Iron-sulphur (Fe-S) clusters have long been recognized as essential and versatile cofactors of proteins involved in catalysis, electron transport and sensing of ambient conditions. Despite the relative simplicity of Fe-S clusters in terms of structure and composition, their synthesis and assembly into apoproteins is a highly complex and coordinated process in living cells. Different biogenesis machineries in both bacteria and eukaryotes have been discovered that assist Fe-S-protein maturation according to uniform biosynthetic principles. The importance of Fe-S proteins for life is documented by an increasing number of diseases linked to these components and their biogenesis.

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Iron-sulfur (Fe/S) proteins are involved in a wide variety of cellular processes such as enzymatic reactions, respiration, cofactor biosynthesis, ribosome biogenesis, regulation of gene expression, and DNA-RNA metabolism. Assembly of Fe/S clusters, small inorganic cofactors, is assisted by complex proteinaceous machineries, which use cysteine as a source of sulfur, combine it with iron to synthesize an Fe/S cluster on scaffold proteins, and finally incorporate the cluster into recipient apoproteins. In eukaryotes, such as yeast and human cells, more than 20 components are known that facilitate the maturation of Fe/S proteins in mitochondria, cytosol, and nucleus. These biogenesis components also perform crucial roles in other cellular pathways, e.g., in the regulation of iron homeostasis or the modification of tRNA. Numerous diseases including several neurodegenerative and hematological disorders have been associated with defects in Fe/S protein biogenesis, underlining the central importance of this process for life.

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Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals.

The eukaryotic replicative DNA polymerases (Pol α, δ, and ε), and the major DNA mutagenesis enzyme Pol ζ contain two conserved cysteine-rich metal-binding motifs (CysA and CysB) in the C-terminal domain (CTD) of their catalytic subunits. Here, we demonstrate by in vivo and in vitro approaches the presence of an essential [4Fe-4S] cluster in the CysB motif of all four yeast B-family DNA polymerases. Loss of the [4Fe-4S] cofactor by cysteine ligand mutagenesis in Pol3 destabilized the CTD and abrogated interaction with the Pol31-Pol32 subunits. Reciprocally, overexpression of accessory subunits increased the amount of CTD-bound Fe-S cluster. This implies an important physiological role of the Fe-S cluster in polymerase complex stabilization. Further, we demonstrate that the Zn-binding CysA motif is required for PCNA-mediated Pol δ processivity. Together, our findings show that the function of eukaryotic replicative DNA polymerases crucially depends on different metallocenters for accessory subunit recruitment and for replisome stability.

The mitochondrial proteins Isu1p and Isu2p play an essential role in the maturation of cellular iron–sulfur (Fe/S) proteins in eukaryotes. By radiolabelling of yeast cells with 55Fe we demonstrate that Isu1p binds an oxygen‐resistant non‐chelatable Fe/S cluster providing in vivo evidence for a scaffolding function of Isu1p during Fe/S cluster assembly. Depletion of the cysteine desulfurase Nfs1p, the ferredoxin Yah1p or the yeast frataxin homologue Yfh1p by regulated gene expression causes a strong decrease in the de novo synthesis of Fe/S clusters on Isu1p. In contrast, depletion of the Hsp70 chaperone Ssq1p, its co‐chaperone Jac1p or the glutaredoxin Grx5p markedly increased the amount of Fe/S clusters bound to Isu1p, even though these mitochondrial proteins are crucial for maturation of Fe/S proteins. Hence Ssq1p/Jac1p and Grx5p are required in a step after Fe/S cluster synthesis on Isu1p, for instance in dissociation of preassembled Fe/S clusters from Isu1p and/or their insertion into apoproteins. We propose a model that dissects Fe/S cluster biogenesis into two major steps and assigns its central components to one of these two steps.

Instability of the nuclear genome is a hallmark of cancer and aging. MMS19 protein has been linked to maintenance of genomic integrity but the molecular basis of this connection is unknown. Here, we identify MMS19 as a member of the cytosolic iron-sulfur protein assembly (CIA) machinery. MMS19 functions as part of the CIA targeting complex that specifically interacts with and facilitates iron-sulfur cluster insertion into apoproteins involved in methionine biosynthesis, DNA replication, DNA repair and telomere maintenance. MMS19 thus serves as an adapter between early-acting CIA components and a subset of cellular iron-sulfur proteins. The function of MMS19 in maturation of crucial components of DNA metabolism may explain the sensitivity of MMS19 mutants to DNA damage and the presence of extended telomeres.