Scaffold protein for the de novo synthesis of iron-sulfur (Fe-S) clusters within mitochondria, which is required for maturation of both mitochondrial and cytoplasmic [2Fe-2S] and [4Fe-4S] proteins. First, a [2Fe-2S] cluster is transiently assembled on the scaffold proteins ISU1 and ISU2. In a second step, the cluster is released from ISU1/ISU2, transferred to glutaredoxin GRX5, followed by the formation of mitochondrial [2Fe-2S] proteins, the synthesis of [4Fe-4S] clusters and their target-specific insertion into the recipient apoproteins. Cluster assembly on ISU1/ISU2 depends on the function of the cysteine desulfurase complex NFS1-ISD11, which serves as the sulfur donor for cluster synthesis, the iron-binding protein frataxin (YFH1) as the putative iron donor, and the electron transfer chain comprised of ferredoxin reductase ARH1 and ferredoxin YAH1, which receive their electrons from NADH. Fe-S cluster release from ISU1/ISU2 is achieved by interaction with the Hsp70 chaperone SSQ1, assisted by the DnaJ-like co-chaperone JAC1 and the nucleotide exchange factor MGE1. ISU1 is the major isoform in yeast, while ISU2 is not detectable in cells grown to stationary phase (PubMed:10588895, PubMed:12970193, PubMed:14741370, PubMed:15123690, PubMed:16341089, PubMed:16431909, PubMed:23615440, PubMed:25358379). Also involved in production of a sulfur precursor required for thiolation of cytoplasmic tRNAs (PubMed:31040179). Binds 1 [2Fe-2S] cluster per subunit. Cofactor biosynthesis; iron-sulfur cluster biosynthesis. Component of the core Fe-S cluster (ISC) assembly machinery. Interacts with frataxin (PubMed:14741370, PubMed:12947415, PubMed:20815377, PubMed:25228696). Interacts with the mitochondrial co-chaperones JAC1 and SSQ1 (PubMed:12756240, PubMed:15123690, PubMed:22306468, PubMed:23946486, PubMed:23615440). Interacts with NFS1 (PubMed:23946486, PubMed:25228696). Interacts with ferredoxin YAH1; interacts with the reduced form (PubMed:25358379). Cells deleted for both ISU1 and ISU2 have decreased activity of several respiratory enzymes that contain Fe-S clusters. As a result, cells grow poorly on carbon sources requiring respiration and also accumulate abnormally high levels of iron in their mitochondria. Knockdown of ISU1 in ISU2 knockout cells decreases cytosolic tRNA thiolation, and increases association between SSQ1 and GRX5 (PubMed:31040179, PubMed:23615440). Present with 10800 molecules/cell in log phase SD medium. Belongs to the NifU family.