Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol.

The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria and cytosol. The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the mitochondrial transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this cytosolic [2Fe-2S] protein maturation pathway defined here was yeast Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or target apoproteins. Finally, the most critical GSH requirement was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.

Fe-S cluster coordination of the chromokinesin KIF4A alters its subcellular localization during mitosis.

Fe-S clusters act as co-factors of proteins with diverse functions, for example, in DNA repair. Downregulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability through the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B (also known as FAM96B) and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, colocalize with components of the mitotic machinery. Downregulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon downregulation of the CIA targeting complex contributes to the mitotic defects.

The diferric-tyrosyl radical cluster of ribonucleotide reductase and cytosolic iron-sulfur clusters have distinct and similar biogenesis requirements.

How each metalloprotein assembles the correct metal at the proper binding site presents challenges to the cell. The di-iron enzyme ribonucleotide reductase (RNR) uses a diferric-tyrosyl radical (FeIII2-Y•) cofactor to initiate nucleotide reduction. Assembly of this cofactor requires O2, FeII, and a reducing equivalent. Recent studies show that RNR cofactor biosynthesis shares the same source of iron, in the form of [2Fe-2S]-GSH2 from the monothiol glutaredoxin Grx3/4, and the same electron source, in the form of the Dre2-Tah18 electron transfer chain, with the cytosolic iron-sulfur protein assembly (CIA) machinery required for maturation of [4Fe-4S] clusters in cytosolic and nuclear proteins. Here, we further investigated the interplay between the formation of the FeIII2-Y• cofactor in RNR and the cellular iron-sulfur (Fe-S) protein biogenesis pathways by examining both the iron loading into the RNR ß subunit and the RNR catalytic activity in yeast mutants depleted of individual components of the mitochondrial iron-sulfur cluster assembly (ISC) and the CIA machineries. We found that both iron loading and cofactor assembly in RNR are dependent on the ISC machinery. We also found that Dre2 is required for RNR cofactor formation but appears to be dispensable for iron loading. None of the CIA components downstream of Dre2 was required for RNR cofactor formation. Thus, the pathways for RNR and Fe-S cluster biogenesis bifurcate after the Dre2-Tah18 step. We conclude that RNR cofactor biogenesis requires the ISC machinery to mature the Grx3/4 and Dre2 Fe-S proteins, which then function in iron and electron delivery to RNR, respectively.

A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation.

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.

Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes.

The eukaryotic replicative DNA polymerases (Pol α, δ and ɛ) 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 and Pol32 subunits. Reciprocally, overexpression of accessory subunits increased the amount of the 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 replisome stability.

Hsp90 and metal-binding J-protein family chaperones are not critically involved in cellular iron-sulfur protein assembly and iron regulation in yeast.

Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions.

Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein biogenesis.

Cytosolic and nuclear iron-sulfur (Fe-S) proteins play key roles in processes such as ribosome maturation, transcription and DNA repair-replication. For biosynthesis of their Fe-S clusters, a dedicated cytosolic Fe-S protein assembly (CIA) machinery is required. Here, we identify the essential flavoprotein Tah18 as a previously unrecognized CIA component and show by cell biological, biochemical and spectroscopic approaches that the complex of Tah18 and the CIA protein Dre2 is part of an electron transfer chain functioning in an early step of cytosolic Fe-S protein biogenesis. Electrons are transferred from NADPH via the FAD- and FMN-containing Tah18 to the Fe-S clusters of Dre2. This electron transfer chain is required for assembly of target but not scaffold Fe-S proteins, suggesting a need for reduction in the generation of stably inserted Fe-S clusters. The pathway is conserved in eukaryotes, as human Ndor1-Ciapin1 proteins can functionally replace yeast Tah18-Dre2.

Depletion of thiol reducing capacity impairs cytosolic but not mitochondrial iron-sulfur protein assembly machineries.

Iron­sulfur (Fe/S) clusters are versatile inorganic cofactors that play central roles in essential cellular functions, from respiration to genome stability. >30 proteins involved in Fe/S protein biogenesis in eukaryotes are known, many of which bind clusters via cysteine residues. This opens up the possibility that the thiol-reducing glutaredoxin and thioredoxin systems are required at both the Fe/S biogenesis and target protein level to counteract thiol oxidation. To address the possible interplay of thiol redox chemistry and Fe/S protein biogenesis, we have characterized the status of the mitochondrial (ISC) and cytosolic (CIA) Fe/S protein assembly machineries in Saccharomyces cerevisiae mutants in which the three partially redundant glutathione (Glr1) and thioredoxin (Trr1 and Trr2) oxidoreductases have been inactivated in either mitochondria, cytosol, or both compartments. Cells devoid of mitochondrial oxidoreductases maintained a functional mitochondrial ISC machinery and showed no altered iron homeostasis despite a non-functional complex II of the respiratory chain due to redox-specific defects. In cells that lack either cytosolic or total cellular thiol reducing capacity, both the ISC system and iron homeostasis were normal, yet cytosolic and nuclear Fe/S target proteins were not matured. This dysfunction could be attributed to a failure in the assembly of [4Fe­4S] clusters in the CIA factor Nar1, even though Nar1 maintained robust protein levels and stable interactions with later-acting CIA components. Overall, our analysis has uncovered a hitherto unknown thiol-dependence of the CIA machinery and has demonstrated the surprisingly varying sensitivity of Fe/S proteins to thiol oxidation.

The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion.

Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery.

The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron-sulfur proteins.

Mitochondria have been derived from alpha-bacterial endosymbionts during the evolution of eukaryotes. Numerous bacterial functions have been maintained inside the organelles including fatty acid degradation, citric acid cycle, oxidative phosphorylation, and the synthesis of heme or lipoic acid cofactors. Additionally, mitochondria have inherited the bacterial iron-sulfur cluster assembly (ISC) machinery. Many of the ISC components are essential for cell viability because they generate a still unknown, sulfur-containing compound for the assembly of cytosolic and nuclear Fe/S proteins that perform important functions in, e.g., protein translation, DNA synthesis and repair, and chromosome segregation. The sulfur-containing compound is exported by the mitochondrial ABC transporter Atm1 (human ABCB7) and utilized by components of the cytosolic iron-sulfur protein assembly (CIA) machinery. An appealing minimal model for the striking compartmentation of eukaryotic Fe/S protein biogenesis is provided by organisms that contain mitosomes instead of mitochondria. Mitosomes have been derived from mitochondria by reductive evolution, during which they have lost virtually all classical mitochondrial tasks. Nevertheless, mitosomes harbor all core ISC components which presumably have been maintained for assisting the maturation of cytosolic-nuclear Fe/S proteins. The current review is centered around the Atm1 export process. We present an overview on the mitochondrial requirements for the export reaction, summarize recent insights into the 3D structure and potential mechanism of Atm1, and explain how the CIA machinery uses the mitochondrial export product for the assembly of cytosolic and nuclear Fe/S proteins.

The Cfd1-Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol.

Biogenesis of iron-sulfur ([Fe-S]) proteins in eukaryotes requires the function of complex proteinaceous machineries in both mitochondria and cytosol. In contrast to the mitochondrial pathway, little is known about [Fe-S] protein assembly in the cytosol. So far, four highly conserved proteins (Cfd1, Nbp35, Nar1 and Cia1) have been identified as members of the cytosolic [Fe-S] protein assembly machinery, but their molecular function is unresolved. Using in vivo and in vitro approaches, we found that the soluble P-loop NTPases Cfd1 and Nbp35 form a complex and bind up to three [4Fe-4S] clusters, one at the N terminus of Nbp35 and one each at a new C-terminal cysteine-rich motif present in both proteins. These labile [Fe-S] clusters can be rapidly transferred and incorporated into target [Fe-S] apoproteins in a Nar1- and Cia1-dependent fashion. Our data suggest that the Cfd1-Nbp35 complex functions as a novel scaffold for [Fe-S] cluster assembly in the eukaryotic cytosol.