Fe-S Cluster Assembly Machinery Research Articles

Human frataxin, the Friedreich ataxia deficient protein, interacts with mitochondrial respiratory chain

Friedreich ataxia (FRDA) is a rare, inherited neurodegenerative disease caused by an expanded GAA repeat in the first intron of the FXN gene, leading to transcriptional silencing and reduced expression of frataxin. Frataxin participates in the mitochondrial assembly of FeS clusters, redox cofactors of the respiratory complexes I, II and III. To date it is still unclear how frataxin deficiency culminates in the decrease of bioenergetics efficiency in FRDA patients’ cells. We previously demonstrated that in healthy cells frataxin is closely attached to the mitochondrial cristae, which contain both the FeS cluster assembly machinery and the respiratory chain complexes, whereas in FRDA patients’ cells with impaired respiration the residual frataxin is largely displaced in the matrix. To gain novel insights into the function of frataxin in the mitochondrial pathophysiology, and in the upstream metabolic defects leading to FRDA disease onset and progression, here we explored the potential interaction of frataxin with the FeS cluster-containing respiratory complexes I, II and III. Using healthy cells and different FRDA cellular models we found that frataxin interacts with these three respiratory complexes. Furthermore, by EPR spectroscopy, we observed that in mitochondria from FRDA patients’ cells the decreased level of frataxin specifically affects the FeS cluster content of complex I. Remarkably, we also found that the frataxin-like protein Nqo15 from T. thermophilus complex I ameliorates the mitochondrial respiratory phenotype when expressed in FRDA patient’s cells. Our data point to a structural and functional interaction of frataxin with complex I and open a perspective to explore therapeutic rationales for FRDA targeted to this respiratory complex.

Interaction between Cfd1 and Nbp35 proteins involved in cytosolic Fe[sbnd]S cluster assembly machinery deciphers a stable complexation in Leishmania donovani

Leishmania donovani is the causative unicellular parasite for visceral leishmaniasis (VL); and FeS proteins are likely to be very essential for their survival and viability. Cytosolic FeS cluster assembly (CIA) machinery is one of the four systems for the biosynthesis and transfer of FeS clusters among eukaryotes; Cfd1 and Nbp35 are the scaffold components for cytosolic FeS cluster biogenesis. We investigated the role of CIA machinery components and purified Cfd1 and Nbp35 proteins of L. donovani. We also investigated the interactive nature between LdCfd1 and LdNbp35 proteins by in silico analysis, in vitro co-purification, pull down assays along with in vivo immuno-precipitation; which inferred that both LdCfd1 and LdNbp35 proteins are interacting with each other. Thus, our collective data revealed the interaction between these two proteins which forms a stable complex that can be attributed to the cellular process of FeS clusters biogenesis, and transfer to target apo-proteins of L. donovani. The expression of Cfd1 and Nbp35 proteins in Amp B resistant parasites is up-regulated leading to increased amount of FeS proteins. Hence, it favors increased tolerance towards ROS level, which helps parasites survival under drug pressure contributing in Amphotericin B resistance.

An early origin of iron-sulfur cluster biosynthesis machineries before Earth oxygenation.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential for life. It is largely thought that the emergence of oxygenic photosynthesis and progressive oxygenation of the atmosphere led to the origin of multiprotein machineries (ISC, NIF and SUF) assisting Fe-S cluster synthesis in the presence of oxidative stress and shortage of bioavailable iron. However, previous analyses have left unclear the origin and evolution of these systems. Here, we combine exhaustive homology searches with genomic context analysis and phylogeny to precisely identify Fe-S cluster biogenesis systems in over 10,000 archaeal and bacterial genomes. We highlight the existence of two additional and clearly distinct 'minimal' Fe-S cluster assembly machineries, MIS (minimal iron-sulfur) and SMS (SUF-like minimal system), which we infer in the last universal common ancestor (LUCA) and we experimentally validate SMS as a bona fide Fe-S cluster biogenesis system. These ancestral systems were kept in archaea whereas they went through stepwise complexification in bacteria to incorporate additional functions for higher Fe-S cluster synthesis efficiency leading to SUF, ISC and NIF. Horizontal gene transfers and losses then shaped the current distribution of these systems, driving ecological adaptations such as the emergence of aerobic lifestyles in archaea. Our results show that dedicated machineries were in place early in evolution to assist Fe-S cluster biogenesis and that their origin is not directly linked to Earth oxygenation.

The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins

Proteins incorporating iron-sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

Exploring iron-binding to human frataxin and to selected Friedreich ataxia mutants by means of NMR and EPR spectroscopies

The neurodegenerative disease Friedreich ataxia results from a deficiency of frataxin, a mitochondrial protein. Most patients have a GAA expansion in the first intron of both alleles of frataxin gene, whereas a minority of them are heterozygous for the expansion and contain a mutation in the other allele. Frataxin has been claimed to participate in iron homeostasis and biosynthesis of FeS clusters, however its role in both pathways is not unequivocally defined. In this work we combined different advanced spectroscopic analyses to explore the iron-binding properties of human frataxin, as isolated and at the FeS clusters assembly machinery. For the first time we used EPR spectroscopy to address this key issue providing clear evidence of the formation of a complex with a low symmetry coordination of the metal ion. By 2D NMR, we confirmed that iron can be bound in both oxidation states, a controversial issue, and, in addition, we were able to point out a transient interaction of frataxin with a N-terminal 6his-tagged variant of ISCU, the scaffold protein of the FeS clusters assembly machinery. To obtain insights on structure/function relationships relevant to understand the disease molecular mechanism(s), we extended our studies to four clinical frataxin mutants. All variants showed a moderate to strong impairment in their ability to activate the FeS cluster assembly machinery in vitro, while keeping the same iron-binding features of the wild type protein. This supports the multifunctional nature of frataxin and the complex biochemical consequences of its mutations.

An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

An Orchestrated Balance between Mitochondria Biogenesis, Iron-Sulfur Cluster Synthesis and Cellular Iron Acquisition

Fe-S clusters are essential cofactors for mitochondria functions, and mitochondria are required for Fe-S cluster synthesis. Additionally, mitochondria biogenesis demands cellular iron uptake, which is negatively regulated by Fe-S clusters. Fe-S clusters are synthesized in the mitochondria and cytosol by two different machineries. However, cytosolic Fe-S cluster synthesis necessitates the mitochondrial Fe-S cluster assembly machinery.PGC-1α is a transcriptional coactivator and a master regulator of mitochondria biogenesis. We confirmed that overexpression of PGC-1α in adipocytes and hepatocytes stimulated mitochondria biogenesis, as measured by Mitotrack Green and Deep Red staining, which label total and alive mitochondria, respectively. We further measured Fe-S cluster synthesis by monitoring the gene expression of Fe-S cluster assembly machinery. By using RT-qPCR and Western Blot analyses, we confirmed that PGC-1α expression increases expression of ABCB7, ISCA1, ISCA2, ISD11, Nfu1 and ISCU, components of the Fe-S assembly machinery, suggesting a coordination between mitochondria biogenesis and Fe-S cluster synthesis.Iron Regulatory Proteins (IRP1 and IRP2) control iron metabolism by binding to specific non-coding sequences within an mRNA, known as iron-responsive elements (IRE). In the absence of Fe-S clusters, IRP1 acts as an aconitase (aka ACO1), while IRP2 is degraded by ubiquitination. Aconitases, represented by the cytosolic form ACO1 and mitochondrial form ACO2, catalyze the isomerization of citrate to isocitrate and require Fe-S clusters to be enzymatically active. PGC-1α overexpression enhanced aconitase activity but not their protein levels, corroborating the notion that Fe-S cluster synthesis was increased.To explore whether this coordination solely depends on PGC-1α, we evaluated the Fe-S cluster synthesis status during brown adipocyte maturation, which is characterized by enhanced mitochondria biogenesis and has been suggested to be PGC-1α-independent. We found that the synthesis of Fe-S cluster assembly machinery increased during maturation in both wild-type and PGC-1α-knockout brown adipocytes, indicating that Fe-S cluster synthesis coordinates with mitochondria biogenesis even in the absence of PGC-1α.To explore the impact of Fe-S cluster synthesis on iron acquisition under enhanced mitochondria biogenesis, we evaluated the expression of the iron importer transferrin receptor 1 (TfR1). TfR1 mRNA contains IREs in the 3' untranslated region (UTR). These 3'UTR IREs can be bound by IRPs and responsible for the subsequent stabilization of TfR1 mRNA. Therefore, if IRP1 associates with Fe-S cluster and converted into ACO1, it is expected that both TfR1 mRNA and protein levels would decrease. In contrast, we found that stimulated Fe-S cluster synthesis increased levels of the TfR1 protein, despite reduced IRP1 activity and destabilized TfR1 mRNA. This suggests that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block iron uptake.Moreover, we extended our work to erythropoiesis by using murine erythroleukemia (MEL) cells. Stimulated mitochondria biogenesis enhanced expression of the Fe-S cluster assembly machinery and Fe-S cluster synthesis in these cells. TfR1 protein levels were increased despite elevated Fe-S cluster synthesis and reduced IRP activity. We also found increases in heme levels and the expression of aminolevulinic acid synthase 2 (ALAS2), the rate-limiting enzyme for erythroid heme synthesis. Of note, the ALAS2 mRNA contains IRE at the 5'UTR; binding of IRPs to the IRE inhibits translation while high Fe-S cluster levels lead to release. Moreover, as α- and β-globins chain expression is stimulated by increased heme availability, we also observed that mitochondria biogenesis was associated with increased synthesis of these two proteins and hemoglobinization. These data suggests that erythroid heme synthesis, hemoglobin expression and hemoglobinization coordinates with mitochondria biogenesis via Fe-S cluster synthesis.In conclusion, our data show that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block cellular iron uptake, thus suggesting a potential unidentified iron regulator to ensure adequate iron for mitochondria biogenesis. Moreover, our work suggests a mechanism underlying the essential role of mitochondria biogenesis in erythropoiesis. DisclosuresRivella:Disc Medicine: Consultancy; MeiraGTx: Other: SAB; Ionis Pharmaceuticals, Inc: Consultancy; Protagonist: Consultancy.

Ferrochelatase activity of plant frataxin

Frataxin plays a key role in cellular iron homeostasis of different organisms. It is engaged in several activities at the FeS cluster assembly machinery and it is also involved in heme biosynthesis. In plants, two genes encoding ferrochelatases (FC1 and FC2) catalyze the incorporation of iron into protoporphyrin IX in the last stage of heme synthesis in chloroplasts. Despite ferrochelatases are absent from other cell compartments, a remaining ferrochelatase activity has been observed in plant mitochondria. Here we analyze the possibility that frataxin acts as the iron donor to protoporphyrin IX for the synthesis of heme groups in plant mitochondria. Our findings show that frataxin catalyzes the formation of heme in vitro when it is incubated with iron and protoporphyrin IX. When frataxin is combined with AtNFS1 and AtISD11 the ferrochelatse activity is increased. These results suggest that frataxin could be the iron donor in the final step of heme synthesis in plant mitochondria, and constitutes an important advance in the elucidation of the mechanisms of heme synthesis in plants.

Cytosolic HSC20 integrates de novo iron-sulfur cluster biogenesis with the CIAO1-mediated transfer to recipients.

Iron-sulfur (Fe-S) clusters are cofactors in hundreds of proteins involved in multiple cellular processes, including mitochondrial respiration, the maintenance of genome stability, ribosome biogenesis and translation. Fe-S cluster biogenesis is performed by multiple enzymes that are highly conserved throughout evolution, and mutations in numerous biogenesis factors are now recognized to cause a wide range of previously uncategorized rare human diseases. Recently, a complex formed of components of the cytoplasmic Fe-S cluster assembly (CIA) machinery, consisting of CIAO1, FAM96B and MMS19, was found to deliver Fe-S clusters to a subset of proteins involved in DNA metabolism, but it was unclear how this complex acquired its fully synthesized Fe-S clusters, because Fe-S clusters have been alleged to be assembled de novo solely in the mitochondrial matrix. Here, we investigated the potential role of the human cochaperone HSC20 in cytosolic Fe-S assembly and found that HSC20 assists Fe-S cluster delivery to cytosolic and nuclear Fe-S proteins. Cytosolic HSC20 (C-HSC20) mediated complex formation between components of the cytosolic Fe-S biogenesis pathway (ISC), including the primary scaffold, ISCU1, and the cysteine desulfurase, NFS1, and the CIA targeting complex, consisting of CIAO1, FAM96B and MMS19, to facilitate Fe-S cluster insertion into cytoplasmic and nuclear Fe-S recipients. Thus, C-HSC20 integrates initial Fe-S biosynthesis with the transfer activities of the CIA targeting system. Our studies demonstrate that a novel cytosolic pathway functions in parallel to the mitochondrial ISC to perform de novo Fe-S biogenesis, and to escort Fe-S clusters to cytoplasmic and nuclear proteins.

Compartmentalisation of [FeFe]-hydrogenase maturation in Chlamydomonas reinhardtii.

Molecular hydrogen (H2 ) can be produced in green microalgae by [FeFe]-hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub-cluster. From invitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub-cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process invivo, we expressed HYDA1 from the C.reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase-deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as invivo H2 production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H2 production comparable to the wild type, as did the transformants expressing full-length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm-targeted HYDA1 produced inactive enzyme, which could only be activated invitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C.reinhardtii chloroplast genome resulted in H2 -producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 invivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.

Iron-Sulfur Protein Assembly in Human Cells.

Iron-sulfur (Fe-S) clusters serve as a fundamental inorganic constituent of living cells ranging from bacteria to human. The importance of Fe-S clusters is underscored by their requirement as a co-factor for the functioning of different enzymes and proteins. The biogenesis of Fe-S cluster is a highly coordinated process which requires specialized cellular machinery. Presently, understanding of Fe-S cluster biogenesis in human draws meticulous attention since defects in the biogenesis process result in development of multiple diseases with unresolved solutions. Mitochondrion is the major cellular compartment of Fe-S cluster biogenesis, although cytosolic biogenesis machinery has been reported in eukaryotes, including in human. The core biogenesis pathway comprises two steps. The process initiates with the assembly of Fe-S cluster on a platform scaffold protein in the presence of iron and sulfur donor proteins. Subsequent process is the transfer and maturation of the cluster to a bonafide target protein. Human Fe-S cluster biogenesis machinery comprises the mitochondrial iron-sulfur cluster (ISC) assembly and export system along with the cytosolic Fe-S cluster assembly (CIA) machinery. Impairment in the Fe-S cluster machinery components results in cellular dysfunction leading to various mitochondrial pathophysiological consequences. The current review highlights recent developments and understanding in the domain of Fe-S cluster assembly biology in higher eukaryotes, particularly in human cells.

Defining the Architecture of the Core Machinery for the Assembly of Fe-S Clusters in Human Mitochondria.

Although Fe-S clusters may assemble spontaneously from elemental iron and sulfur in protein-free systems, the potential toxicity of free Fe2+, Fe3+, and S2- ions in aerobic environments underscores the requirement for specialized proteins to oversee the safe assembly of Fe-S clusters in living cells. Prokaryotes first developed multiprotein systems for Fe-S cluster assembly, from which mitochondria later derived their own system and became the main Fe-S cluster suppliers for eukaryotic cells. Early studies in yeast and human mitochondria indicated that Fe-S cluster assembly in eukaryotes is centered around highly conserved Fe-S proteins (human ISCU) that serve as scaffolds upon which new Fe-S clusters are assembled from (i) elemental sulfur, provided by a pyridoxal phosphate-dependent cysteine desulfurase (human NFS1) and its stabilizing-binding partner (human ISD11), and (ii) elemental iron, provided by an iron-binding protein of the frataxin family (human FXN). Further studies revealed that all of these proteins could form stable complexes that could reach molecular masses of megadaltons. However, the protein-protein interaction surfaces, catalytic mechanisms, and overall architecture of these macromolecular machines remained undefined for quite some time. The delay was due to difficulties inherent in reconstituting these very large multiprotein complexes in vitro or isolating them from cells in sufficient quantities to enable biochemical and structural studies. Here, we describe approaches we developed to reconstitute the human Fe-S cluster assembly machinery in Escherichia coli and to define its remarkable architecture.

Minimal cytosolic iron‐sulfur cluster assembly machinery of Giardia intestinalis is partially associated with mitosomes

Iron-sulfur (Fe-S) clusters are essential cofactors that enable proteins to transport electrons, sense signals, or catalyze chemical reactions. The maturation of dozens of Fe-S proteins in various compartments of every eukaryotic cell is driven by several assembly pathways. The ubiquitous cytosolic Fe-S cluster assembly (CIA) pathway, typically composed of eight highly conserved proteins, depends on mitochondrial Fe-S cluster assembly (ISC) machinery. Giardia intestinalis contains one of the smallest eukaryotic genomes and the mitosome, an extremely reduced mitochondrion. Because the only pathway known to be retained within this organelle is the synthesis of Fe-S clusters mediated by ISC machinery, a likely function of the mitosome is to cooperate with the CIA pathway. We investigated the cellular localization of CIA components in G. intestinalis and the origin and distribution of CIA-related components and Tah18-like proteins in other Metamonada. We show that orthologs of Tah18 and Dre2 are missing in these eukaryotes. In Giardia, all CIA components are exclusively cytosolic, with the important exception of Cia2 and two Nbp35 paralogs, which are present in the mitosomes. We propose that the dual localization of Cia2 and Nbp35 proteins in Giardia might represent a novel connection between the ISC and the CIA pathways.

Emerging links between iron-sulfur clusters and 5-methylcytosine base excision repair in plants.

Iron-sulfur (Fe-S) clusters are ancient cofactors present in all kingdoms of life. Both the Fe-S cluster assembly machineries and target apoproteins are distributed across different subcellular compartments. The essential function of Fe-S clusters in nuclear enzymes is particularly difficult to study. The base excision repair (BER) pathway guards the integrity of DNA; enzymes from the DEMETER family of DNA glycosylases in plants are Fe-S cluster-dependent and extend the BER repertowere to excision of 5-methylcytosine (5mC). Recent studies in plants genetically link the majority of proteins from the cytosolic Fe-S cluster biogenesis (CIA) pathway with 5mC BER and DNA repair. This link can now be further explored. First, it opens new possibilities for understanding how Fe-S clusters participate in 5mC BER and related processes. I describe DNA-mediated charge transfer, an Fe-S cluster-based mechanism for locating base lesions with high efficiency, which is used by bacterial DNA glycosylases encoding Fe-S cluster binding domains that are also conserved in the DEMETER family. Second, because detailed analysis of the mutant phenotype of CIA proteins relating to 5mC BER revealed that they formed two groups, we may also gain new insights into both the composition of the Fe-S assembly pathway and the biological contexts of Fe-S proteins.

Turning Saccharomyces cerevisiae into a Frataxin-Independent Organism.

Frataxin (Yfh1 in yeast) is a conserved protein and deficiency leads to the neurodegenerative disease Friedreich’s ataxia. Frataxin is a critical protein for Fe-S cluster assembly in mitochondria, interacting with other components of the Fe-S cluster machinery, including cysteine desulfurase Nfs1, Isd11 and the Isu1 scaffold protein. Yeast Isu1 with the methionine to isoleucine substitution (M141I), in which the E. coli amino acid is inserted at this position, corrected most of the phenotypes that result from lack of Yfh1 in yeast. This suppressor Isu1 behaved as a genetic dominant. Furthermore frataxin-bypass activity required a completely functional Nfs1 and correlated with the presence of efficient scaffold function. A screen of random Isu1 mutations for frataxin-bypass activity identified only M141 substitutions, including Ile, Cys, Leu, or Val. In each case, mitochondrial Nfs1 persulfide formation was enhanced, and mitochondrial Fe-S cluster assembly was improved in the absence of frataxin. Direct targeting of the entire E. coli IscU to ∆yfh1 mitochondria also ameliorated the mutant phenotypes. In contrast, expression of IscU with the reverse substitution i.e. IscU with Ile to Met change led to worsening of the ∆yfh1 phenotypes, including severely compromised growth, increased sensitivity to oxygen, deficiency in Fe-S clusters and heme, and impaired iron homeostasis. A bioinformatic survey of eukaryotic Isu1/prokaryotic IscU database entries sorted on the amino acid utilized at the M141 position identified unique groupings, with virtually all of the eukaryotic scaffolds using Met, and the preponderance of prokaryotic scaffolds using other amino acids. The frataxin-bypassing amino acids Cys, Ile, Leu, or Val, were found predominantly in prokaryotes. This amino acid position 141 is unique in Isu1, and the frataxin-bypass effect likely mimics a conserved and ancient feature of the prokaryotic Fe-S cluster assembly machinery.

Impaired mitochondrial Fe-S cluster biogenesis activates the DNA damage response through different signaling mediators.

Fe-S cluster biogenesis machinery is required for multiple DNA metabolism processes. In this work, we show that, in Saccharomyces cerevisiae, defects at different stages of the mitochondrial Fe-S cluster assembly machinery (ISC) result in increased spontaneous mutation rate and hyper-recombination, accompanied by an increment in Rad52-associated DNA repair foci and a higher phosphorylated state of γH2A histone, altogether supporting the presence of constitutive DNA lesions. Furthermore, ISC assembly machinery deficiency elicits a DNA damage response that upregulates ribonucleotide reductase activity by promoting the reduction of Sml1 levels and the cytosolic redistribution of Rnr2 and Rnr4 enzyme subunits. Depending on the impaired stage of the ISC machinery, different signaling pathway mediators contribute to such a response, converging on Dun1. Thus, cells lacking the glutaredoxin Grx5, which are compromised at the core ISC system, show Mec1- and Rad53-independent Dun1 activation, whereas both Mec1 and Chk1 are required when the non-core ISC member Iba57 is absent. Grx5-null cells exhibit a strong dependence on the error-free post-replication repair and the homologous recombination pathways, demonstrating that a DNA damage response needs to be activated upon ISC impairment to preserve cell viability.

Genes for iron–sulphur cluster assembly are targets of abiotic stress in rice, Oryza sativa

Iron-sulphur (Fe-S) cluster assembly occurs in chloroplasts, mitochondria and cytosol, involving dozens of genes in higher plants. In this study, we have identified 41 putative Fe-S cluster assembly genes in rice (Oryza sativa) genome, and the expression of all genes was verified. To investigate the role of Fe-S cluster assembly as a metabolic pathway, we applied abiotic stresses to rice seedlings and analysed Fe-S cluster assembly gene expression by qRT-PCR. Our data showed that genes for Fe-S cluster assembly in chloroplasts of leaves are particularly sensitive to heavy metal treatments, and that Fe-S cluster assembly genes in roots were up-regulated in response to iron toxicity, oxidative stress and some heavy metal assault. The effect of each stress treatment on the Fe-S cluster assembly machinery demonstrated an unexpected tissue or organelle specificity, suggesting that the physiological relevance of the Fe-S cluster assembly is more complex than thought. Furthermore, our results may reveal potential candidate genes for molecular breeding of rice.

Copper Efflux Is Induced during Anaerobic Amino Acid Limitation in Escherichia coli To Protect Iron-Sulfur Cluster Enzymes and Biogenesis

Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the P(cusC)-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli.

Tissue Specificity of a Human Mitochondrial Disease: DIFFERENTIATION-ENHANCED MIS-SPLICING OF THE Fe-S SCAFFOLD GENE ISCU RENDERS PATIENT CELLS MORE SENSITIVE TO OXIDATIVE STRESS IN ISCU MYOPATHY

ISCU myopathy is a disease caused by muscle-specific deficiency of the Fe-S cluster scaffold protein ISCU. MyoD expression enhanced ISCU mRNA mis-splicing, and oxidative stress exacerbated ISCU depletion in patient cells. ISCU protein deficiency in patients results from muscle-specific mis-splicing as well as oxidative stress. Oxidative stress negatively influences the mammalian Fe-S cluster assembly machinery by destabilization of ISCU. Iron-sulfur (Fe-S) cluster cofactors are formed on the scaffold protein ISCU. ISCU myopathy is a disease caused by an intronic mutation that leads to abnormally spliced ISCU mRNA. We found that two predominant mis-spliced ISCU mRNAs produce a truncated and short-lived ISCU protein product in multiple patient cell types. Expression of the muscle-specific transcription factor MyoD further diminished normal splicing of ISCU mRNA in patient myoblasts, demonstrating that the process of muscle differentiation enhances the loss of normal ISCU mRNA splicing. ISCU protein was nearly undetectable in patient skeletal muscle, but was higher in patient myoblasts, fibroblasts, and lymphoblasts. We next treated patient cells with pro-oxidants to mimic the oxidative stress associated with muscle activity. Brief hydrogen peroxide treatment or incubation in an enriched oxygen atmosphere led to a marked further reduction of ISCU protein levels, which could be prevented by pretreatment with the antioxidant ascorbate. Thus, we conclude that skeletal muscle differentiation of patient cells causes a higher degree of abnormal ISCU splicing and that oxidative stress resulting from skeletal muscle work destabilizes the small amounts of normal ISCU protein generated in patient skeletal muscles.

The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.