Biophysical Characterization of the Iron in Mitochondria from Atm1p-Depleted Saccharomyces cerevisiae (original) (raw)

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 hyperaccumulate 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. Mitochondria play a major role in cellular iron homeostasis, as they are sites where iron is inserted for heme biosynthesis (1) and where iron-sulfur clusters (ISCs) are assembled (2). The ferrous ions used in these processes are imported from the cytosol through the Mrs3p/4p highaffinity inner-membrane (IM) transporters. Some such centers are installed into mitochondrial apo-proteins, and some heme centers are exported to cytosolic targets. The Cytosolic Iron-Sulfur Protein Assembly (CIA) machinery is thought to depend on the mitochondrial ISC machinery, via an arrangement involving the IM protein Atm1p. Atm1p is an ATP Binding Cassette (ABC) "half-transporter" (3) that uses the free energy of ATP hydrolysis to transport an unknown molecular species, termed "X", from the matrix to the intermembrane space (4,