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 in vitro 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.