Affinage

NUBP1

Cytosolic Fe-S cluster assembly factor NUBP1 · UniProt P53384

Length
320 aa
Mass
34.5 kDa
Annotated
2026-06-10
25 papers in source corpus 16 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: tie faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NUBP1 (yeast Nbp35) is a cytosolic P-loop ATPase that serves as a core scaffold of the cytosolic iron-sulfur protein assembly (CIA) machinery, required for maturation of cytosolic and nuclear [4Fe-4S] proteins but not mitochondrial Fe/S enzymes (PMID:15728363, PMID:18573874). It heterodimerizes with NUBP2 (Cfd1) to form a heterotetrameric scaffold that coordinates Fe/S clusters at the N terminus of NUBP1 and at C-terminal CPXC motifs, where two central cysteines coordinate a bridging [4Fe-4S] cluster shared between subunits and are essential for tetramer formation and viability (PMID:17401378, PMID:22362766). Cluster assembly proceeds by transfer of [2Fe-2S] clusters from dimeric GLRX3 to apo-NUBP1, followed by reductive coupling into [4Fe-4S] clusters using electrons supplied specifically by the anamorsin [2Fe-2S] cluster, generating a tightly bound N-terminal cluster and a labile, transferable C-terminal cluster (PMID:32429669, PMID:36916754). The scaffold is an ATPase active as the Nbp35 homodimer and Nbp35-Cfd1 heterodimer but not the Cfd1 homodimer, and nucleotide binding/hydrolysis is allosterically coupled to cluster loading and release, with heterocomplex formation increasing cluster lability to drive transfer to apo-target proteins via Nar1/Cia1 (PMID:23798678, PMID:26195633, PMID:30865432, PMID:30785732). In mammalian cells this activity underlies IRP1 maturation and cellular iron homeostasis (PMID:18573874). Independently of its CIA role, NUBP1 is an integral centriolar and basal-body component that acts with NUBP2 as a negative regulator of ciliogenesis and a regulator of centriole/centrosome duplication, functioning in pathways with KIFC5A and KATNAL2 and physically associating with the CCT/TRiC chaperone complex (PMID:23807208, PMID:26153462, PMID:16638812).

Mechanistic history

Synthesis pass · year-by-year structured walk · 15 steps
  1. 1996 Medium

    Established the essential functional architecture of Nbp35 by showing its P-loop nucleotide-binding motif and N-terminal cysteine cluster are both required for viability, framing it as a nucleotide-binding, metal-coordinating protein before its pathway was known.

    Evidence Point mutagenesis and yeast complementation with immunofluorescence localization

    PMID:8921898

    Open questions at the time
    • Did not identify the cofactor coordinated by the cysteine cluster
    • No pathway or cellular function assigned
  2. 2005 High

    Placed Nbp35 in the cytosolic Fe/S protein assembly pathway by showing it carries an N-terminal Fe/S cluster dependent on mitochondrial ISC machinery and is required for cytosolic but not mitochondrial Fe/S protein maturation.

    Evidence Subcellular fractionation, depletion, enzyme activity assays and genetic interaction analysis in S. cerevisiae

    PMID:15728363

    Open questions at the time
    • Mechanism of cluster assembly on the scaffold not resolved
    • Role of nucleotide binding in cluster handling not addressed
  3. 2006 Medium

    Revealed a CIA-independent role by linking NUBP1 to centrosome duplication control via direct interaction with KIFC5A and NUBP2.

    Evidence Co-immunoprecipitation, RNAi knockdown and centrosome counting in mammalian cells

    PMID:16638812

    Open questions at the time
    • Molecular mechanism connecting NUBP1 to centrosome number unknown
    • Relationship of this function to its Fe/S activity unresolved
  4. 2007 High

    Defined the Cfd1-Nbp35 heterotetramer as the cytosolic Fe/S cluster assembly scaffold, showing it binds up to three [4Fe-4S] clusters and transfers labile clusters to apoproteins in a Nar1/Cia1-dependent manner.

    Evidence In vivo and in vitro reconstitution with Mössbauer/EPR spectroscopy and cluster transfer assays

    PMID:17401378

    Open questions at the time
    • Source of clusters delivered to the scaffold not identified
    • Role of ATP in the transfer cycle not yet defined
  5. 2008 High

    Extended the scaffold model to humans, demonstrating NUBP1 is required for cytosolic Fe/S protein and IRP1 maturation, forms a complex with NUBP2, and controls cellular iron homeostasis.

    Evidence RNAi depletion in HeLa cells, enzyme activity, Co-IP, iron uptake and western blotting

    PMID:18573874

    Open questions at the time
    • Direct biochemical demonstration of human cluster transfer not shown
    • Link between iron homeostasis defect and IRP1 maturation not mechanistically dissected
  6. 2012 High

    Identified the structural basis of inter-subunit cluster bridging, showing the C-terminal CPXC cysteines coordinate a bridging [4Fe-4S] cluster essential for tetramer formation and that nucleotide binding is required for cluster loading.

    Evidence Cysteine mutagenesis, yeast complementation and EPR/Mössbauer spectroscopy

    PMID:22362766

    Open questions at the time
    • Quantitative allosteric coupling between nucleotide and cluster not defined
    • Transfer step from bridging cluster to targets not resolved
  7. 2012 Medium

    Demonstrated an in vivo developmental requirement for Nubp1 in lung epithelial progenitors, tying its centrosome/microtubule function to cell polarity and survival.

    Evidence Forward genetic screen, mouse mutant analysis, immunofluorescence and RNAi knockdown

    PMID:23028652

    Open questions at the time
    • Whether phenotypes stem from CIA or centrosome function not separated
    • Direct partners mediating polarity effects not identified
  8. 2013 High

    Resolved the functional asymmetry of the heterocomplex, showing Nbp35 binds iron readily while Cfd1 does not, and that heterocomplex formation increases cluster lability to enable transfer.

    Evidence In vivo 55Fe radiolabeling, Co-IP and yeast mutant analysis

    PMID:23798678

    Open questions at the time
    • Structural basis of lability change not defined
    • Identity of immediate cluster acceptor in vivo not shown
  9. 2013 High

    Established a centriole/basal-body localization and a conserved role for NUBP1/2 as negative regulators of ciliogenesis, and identified CCT/TRiC as a physical partner.

    Evidence Immunofluorescence, RNAi in C. elegans and NIH 3T3 cells, cilia quantification and Co-IP

    PMID:23807208

    Open questions at the time
    • Mechanism by which NUBP1 restrains ciliogenesis unknown
    • Whether CCT/TRiC binding reflects folding clientship or a functional partnership unresolved
  10. 2015 High

    Characterized the scaffold ATPase activity, showing Nbp35 homodimer and heterodimer hydrolyze ATP while the Cfd1 homodimer does not, and quantified stoichiometric ATP binding to Nbp35.

    Evidence In vitro ATPase assays, site-directed mutagenesis and mantATP binding assays

    PMID:26195633

    Open questions at the time
    • Coupling of hydrolysis to specific cluster handling steps not yet defined
    • Effect of clusters on ATPase rate not measured here
  11. 2015 Medium

    Linked KATNAL2 directly to NUBP1/NUBP2 in a shared pathway governing cytokinesis and ciliogenesis through reciprocal interaction and epistasis.

    Evidence Co-IP, shRNAi, immunofluorescence and overexpression in mouse cells

    PMID:26153462

    Open questions at the time
    • Single-lab evidence without independent replication
    • Biochemical role of KATNAL2-NUBP1 complex unresolved
  12. 2019 High

    Defined allosteric coupling between nucleotide and cluster scaffolding, showing all four ATPase motifs are needed for assembly and transfer and that bridging-cluster occupancy lowers nucleotide affinity.

    Evidence Site-directed mutagenesis, yeast complementation, in vitro nucleotide binding and 55Fe radiolabeling

    PMID:30865432

    Open questions at the time
    • Directionality of the catalytic cycle not fully ordered
    • Structural conformations underlying allostery not visualized
  13. 2019 High

    Established ordered nucleotide loading within the heterodimer, showing Cfd1 must bind nucleotide before Nbp35 and that Cfd1 becomes hydrolysis-competent only in complex with Nbp35.

    Evidence Fluorescence nucleotide titrations, mutagenesis and in vitro ATPase assays

    PMID:30785732

    Open questions at the time
    • Coupling of this ordered cycle to cluster transfer timing not determined
    • Structural intermediates not captured
  14. 2020 High

    Identified GLRX3 as the cluster donor, showing dimeric GLRX3 transfers [2Fe-2S] clusters to apo-NUBP1 that are reductively coupled into [4Fe-4S] clusters, with C-terminal cluster binding promoting dimerization.

    Evidence In vitro cluster transfer with NMR, UV-vis/CD spectroscopy and analytical ultracentrifugation

    PMID:32429669

    Open questions at the time
    • Electron source for reductive coupling not yet defined at this stage
    • In vivo relevance of the reconstituted pathway not confirmed
  15. 2023 High

    Completed the electron-donation model by showing a hetero-complex of anamorsin and GLRX3 synergistically delivers two [2Fe-2S] clusters and two electrons to assemble the N-terminal [4Fe-4S] cluster of NUBP1.

    Evidence In vitro reconstitution with NMR, UV-vis/CD spectroscopy and analytical ultracentrifugation

    PMID:36916754

    Open questions at the time
    • Assembly mechanism of the C-terminal/bridging cluster by this system not addressed
    • In vivo confirmation of the synergistic complex not shown

Open questions

Synthesis pass · forward-looking unresolved questions
  • How NUBP1's cytosolic Fe/S cluster assembly activity is mechanistically connected to its independent centriole/ciliogenesis functions remains unresolved.
  • No evidence whether Fe/S cluster status influences centriolar function
  • Structural basis of NUBP1 association with KIFC5A, KATNAL2 and CCT/TRiC not defined
  • No structure of the full scaffold cluster-transfer cycle

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140657 ATP-dependent activity 3 GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0005634 nucleus 3 GO:0005815 microtubule organizing center 3 GO:0005829 cytosol 2 GO:0005929 cilium 1
Pathway
R-HSA-1852241 Organelle biogenesis and maintenance 2
Partners
ANAMORSINCCT/TRICGLRX3KATNAL2KIFC5ANUBP2
Complex memberships
CCT/TRiC chaperonin (associated)NUBP1-NUBP2 (Nbp35-Cfd1) CIA scaffold heterotetramer

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2005 Yeast Nbp35p resides in the cytosol and nucleus, carries an Fe/S cluster at its N terminus whose assembly requires the mitochondrial ISC machinery, and its depletion impairs cytosolic/nuclear Fe/S protein maturation (e.g., Leu1p) without affecting mitochondrial Fe/S enzymes. Nbp35p genetically interacts with Cfd1p and Nar1p. Subcellular fractionation, RNAi/depletion, enzyme activity assays, genetic interaction analysis (S. cerevisiae) Proceedings of the National Academy of Sciences of the United States of America High 15728363
2007 Cfd1 and Nbp35 form a heterotetrameric complex that binds up to three [4Fe-4S] clusters: one at the N terminus of Nbp35 and one each at the C-terminal CXXC motifs of both proteins. These labile clusters can be transferred to Fe/S apoproteins in a Nar1- and Cia1-dependent manner, establishing the Cfd1-Nbp35 complex as a scaffold for cytosolic Fe/S cluster assembly. In vivo and in vitro reconstitution, Mössbauer/EPR spectroscopy, co-purification, Fe/S cluster transfer assay Nature chemical biology High 17401378
2008 Human NUBP1 (huNbp35) is a cytosolic Fe/S protein whose depletion by RNAi impairs maturation of cytosolic Fe/S proteins (glutamine PRPP amidotransferase and IRP1) but not mitochondrial Fe/S proteins. Loss of NUBP1 consequently dysregulates iron homeostasis (decreased H-ferritin, increased transferrin receptor, higher transferrin uptake). NUBP1 forms a complex with huCfd1/NUBP2 in vivo. RNAi depletion in HeLa cells, enzyme activity assays, co-immunoprecipitation, iron uptake assays, western blotting Molecular and cellular biology High 18573874
2012 The two central cysteine residues (CPXC motif) of the C-terminal domain of Nbp35 and Cfd1 are essential for cell viability, Fe/S cluster coordination, and Cfd1-Nbp35 hetero-tetramer formation. These CPXC motifs coordinate a bridging [4Fe-4S] cluster between the two subunits. Mutation of the nucleotide-binding motif prevents Fe/S cluster loading on the scaffold unless wild-type copies are present in trans, demonstrating nucleotide binding is required for cluster assembly. Cysteine mutagenesis, yeast complementation, EPR/Mössbauer spectroscopy, biochemical analysis The Journal of biological chemistry High 22362766
2013 Interaction of Nbp35 with Cfd1 increases the kinetic lability of assembled FeS clusters on Nbp35, facilitating cluster transfer to target apo-FeS proteins. Free Nbp35 binds 55Fe readily, whereas Cfd1 alone shows no detectable 55Fe binding. A Cfd1 mutation impairing heterocomplex stability supports iron binding to Nbp35 but impairs iron release. 55Fe radiolabeling in vivo, co-immunoprecipitation, yeast genetics and mutant analysis The Journal of biological chemistry High 23798678
2013 NUBP1 and NUBP2 are integral components of centrioles throughout the cell cycle and localize to the basal body of primary cilia. RNAi silencing of Nubp1 in C. elegans causes morphologically aberrant and supernumerary cilia. Downregulation of Nubp1 or Nubp2 in mammalian cells increases ciliogenesis, establishing them as negative regulators of ciliogenesis. NUBP1 interacts with members of the CCT/TRiC chaperone complex. Immunofluorescence/localization, RNAi in C. elegans and NIH 3T3 cells, cilia quantification, co-immunoprecipitation with CCT/TRiC Cellular and molecular life sciences : CMLS High 23807208
2015 The Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases in vitro; the Cfd1 homodimer shows no or very low ATPase activity. Mutation of key active-site residues reduces ATP hydrolysis to background. The fluorescent ATP analog mantATP binds stoichiometrically to Nbp35 (KD = 15.6 μM), and an Nbp35 mutant deficient in ATP hydrolysis shows increased KD for mantATP. In vitro ATPase assay, site-directed mutagenesis, fluorescent nucleotide binding assay (mantATP) The Journal of biological chemistry High 26195633
2015 KATNAL2 isoforms interact directly and independently with NUBP1 and NUBP2 in vivo. Nubp1 and Nubp2 are integral components of centrioles, act as negative regulators of ciliogenesis, and are implicated in centriole duplication; shRNAi of Katnal2 phenocopies and genetic interactions link KATNAL2 and NUBP1/2 to the same regulatory pathway for cytokinesis and ciliogenesis. Co-immunoprecipitation, shRNAi, immunofluorescence, overexpression in mouse cells Cellular and molecular life sciences : CMLS Medium 26153462
2006 Mouse KIFC5A interacts with NUBP1 and NUBP2 (which also interact with each other). NUBP1 and NUBP2 knockdown phenocopies KIFC5A silencing, causing centrosome amplification, implicating NUBP1 in a pathway with KIFC5A that controls centrosome duplication in mammalian cells. Co-immunoprecipitation, RNAi knockdown, immunofluorescence, centrosome counting Journal of cell science Medium 16638812
2020 Dimeric cluster-bridged GLRX3 transfers its [2Fe-2S]2+ clusters to monomeric apo NUBP1; these clusters are reductively coupled (with glutathione as reductant) to form [4Fe-4S]2+ clusters at both the N-terminal CX13CX2CX5C and C-terminal CPXC motifs of NUBP1. Cluster binding at the C-terminal motif promotes NUBP1 dimerization, while the N-terminal [4Fe-4S] cluster is tightly bound and the C-terminal cluster is labile. In vitro cluster transfer assay, NMR spectroscopy, UV-visible/CD spectroscopy, analytical ultracentrifugation Journal of the American Chemical Society High 32429669
2023 A hetero-tetrameric complex of two molecules of cluster-reduced [2Fe-2S]+-anamorsin and one molecule of dimeric [2Fe-2S]2+-GLRX3 synergistically provides two [2Fe-2S]2+ clusters from GLRX3 and two electrons from anamorsin to assemble a [4Fe-4S]2+ cluster on the N-terminal binding site of NUBP1. Only the anamorsin [2Fe-2S] cluster bound to the CX8CX2CXC motif provides the required electrons. In vitro reconstitution, NMR spectroscopy, UV-visible/CD spectroscopy, analytical ultracentrifugation Protein science : a publication of the Protein Society High 36916754
2019 Mutations in all four conserved ATPase site motifs of Nbp35 diminish both FeS cluster assembly and transfer in vivo. Four phenotypic classes correspond to effects on ATP binding vs. hydrolysis. In vitro, occupancy of the bridging FeS cluster binding site decreases scaffold affinity for nucleotide, revealing allosteric coupling between ATP binding/hydrolysis and cluster scaffolding. Site-directed mutagenesis, in vivo complementation assays (yeast), in vitro nucleotide binding assays, 55Fe radiolabeling Biochemistry High 30865432
2019 In the Nbp35-Cfd1 heterodimer, nucleotide must bind to the Cfd1 subunit before it can bind to Nbp35; the Cfd1 subunit is hydrolysis competent only when bound to Nbp35. The Cfd1 homodimer binds ATP but has no detectable ATPase activity. All forms of the CIA scaffold are specific for adenosine nucleotides. Fluorescence nucleotide titrations, site-directed mutagenesis, in vitro ATPase assays Biochemistry High 30785732
2012 Nubp1 is expressed in distal epithelial progenitor cells of the mouse lung. Loss-of-function mutation of Nubp1 increases apoptosis in these cells, disrupts localization of polarity protein Par3 and mitosis-relevant protein Numb, and impairs centrosome dynamics and microtubule organization. Nubp1 knockdown in lung epithelial cells also disrupts centrosome dynamics. Forward genetic screen, in vivo mouse mutant analysis, immunofluorescence, RNAi knockdown in lung epithelial cells PloS one Medium 23028652
2009 In HeLa cells, GFP fused to the N-terminus of NUBP1 accumulates in the nucleus, whereas C-terminal GFP fusion does not show nuclear transfer, indicating that the C-terminal region of NUBP1 is important for nuclear localization. GFP fusion live imaging in HeLa cells Molecular biology reports Low 19263241
1996 A single amino-acid substitution in the conserved nucleotide-binding P-loop motif of yeast Nbp35p renders the protein non-functional, and a conserved cluster of four cysteines at the N-terminal end is also required for its essential function. Nbp35p localizes to the nucleus by indirect immunofluorescence. Point mutagenesis, yeast complementation, indirect immunofluorescence Gene Medium 8921898

Source papers

Stage 0 corpus · 25 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
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 87 22362766
2008 Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis. Molecular and cellular biology 87 18573874
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 31 26153462
2013 Interaction with Cfd1 increases the kinetic lability of FeS on the Nbp35 scaffold. The Journal of biological chemistry 30 23798678
2011 Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication. The EMBO journal 30 21785410
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
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
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 19 32429669
2015 The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase. The Journal of biological chemistry 19 26195633
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 13 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

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