Biogenesis of iron- sulfur ([Fe- S]) proteins in eukaryotes requires the function of complex proteinaceous machineries in both mitochondria and cytosol. In contrast to the mitochondrial pathway, little is known about [ Fe- S] protein assembly in the cytosol. So far, four highly conserved proteins ( Cfd1, Nbp35, Nar1 and Cia1) have been identified as members of the cytosolic [ Fe- S] protein assembly machinery, but their molecular function is unresolved. Using in vivo and in vitro approaches, we found that the soluble P -loop NTPases Cfd1 and Nbp35 form a complex and bind up to three [ 4Fe- 4S] clusters, one at the N terminus of Nbp35 and one each at a new C- terminal cysteine- rich motif present in both proteins. These labile [ Fe- S] clusters can be rapidly transferred and incorporated into target [ Fe- S] apoproteins in a Nar1- and Cia1- dependent fashion. Our data suggest that the Cfd1 - Nbp35 complex functions as a novel scaffold for [ Fe- S] cluster assembly in the eukaryotic cytosol.
