Affinage

NUBP1

Cytosolic Fe-S cluster assembly factor NUBP1 · UniProt P53384

Length
320 aa
Mass
34.5 kDa
Annotated
2026-04-29
27 papers in source corpus 15 papers cited in narrative 15 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NUBP1 is a cytosolic P-loop ATPase that serves as the primary scaffold for [4Fe-4S] cluster assembly and delivery to cytosolic and nuclear iron-sulfur proteins, and independently functions at centrioles and basal bodies to regulate centrosome duplication and ciliogenesis. NUBP1 forms a heterotetrameric complex with NUBP2 (Cfd1) in which bridging [4Fe-4S] clusters are coordinated at C-terminal CPXC motifs across the subunit interface; the [2Fe-2S] clusters donated by GLRX3 are reductively coupled—using electrons supplied by anamorsin—to generate [4Fe-4S] clusters on both the N-terminal (CX13CX2CX5C) and C-terminal cysteine motifs of NUBP1, with the labile C-terminal cluster serving as the transferable species (PMID:17401378, PMID:32429669, PMID:36916754). ATPase activity resides in the Nbp35 subunit, is allosterically coupled to Fe-S cluster occupancy, and nucleotide binding/hydrolysis cycles drive conformational changes essential for both cluster assembly and CIA pathway-dependent transfer to apo-targets (PMID:26195633, PMID:30865432, PMID:30785732). At centrioles, NUBP1 acts together with NUBP2 and the kinesin KIFC5A as a negative regulator of ciliogenesis and a positive regulator of normal centrosome duplication, and its loss causes centrosome amplification, multipolar spindles, and ectopic cilia formation (PMID:16638812, PMID:23807208).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 2005 High

    Establishing NUBP1 as an essential component of cytosolic/nuclear Fe-S protein maturation resolved the question of whether a dedicated scaffold exists downstream of the mitochondrial ISC export machinery for extra-mitochondrial Fe-S biogenesis.

    Evidence Genetic depletion of Nbp35 in yeast with enzyme activity assays and subcellular fractionation

    PMID:15728363

    Open questions at the time
    • The biochemical nature of the Fe-S cluster on Nbp35 was undefined
    • How Nbp35 cooperates with Cfd1 was unclear
    • No reconstituted in vitro system existed
  2. 2006 High

    Discovery that NUBP1 interacts with NUBP2 and the kinesin KIFC5A at centrosomes, and that NUBP1 loss causes centrosome amplification, revealed an unexpected second cellular role distinct from Fe-S biogenesis.

    Evidence Co-immunoprecipitation, RNAi knockdown, and immunofluorescence in mouse cells

    PMID:16638812

    Open questions at the time
    • Whether the centrosome function requires ATPase or Fe-S cluster activity was unknown
    • Mechanism of centrosome duplication control was not defined
  3. 2007 High

    Reconstitution of the Cfd1-Nbp35 heterotetramer with spectroscopic characterization of its three [4Fe-4S] clusters and demonstration of cluster transfer to apo-proteins established this complex as the central Fe-S scaffold in the CIA pathway.

    Evidence In vitro reconstitution with UV-visible and Mössbauer spectroscopy, 55Fe radiolabeling, and genetic epistasis in yeast

    PMID:17401378

    Open questions at the time
    • Source of electrons and [2Fe-2S] precursors feeding the scaffold was unknown
    • Role of ATP hydrolysis in cluster assembly was not tested
  4. 2008 High

    Demonstrating that human NUBP1 depletion specifically impairs cytosolic Fe-S protein maturation (IRP1, GPAT) with downstream iron regulatory consequences validated the yeast paradigm in human cells.

    Evidence RNAi in HeLa cells with enzyme activity assays, co-immunoprecipitation, and iron metabolism readouts

    PMID:18573874

    Open questions at the time
    • In vitro reconstitution with human proteins had not been performed
    • Whether the human system uses the same cluster transfer mechanism was unproven
  5. 2012 High

    Mutagenesis of the CPXC motif showed these cysteines are essential for [4Fe-4S] bridging across the heterodimer interface, for complex stability, and for viability, defining the chemical architecture of the transferable cluster site and linking nucleotide binding to cluster loading.

    Evidence Site-directed mutagenesis with Mössbauer/EPR spectroscopy and genetic complementation in yeast

    PMID:22362766

    Open questions at the time
    • Structural basis of bridging coordination was inferred, not crystallographically resolved
    • How ATP hydrolysis mechanistically couples to cluster loading remained unclear
  6. 2012 Medium

    Identification of Nubp1 as required for lung branching morphogenesis and distal progenitor survival in mice linked its centrosome/polarity functions to an in vivo developmental phenotype.

    Evidence Forward genetic screen and mouse mutant analysis with immunofluorescence

    PMID:23028652

    Open questions at the time
    • Whether the lung phenotype reflects Fe-S or centrosome dysfunction was not distinguished
    • Single genetic screen without independent allelic confirmation
  7. 2013 High

    Localization of NUBP1 to centrioles throughout the cell cycle and basal bodies, combined with evidence that its silencing increases ciliation in mammalian cells and causes aberrant cilia in C. elegans, established NUBP1 as a negative regulator of ciliogenesis.

    Evidence RNAi in NIH 3T3 cells and C. elegans, immunofluorescence, live imaging, co-immunoprecipitation

    PMID:23807208

    Open questions at the time
    • Mechanism by which NUBP1 suppresses ciliogenesis was not defined
    • Whether CCT/TRiC interaction is functionally relevant to ciliogenesis was not tested
  8. 2013 High

    Showing that Cfd1 interaction increases the kinetic lability of Nbp35-bound Fe-S clusters clarified the functional asymmetry: NUBP2 acts as a modulator that promotes cluster release rather than a direct Fe-S carrier.

    Evidence 55Fe radiolabeling and mutant analysis of heterocomplex stability in yeast

    PMID:23798678

    Open questions at the time
    • Structural basis for increased lability upon heterocomplex formation was unknown
    • Whether the same asymmetry applies to the human complex was not shown
  9. 2015 High

    Biochemical demonstration that ATPase activity resides in the Nbp35 subunit (not Cfd1 alone) defined the catalytic center and showed that hydrolysis-deficient mutations alter nucleotide affinity.

    Evidence In vitro ATPase assays with purified proteins and fluorescent nucleotide binding

    PMID:26195633

    Open questions at the time
    • Connection between ATPase cycle and cluster assembly/transfer was not yet established
    • No structural information on the ATP-bound state
  10. 2019 High

    Systematic mutagenesis of all four ATPase motifs demonstrated that nucleotide binding and hydrolysis are required for both cluster assembly and transfer in vivo, and that Fe-S cluster occupancy allosterically reduces nucleotide affinity, establishing a bidirectional coupling mechanism.

    Evidence Site-directed mutagenesis of ATPase motifs combined with in vivo/in vitro cluster assembly, transfer assays, and nucleotide binding measurements in yeast

    PMID:30785732 PMID:30865432

    Open questions at the time
    • No structural snapshot of the allosteric conformational change
    • Whether ATP hydrolysis drives cluster release or cluster loading (or both) could not be kinetically separated
  11. 2020 High

    Reconstitution of GLRX3-to-NUBP1 [2Fe-2S] cluster transfer with reductive coupling to [4Fe-4S] identified the immediate upstream donor and the chemical mechanism of cluster maturation on human NUBP1.

    Evidence In vitro cluster transfer with NMR, UV-visible, Mössbauer spectroscopy, and analytical ultracentrifugation using purified human proteins

    PMID:32429669

    Open questions at the time
    • Role of NUBP2 in the transfer from GLRX3 was not addressed
    • Electron source for reductive coupling in this system was glutathione, not anamorsin
  12. 2023 High

    Demonstration that an anamorsin-GLRX3 heterotetrameric complex synergistically provides both [2Fe-2S] clusters and electrons specifically to the N-terminal site of NUBP1 resolved the physiological electron donor and the site-specificity of the initial cluster assembly step.

    Evidence In vitro reconstitution with NMR, UV-visible, and Mössbauer spectroscopy using purified human proteins

    PMID:36916754

    Open questions at the time
    • How the C-terminal CPXC site acquires its cluster from GLRX3/anamorsin was not resolved
    • Full reconstitution including NUBP2, downstream CIA factors, and apo-target proteins has not been achieved

Open questions

Synthesis pass · forward-looking unresolved questions
  • The mechanistic link between NUBP1's Fe-S scaffold function and its centriole/ciliogenesis role remains unresolved: it is unknown whether these are independent functions or whether Fe-S cluster chemistry is required at centrosomes.
  • No separation-of-function mutations distinguishing Fe-S and centrosome roles
  • No structural model of full-length NUBP1 or the NUBP1-NUBP2 heterotetramer
  • Mechanism by which NUBP1 negatively regulates ciliogenesis is undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140657 ATP-dependent activity 3 GO:0016787 hydrolase activity 2
Localization
GO:0005829 cytosol 3 GO:0005634 nucleus 2 GO:0005815 microtubule organizing center 2 GO:0005929 cilium 1
Pathway
R-HSA-1430728 Metabolism 5 R-HSA-1640170 Cell Cycle 2 R-HSA-1852241 Organelle biogenesis and maintenance 2
Partners
CYBC1GLRX3KATNAL2KIFC5ANUBP2
Complex memberships
NUBP1-NUBP2 (Nbp35-Cfd1) heterotetramer

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2007 Yeast Cfd1 (NUBP2 ortholog) and Nbp35 (NUBP1 ortholog) form a heterotetrameric complex that binds up to three [4Fe-4S] clusters—one at the N-terminus of Nbp35 and one each at a C-terminal cysteine-rich motif in both proteins—and these labile clusters can be transferred to target Fe-S apoproteins in a Nar1- and Cia1-dependent manner, establishing the Cfd1-Nbp35 complex as a scaffold for Fe-S cluster assembly in the eukaryotic cytosol. In vitro reconstitution, UV-visible and Mössbauer spectroscopy, in vivo 55Fe radiolabeling, genetic epistasis Nature Chemical Biology High 17401378
2005 Yeast Nbp35 (NUBP1 ortholog) resides in the cytosol and nucleus, carries an Fe/S cluster at its N-terminus whose assembly requires the mitochondrial ISC machinery and export machinery, and its depletion specifically impairs cytosolic/nuclear Fe-S protein maturation (e.g., isopropylmalate isomerase) without affecting mitochondrial Fe-S enzymes; Nbp35 genetically interacts with Cfd1 and Nar1. Genetic depletion, enzyme activity assays, subcellular fractionation, genetic interaction analysis PNAS High 15728363
2008 Human NUBP1 (huNbp35) is a cytosolic Fe-S protein whose RNAi-mediated depletion in HeLa cells specifically impairs maturation of cytosolic Fe-S proteins (GPAT/IRP1) but not mitochondrial Fe-S proteins, and impaired IRP1 maturation causes iron metabolic dysregulation (decreased H-ferritin, increased transferrin receptor); NUBP1 forms a complex with huCfd1 (NUBP2) in vivo. RNA interference, enzyme activity assays, co-immunoprecipitation, iron metabolism assays in HeLa cells Molecular and Cellular Biology High 18573874
2012 The two central cysteine residues (CPXC motif) of the Nbp35 (NUBP1 ortholog) C-terminal domain are essential for cell viability, [4Fe-4S] cluster coordination, and Cfd1-Nbp35 hetero-tetramer formation; Mössbauer spectroscopy and EPR indicate the C-terminal [4Fe-4S] cluster bridges across the CPXC motifs of both subunits; nucleotide binding is required for Fe-S cluster loading onto the scaffold proteins. Site-directed mutagenesis, Mössbauer spectroscopy, EPR, genetic complementation, yeast two-hybrid Journal of Biological Chemistry High 22362766
2013 Interaction of Cfd1 (NUBP2 ortholog) with Nbp35 (NUBP1 ortholog) increases the kinetic lability of assembled Fe-S clusters on the Nbp35 scaffold, facilitating transfer to target apo-proteins; Nbp35 readily binds 55Fe in cells whereas free Cfd1 does not; a Cfd1 mutant defective in heterocomplex formation supports iron binding to Nbp35 but impairs iron release. 55Fe radiolabeling in yeast, mutant analysis of heterocomplex stability, iron release kinetics Journal of Biological Chemistry High 23798678
2015 The Nbp35 (NUBP1 ortholog) homodimer and the Nbp35-Cfd1 heterodimer are ATPases in vitro (whereas Cfd1 homodimer has no ATPase activity); mutation of key ATPase active-site residues abolishes hydrolysis; mantATP binds stoichiometrically to Nbp35 with KD = 15.6 μM; hydrolysis-deficient mutant shows increased KD for mantATP. In vitro ATPase assay, site-directed mutagenesis, fluorescent nucleotide analog binding Journal of Biological Chemistry High 26195633
2019 Mutation of conserved residues in all four ATPase motifs of Nbp35 (NUBP1 ortholog) impairs both Fe-S cluster assembly and transfer in vivo; occupancy of the bridging Fe-S cluster site decreases the scaffold's affinity for nucleotide; nucleotide binding and hydrolysis drive conformational changes that regulate protein interactions and cluster transfer within the CIA pathway. Site-directed mutagenesis, in vivo and in vitro Fe-S cluster assembly/transfer assays, nucleotide binding measurements Biochemistry High 30865432
2019 In the Nbp35-Cfd1 heterodimer, nucleotide must bind to the Cfd1 subunit before it can bind to Nbp35; Cfd1 controls nucleotide binding order and becomes hydrolysis-competent only when bound to Nbp35; determined by titration of nucleotide binding sites combined with site-directed mutagenesis. Fluorescent nucleotide titration, site-directed mutagenesis, ATPase assays Biochemistry High 30785732
2020 Human cytosolic GLRX3 (glutaredoxin-3) transfers its [2Fe-2S]2+ clusters to monomeric apo-NUBP1; these clusters are reductively coupled (using glutathione as reductant) to form [4Fe-4S]2+ clusters on both the N-terminal CX13CX2CX5C and C-terminal CPXC motifs of NUBP1; cluster binding to the C-terminal motif promotes NUBP1 dimerization, while the [4Fe-4S]2+ cluster at the C-terminal motif is labile and at the N-terminal motif is tightly bound. In vitro cluster transfer assays, NMR spectroscopy, UV-visible and Mössbauer spectroscopy, analytical ultracentrifugation Journal of the American Chemical Society High 32429669
2023 A hetero-tetrameric complex formed by two molecules of cluster-reduced [2Fe-2S]+-anamorsin and one molecule of dimeric cluster-oxidized [2Fe-2S]2+-GLRX3 synergically provides two [2Fe-2S]2+ clusters from GLRX3 and two electrons from anamorsin to assemble a [4Fe-4S]2+ cluster specifically on the N-terminal cluster-binding site of NUBP1; only the anamorsin [2Fe-2S] cluster bound to the CX8CX2CXC motif provides the electrons. In vitro reconstitution, NMR, UV-visible spectroscopy, Mössbauer spectroscopy Protein Science High 36916754
2006 Mouse NUBP1 and NUBP2 interact with each other and with the minus-end-directed kinesin motor KIFC5A; knockdown of Nubp1 or double knockdown of Nubp1 and Nubp2 causes centrosome amplification (reduplication and cytokinesis defects) and multipolar spindles, phenocopying KIFC5A silencing, placing NUBP1 in a pathway with KIFC5A regulating centrosome duplication. Co-immunoprecipitation, RNAi knockdown, immunofluorescence microscopy, in vitro motor assays Journal of Cell Science High 16638812
2013 NUBP1 and NUBP2 are integral components of centrioles throughout the cell cycle and localize to the basal body of the primary cilium; RNAi silencing of Nubp1 in C. elegans causes morphologically aberrant and additional sensory cilia; downregulation of Nubp1 or Nubp2 in quiescent mouse NIH 3T3 cells markedly increases the number of ciliated cells (i.e., NUBP1 is a negative regulator of ciliogenesis); NUBP1 interacts with members of the CCT/TRiC chaperone complex. RNAi, immunofluorescence, live imaging, Co-immunoprecipitation, C. elegans genetics Cellular and Molecular Life Sciences High 23807208
2015 KATNAL2 isoforms (katanin-like microtubule-severing AAA proteins) directly and independently interact with NUBP1 and NUBP2 in vivo; shRNAi of Katnal2 causes increased centriole numbers, multipolar spindles, and reduced ciliogenesis; overexpression reduces ciliogenesis, consistent with NUBP1 and NUBP2 acting as integral centriole components and negative regulators of ciliogenesis. Co-immunoprecipitation, shRNAi, immunofluorescence, cell cycle analysis Cellular and Molecular Life Sciences Medium 26153462
2012 Nubp1 is required for lung branching morphogenesis and distal progenitor cell survival in mice; Nubp1 mutant mice show increased apoptosis and loss of distal progenitor markers; Nubp1 mutation disrupts localization of polarity protein Par3 and mitosis-relevant protein Numb; Nubp1 knockdown in lung epithelial cells impairs centrosome dynamics and microtubule organization. Forward genetic screen, mouse mutant analysis, immunofluorescence, siRNA knockdown in lung epithelial cells PLoS One Medium 23028652
2009 The C-terminal region of human NUBP1 is important for nuclear localization, as GFP fused to the N-terminus of NUBP1 accumulates in the nucleus whereas GFP fused to the C-terminus does not; N-terminal GFP fusion to NUBP2 also induces nuclear localization. GFP fusion live-cell imaging in HeLa cells Molecular Biology Reports Low 19263241

Source papers

Stage 0 corpus · 27 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2007 The Cfd1-Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol. Nature chemical biology 143 17401378
2005 The eukaryotic P loop NTPase Nbp35: an essential component of the cytosolic and nuclear iron-sulfur protein assembly machinery. Proceedings of the National Academy of Sciences of the United States of America 129 15728363
2008 Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis. Molecular and cellular biology 87 18573874
2012 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 Journal of biological chemistry 86 22362766
2008 The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage. The Journal of biological chemistry 54 18957412
2008 Archaeal ApbC/Nbp35 homologs function as iron-sulfur cluster carrier proteins. Journal of bacteriology 37 19114487
1996 NBP35 encodes an essential and evolutionary conserved protein in Saccharomyces cerevisiae with homology to a superfamily of bacterial ATPases. Gene 33 8921898
2015 A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells. Cellular and molecular life sciences : CMLS 30 26153462
2011 Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication. The EMBO journal 30 21785410
2013 Interaction with Cfd1 increases the kinetic lability of FeS on the Nbp35 scaffold. The Journal of biological chemistry 29 23798678
2006 Motor protein KIFC5A interacts with Nubp1 and Nubp2, and is implicated in the regulation of centrosome duplication. Journal of cell science 29 16638812
2013 The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis. Cellular and molecular life sciences : CMLS 24 23807208
2008 Arabidopsis cytosolic Nbp35 homodimer can assemble both [2Fe-2S] and [4Fe-4S] clusters in two distinct domains. Biochemical and biophysical research communications 21 19084504
2000 NBP1 (Nap1 binding protein 1), an essential gene for G2/M transition of Saccharomyces cerevisiae, encodes a protein of distinct sub-nuclear localization. Gene 20 10767562
2015 The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase. The Journal of biological chemistry 19 26195633
2020 GLRX3 Acts as a [2Fe-2S] Cluster Chaperone in the Cytosolic Iron-Sulfur Assembly Machinery Transferring [2Fe-2S] Clusters to NUBP1. Journal of the American Chemical Society 18 32429669
2017 NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana. The Plant journal : for cell and molecular biology 18 27801963
2012 Nubp1 is required for lung branching morphogenesis and distal progenitor cell survival in mice. PloS one 17 23028652
1999 Two novel mouse genes--Nubp2, mapped to the t-complex on chromosome 17, and Nubp1, mapped to chromosome 16--establish a new gene family of nucleotide-binding proteins in eukaryotes. Genomics 17 10486206
2014 Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica. PloS one 14 25271645
2019 Coupling Nucleotide Binding and Hydrolysis to Iron-Sulfur Cluster Acquisition and Transfer Revealed through Genetic Dissection of the Nbp35 ATPase Site. Biochemistry 12 30865432
2009 Comparison of intracellular localization of Nubp1 and Nubp2 using GFP fusion proteins. Molecular biology reports 11 19263241
2019 The bacterial MrpORP is a novel Mrp/NBP35 protein involved in iron-sulfur biogenesis. Scientific reports 7 30679587
2023 Unraveling the mechanism of [4Fe-4S] cluster assembly on the N-terminal cluster binding site of NUBP1. Protein science : a publication of the Protein Society 5 36916754
2019 The Cfd1 Subunit of the Nbp35-Cfd1 Iron Sulfur Cluster Scaffolding Complex Controls Nucleotide Binding. Biochemistry 3 30785732
2019 The Nbp35/ApbC homolog acts as a nonessential [4Fe-4S] transfer protein in methanogenic archaea. FEBS letters 3 31709520
2023 Interaction between Cfd1 and Nbp35 proteins involved in cytosolic FeS cluster assembly machinery deciphers a stable complexation in Leishmania donovani. International journal of biological macromolecules 2 37774824