Abstract
Iron-sulfur cluster (ISC) assembly occurs in both mitochondria and cytosol. Mitochondria are thought to export a low-molecular-mass (LMM) iron and/or sulfur species which is used as a substrate for cytosolic ISC assembly. This species, called X-S or (Fe-S)(int), has not been directly detected. Here, an assay was developed in which mitochondria were isolated from (57)Fe-enriched cells and incubated in various buffers. Thereafter, mitochondria were separated from the supernatant, and both fractions were investigated by ICP-MS-detected size exclusion liquid chromatography. Aqueous (54)Fe(II) in the buffer declined upon exposure to intact (57)Fe-enriched mitochondria. Some (54)Fe was probably surface-absorbed but some was incorporated into mitochondrial iron-containing proteins when mitochondria were activated for ISC biosynthesis. When activated, mitochondria exported/released two LMM nonproteinaceous iron complexes. One species, which comigrated with an Fe-ATP complex, developed faster than the other Fe species, which also comigrated with phosphorus. Both were enriched in (54)Fe and (57)Fe, suggesting that the added (54)Fe entered a pre-existing pool of (57)Fe, which was also the source of the exported species. When (54)Fe-loaded (57)Fe-enriched mitochondria were mixed with isolated cytosol and activated, multiple cytosolic proteins became enriched with Fe. No incorporation was observed when (54)Fe was added directly to the cytosol in the absence of mitochondria. This suggests that a different Fe source in mitochondria, the one enriched mainly with (57)Fe, was used to export a species that was ultimately incorporated into cytosolic proteins. Iron from buffer was imported into mitochondria fastest, followed by mitochondrial ISC assembly, LMM iron export, and cytosolic ISC assembly.