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

TL;DR

This study shows that the Mak16 protein uses an iron-sulfur cluster to help build ribosomes, and this cluster is sensitive to stress and crucial for proper function.

Contribution

The discovery of a redox-active [4Fe-4S] cluster in Mak16 and its role in ribosome biogenesis is novel.

Findings

The [4Fe-4S] cluster in Mak16 is essential for its interaction with Rpf1 and 25S rRNA maturation.

Oxidative stress destabilizes Mak16 and disrupts its function in ribosome assembly.

The Fe/S cluster in Mak16 acts as a redox sensor, potentially regulating cellular homeostasis under stress.

Abstract

This study identifies an iron–sulfur (Fe/S) cluster in the 60S ribosomal assembly factor Mak16 as essential for maintaining complex stability with its interacting partner Rpf1, which is crucial for the maturation of 25S ribosomal RNA (rRNA) during nuclear ribosome biogenesis. The [4Fe-4S] cluster of Mak16 displays redox properties typical of low-potential centers and is highly labile in the presence of oxygen and oxidative stress-inducing agents. Together, these features suggest that the Fe/S cluster of Mak16 plays a structural and potentially regulatory role in ribosome assembly. Our findings uncover an unappreciated requirement for an Fe/S cofactor in a key process in eukaryotic cells and expand the importance of Fe/S protein biosynthesis.

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.

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