Mitochondrial iron transporter ClMrs3/4 regulates iron homeostasis to modulate nitric oxide balance facilitating appressorial development in Curvularia lunata.

The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.

The role of hypoxia and nitrogen monoxide in the regulation of cellular iron metabolism

The role of mitochondria in the recovery of neurons after injury.

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans, a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this study, we identified and functionally characterized Mrs3/4, a homolog of the Saccharomyces cerevisiae mitochondrial iron transporter, in C. neoformans var. grubii. A strain expressing an Mrs3/4-GFP fusion protein was generated, and the mitochondrial localization of the fusion protein was confirmed. Moreover, a mutant lacking the MRS3/4 gene was constructed; this mutant displayed significantly reduced mitochondrial iron and cellular heme accumulation. In addition, impaired mitochondrial iron-sulfur cluster metabolism and altered expression of genes required for iron uptake at the plasma membrane were observed in the mrs3/4 mutant, suggesting that Mrs3/4 is involved in iron import and metabolism in the mitochondria of C. neoformans. Using a murine model of cryptococcosis, we demonstrated that an mrs3/4 mutant is defective in survival and virulence. Taken together, our study suggests that Mrs3/4 is responsible for iron import in mitochondria and reveals a link between mitochondrial iron metabolism and the virulence of C. neoformans.

Mitochondrial Iron Transport Via MFRN1 Is Required for Erythroid Cell Cycle Progression Even Under Iron-Replete Conditions Sufficient for Hemoglobinization

Background: Iron in the mitochondria is regulated to maintain mitochondrial physiological function. A loss of mitoNEET protein located in the outer mitochondrial membrane has been shown to increase mitochondrial iron content in adipocytes. However, the role of mitoNEET in iron homeostasis has not been determined in the heart. Methods and Results: MitoNEET flox/flox (mNT f/f ) mice were generated with lox-P and homologous recombination strategies. Cardiac-specific deletion of mitoNEET was achieved using αMHC-Cre (mNT -/- ). Mice, mNT -/- (n=11) and mNT f/f (n=10), were bred for 3 months. Mitochondrial iron content, measured by colorimetric method, was significantly increased in mNT -/- mice compared to mNT f/f mice (1.13±0.09 vs. 0.58±0.07 μg/dl/μg mitochondrial protein, P<0.001). In parallel, immunoblot analysis showed that mitochondrial ferritin, mitochondrial iron transporter, was significantly higher in mNT -/- than mNT f/f by 45%. Mitoferrin2, mitochondrial iron importer, and ATP-binding cassette transporter type B8, mitochondrial iron exporter, protein did not differ between groups. The activity of electron transport chain complex V, mitochondrial iron importer, was also higher in mNT -/- than mNT f/f (617±22 vs. 496±17 mM/min/mg mitochondrial protein, P<0.05). In contrast, frataxin, involved in the synthesis of iron-sulfur cluster (ISC), was significantly lower in mNT -/- by 49%. ATP-binding cassette transporter type B7, mitochondrial ISC exporter, did not differ. Body weight and heart weight were comparable between groups. Left ventricular end-diastolic diameter (2.9±0.1 vs. 2.9±0.1 mm), percent fractional shortening (55±3 vs. 55±3 %), and wall thickness (0.71±0.01 vs. 0.73±0.01 mm) measured by echocardiography were also similar. Conclusions: Cardiac specific deletion of mitoNEET protein increased mitochondrial iron content in mice, in association with the increase in iron importer and transporter and the decrease in frataxin, suggesting that mitoNEET may play an important role in the regulation of mitochondrial function via iron homeostasis in the heart.

Mitochondrial iron transporter (MIT) genes are essential for mitochondrial acquisition/import of iron and vital to proper functioning of mitochondria. Unlike other organisms, research on the MITs in plants is limited. The present study provides comparative bioinformatics assays for the potato MIT gene (StMIT) as well as gene expression analyses. The phylogenetic analyses revealed monocots-dicot divergence in MIT proteins and it was also found clade specific motif diversity. In addition, docking analyses indicated that Asp172 and Gly100 residues to be identified as the closest residues binding to ferrous iron. The percentage of structure overlap of the StMIT 3D protein model with Arabidopsis, maize and rice MIT proteins was found between 80.18% and 85.71%. The transcript analyses exhibited that the expression of StMIT was triggered under drought and salinity stresses. The findings of the present study would provide valuable leads for further studies targeting specifically the MIT gene and generally the plant iron metabolism.

The BolA homologue Fra2 and the cytosolic monothiol glutaredoxins Grx3 and Grx4 together play a key role in regulating iron homeostasis in Saccharomyces cerevisiae. Genetic studies indicate that Grx3/4 and Fra2 regulate activity of the iron-responsive transcription factors Aft1 and Aft2 in response to mitochondrial Fe-S cluster biosynthesis. We have previously shown that Fra2 and Grx3/4 form a [2Fe-2S](2+)-bridged heterodimeric complex with iron ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. To further characterize this unusual Fe-S-binding complex, site-directed mutagenesis was used to identify specific residues in Fra2 that influence Fe-S cluster binding and regulation of Aft1 activity in vivo. Here, we present spectroscopic evidence that His-103 in Fra2 is an Fe-S cluster ligand in the Fra2-Grx3 complex. Replacement of this residue does not abolish Fe-S cluster binding, but it does lead to a change in cluster coordination and destabilization of the [2Fe-2S] cluster. In vivo genetic studies further confirm that Fra2 His-103 is critical for control of Aft1 activity in response to the cellular iron status. Using CD spectroscopy, we find that ∼1 mol eq of apo-Fra2 binds tightly to the [2Fe-2S] Grx3 homodimer to form the [2Fe-2S] Fra2-Grx3 heterodimer, suggesting a mechanism for formation of the [2Fe-2S] Fra2-Grx3 heterodimer in vivo. Taken together, these results demonstrate that the histidine coordination and stability of the [2Fe-2S] cluster in the Fra2-Grx3 complex are essential for iron regulation in yeast.

Predicted iron metabolism genes in hard ticks and their response to iron reduction in Dermacentor andersoni cells

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic β-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

Nitric oxide (NO), once regarded as a poisonous air pollutant, is now understood as a regulatory molecule essential for several biological functions in plants. In this review, we summarize NO generation in different plant organs and cellular compartments, and also discuss the role of NO in iron (Fe) homeostasis, particularly in Fe-deficient plants. Fe is one of the most limiting essential nutrient elements for plants. Plants often exhibit Fe deficiency symptoms despite sufficient tissue Fe concentrations. NO appears to not only up-regulate Fe uptake mechanisms but also makes Fe more bioavailable for metabolic functions. NO forms complexes with Fe, which can then be delivered into target cells/tissues. NO generated in plants can alleviate oxidative stress by regulating antioxidant defense processes, probably by improving functional Fe status and by inducing post-translational modifications in the enzymes/proteins involved in antioxidant defense responses. It is hypothesized that NO acts in cooperation with transcription factors such as bHLHs, FIT, and IRO to regulate the expression of enzymes and proteins essential for Fe homeostasis. However, further investigations are needed to disentangle the interaction of NO with intracellular target molecules that leads to enhanced internal Fe availability in plants.

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.

Patients with iron overload disorders often develop lymphocyte deficiencies, indicating iron homeostasis is critical for lymphopoiesis. Loss of ABCB7, a mitochondrial iron transporter, leads to mitochondrial iron accumulation and rapid hematopoietic failure. We found that ABCB7 is required for lymphopoiesis, as CD2-icre ABCB7 cKO mice have a severe block in both T and B cell development. The block in T cell development occurs at the DN3 stage. This DN3 block is not due to aberrant β-selection, proliferation, or viability. The block in B cell development occurs at the Fr. C to Fr. C′ transition. Pre-BCR expression was reduced in developing B cells and they exhibited reduced proliferation compared to WT B cells. Because loss of ABCB7 causes mitochondrial iron accumulation, which is toxic to cells, we hypothesized that lymphoid cells increase heme synthesis in order to detoxify the accumulating iron. We found that HO-1, a surrogate marker for intracellular heme levels, was elevated in these lymphocytes. Currently, we are examining the mechanism responsible for the block in lymphocyte development upon loss of ABCB7.

Mitochondria are one of the major iron sinks in plant cells. Mitochondrial iron accumulation involves the action of ferric reductase oxidases (FRO) and carriers located in the inner mitochondrial membrane. It has been suggested that among these transporters, mitoferrins (mitochondrial iron transporters, MITs) belonging to the mitochondrial carrier family (MCF) function as mitochondrial iron importers. In this study, two cucumber proteins, CsMIT1 and CsMIT2, with high homology to Arabidopsis, rice and yeast MITs were identified and characterized. CsMIT1 and CsMIT2 were expressed in all organs of the two-week-old seedlings. Under Fe-limited conditions as well as Fe excess, the mRNA levels of CsMIT1 and CsMIT2 were altered, suggesting their regulation by iron availability. Analyses using Arabidopsis protoplasts confirmed the mitochondrial localization of cucumber mitoferrins. Expression of CsMIT1 and CsMIT2 restored the growth of the Δmrs3Δmrs4 mutant (defective in mitochondrial Fe transport), but not in mutants sensitive to other heavy metals. Moreover, the altered cytosolic and mitochondrial Fe concentrations, observed in the Δmrs3Δmrs4 strain, were recovered almost to the levels of WT yeast by expressing CsMIT1 or CsMIT2. These results indicate that cucumber proteins are involved in the iron transport from the cytoplasm to the mitochondria.

For their function, many proteins depend on cofactor binding to specific sequence motifs. Fe‐S clusters are conserved and versatile inorganic cofactors that participate in a variety of processes and functions. In eukaryotes, biogenesis and recycling of Fe‐S clusters play important roles in the mechanisms of iron homeostasis involved in mitochondrial function. These Fe‐S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The present study aimed to determine the effects of mutations in the ISC genes on mitochondrial functionality and iron homeostasis under oxidative stress in S. cerevisiae BY4741 and its KanMX4 interruption ISC genes mutants: ssq1Δ, grx5Δ and isa1Δ. Intracellular ROS, Fe2+ and mitochondrial membrane potential in yeast cultures were determined using oxidant‐sensitive, cell‐permeant fluorescent probes (DHE, PGFL and Rho123). Fluorescence was quantified by flow cytometry, viewed and colocalized by confocal microscopy. Mitochondria were isolated and permeabilized for spectrophotometric determination of respiratory complexes activities. Oxygen consumption rate (OCR) was measured with a Clark‐type oxygen electrode coupled to a biological oxygen monitor. Electron transport chain (ETC) supercomplexes were identified by BN‐PAGE and Raman spectroscopy was performed to detect mitochondrial Fe‐S content. The results indicate that ROS generation caused by oxidizing agents like menadione is increased by ISC system dysfunction, due to loss of iron homeostasis (i.e. augmented free Fe2+), suggesting that ROS and Fe2+ from Fe‐S cluster proteins react creating a strong oxidative status in mitochondria. This was associated to an impairment on the activity of the complexes II, III and IV from the ETC in the ISC mutants. Indeed, increased oxygen generation instead consumption was observed in SSQ1 and ISA1 mutants suggesting a massive generation of ROS and its detoxification by superoxide dismutase and catalase. In mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased along with III2IV2 respiratory supercomplex formation, but not in the iron‐deficient ATX1 (cytosolic copper metallochaperone) and MRS4 (mitochondrial iron transporter) mutants, used as controls. In conclusion these results indicate that the ISC system is important in iron‐homeostasis, ROS stress and in the assembly of the supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.