Yeast Mitochondria Import Aqueous Fe^II and, When Activated for Iron-Sulfur Cluster Assembly, Export or Release Low-Molecular-Mass Iron and Also Export Iron That Incorporates into Cytosolic Proteins

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