Genetic and biochemical analysis of high iron toxicity in yeast: iron toxicity is due to the accumulation of cytosolic iron and occurs under both aerobic and anaerobic conditions

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.     

Frataxin and mitochondrial carrier proteins, Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.     

YFH1-mediated iron homeostasis is independent of mitochondrial respiration.

The human gene frataxin and its yeast homolog YFH1 affect mitochondrial function. Deficits in frataxin result in Friedreich ataxia, while deletion of YFH1 results in respiratory incompetence. We determined that as long as respiratory incompetent yeast express Yfh1p they do not accumulate excessive mitochondrial iron. Deletion of YFH1 in respiratory incompetent yeast results in mitochondrial iron accumulation, while the reintroduction of Yfh1p results in mitochondrial iron export. Further, overexpression of Yfh1p has no effect on oxygen consumption in wild-type yeast grown in either fermentative or respiratory carbon sources. We conclude that the effect of Yfh1p on mitochondrial iron metabolism is independent of respiratory activity.     

Major targets of iron-induced protein oxidative damage in frataxin-deficient yeasts are magnesium-binding proteins

Iron accumulation has been associated with several pathological conditions such as Friedreich ataxia. This human disorder is caused by decreased expression of frataxin. Iron-overload triggers oxidative stress, but the main targets of such stress are not known. In yeast cells lacking the frataxin ortholog YFH1, we have identified a set of 14 carbonylated proteins, which include mitochondrial ATP synthase, phosphoglycerate kinase, pyruvate kinase, and molecular chaperones. Interestingly, most of the target proteins are magnesium- and/or nucleotide-binding proteins. This key feature leads us to postulate that when iron accumulates, chelatable iron replaces magnesium at the corresponding metal-binding site, promoting selective damage to these proteins. Consistent with this hypothesis, in vitro experiments performed with pure pyruvate kinase and phosphoglycerate kinase showed that oxidation of these proteins can be prevented by magnesium and increased by the presence of ATP. Also, chelatable iron, which forms complexes with nucleotides, showed a sevenfold increase in Deltayfh1 cells. Moreover, lowering chelatable iron in Deltayfh1 cells by desferrioxamine prevented enzyme inactivation. As a general conclusion, we propose that magnesium bound to proteins is replaced by chelatable iron when this metal accumulates. This mechanism explains selective protein oxidation and provides clues for better understanding of iron-overloading pathologies.     

Deficiency of the mitochondrial electron transport chain in muscle does not cause insulin resistance

Background

It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis.

Methodology/Principal Findings

Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance.

Conclusions/Significance

The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.     

The role of the mitochondrion in cellular iron homeostasis

The yeast ATM1 protein is essential for normal mitochondrial iron homeostasis. Deletion of ATM1 results in mitochondrial iron accumulation and oxidative mitochondrial damage. Mutations in ABC7, the human homolog of ATM1, result in X-linked sideroblastic anemia and ataxia. Here we show that a deletion of ATM1 also has effects on extra-mitochondrial iron metabolism. ATM1-deficient cells have an increased iron requirement for growth. When grown in iron-rich medium, mutant cells accumulate excess mitochondrial iron and have increased expression of the genes required for both high and low affinity iron uptake. Thus, ATM1 mutant cells simultaneously demonstrate features of both iron overload and iron starvation. Yfh1p is the yeast homolog of the human frataxin protein, which is deficient in Friedreich's ataxia. As in atm1 cells, a yfh1 deletion results in both mitochondrial iron accumulation and cytosolic iron starvation. In spite of their apparent roles in cellular iron homeostasis, we find that the expression of neither ATM1 nor YFH1 is responsive to cellular iron status. Based on these observations, we propose a model in which cellular iron is prioritized for use by the mitochondrion, and available to the remainder of the cell only after mitochondrial needs have been fulfilled.     

Oxidative stress and protease dysfunction in the yeast model of Friedreich ataxia

Friedreich ataxia has frequently been associated with an increased susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich ataxia to study the physiological consequences of a shift from anaerobiosis to aerobiosis. Cells lacking frataxin (Deltayfh1) showed no growth defect when cultured anaerobically. Under these conditions, a significant amount of aconitase was functional, with an intact 4 Fe/4 S cluster. When shifted to aerobic conditions, aconitase was rapidly degraded, and oxidatively modified proteins (carbonylated and HNE-modified proteins) accumulated in both the cytosol and the mitochondria. The ATP-dependent mitochondrial protease Pim1 (Lon) was strongly activated, although its expression level remained unchanged, and the cytosolic activity of the 20S proteasome was greatly decreased, compared to that in wild-type cells. Analysis of the purified proteasome revealed that the decrease in proteasome activity was likely due to both direct inactivation of the enzyme and inhibition by cytosolic oxidized proteins. These features indicate that the cells were subjected to major oxidative stress triggered by oxygen. Accumulation of oxidatively modified proteins, activation of Pim1, and proteasome inhibition did not directly depend on the amount of mitochondrial iron, because these phenotypes remained unchanged when the cells were grown under iron-limiting conditions, and these phenotypes were not observed in another mutant (Deltaggc1) which overaccumulates iron in its mitochondrial compartment. We conclude that oxygen is primarily involved in generating the deleterious phenotypes that are observed in frataxin-deficient yeast cells.     

CCC1 is a transporter that mediates vacuolar iron storage in yeast

The budding yeast Saccharomyces cerevisiae can grow for generations in the absence of exogenous iron, indicating a capacity to store intracellular iron. As cells can accumulate iron by endocytosis we studied iron metabolism in yeast that were defective in endocytosis. We demonstrated that endocytosis-defective yeast (Delta end4) can store iron in the vacuole, indicating a transfer of iron from the cytosol to the vacuole. Using several different criteria we demonstrated that CCC1 encodes a transporter that effects the accumulation of iron and Mn(2+) in vacuoles. Overexpression of CCC1, which is localized to the vacuole, lowers cytosolic iron and increases vacuolar iron content. Conversely, deletion of CCC1 results in decreased vacuolar iron content and decreased iron stores, which affect cytosolic iron levels and cell growth. Furthermore Delta ccc1 cells show increased sensitivity to external iron. The sensitivity to iron is exacerbated by ectopic expression of the iron transporter FET4. These results indicate that yeast can store iron in the vacuole and that CCC1 is involved in the transfer of iron from the cytosol to the vacuole.