Cytosolic iron–sulfur protein assembly system identifies clients by a C-terminal tripeptide

The eukaryotic cytosolic Fe–S protein assembly (CIA) machinery inserts iron–sulfur (Fe–S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe–S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO(–) tripeptide is present at the C-terminus of more than a quarter of clients or their adaptors. When present, this targeting complex recognition (TCR) motif is necessary and sufficient for binding to the CTC in vitro and for directing Fe–S cluster delivery in vivo. Remarkably, fusion of this TCR signal enables engineering of cluster maturation on a nonnative protein via recruitment of the CIA machinery. Our study advances our understanding of Fe–S protein maturation and paves the way for bioengineering novel pathways containing Fe–S enzymes.