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Iron that is stored in macrophages as ferritin can be made bioavailable by degrading ferritin in the lysosome and releasing iron back into the cytosol. Iron stored in ferritin is found as Fe3+ and must be reduced to Fe2+ before it can be exported from the lysosome. Here we report that the lysosomal reductase Cyb561a3 (LcytB) and the endosomal reductase six-transmembrane epithelial antigen of prostate 3 (Steap3) act as lysosomal ferrireductases in the mouse macrophage cell line RAW264.7 converting Fe3+ to Fe2+ for iron recycling. We determined that when lysosomes were loaded with horse cationic ferritin, reductions or loss of LcytB or Steap3 using CRISPR/Cas9-mediated knockout technology resulted in decreased lysosomal iron export. Loss of both reductases was additive in decreasing lysosomal iron export. Decreased reductase activity resulted in increased transcripts for iron acquisition proteins DMT1 and transferrin receptor 1 (Tfrc1) suggesting that cells were iron limited. We show that transcript expression of LcytB and Steap3 is decreased in macrophages exposed to Escherichia coli pathogen UTI89, which supports a role for these reductases in regulating iron availability for pathogens. We further show that loss of LcytB and Steap3 in macrophages infected with UTI89 led to increased proliferation of intracellular UTI89 suggesting that the endolysosomal system is retaining Fe3+ that can be used for proliferation of intravesicular pathogens. Together, our findings reveal an important role for both LcytB and Steap3 in macrophage iron recycling and suggest that limiting iron recycling by decreasing expression of endolysosomal reductases is an innate immune response to protect against pathogen proliferation and sepsis.

OBJECTIVE To determine why serum ferritin and reticulocyte hemoglobin (RET-He), drawn to assess neonatal iron sufficiency, sometimes have markedly discordant results. STUDY DESIGN Retrospective records review of five NICUs over 28 months, identifying all patients with a ferritin and RET-He within 48 h. We examined records of all who had marked discordance (one value >95th % reference interval, the other

Building on our previous study, showing a correlation between ferritin in serum and urine, we conducted a feasibility evaluation, measuring urinary ferritin as a potential noninvasive screening test for iron deficiency among NICU patients. This was a prospective analysis of paired serum/urine ferritin levels. We defined iron-limited erythropoiesis by a RET-He

T cells must shift their metabolism to respond to infections and tumors and to undergo memory formation. The ATP-binding cassette transporter ABCB10 localizes to the mitochondrial inner membrane, where it is thought to export a substrate important in heme biosynthesis and metabolism, but its role in T cell development and activation is unknown. In this article, we use a combination of methods to study the effect of ABCB10 loss in primary and malignantly transformed T cells. Although Abcb10 is dispensable for development of both CD4+ and CD8+ T cells, it is required for expression of specific cytokines in CD4+, but not CD8+, T cells activated in vitro. These defects in cytokine expression are magnified on repeated stimulation. In vivo, CD8+ cells lacking ABCB10 expand more in response to viral infection than their control counterparts, while CD4+ cells show reductions in both number and percentage. CD4+ cells lacking ABCB10 show impairment in Ag-specific memory formation and recall responses that become more severe with time. In malignant human CD4+ Jurkat T cells, we find that CRISPR-mediated ABCB10 disruption recapitulates the same cytokine expression defects upon activation as observed in primary mouse T cells. Mechanistically, ABCB10 deletion in Jurkat T cells disrupts the ability to switch to aerobic glycolysis upon activation. Cumulatively, these results show that ABCB10 is selectively required for specific cytokine responses and memory formation in CD4+ T cells, suggesting that targeting this molecule could be used to mitigate aberrant T cell activation.

Budding yeast (Saccharomyces cerevisiae) responds to low cytosolic iron by up-regulating the expression of iron import genes; iron import can reflect iron transport into the cytosol or mitochondria. Mmt1 and Mmt2 are nuclearly encoded mitochondrial proteins that export iron from the mitochondria into the cytosol. Here we report that MMT1 and MMT2 expression is transcriptionally regulated by two pathways: the low-iron-sensing transcription factor Aft1 and the oxidant-sensing transcription factor Yap1. We determined that MMT1 and MMT2 expression is increased under low-iron conditions and decreased when mitochondrial iron import is increased through overexpression of the high-affinity mitochondrial iron importer Mrs3. Moreover, loss of iron-sulfur cluster synthesis induced expression of MMT1 and MMT2. We show that exposure to the oxidant H2O2 induced MMT1 expression but not MMT2 expression and identified the transcription factor Yap1 as being involved in oxidant-mediated MMT1 expression. We defined Aft1- and Yap1-dependent transcriptional sites in the MMT1 promoter that are necessary for low-iron- or oxidant-mediated MMT1 expression. We also found that the MMT2 promoter contains domains that are important for regulating its expression under low-iron conditions, including an upstream region that appears to partially repress expression under low-iron conditions. Our findings reveal that MMT1 and MMT2 are induced under low-iron conditions and that the low-iron regulator Aft1 is required for this induction. We further uncover an Aft1-binding site in the MMT1 promoter sufficient for inducing MMT1 transcription and identify an MMT2 promoter region required for low iron induction.

Friedreich's ataxia (FRDA) is a degenerative disease caused by a decrease in the mitochondrial protein frataxin (Fxn), which is involved in iron-sulfur cluster (ISC) synthesis. Diminutions in Fxn result in decreased ISC synthesis, increased mitochondrial iron accumulation, and impaired mitochondrial function. Here, we show that conditions that result in increased mitochondrial reactive oxygen species in yeast or mammalian cell culture give rise to increased turnover of Fxn but not of other ISC synthesis proteins. We demonstrate that the mitochondrial Lon protease is involved in Fxn degradation and that iron export through the mitochondrial metal transporter Mmt1 protects yeast Fxn from degradation. We also determined that when FRDA fibroblasts were grown in media containing elevated iron, mitochondrial reactive oxygen species increased and Fxn decreased compared to WT fibroblasts. Furthermore, we screened a library of FDA-approved compounds and identified 38 compounds that increased yeast Fxn levels, including the azole bifonazole, antiparasitic fipronil, antitumor compound dibenzoylmethane, antihypertensive 4-hydroxychalcone, and a nonspecific anion channel inhibitor 4,4-diisothiocyanostilbene-2,2-sulfonic acid. We show that top hits 4-hydroxychalcone and dibenzoylmethane increased mRNA levels of transcription factor nuclear factor erythroid 2-related factor 2 in FRDA patient-derived fibroblasts, as well as downstream antioxidant targets thioredoxin, glutathione reductase, and superoxide dismutase 2. Taken together, these findings reveal that FRDA progression may be in part due to oxidant-mediated decreases in Fxn and that some approved compounds may be effective in increasing mitochondrial Fxn in FRDA, delaying disease progression.

In order to reduce iron deficiency in neonates at-risk for iron deficiency, we implemented a guideline to increase the consistency of early iron supplementation in infants of diabetic mothers, small for gestational age neonates and very low birth-weight premature neonates. Three years following implementation we performed a retrospective analysis in order to assess adherence to the guideline and to compare timing of early iron supplementation and reticulocyte-hemoglobin (RET-He) values at one month of life in at-risk infants. Adherence with early iron supplementation guidelines was 73.4% (399/543) with 51% (275/543) having RET-He values obtained at one month. Despite good adherence, 16% (44/275) had RET-He

In a two-part process, we assessed elements of the principal hormonal pathway regulating iron homeostasis in human neonates. Part 1: Quantifying erythropoietin (Epo), erythroferrone (ERFE), hepcidin, and relevant serum and erythrocytic iron-related metrics in umbilical cord blood from term (n = 13) and preterm (n = 10) neonates, and from neonates born to mothers with diabetes and obesity (n = 13); Part 2: Quantifying serum Epo, ERFE, and hepcidin before and following darbepoetin administration. Part 1: We measured Epo, ERFE and hepcidin in all cord blood samples. Epo and ERFE levels did not differ between the three groups. Preterm neonates had the lowest hepcidin levels, while neonates born to diabetic women with a very high BMI had the lowest ferritin and RET-He levels. Part 2: Following darbepoetin dosing, ERFE levels generally increased (p < 0.05) and hepcidin levels generally fell (p < 0.05). Our observations suggest that the Epo/ERFE/hepcidin axis is intact in the newborn period.

Background Immature reticulocytes (IRC) are the first cells to respond to changes in erythropoiesis. For antidoping applications, measurement of IRC may improve detection of blood doping practices. Unfortunately, this small cell population has limited stability in liquid blood samples and is difficult to measure with optimal precision. We developed a method to measure 3 IRC membrane proteins in dried blood spots (DBS) to monitor changes in erythropoiesis. Methods DBS spots were washed with buffers to remove soluble proteins, membrane proteins remaining in the spot were digested with trypsin, and one peptide for each protein was measured by LC-MS/MS. IRC protein concentration was determined using a DBS single point calibrator. Results Intraassay precision for IRC proteins was between 5%–15%. IRC proteins were stable in DBS for 29 days at room temperature. In a longitudinal study of 25 volunteers, the mean intraindividual variation for 3 IRC proteins was 17%, 20%, and 24% from capillary blood DBS. In comparison, the mean longitudinal variation for IRC counts measured on an automated hematology analyzer was 38%. IRC protein concentration from capillary blood DBS correlated well with venous blood DBS protein concentrations. Conclusions Measurement of IRC proteins in DBS samples provides a method to measure changes in erythropoiesis with improved analytical sensitivity, stability, and precision. When combined with the inherent advantages of capillary blood collection in the field, this method may substantially improve the detection of blood doping practices.

International audience; Mitochondrial iron import is essential for iron-sulfur cluster formation and heme biosynthesis. Two nuclear-encoded vertebrate mitochondrial high-affinity iron importers, mitoferrin1 (Mfrn1) and Mfrn2, have been identified in mammals. In mice, the gene encoding Mfrn1, solute carrier family 25 member 37 (Slc25a37), is highly expressed in sites of erythropoiesis, and whole-body Slc25a37 deletion leads to lethality. Here, we report that mice with a deletion of Slc25a28 (encoding Mfrn2) are born at expected Mendelian ratios, but show decreased male fertility due to reduced sperm numbers and sperm motility. Mfrn2 -/- mice placed on a low-iron diet exhibited reduced mitochondrial manganese, cobalt, and zinc levels, but not reduced iron. Hepatocyte-specific loss of Slc25a37 (encoding Mfrn1) in Mfrn2 -/- mice did not affect animal viability, but resulted in a 40% reduction in mitochondrial iron and reduced levels of oxidative phosphorylation proteins. Placing animals on a low-iron diet exaggerated the reduction in mitochondrial iron observed in liver-specific Mfrn1/2-knockout animals. Mfrn1 -/-/Mfrn2 -/- bone marrow-derived macrophages or skin fibroblasts in vitro were unable to proliferate, and overexpression of Mfrn1-GFP or Mfrn2-GFP prevented this proliferation defect. Loss of both mitoferrins in hepatocytes dramatically reduced regeneration in the adult mouse liver, further supporting the notion that both mitoferrins transport iron and that their absence limits proliferative capacity of mammalian cells. We conclude that Mfrn1 and Mfrn2 contribute to mitochondrial iron homeostasis and are required for high-affinity iron import during active proliferation of mammalian cells.

Building on our previous study, showing a correlation between ferritin in serum and urine, we conducted a feasibility evaluation, measuring urinary ferritin as a potential noninvasive screening test for iron deficiency among NICU patients.This was a prospective analysis of paired serum/urine ferritin levels. We defined iron-limited erythropoiesis by a RET-He5th percentile lower reference interval (28 pg).We obtained 49 paired serum/urine samples from neonates judged as at-risk for iron deficiency. Urine ferritin ("corrected" for urine creatinine and specific gravity) correlated with serum ferritin (correlation coefficient of logMeasuring urinary ferritin in NICU patients is feasible. Since low values identify iron-limitation, this could become a useful noninvasive screen.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.

OBJECTIVE: To create neonatal reference intervals for the MicroR and HYPO-He complete blood count (CBC) parameters and to test whether these parameters are sensitive early markers of disease at early stages of microcytic/hypochromic disorders while the CBC indices are still normal. STUDY DESIGN: We retrospectively collected the CBC parameters MicroR and HYPO-He, along with the standard CBC parameters, from infants aged 0–90 days at Intermountain Healthcare hospitals using Sysmex hematology analyzers. We created reference intervals for these parameters by excluding values from neonates with proven microcytic disorders (ie, iron deficiency or alpha thalassemia) from the dataset. RESULT: From >11 000 CBCs analyzed, we created reference intervals for MicroR and HYPO-He in neonates aged 0–90 days. The upper intervals are considerably higher in neonates than in adults, validating increased anisocytosis and polychromasia among neonates. Overall, 52% of neonates with iron deficiency (defined by reticulocyte hemoglobin equivalent 90% upper interval (relative risk, 4.14; 95% CI, 3.80–4.53; P < .001), and 68% had an HYPO-He >90% upper interval (relative risk, 6.64; 95% CI, 6.03–7.32; P < .001). These 2 new parameters were more sensitive than the red blood cell (RBC) indices (P < .001) in identifying 24 neonates with iron deficiency at birth. CONCLUSIONS: We created neonatal reference intervals for MicroR and HYPO-He. Although Sysmex currently designates these as research use only in the US, they can be measured as part of a neonate’s CBC with no additional phlebotomy volume or run time and can identify microcytic and hypochromic disorders even when the RBC indices are normal.

The budding yeast Saccharomyces cerevisiae stores iron in the vacuole, which is a major resistance mechanism against iron toxicity. One key protein involved in vacuolar iron storage is the iron importer Ccc1, which facilitates iron entry into the vacuole. Transcription of the CCC1 gene is largely regulated by the binding of iron–sulfur clusters to the activator domain of the transcriptional activator Yap5. Additional evidence, however, suggests that Yap5-independent transcriptional activation of CCC1 also contributes to iron resistance. Here, we demonstrate that components of the signaling pathway involving the low-glucose sensor Snf1 regulate CCC1 transcription and iron resistance. We found that SNF1 deletion acts synergistically with YAP5 deletion to regulate CCC1 transcription and iron resistance. A kinase-dead mutation of Snf1 lowered iron resistance as did deletion of SNF4, which encodes a partner protein of Snf1. Deletion of all three alternative partners of Snf1 encoded by SIT1, SIT2, and GAL83 decreased both CCC1 transcription and iron resistance. The Snf1 complex is known to activate the general stress transcription factors Msn2 and Msn4. We show that Msn2 and Msn4 contribute to Snf1-mediated CCC1 transcription. Of note, SNF1 deletion in combination with MSN2 and MSN4 deletion resulted in additive effects on CCC1 transcription, suggesting that other activators contribute to the regulation of CCC1 transcription. In conclusion, we show that yeast have developed multiple transcriptional mechanisms to regulate Ccc1 expression and to protect against high cytosolic iron toxicity.

ATP-binding cassette subfamily B member 10 (Abcb10) is a mitochondrial ATP-binding cassette (ABC) transporter that complexes with mitoferrin1 and ferrochelatase to enhance heme biosynthesis in developing red blood cells. Reductions in Abcb10 levels have been shown to reduce mitoferrin1 protein levels and iron import into mitochondria, resulting in reduced heme biosynthesis. As an ABC transporter, Abcb10 binds and hydrolyzes ATP, but its transported substrate is unknown. Here, we determined that decreases in Abcb10 did not result in protoporphyrin IX accumulation in morphant-treated zebrafish embryos or in differentiated Abcb10-specific shRNA murine Friend erythroleukemia (MEL) cells in which Abcb10 was specifically silenced with shRNA. We also found that the ATPase activity of Abcb10 is necessary for hemoglobinization in MEL cells, suggesting that the substrate transported by Abcb10 is important in mediating increased heme biosynthesis during erythroid development. Inhibition of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) with succinylacetone resulted in both 5-aminolevulinic acid (ALA) accumulation in control and Abcb10-specific shRNA MEL cells, demonstrating that reductions in Abcb10 do not affect ALA export from mitochondria and indicating that Abcb10 does not transport ALA. Abcb10 silencing resulted in an alteration in the heme biosynthesis transcriptional profile due to repression by the transcriptional regulator Bach1, which could be partially rescued by overexpression of Alas2 or Gata1, providing a mechanistic explanation for why Abcb10 shRNA MEL cells exhibit reduced hemoglobinization. In conclusion, our findings rule out that Abcb10 transports ALA and indicate that Abcb10's ATP-hydrolysis activity is critical for hemoglobinization and that the substrate transported by Abcb10 provides a signal that optimizes hemoglobinization.

Background Vacuolated amniotic epithelium with lipid droplets in gastroschisis placentas is an unusual finding. Mass spectrometry of lipid droplets identified triglycerides, ester-linked to an unusual pattern of fatty acids. We hypothesize that these findings result from a Chlamydia trachomatis infection during the periconceptional period. The rising incidence of chlamydia infections has paralleled the increasing prevalence of gastroschisis among women less than 25 years of age. Histologically, young women are at greatest risk for a chlamydia infection due to their immature columnar epithelium, the preferential site for attachment of Chlamydia trachomatis infectious particle (elementary body). Methods Chlamydia trachomatis survive in an inclusion, relying on its host to acquire essential nutrients, amino acids, and nucleotides for survival and replication. If essential nutrients are not available, the bacteria cannot replicate and may be trafficked to the lysosome for degradation or remain quiescent, within the inclusion, subverting innate immunologic clearance. Results Chlamydiae synthesize several lipids (phosphatidylethanolamine, phosphatidylserine, and phosphoatidylglycerol); however, their lipid content reveal eukaryotic lipids (sphingomyelin, cholesterol, phosphatidylcholine, and phosphatidylinositol), evidence that chlamydiae “hijack” host lipids for expansion and replication. Conclusion The abnormal amniotic epithelial findings are supported by experimental evidence of the trafficking of host lipids into the chlamydiae inclusion. If not lethal, what harm will elementary bodies inflict to the developing embryo? Do these women have a greater pro-inflammatory response to an environmental exposure, whether cigarette smoking, change in partner, or a pathogen? Testing the hypothesis that Chlamydia trachomatis is responsible for amniotic epithelium vacuoles will be a critical first step. Birth Defects Research 109:1003–1010, 2017. © 2017 Wiley Periodicals, Inc.

Budding yeast (Saccharomyces cerevisiae) responds to low cytosolic iron by up-regulating the expression of iron import genes; iron import can reflect iron transport into the cytosol or mitochondria. Mmt1 and Mmt2 are nuclearly encoded mitochondrial proteins that export iron from the mitochondria into the cytosol. Here we report that MMT1 and MMT2 expression is transcriptionally regulated by two pathways: the low-iron-sensing transcription factor Aft1 and the oxidant-sensing transcription factor Yap1. We determined that MMT1 and MMT2 expression is increased under low-iron conditions and decreased when mitochondrial iron import is increased through overexpression of the high-affinity mitochondrial iron importer Mrs3. Moreover, loss of iron-sulfur cluster synthesis induced expression of MMT1 and MMT2. We show that exposure to the oxidant H(2)O(2) induced MMT1 expression but not MMT2 expression and identified the transcription factor Yap1 as being involved in oxidant-mediated MMT1 expression. We defined Aft1- and Yap1-dependent transcriptional sites in the MMT1 promoter that are necessary for low-iron- or oxidant-mediated MMT1 expression. We also found that the MMT2 promoter contains domains that are important for regulating its expression under low-iron conditions, including an upstream region that appears to partially repress expression under low-iron conditions. Our findings reveal that MMT1 and MMT2 are induced under low-iron conditions and that the low-iron regulator Aft1 is required for this induction. We further uncover an Aft1-binding site in the MMT1 promoter sufficient for inducing MMT1 transcription and identify an MMT2 promoter region required for low iron induction.

Objectives Small for gestational age infants (SGA), infants of diabetic mothers (IDM), and very low birth weight infants (VLBW) are at risk for congenital iron deficiency. We evaluated the iron status of SGA, IDM, and VLBW neonates at birth and sought mechanistic explanations in those with iron deficiency. Methods This was a prospective study. If congenital iron deficiency was present, maternal iron studies were obtained. When neonates were two weeks old, their iron status was reevaluated. Results Sixteen of 180 neonates screened were iron deficient at birth. The Body Mass Index of the 16 mothers was high. These mothers often had mild iron deficiency and measurable hepcidin levels. Two weeks after birth, neonates had improved iron measurements. Conclusions Among SGA, IDM, and VLBW neonates, maternal obesity is a risk factor for congenital iron deficiency. We speculate that elevated hepcidin levels in obese pregnant women impede iron absorption and interfere with transplacental iron transfer.

In an iron deficient child, oral iron repeatedly failed to improve the condition. Whole exome sequencing identified one previously reported plus two novel mutation in the TMPRSS6 gene, with no mutations in other iron-associated genes. We propose that these mutations result in a novel variety of iron-refractory iron deficiency anemia.

Serum ferritin reflects total body iron stores, thus a low serum ferritin is used as a parameter of iron deficiency. In healthy adults in Japan, urine ferritin levels were about 5% of serum ferritin levels, with a correlation coefficient of 0.79. It is not known whether a low urine ferritin could serve as a non-invasive screen for iron deficiency. If so, this might be useful for neonates and young children, avoiding phlebotomy to screen for iron deficiency. However, for urinary ferritin screening to be feasible, ferritin must be measurable in the urine and correlate with serum ferritin. Testing should also clarify whether the iron content of ferritin in serum and urine are similar. In this pilot feasibility study we measured ferritin in paired serum and urine samples of healthy adult males, healthy term neonates, growing preterm neonates, and children who had very high serum ferritin levels from liver disorders or iron overload. We detected ferritin in every urine sample, and found a correlation with paired serum ferritin (Spearman correlation coefficient 0.78 of log(10)-transformed values). These findings suggest merit in further studying urinary ferritin in select populations, as a potential non-invasive screen to assess iron stores.

Mitochondria are an iconic distinguishing feature of eukaryotic cells. Mitochondria encompass an active organellar network that fuses, divides, and directs a myriad of vital biological functions, including energy metabolism, cell death regulation, and innate immune signaling in different tissues. Another crucial and often underappreciated function of these dynamic organelles is their central role in the metabolism of the most abundant and biologically versatile transition metals in mammalian cells, iron. In recent years, cellular and animal models of mitochondrial iron dysfunction have provided vital information in identifying new proteins that have elucidated the pathways involved in mitochondrial homeostasis and iron metabolism. Specific signatures of mitochondrial iron dysregulation that are associated with disease pathogenesis and/or progression are becoming increasingly important. Understanding the molecular mechanisms regulating mitochondrial iron pathways will help better define the role of this important metal in mitochondrial function and in human health and disease.

The congenital dyserythropoietic anemias are a heterogeneous group of disorders characterized by anemia and ineffective erythropoiesis. Congenital dyserythropoietic anemia type I (CDA1) can present in utero with hydrops fetalis, but more often it presents in childhood or adulthood with moderate macrocytic anemia, jaundice, and progressive iron-overload. CDA1 is inherited in an autosomal recessive manner, with biallelic pathogenic variants in CDAN1 or C15orf41. This case report documents a severe fetal presentation of CDA1 where we identified two novel compound heterozygous mutations in CDAN1 and describes the associated pathologic findings and levels of iron-regulatory proteins hepcidin, erythroferrone, and GDF15.

Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho(−) or Rho(0)) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron–sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial iron metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29. Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increases mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism.

Mitochondrial are a key organelle in iron metabolism and many metabolic processes involved in iron homeostasis occur in the mitochondria. Eukaryotic cells have developed different transport mechanisms to deal with coordinating movement of iron and iron-related molecules across membranes. Some of those transport mechanisms involve ATP-binding cassette (ABC) transporters. There are four mitochondrial ABC transporters Abcb6, Abcb7, Abcb8 and Abcb10. Abcb6 is localized to the outer membrane of mitochondria where it is involved in porphyrin transport. Abcb7, Abcb8 and Abcb10 are localized to the inner mitochondrial membrane and the exact molecule transported by each is still unclear. Here, we provide a brief review of what is known about each transporter and its role in mitochondrial iron homeostasis. We describe the human diseases associated with known mutations in the genes encoding these proteins and discuss the possible importance of these transporters in immune cell function.

All eukaryotes require the transition metal, iron, a redox active element that is an essential cofactor in many metabolic pathways, as well as an oxygen carrier. Iron can also react to generate oxygen radicals such as hydroxyl radicals and superoxide anions, which are highly toxic to cells. Therefore, organisms have developed intricate mechanisms to acquire iron as well as to protect themselves from the toxic effects of excess iron. In fungi and plants, iron is stored in the vacuole as a protective mechanism against iron toxicity. Iron storage in the vacuole is mediated predominantly by the vacuolar metal importer Ccc1 in yeast and the homologous transporter VIT1 in plants. Transcription of yeast CCC1 expression is tightly controlled primarily by the transcription factor Yap5, which sits on the CCC1 promoter and activates transcription through the binding of Fe-S clusters. A second mechanism that regulates CCC1 transcription is through the Snf1 signaling pathway involved in low-glucose sensing. Snf1 activates stress transcription factors Msn2 and Msn4 to mediate CCC1 transcription. Transcriptional regulation by Yap5 and Snf1 are completely independent and provide for a graded response in Ccc1 expression. The identification of multiple independent transcriptional pathways that regulate the levels of Ccc1 under high iron conditions accentuates the importance of protecting cells from the toxic effects of high iron.

Vacuolated amniotic epithelium with lipid droplets in gastroschisis placentas is an unusual finding. Mass spectrometry of lipid droplets identified triglycerides, ester-linked to an unusual pattern of fatty acids. We hypothesize that these findings result from a Chlamydia trachomatis infection during the periconceptional period. The rising incidence of chlamydia infections has paralleled the increasing prevalence of gastroschisis among women less than 25 years of age. Histologically, young women are at greatest risk for a chlamydia infection due to their immature columnar epithelium, the preferential site for attachment of Chlamydia trachomatis infectious particle (elementary body).Chlamydia trachomatis survive in an inclusion, relying on its host to acquire essential nutrients, amino acids, and nucleotides for survival and replication. If essential nutrients are not available, the bacteria cannot replicate and may be trafficked to the lysosome for degradation or remain quiescent, within the inclusion, subverting innate immunologic clearance.Chlamydiae synthesize several lipids (phosphatidylethanolamine, phosphatidylserine, and phosphoatidylglycerol); however, their lipid content reveal eukaryotic lipids (sphingomyelin, cholesterol, phosphatidylcholine, and phosphatidylinositol), evidence that chlamydiae "hijack" host lipids for expansion and replication.The abnormal amniotic epithelial findings are supported by experimental evidence of the trafficking of host lipids into the chlamydiae inclusion. If not lethal, what harm will elementary bodies inflict to the developing embryo? Do these women have a greater pro-inflammatory response to an environmental exposure, whether cigarette smoking, change in partner, or a pathogen? Testing the hypothesis that Chlamydia trachomatis is responsible for amniotic epithelium vacuoles will be a critical first step. Birth Defects Research 109:1003-1010, 2017. © 2017 Wiley Periodicals, Inc.

The budding yeast Saccharomyces cerevisiae stores iron in the vacuole, which is a major resistance mechanism against iron toxicity. One key protein involved in vacuolar iron storage is the iron importer Ccc1, which facilitates iron entry into the vacuole. Transcription of the CCC1 gene is largely regulated by the binding of iron–sulfur clusters to the activator domain of the transcriptional activator Yap5. Additional evidence, however, suggests that Yap5-independent transcriptional activation of CCC1 also contributes to iron resistance. Here, we demonstrate that components of the signaling pathway involving the low-glucose sensor Snf1 regulate CCC1 transcription and iron resistance. We found that SNF1 deletion acts synergistically with YAP5 deletion to regulate CCC1 transcription and iron resistance. A kinase-dead mutation of Snf1 lowered iron resistance as did deletion of SNF4, which encodes a partner protein of Snf1. Deletion of all three alternative partners of Snf1 encoded by SIT1, SIT2, and GAL83 decreased both CCC1 transcription and iron resistance. The Snf1 complex is known to activate the general stress transcription factors Msn2 and Msn4. We show that Msn2 and Msn4 contribute to Snf1-mediated CCC1 transcription. Of note, SNF1 deletion in combination with MSN2 and MSN4 deletion resulted in additive effects on CCC1 transcription, suggesting that other activators contribute to the regulation of CCC1 transcription. In conclusion, we show that yeast have developed multiple transcriptional mechanisms to regulate Ccc1 expression and to protect against high cytosolic iron toxicity.

Mmt1 and Mmt2 are highly homologous yeast members of the cation diffusion facilitator transporter family localized to mitochondria. Overexpression of MMT1/2 led to changes in cellular metal homeostasis (increased iron sensitivity, decreased cobalt sensitivity, increased sensitivity to copper), oxidant generation, and increased sensitivity to H2O2. The phenotypes due to overexpression of MMT1&2 were similar to that seen in cells with deletions in MRS3 and MRS4, genes that encode the mitochondrial iron importers. Overexpression of MMT1&2 resulted in induction of the low iron transcriptional response, similar to that seen in Δmrs3Δmr4 cells. This low iron transcriptional response was suppressed by deletion of CCC1, the gene that encodes the vacuolar iron importer. Measurement of the activity of the iron-dependent gentisate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans expressed in yeast cytosol, showed that changes in Mmt1/2 levels affected cytosol iron concentration even in the absence of Ccc1. Overexpression of MMT1 resulted in increased cytosolic iron whereas deletion of MMT1/MMT2 led to decreased cytosolic iron. These results support the hypothesis that Mmt1/2 function as mitochondrial iron exporters.

Disturbed iron (Fe) ion homeostasis and mitochondrial dysfunction have been implicated in neurodegeneration. Both processes are related, because central Fe ion consuming biogenetic pathways take place in mitochondria and affect their oxidative energy metabolism. Iron is imported into mitochondria by the two homologous Fe ion importers mitoferrin-1 and mitoferrin-2. To elucidate more specifically the role of mitochondrial Fe ions for brain energy metabolism and for proper neuronal function, we generated mice with a neuron-specific knockout of mitoferrin-1 (Slc25a37(−/−) or mfrn-1(−/−)) and compared them with corresponding control littermates (mfrn-1(flox/flox)). Mice lacking neuronal mfrn-1 exhibited no obvious anatomical or behavioral abnormalities as neonates, young or adult animals. However, they exhibited a moderate decrease in brain mitochondrial O(2)-consumption with complex-I substrates of the electron transport chain (p < 0.05), indicating a moderate suppression of electron transport. While these mice did not exhibit altered basal fear levels, inquisitiveness or motor skills in specific neurobiological test batteries, they clearly exhibited decreased spatial learning skills and missing establishment of stable spatial memory in Morris water maze, as compared to floxed controls (p < 0.05). We thus conclude that mitochondrial Fe ion supply is an important player in neuronal energy metabolism and proper brain function and that the carrier mitoferrin-1 cannot be completely replaced by mitoferrin-2 or other as yet unknown Fe ion carriers.

Extracellular vesicles, including exosomes, have recently been implicated as novel mediators of immune cell communication in mammals. However, roles for endogenously produced exosomes in regulating immune cell functions in vivo are just beginning to be identified. In this article, we demonstrate that Rab27a and Rab27b double-knockout (Rab27DKO) mice that are deficient in exosome secretion have a chronic, low-grade inflammatory phenotype characterized by elevated inflammatory cytokines and myeloproliferation. Upon further investigation, we found that some of these phenotypes could be complemented by wild-type (WT) hematopoietic cells or administration of exosomes produced by GM-CSF–expanded bone marrow cells. In addition, chronically inflamed Rab27DKO mice had a blunted response to bacterial LPS, resembling endotoxin tolerance. This defect was rescued by bone marrow exosomes from WT, but not miR-155−/−, cells, suggesting that uptake of miR-155–containing exosomes is important for a proper LPS response. Further, we found that SHIP1 and IRAK-M, direct targets of miR-155 that are known negative regulators of the LPS response, were elevated in Rab27DKO mice and decreased after treatment with WT, but not miR-155−/−, exosomes. Together, our study finds that Rab27-dependent exosome production contributes to homeostasis within the hematopoietic system and appropriate responsiveness to inflammatory stimuli.

Small-for-gestational-age (SGA) neonates, infants of diabetic mothers (IDM) and very-low-birth weight premature neonates (VLBW) are reported to have increased risk for developing iron deficiency and possibly associated neurocognitive delays.We conducted a pilot study to assess iron status at birth in at-risk neonates by measuring iron parameters in umbilical cord blood from SGA, IDM, VLBW and comparison neonates.Six of the 50 infants studied had biochemical evidence of iron deficiency at birth. Laboratory findings consistent with iron deficiency were found in one SGA, one IDM, three VLBW, and one comparison infant. None of the infants had evidence of iron deficiency anemia.Evidence of biochemical iron deficiency at birth was found in 17% of screened neonates. Studies are needed to determine whether these infants are at risk for developing iron-limited erythropoiesis, iron deficiency anemia or iron-deficient neurocognitive delay.

Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias1,2. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt tq209). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe–2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe–2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe–2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.

Mon1a was originally identified as a modifier gene of vesicular traffic, as a mutant Mon1a allele resulted in increased localization of cell surface proteins, whereas reduced levels of Mon1a showed decreased secretory activity. Here we show that Mon1a affects different steps in the secretory pathway including endoplasmic reticulum-to-Golgi traffic. siRNA-dependent reduction of Mon1a levels resulted in a delay in the reformation of the Golgi apparatus after Brefeldin A treatment. Endoglycosidase H treatment of ts045VSVG-GFP confirmed that knockdown of Mon1a delayed endoplasmic reticulum-to-Golgi trafficking. Reductions in Mon1a also resulted in delayed trafficking from Golgi to the plasma membrane. Immunoprecipitation and mass spectrometry analysis showed that Mon1a associates with dynein intermediate chain. Reductions in Mon1a or dynein altered steady state Golgi morphology. Reductions in Mon1a delayed formation of ERGIC-53-positive vesicles, whereas reductions in dynein did not affect vesicle formation. These data provide strong evidence for a role for Mon1a in anterograde trafficking through the secretory apparatus.

The lysosome is considered the terminal point in the endocytic pathway, as molecules are delivered to the lysosome for degradation (for review see (1)). Fusion of endosomes with lysosomes leads to the intermixing of vesicular contents with lysosomal hydrolases. The addition of vesicular contents and membrane would be expected to increase lysosome size. The steady state median size of lysosomes is constant, however, indicating that lysosomal surface area is regulated by lysosomal membrane efflux. It has been suggested that endocytic membrane influx is transient and that once endosomal contents are transferred to lysosomes, endosomes fission off, a process referred to as “kiss-and-run” (2). Constant fusion and fission of membrane along the endocytic pathway results in remodeling of organelles, which may eventually become lysosomes, a process termed maturation (3). Lysosomes are also capable of homotypic fusion (4–13) yet the number of lysosomes is constant. Therefore, lysosomes must be capable of fission or some other form of membrane recycling, although the machinery required for lysosome fission has not been identified. Malregulation of fusion or fission will result in alterations in lysosome size. This is a hallmark of Chediak Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by enlarged lysosomes or lysosome-related organelles present within all cells (14–17). The mutated gene responsible for CHS (LYST) and the orthologous mouse disorder beige (Lyst) were identified almost 15 years ago (18, 19). The identification of Chs1/Lyst defined a family of proteins termed the BEige And CHediak (BEACH) family (15, 20). The BEACH family protein LvsA in Dictyostelium discoideum regulates the size of the contractile vacuole and is involved in cytokinesis (21–23). Neurobeachin has been characterized to be involved in central synapse formation (24). FAN, the smallest member of the BEACH family, is thought to be an adapter protein linking TNFa signaling to neutral sphingomyelinase. Bph1, the only Saccharomyces cerevisiae homologue is involved in vesicle trafficking, but loss of Bph1 does not affect vacuole morphology (25). The functions of many members of the BEACH family are still unclear. Previously, we suggested that loss of Chs1/Lyst resulted in decreased lysosomal fission based upon the observation that overexpression of Lyst resulted in smaller than wild type lysosomes (26). Fusion of wild type and beigej cells led to complementation of the large lysosome size (27). We noted, however, that complementation of large beigej lysosomes required their fusion with wild type lysosomes, suggesting that the wild type lysosome provided a factor promoting decreased lysosome size. Studies on the D. discoideum homologue LvsB suggested that LvsB was either a negative regulator of lysosome fusion (28, 29) or a positive regulator of “post-lysosome” fission (30). In this study we examine whether the loss of Chs1/Lyst changes the rate of lysosome fusion or lysosome fission by measuring the rate of reformation of lysosomes following treatments that increase or decrease lysosome size. We show that the loss of Chs1/Lyst does not affect lysosome fusion rates but affects the rate of lysosome fission and that it is the defect in fission that gives rise to enlarged lysosomes associated with the loss of Chs1/Lyst.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the Authors.We, the authors, wish to retract “Decoupling Ferritin Synthesis from Free Cytosolic Iron Results in Ferritin Secretion” by De Domenico et al. (Cell Metab., 13 (2011) 57–67, http://dx.doi.org/10.1016/j.cmet.2010.12.003 and “The Role of Ubiquitination in Hepcidin-Independent and Hepcidin-Dependent Degradation of Ferroportin” by De Domenico et al. (Cell Metab., 14 (2011) 635–646, http://dx.doi.org/10.1016/j.cmet.2011.09.008) because a number of errors have been detected in the assembly of the figures, and some of the original data were inappropriately removed from the laboratory. We stand by the validity of our studies; the data are reproducible, and the conclusions were not affected by the errors. However, we believe that the most responsible course of action is to retract the paper. We are preparing a new expanded version of this study for future submission. We deeply apologize to the community for the inconvenience.

The budding yeast Saccharomyces cerevisiae responds to high cytosolic iron by inducing Yap5-mediated transcription. We identified genes regulated by Yap5 in response to iron and show that one of the genes induced is TYW1, which encodes an iron-sulfur cluster enzyme that participates in the synthesis of wybutosine-modified tRNA. Strains deleted for TYW1 do not show a phenotype in standard yeast medium. In contrast, overexpression of TYW1 results in decreased cell growth and induction of the iron regulon, leading to increased expression of the high affinity iron transporters. We identified a minimal domain of S. cerevisiae Tyw1 that is sufficient to induce the iron regulon. CCC1, a vacuolar iron importer, is a Yap5-regulated gene, and deletion of either CCC1 or YAP5 resulted in high iron sensitivity. Deletion of TYW1 in a Δccc1 strain led to increased iron sensitivity. The increased iron sensitivity of Δccc1Δtyw1 could be suppressed by overexpression of iron-sulfur cluster enzymes. We conclude that the Yap5-mediated induction of TYW1 provides protection from high iron toxicity by the consumption of free cytosolic iron through the formation of protein-bound iron-sulfur clusters.

Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity, but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in Δccc1 cells to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown Δccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4, and RIM2. Overexpression of MRS3 suppressed high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions, Δccc1 cells were still sensitive to high iron toxicity, but overexpression of MRS3 did not suppress iron toxicity and did not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in Δccc1 cells occurred under both aerobic and anaerobic conditions. Microarray analysis showed no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1, which encodes a peroxiredoxin, exacerbated iron toxicity in Δccc1 cells under both aerobic and anaerobic conditions, suggesting a unique role for Tsa1 in iron toxicity.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the Authors.We, the authors, wish to retract “Decoupling Ferritin Synthesis from Free Cytosolic Iron Results in Ferritin Secretion” by De Domenico et al. (Cell Metab., 13 (2011) 57–67, http://dx.doi.org/10.1016/j.cmet.2010.12.003 and “The Role of Ubiquitination in Hepcidin-Independent and Hepcidin-Dependent Degradation of Ferroportin” by De Domenico et al. (Cell Metab., 14 (2011) 635–646, http://dx.doi.org/10.1016/j.cmet.2011.09.008) because a number of errors have been detected in the assembly of the figures, and some of the original data were inappropriately removed from the laboratory. We stand by the validity of our studies; the data are reproducible, and the conclusions were not affected by the errors. However, we believe that the most responsible course of action is to retract the paper. We are preparing a new expanded version of this study for future submission. We deeply apologize to the community for the inconvenience.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well understood. To better understand these regulatory mechanisms, we profiled gene expression in EPO-treated fetal liver cells to identify novel iron regulatory genes (Figure A). We determined that FAM210B, a mitochondrial inner membrane protein, was essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation (Figure B). Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation (Figure C). These defects were corrected with a lipid-soluble small molecule iron transporter in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter, but is required for optimal mitochondrial iron import during erythroid differentiation (Figure D). FAM210B is also required for optimal FECH activity in differentiating erythroid cells. As FAM210B interacts with the terminal enzymes of the heme synthesis pathway, we propose that FAM210B functions as an adaptor protein to facilitate the formation of an oligomeric mitochondrial iron transport complex, which is required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis (Figure E). Collectively, our data reveal a novel mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism. Figure. Figure. Disclosures Palis: Rubies Therapeutics: Consultancy.

Mutations in the iron exporter ferroportin (Fpn) result in iron overload in macrophages or hepatocytes depending upon the mutation. Patients with Fpn mutation D157G show high serum ferritin and normal to slightly elevated transferrin saturation. Here, we show that Fpn(D157G)–green fluorescent protein (GFP) is down-regulated independent of hepcidin, and that this down-regulation is due to the constitutive binding of Jak2 and Fpn phosphorylation. Expression of Fpn(D157G)-GFP in Danio rerio results in a severe growth defect, which can be rescued by iron supplementation. These results identify a hepcidin-independent regulation of Fpn that can result in alterations in iron homeostasis.

Deletion of two homologous genes, MRS3 and MRS4, that encode mitochondrial iron transporters affects the activity of the vacuolar iron importer Ccc1. Ccc1 levels are decreased in Deltamrs3Deltamrs4 cells, but the activity of the transporter is increased, resulting is reduced cytosolic iron. Overexpression of CCC1 in Deltamrs3Deltamrs4 cells results in a severe growth defect due to decreased cytosolic iron, referred to as the mitochondria-vacuole signaling (MVS) phenotype. Mutants were identified that suppress the MVS growth defect, and FRA1 was identified as a gene that suppresses the MVS phenotype. Overexpression of FRA1 suppresses altered transition metal metabolism in Deltamrs3Deltamrs4 cells, whereas deletion of FRA1 is synthetically lethal with Deltamrs3Deltamrs4. Fra1 binds to Tsa1, which encodes a thioredoxin-dependent peroxidase. Deletion of TSA1 or TRR1 is synthetically lethal in Deltamrs3Deltamrs4 cells, suggesting that Deltamrs3Deltamrs4 cells generate reactive oxygen metabolites. The generation of reactive oxygen metabolites in Deltamrs3Deltamrs4 cells was confirmed by use of the reporter molecule 2',7'-dichlorodihydrofluorescein diacetate. These results suggest that mitochondria-induced oxidant damage is responsible for activating Ccc1 and that Fra1 and Tsa1 can reduce oxidant damage.

Mitoferrin-1 (Mfrn1; Slc25a37), a member of the solute carrier family localized in the mitochondrial inner membrane, functions as an essential iron importer for the synthesis of mitochondrial heme and iron–sulfur clusters in erythroblasts. The biochemistry of Mfrn1-mediated iron transport into the mitochondria, however, is poorly understood. Here, we used the strategy of in vivo epitope-tagging affinity purification and mass spectrometry to investigate Mfrn1-mediated mitochondrial iron homeostasis. Abcb10, a mitochondrial inner membrane ATP-binding cassette transporter highly induced during erythroid maturation in hematopoietic tissues, was found as one key protein that physically interacts with Mfrn1 during mouse erythroleukemia (MEL) cell differentiation. Mfrn1 was shown previously to have a longer protein half-life in differentiated MEL cells compared with undifferentiated cells. In this study, Abcb10 was found to enhance the stabilization of Mfrn1 protein in MEL cells and transfected heterologous COS7 cells. In undifferentiated MEL cells, cotransfected Abcb10 specifically interacts with Mfrn1 to enhance its protein stability and promote Mfrn1-dependent mitochondrial iron importation. The structural stabilization of the Mfrn1–Abcb10 complex demonstrates a previously uncharacterized function for Abcb10 in mitochondria. Furthermore, the binding domain of Mfrn1–Abcb10 interaction maps to the N terminus of Mfrn1. These results suggest the tight regulation of mitochondrial iron acquisition and heme synthesis in erythroblasts is mediated by both transcriptional and posttranslational mechanisms, whereby the high level of Mfrn1 is stabilized by oligomeric protein complexes.

Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865–33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal- binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains.

Iron is an essential nutrient but in excess may damage cells by generating reactive oxygen species due to Fenton reaction or by substituting for other transition metals in essential proteins. The budding yeast Saccharomyces cerevisiae detoxifies cytosolic iron by storage in the vacuole. Deletion of CCC1, which encodes the vacuolar iron importer, results in high iron sensitivity due to increased cytosolic iron. We selected mutants that permitted Δccc1 cells to grow under high iron conditions by UV mutagenesis. We identified a mutation (N44I) in the vacuolar zinc transporter ZRC1 that changed the substrate specificity of the transporter from zinc to iron. COT1, a vacuolar zinc and cobalt transporter, is a homologue of ZRC1 and both are members of the cation diffusion facilitator family. Mutation of the homologous amino acid (N45I) in COT1 results in an increased ability to transport iron and decreased ability to transport cobalt. These mutations are within the second hydrophobic domain of the transporters and show the essential nature of this domain in the specificity of metal transport.

Many intracellular pathogens infect macrophages and these pathogens require iron for growth. Here we demonstrate in vitro that the intracellular growth of Chlamydia psittaci, trachomatis, and Legionella pneumophila is regulated by the levels of intracellular iron. Macrophages that express cell surface ferroportin, the only known cellular iron exporter, limit the intracellular growth of these bacteria. Hepcidin is an antimicrobial peptide secreted by the liver in response to inflammation. Hepcidin binds to ferroportin mediating its internalization and degradation. Addition of hepcidin to infected macrophages enhanced the intracellular growth of these pathogens. Macrophages from flatiron mice, a strain heterozygous for a loss-of-function ferroportin mutation, showed enhanced intracellular bacterial growth independent of the presence of exogenous hepcidin. Macrophages, from wild-type or flatiron mice, incubated with the oral iron chelator deferriprone or desferasirox showed reduced intracellular bacterial growth suggesting that these chelators might be therapeutic in chronic intracellular bacterial infections.

SummaryThe ESCRT pathway mediates membrane remodeling during enveloped virus budding, cytokinesis, and intralumenal endosomal vesicle formation. Late in the pathway, a subset of membrane-associated ESCRT-III proteins display terminal amphipathic “MIM1” helices that bind and recruit VPS4 ATPases via their MIT domains. We now report that VPS4 MIT domains also bind a second, “MIM2” motif found in a different subset of ESCRT-III subunits. The solution structure of the VPS4 MIT-CHMP6 MIM2 complex revealed that MIM2 elements bind in extended conformations along the groove between the first and third helices of the MIT domain. Mutations that block VPS4 MIT-MIM2 interactions inhibit VPS4 recruitment, lysosomal protein targeting, and HIV-1 budding. MIT-MIM2 interactions appear to be common throughout the ESCRT pathway and possibly elsewhere, and we suggest how these interactions could contribute to a mechanism in which VPS4 and ESCRT-III proteins function together to constrict the necks of budding vesicles.