Biophysical Characterization of the Iron in Mitochondria from Atm1p-Depleted Saccharomyces cerevisiae

Abstract

Atm1p is an ABC transporter localized in the mitochondrial inner membrane; it functions to export an unknown species into the cytosol and is involved in cellular iron metabolism. Depletion or deletion of Atm1p causes Fe accumulation in mitochondria and a defect in cytosolic Fe/S cluster assembly, but reportedly not a defect in mitochondrial Fe/S cluster assembly. In this study the nature of the accumulated Fe was examined using Mössbauer spectroscopy, EPR, electronic absorption spectroscopy, X-ray absorption spectroscopy, and electron microscopy. The Fe that accumulated in aerobically grown cells was in the form of Fe(III) phosphate nanoparticles similar to that which accumulates in yeast frataxin Yfh1p-deleted or yeast ferredoxin Yah1p-depleted cells. Relative to WT mitochondria, Fe/S cluster and heme levels in Atm1p-depleted mitochondria from aerobic cells were significantly diminished. Atm1p-depletion also caused a build-up of nonheme Fe(II) ions in the mitochondria and an increase in oxidative damage. Atm1p-depleted mitochondria isolated from anaerobically grown cells exhibited WT levels of Fe/S clusters and hemes, and they did not hyper-accumulate Fe. Atm1p-depleted cells lacked Leu1p activity, regardless of whether they were grown aerobically or anaerobically. These results indicate that Atm1p does not participate in mitochondrial Fe/S cluster assembly, and that the species exported by Atm1p is required for cytosolic Fe/S cluster assembly. The Fe/S cluster defect and the Fe-accumulation phenotype, resulting from the depletion of Atm1p in aerobic cells (but not in anaerobic cells), may be secondary effects that are observed only when cells are exposed to oxygen during growth. Reactive oxygen species generated under these conditions might degrade iron-sulfur clusters and lower heme levels in the organelle.

Probing in vivo Mn2+speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy

“…The in vivo study of low molecular-weight complexes of Mn 2þ can be profitably compared and contrasted with the study of biological Fe speciation. Fe is readily examined with multiple tools, including Mossbauer, EPR, X-ray absorption, and optical spectroscopies (38,39), whereas it is likely that only X-ray absorption methods (6,18,40) can complement ENDOR studies of cellular Mn 2þ .…”

Co-precipitation of Phosphate and Iron Limits Mitochondrial Phosphate Availability in Saccharomyces cerevisiae Lacking the Yeast Frataxin Homologue (YFH1)

“…Mitochondrial Matrix-Mutants affected in [Fe-S] cluster biogenesis accumulate iron in the mitochondria essentially in an insoluble form, as ferric phosphate nanoparticles (6,13,20,21). We tested whether the apparent higher rate of iron uptake by mitochondria isolated from ⌬yfh1 cells was associated with precipitation of iron in vitro in the mitochondrial matrix.…”

“…Mitochondria-Frataxin-deficient yeast cells accumulate iron in the mitochondria in the form of amorphous nanoparticles of ferric phosphate, as do other mutants defective in [Fe-S] cluster biogenesis or export (6,13,20,21). This observation has several implications.…”

“…However, the mechanism of uptake is still completely unknown and will probably remain so as long as the iron substrate used by these proteins in vivo is not known. In ⌬yfh1 mitochondria, iron accumulates in the form of amorphous nanoparticles of ferric phosphate (6), and there is now evidence that the same is true for other mutants defective in [Fe-S] cluster biogenesis or export (13,20,21). Iron precipitated in this form is unavailable for biological processes, and thus, ⌬yfh1 cells might paradoxically suffer from iron deficiency despite being overloaded with iron (13).…”

Mitochondrial Iron-Sulfur Cluster Activity and Cytosolic Iron Regulate Iron Traffic in Saccharomyces cerevisiae

“…These effects are absent when ISC mutant cells are grown anaerobically (21,22 [Fe cyt ] is not low in ISC mutant cells (23) raised the possibility that Fe cyt may not be sensed in cellular iron regulation.…”