The function of Mak16 in ribosome biogenesis depends on its [4Fe-4S] cluster

Mak16 and its interacting partner Rpf1 play a critical role at an early step in the maturation of the ribosomal 60S subunit of eukaryotes, as revealed by cryoelectron microscopy structures. While these studies suggested no metal participation or the presence of a Zn²⁺ ion in Mak16, we identify a previously unexplored iron-sulfur (Fe/S) cluster in yeast Mak16 through both in vivo and in vitro methods. We demonstrate a functional link between mitochondrial and cytosolic Fe/S protein biogenesis and ribosome assembly, highlighting an overlooked aspect of 60S ribosomal biogenesis. Characterization of human and yeast Mak16 revealed a redox-active [4Fe-4S]^2+/1+ cluster with a midpoint potential below -500 mV. Oxidative stress destabilizes Mak16 and disrupts its interaction with Rpf1 in vivo, while in vitro H₂O₂ causes [3Fe-4S]¹⁺ cluster formation. Our findings also reveal that upon binding to rRNA expansion segment 7 the redox properties of the nearby Fe/S cluster largely remain unchanged. However, disruption of Fe/S cluster coordination destabilized Mak16, impaired the Mak16-Rpf1 complex formation and decreased the 25S rRNA level. The critical role of Fe/S proteins in eukaryotic DNA replication and repair, mitoribosomal function, and maturation has now been extended to nuclear ribosomal assembly. Relying on a vulnerable cofactor comes at a cost, as cluster loss can severely disrupt essential cellular processes. The inherent biosynthetic complexity and instability of the Fe/S cluster of Mak16 allows it to function as sensor for redox imbalance, creating the possibility to regulate cellular homeostasis under stress.