Function and crystal structure of the dimeric P-loop ATPase CFD1 coordinating an exposed [4Fe-4S] cluster for transfer to apoproteins.

Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires complex machineries in mitochondria and cytosol. Initially, Fe-S clusters are assembled on dedicated scaffold proteins and then are trafficked to target apoproteins. Within the cytosolic Fe-S protein assembly (CIA) machinery, the conserved P-loop nucleoside triphosphatase Nbp35 performs a scaffold function. In yeast, Nbp35 cooperates with the related Cfd1, which is evolutionary less conserved and is absent in plants. Here, we investigated the potential scaffold function of human CFD1 (NUBP2) in CFD1-depleted HeLa cells by measuring Fe-S enzyme activities or 55Fe incorporation into Fe-S target proteins. We show that CFD1, in complex with NBP35 (NUBP1), performs a crucial role in the maturation of all tested cytosolic and nuclear Fe-S proteins, including essential ones involved in protein translation and DNA maintenance. CFD1 also matures iron regulatory protein 1 and thus is critical for cellular iron homeostasis. To better understand the scaffold function of CFD1-NBP35, we resolved the crystal structure of Chaetomium thermophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 protein. Importantly, two ctCfd1 monomers coordinate a bridging [4Fe-4S] cluster via two conserved cysteine residues. The surface-exposed topology of the cluster is ideally suited for both de novo assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaffold components during Fe-S cluster assembly and/or release.

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.

Biochemical Analyses of Human Iron-Sulfur Protein Biogenesis and of Related Diseases.

Maturation of Fe/S proteins in mammals is an intricate process mediated by two assembly systems located in the mitochondrial and cytosolic-nuclear compartments. Malfunction particularly of the mitochondrial system gives rise to severe neurological, metabolic, or hematological disorders, often with fatal outcome. In this chapter, we describe approaches for the differential biochemical investigation of cellular Fe/S protein maturation in mitochondria, cytosol, and nucleus. The analyses may also facilitate the identification of the affected Fe/S protein assembly step in diseased state. As Fe/S cluster insertion into target apoproteins is a frequent determinant of protein stability, examination of protein steady-state levels in biological samples frequently permits reliable first clues about the maturation process. In some specific cases, this approach allows the assessment of enzymatic or regulatory functions of Fe/S proteins, including the formation of lipoate cofactor by mitochondrial lipoic acid synthase or the posttranscriptional regulation of transferrin receptor and ferritin expression by the cytosolic iron regulatory proteins. More direct Fe/S protein maturation assays like enzymatic analyses may further validate the observed maturation defects. Here, we present a simple protocol for the determination of dihydropyrimidine dehydrogenase enzyme activity by thin-layer chromatography. In order to directly monitor Fe/S cluster insertion into target apoproteins, we have developed a 55Fe radiolabeling technique tracing the in vivo Fe/S cofactor formation in mammalian tissue culture. The combination of the presented techniques represents a comprehensive strategy to assess the multiple facets of Fe/S protein assembly for both mechanistic analyses and for the elucidation of specific defects in Fe/S diseases.

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.