Eunsook Song

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots.

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and β-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and β-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and β-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.

Iron transport by proteoliposomes containing mitochondrial F1Fo ATP synthase isolated from rat heart

In this work, we present evidence of Fe(2+) transport by rat heart mitochondrial F(1)F(0) ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 degrees C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the alpha, beta, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg(+2), Ca(+2), Mn(+2), Zn(+2), Cu(+2), Fe(+3), and Al(+3) was observed. Moreover, Cu(+2), even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H(+).

ATP5B regulates mitochondrial fission and fusion in mammalian cells

ABSTRACT Mitochondria are essential organelles that produce ATP and regulate cell growth, proliferation, and cell death. To maintain homeostasis, fusion and fission of mitochondria must be strictly regulated. Even though oligomerization of ATP synthase could affect the mitochondrial morphology, the exact mechanism is not clear. We confirmed that structure and function of ATP5B, which is a major component of the catalytic center of ATP synthase complexes, are closely connected to the mitochondrial morphology. ATP5B itself can enhance elongation of mitochondria. Moreover, mutations of the threonine residue at β-barrel domain, and the serine residue at nucleotide-binding domain of ATP5B, produce the opposite effect on the fission and fusion of mitochondrial networks. Here, we demonstrate that ATP5B is clearly involved in the mechanism of regulation for mitochondrial fusion and fission in mammalian cells.

Phosphorylation of β subunit in F1F0 ATP synthase is associated with increased iron uptake in iron-overloaded heart mitochondria

F1F0 ATP synthase was prepared from iron-overloaded heart mitochondria to study the effect of iron on mitochondria. As F1F0 ATP synthase was able to transport iron, proteoliposomes containing F1F0 ATP synthase were prepared to compare iron uptake between control and iron-overloaded mitochondria. A threefold increase in Vmax (nmol/min/mg) (6.35 ± 0.17 vs. 2.08 ± 0.06) and an eightfold increase in Km (µM) (7.5 ± 0.7 vs. 0.85 ± 0.5) by F1F0 ATP synthase for iron uptake were observed in iron-overloaded mitochondria. Mitochondrial ATP synthase prepared from iron-overloaded heart has a canonical subunit composition in an altered stoichiometry compared to the control enzyme: the α, β, γ, OSCP, d, a, δ, and c subunits increased, but the b, e, A6L, and F6 subunits decreased significantly. In addition, the pattern of β subunit isomers with different pIs changed. These isomers appeared to be associated with augmented phosphorylation by excess iron.

Effects of ATP and ADP on iron uptake in rat heart mitochondria

Abstract Iron uptake in mitochondria and fractionated mitochondria compartments was studied to understand iron transport in heart mitochondria. The inner membrane is most active in iron uptake. Mitochondrial uptake was dependent on iron concentration and the amount of mitochondria. Iron transport was inversely proportional to pH in the range of 6.0 to 8.0. Iron transport reached a maximum after 30 min of incubation at 37°C. Iron uptake was inhibited by 1 mM ATP and stimulated by 1 mM ADP. The oxidative phosphorylation inhibitor oligomycin inhibited iron uptake, but rotenone and antimycin A did not. The divalent ions Mg2+, Cu2+, Mn2+, and Zn2+ suppressed iron uptake at 10 µM and stimulated it at 1 mM. The divalent ion Ca2+ stimulated iron uptake at 10 µM and suppressed it at 1 mM, competing with iron. The uptake of calcium was stimulated by 10 to 1000 µM ATP, while iron uptake was stimulated reciprocally by 10 to 1000 µM ADP, suggesting that these ions have movements similar to those of ATP and ADP.

Changes in cytochrome c oxidase and NO in rat lung mitochondria following iron overload

Abstract In this study, the effects of iron on cytochrome c oxidase (CcO) in rat lung mitochondria were examined. Similar to liver mitochondria, iron accumulated considerably in lung mitochondria (more than 2‐fold). Likewise, the reactive oxygen species and nitric oxide (NO) content of mitochondria were increased by more than 50% and 100%, respectively. NO might be produced by nitric oxide synthase (NOS), eNOS and iNOS type, with particular contribution by NOS in mitochondria. The respiratory control ratio of ironoverloaded lung mitochondria dropped to nearly 50% due to increased state 4. Likewise, cytochrome c oxidase activity was lowered significantly to approximately 50% due to excess iron. Real‐time PCR revealed that the expression of isoforms 1 and 2 of subunit IV of CcO was enhanced greatly under excess iron conditions. Taken together, these results show that oxidative phosphorylation within lung mitochondria may be influenced by iron overload through changes in cytochrome c oxidase and NO.

Temporal changes in mitochondrial activities of rat heart after a single injection of iron, including increased complex II activity

Abstract Male rats were given a single injection of iron, and temporal changes in iron content and iron-induced effects were examined in heart cellular fractions. Over a period of 72 h, the contents of total and labile iron, reactive oxygen species, and NO in tissue homogenate, nuclear debris, and postmitochondrial fractions were mostly constant, but in mitochondria they continuously increased. An abrupt decrease in membrane potential and NAD(P)H at 12 h was also found in mitochondria. The respiratory control ratio was reduced slowly with a slight recovery at 72 h, suggesting uncoupling by iron. While the ATP content of tissue homogenate decreased steadily until 72 h, it showed a prominent increase in mitochondria at 12 h. Total iron and calcium concentration also progressively increased in mitochondria over 72 h. Enzyme activity of the oxidative phosphorylation system was significantly altered by iron injection: activities of complexes I, III, and IV were reduced considerably, but complex II activity and the ATPase activity of complex V were enhanced. A reversal of activity in complexes I and II at 12 h suggested reverse electron transfer due to iron overload. These results support the argument that mitochondrial activities including oxidative phosphorylation are modulated by excessive iron.

Iron toxicity to peritoneal macrophage due to alteration of Mitochondria by NO

The cytotoxic effect of iron was examined in peritoneal macrophage to determine contributing factors by iron injection to rat. Viability was reduced by 24% by the iron‐overload and by 30% by short‐term iron addition. Total iron was increased by 45% in the iron‐overloaded with remarkable elevation (9 to 14 fold) in the presence of FeSO4. Free calcium was also increased by 19% in control and 44% in iron‐overloaded group due to additional FeSO4. NO and MDA were increased by 40% and 136%, respectively, with significant reduction (37%) of NAD(P)H. RCR and cytochrome c oxidase activity were lowered approximately by 10% with reduction of mitochondrial membrane potential. Addition of iron was frequently associated with altered distribution of mitochondria of high membrane potential in the iron‐overloaded macrophage. These results suggest altered mitochondria with high NO and low NAD(P)H due to iron.