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 present at the C-terminus of clients is necessary and sufficient for binding to the CTC in vitro and directing Fe-S cluster delivery in vivo. Remarkably, fusion of this TCR (target complex recognition) signal enables engineering of cluster maturation on a non-native protein via recruitment of the CIA machinery. Our study significantly advances our understanding of Fe-S protein maturation and paves the way for bioengineering applications.