiency to mature cytosolic and nuclear Fe/S proteins.

The diferric-tyrosyl radical cluster of ribonucleotide reductase and cytosolic iron-sulfur clusters have distinct and similar biogenesis requirements

How each metalloprotein assembles the correct metal at the proper binding site presents challenges to the cell. The di-iron enzyme ribonucleotide reductase (RNR) uses a diferric-tyrosyl radical (FeIII2-Y•) cofactor to initiate nucleotide reduction. Assembly of this cofactor requires O2, FeII, and a reducing equivalent. Recent studies show that RNR cofactor biosynthesis shares the same source of iron, in the form of [2Fe-2S]-GSH2 from the monothiol glutaredoxin Grx3/4, and the same electron source, in the form of the Dre2-Tah18 electron transfer chain, with the cytosolic iron-sulfur protein assembly (CIA) machinery required for maturation of [4Fe-4S] clusters in cytosolic and nuclear proteins. Here, we further investigated the interplay between the formation of the FeIII2-Y• cofactor in RNR and the cellular iron-sulfur (Fe-S) protein biogenesis pathways by examining both the iron loading into the RNR β subunit and the RNR catalytic activity in yeast mutants depleted of individual components of the mitochondrial iron-sulfur cluster assembly (ISC) and the CIA machineries. We found that both iron loading and cofactor assembly in RNR are dependent on the ISC machinery. We also found that Dre2 is required for RNR cofactor formation but appears to be dispensable for iron loading. None of the CIA components downstream of Dre2 was required for RNR cofactor formation. Thus, the pathways for RNR and Fe-S cluster biogenesis bifurcate after the Dre2-Tah18 step. We conclude that RNR cofactor biogenesis requires the ISC machinery to mature the Grx3/4 and Dre2 Fe-S proteins, which then function in iron and electron delivery to RNR, respectively.

Evolutionary conservation and in vitro reconstitution of microsporidian iron\u2013sulfur cluster biosynthesis

Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (mitosomes) of Trachipleistophora hominis are the functional site of iron–sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe–2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron–sulfur protein assembly (CIA) pathway includes the essential Cfd1–Nbp35 scaffold complex that assembles a [4Fe–4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides compelling evidence for the ancient chimeric ancestry of eukaryotes.

MET18 Deficiency Increases the Sensitivity of Yeast to Oxidative Stress and Shortens Replicative Lifespan by Inhibiting Catalase Activity.

Yeast MET18, a subunit of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery which is responsible for the maturation of Fe/S proteins, has been reported to participate in the oxidative stress response. However, the underlying molecular mechanisms remain unclear. In this study, we constructed a MET18/met18Δ heterozygous mutant yeast strain and found that MET18 deficiency in yeast cells impaired oxidative stress resistance as evidenced by increased sensitivity to hydrogen peroxide (H2O2) and cumene hydroperoxide (CHP). Mechanistically, the mRNA levels of catalase A (CTA1) and catalase T (CTT1) as well as the total catalase activity were significantly reduced in MET18-deficient cells. In contrast, overexpression of CTT1 or CTA1 in MET18-deficient cells significantly increased the intracellular catalase activity and enhanced the resistance ability against H2O2 and CHP. In addition, MET18 deficiency diminished the replicative capacity of yeast cells as evidenced by the shortened replicative lifespan, which can be restored by CTT1 overexpression, but not by CTA1, in the MET18-deficient cells. These results suggest that MET18, in a catalase-dependent manner, plays an essential role in enhancing the resistance of yeast cells to oxidative stress and increasing the replicative capacity of yeast cells.

The conserved protein Dre2 uses essential [2Fe–2S] and [4Fe–4S] clusters for its function in cytosolic iron–sulfur protein assembly

The cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery comprises 11 essential components and matures Fe-S proteins involved in translation and genome maintenance. Maturation is initiated by the electron transfer chain NADPH-diflavin reductase Tah18-Fe-S protein Dre2 that facilitates the de novo assembly of a [4Fe-4S] cluster on the scaffold complex Cfd1-Nbp35. Tah18-Dre2 also play a critical role in the assembly of the diferric tyrosyl radical cofactor of ribonucleotide reductase. Dre2 contains eight conserved cysteine residues as potential co-ordinating ligands for Fe-S clusters but their functional importance and the typeof bound clusters is unclear. In the present study, we use a combination of mutagenesis, cell biological and biochemical as well as UV-visible, EPR and Mössbauer spectroscopic approaches to show that the yeast Dre2 cysteine residues Cys(252), Cys(263), Cys(266) and Cys(268) (motif I) bind a [2Fe-2S] cluster, whereas cysteine residues Cys(311), Cys(314), Cys(322) and Cys(325) (motif II) co-ordinate a [4Fe-4S] cluster. All of these residues with the exception of Cys(252) are essential for cell viability, cytosolic Fe-S protein activity and invivo (55)Fe-S cluster incorporation. The N-terminal methyltransferase-like domain of Dre2 is important for proper Fe-S cluster assembly at motifs I and II, which occurs in an interdependent fashion. Our findings further resolve why recombinant Dre2 from Arabidopsis, Trypanosoma or humans has previously been isolated with a single [2Fe-2S] instead of native [2Fe-2S] plus [4Fe-4S] clusters. In the presence of oxygen, the motif I-bound [2Fe-2S] cluster is labile and the motif II-bound [4Fe-4S] cluster is readily converted into a [2Fe-2S] cluster.

The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron–sulfur proteins

Mitochondria have been derived from alpha-bacterial endosymbionts during the evolution of eukaryotes. Numerous bacterial functions have been maintained inside the organelles including fatty acid degradation, citric acid cycle, oxidative phosphorylation, and the synthesis of heme or lipoic acid cofactors. Additionally, mitochondria have inherited the bacterial iron–sulfur cluster assembly (ISC) machinery. Many of the ISC components are essential for cell viability because they generate a still unknown, sulfur-containing compound for the assembly of cytosolic and nuclear Fe/S proteins that perform important functions in, e.g., protein translation, DNA synthesis and repair, and chromosome segregation. The sulfur-containing compound is exported by the mitochondrial ABC transporter Atm1 (human ABCB7) and utilized by components of the cytosolic iron–sulfur protein assembly (CIA) machinery. An appealing minimal model for the striking compartmentation of eukaryotic Fe/S protein biogenesis is provided by organisms that contain mitosomes instead of mitochondria. Mitosomes have been derived from mitochondria by reductive evolution, during which they have lost virtually all classical mitochondrial tasks. Nevertheless, mitosomes harbor all core ISC components which presumably have been maintained for assisting the maturation of cytosolic-nuclear Fe/S proteins. The current review is centered around the Atm1 export process. We present an overview on the mitochondrial requirements for the export reaction, summarize recent insights into the 3D structure and potential mechanism of Atm1, and explain how the CIA machinery uses the mitochondrial export product for the assembly of cytosolic and nuclear Fe/S proteins.

Maturation of cytosolic and nuclear iron–sulfur proteins

Eukaryotic cells contain numerous cytosolic and nuclear iron-sulfur (Fe/S) proteins that perform key functions in metabolic catalysis, iron regulation, protein translation, DNA synthesis, and DNA repair. Synthesis of Fe/S clusters and their insertion into apoproteins are essential for viability and are conserved in eukaryotes. The process is catalyzed in two major steps by the CIA (cytosolic iron-sulfur protein assembly) machinery encompassing nine known proteins. First, a [4Fe-4S] cluster is assembled on a scaffold complex. This step requires a sulfur-containing compound from mitochondria and reducing equivalents from an electron transfer chain. Second, the Fe/S cluster is transferred from the scaffold to specific apoproteins by the CIA targeting complex. This review summarizes our molecular knowledge on CIA protein function during the assembly process.

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

Biogenesis of iron-sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron-sulfur protein assembly machinery, two human key proteins--NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsin--form a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron-sulfur cluster proteins. The Ndor1-anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells.