The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase

Camire, Eric J.; Grossman, John D.; Thole, Grace J.; Fleischman, Nicholas M.; Perlstein, Deborah L.

doi:10.1074/jbc.m115.667022

Cited by 26 publications

(75 citation statements)

References 30 publications

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“…Nbp35 and Cfd1 belong to the SIMIBI (signal recognition, MinD and BioD) (43) family of P-loop nucleoside triphosphatases (NTPases), forming a subgroup with several metal-binding and -trafficking proteins. The yeast Nbp35-Cfd1 complex possesses ATPase activity in vitro (18), and mutation of the Walker nucleotide-binding motifs impairs de novo [4Fe-4S] cluster assembly on this complex in vivo (13). Site-directed mutagenesis revealed cluster coordination via two essential cysteine ligands present in the conserved C-terminal CPxC motifs of both proteins.…”

Section: Significancementioning

confidence: 99%

“…Better mechanistic understanding of the eukaryotic CIA scaffold complex function will benefit from structural insights into the 3D architecture of these proteins, in particular the coordination of the transiently bound [4Fe-4S] cluster. We succeeded in crystallizing Cfd1 from the thermophilic fungus C. thermophilum (ct), which has a higher stability than Saccharomyces cerevisiae or human counterparts (13,18). The crystal structure [2.57-Å resolution; Protein Data Bank (PDB) ID code 6G2G] showed a ctCfd1 homodimer with a bound [4Fe-4S] cluster (Fig.…”

Section: Depletion Of Cfd1 Impairs Maturation Of Nuclear Fe-s Proteinsmentioning

confidence: 99%

“…Insights into the composition and function of the CIA machinery were initially obtained in yeast. Two homologous P-loop ATPases termed "Cfd1" and "Nbp35" were shown to serve as a scaffold complex that de novo assembles a [4Fe-4S] cluster (12)(13)(14)(15)(16)(17)(18). This synthesis reaction requires the electron transport chain composed of the diflavin oxidoreductase Tah18 and the Fe-S protein Dre2 (19,20).…”

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confidence: 99%

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

Stehling

Jeoung

Freibert

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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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 Fe 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 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.

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Section: Significancementioning

confidence: 99%

Section: Depletion Of Cfd1 Impairs Maturation Of Nuclear Fe-s Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Stehling

Jeoung

Freibert

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…In Saccharomyces cerevisiae it was identified as cytosolic Fe-S cluster deficient ( Cfd1 ) in a screen for genes able to convert Iron regulatory protein 1 into c-aconitase [20] [21]. These and subsequent studies in yeast and animal cell lines established that Nubp2 is an integral component of the cytosolic iron-Sulphur (Fe-S) cluster assembly (CIA) pathway where it acts along with its homologous binding partner Nubp1 as a scaffold for transfer of the Fe-S cofactor to non-mitochondrial apoproteins [22–24]. Later studies revealed that knockdown of Nubp1 alone, or of both Nubp1 and Nubp2 , led to excessive centrosome duplication in vitro [25].…”

Section: Introductionmentioning

confidence: 99%

Nubp2is required for cranial neural crest survival in the mouse

DiStasio

Paulding

Chatuverdi

et al. 2019

Preprint

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The N-ethyl-N-nitrosourea (ENU) forward genetic screen is a useful tool for the unbiased discovery of novel mechanisms regulating developmental processes. We recovered the dorothy mutation in such a screen designed to recover recessive mutations affecting craniofacial development in the mouse. Dorothy embryos die prenatally and exhibit many striking phenotypes commonly associated with ciliopathies, including a severe midfacial clefting phenotype. We used exome sequencing to discover a missense mutation in Nucleotide Binding Protein 2 (Nubp2) to be causative. This finding was confirmed with a complementation analysis between the dorothy allele and a Nubp2 null allele (Nubp2Null). We demonstrate that Nubp2 is indispensable for embryogenesis. NUBP2 is implicated in both the Cytosolic Iron/Sulfur cluster Assembly (CIA) pathway and in the negative regulation of ciliogenesis. Conditional ablation of Nubp2 in the neural crest lineage with Wnt1-cre recapitulates the dorothy craniofacial phenotype. Using this model, we found that the proportion of ciliated cells in the craniofacial mesenchyme was unchanged, and that markers of the Shh, Fgf, and Bmp signaling pathways are unaltered. Finally, we show that the phenotype results from a marked increase in apoptosis within the craniofacial mesenchyme.Summary StatementAn ENU screen identifies a novel allele of Nubp2 which is then demonstrated to be required for cranial neural crest survival and proper midfacial development.

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“…The bridging [4Fe‐4S] cluster is transiently assembled and can be transferred to apoproteins . The deviant Walker A motif is required for ATP binding and hydrolysis, and the ATPase activity is essential for in vivo Fe‐S cluster assembly and transfer .…”

mentioning

confidence: 99%

The Nbp35/ApbC homolog acts as a nonessential [4Fe‐4S] transfer protein in methanogenic archaea

et al. 2019

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The nucleotide binding protein 35 (Nbp35)/cytosolic Fe‐S cluster deficient 1 (Cfd1)/alternative pyrimidine biosynthetic protein C (ApbC) protein homologs have been identified in all three domains of life. In eukaryotes, the Nbp35/Cfd1 heterocomplex is an essential Fe‐S cluster assembly scaffold required for the maturation of Fe‐S proteins in the cytosol and nucleus, whereas the bacterial ApbC is an Fe‐S cluster transfer protein only involved in the maturation of a specific target protein. Here, we show that the Nbp35/ApbC homolog MMP0704 purified from its native archaeal host Methanococcus maripaludis contains a [4Fe‐4S] cluster that can be transferred to a [4Fe‐4S] apoprotein. Deletion of mmp0704 from M. maripaludis does not cause growth deficiency under our tested conditions. Our data indicate that Nbp35/ApbC is a nonessential [4Fe‐4S] cluster transfer protein in methanogenic archaea.

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The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase

Cited by 26 publications

References 30 publications

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

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

Nubp2is required for cranial neural crest survival in the mouse

The Nbp35/ApbC homolog acts as a nonessential [4Fe‐4S] transfer protein in methanogenic archaea

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