The mitochondrial Fe-S cluster assembly machinery plays a central role in the maturation of both mitochondrial and extra-mitochondrial Fe-S proteins. In mammals, Fe-S cluster proteins are involved in crucial biological processes including enzymatic catalysis, DNA synthesis and repair, ribosome biogenesis, iron homeostasis and heme synthesis. Mutation in the iron-sulfur cluster assembly genes is associated with severe human diseases such as infantile encephalopathy, myopathies, neurodegenerative and anemia-related diseases. It is therefore particularly relevant to investigate the mechanisms by which Fe-S are delivered to cytosolic and nuclear recipient in mammals. Recently, mitoNEET has been identified as the first Fe-S protein of the outer mitochondrial membrane in mammalian cells. mitoNEET function is unknown but its location and the pH-lability of its Fe-S, which is coordinated to three cysteines and one histidine, makes it a high priority candidate to participate in Fe-S transfer from mitochondria to the cytosol in mammalian cells. Very recently, in vitro studies demonstrated that human mitoNEET can deliver its Fe-S to the bacterial acceptor ferredoxin. Our preliminary data, showing that mitoNEET deficiency affects the maturation of the Iron Regulatory Protein-1 (IRP1), a cytosolic Fe-S protein involved in the control of iron homeostasis, support a role of mitoNEET in Fe-S biogenesis. The fact that mitoNEET is a diabetes drug target is another spur to identify its specific function. Recent studies indicate that pioglitazone, a member of the thiazolidinedione class, stabilizes mitoNEET Fe-S, implying a direct action of this diabetes drug on the protein. The proposal is organized in three main work packages with the following specific objectives: i) specifying mitoNEET function in cytosolic Fe-S biogenesis and its impact on cellular iron metabolism controlled by IRP1, ii) characterizing the mitoNEET function in a Fe-S transfer process using physiological acceptor proteins, in particular IRP1, and iii) defining the mechanism by which pioglitazone prevents Fe-S release from mitoNEET (or stabilizes mitoNEET Fe-S). This project is based on a multidisciplinary approach including biophysics (Raman, NMR, EPR and Mössbauer spectroscopies) and biochemistry, molecular and cell biology as well as studies in living cells and animal models, to validate the in vitro approach in a physiological context.