A Conserved Cia1–Cia2 Interface Mediates Client Recruitment in the Cytosolic Iron–Sulfur Cluster Assembly Pathway

The cytosolic iron-sulfur cluster assembly (CIA) targeting complex maturates over 30 cytosolic and nuclear Fe-S proteins, raising the question of how a single complex recognizes such a diverse set of clients. The discovery of a C-terminal targeting complex recognition (TCR) peptide in up to 25% of CIA clients provided a clue to substrate specificity, yet the molecular and energetic basis for this interaction remained unresolved. By integrating computational and biochemical approaches, we show that the TCR peptide binds a conserved interface between the Cia1 and Cia2 subunits of the targeting complex, even in the absence of the Fe-S cluster. Since this same site also mediates binding of predominantly apo-Nar1, the proposed Fe-S cluster carrier, we provide <i>in vitro</i> evidence for Nar1's role as a cluster trafficking protein in the CIA pathway. We further show that Cia1-Cia2 complex formation is essential for CIA function as substitutions disrupting this interface, including the disease-linked R65W Cia1 variant, impair TCR peptide-dependent client recruitment. Our findings also clarify the role of the poorly characterized human paralog Cia2a, proposed to act solely in iron regulatory protein 1 (IRP1) maturation. We find that a Cia1-Cia2a complex can bind the TCR peptide, suggesting a broader role for Cia2a in Fe-S protein biogenesis, as IRP1 lacks a TCR motif. Together, these findings define a well-conserved molecular mechanism for client recognition in the CIA pathway and uncover how CIA targeting complex assembly and client identification are mechanistically linked to human disease.