{"gene":"NUBP1","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":2007,"finding":"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.","method":"In vitro reconstitution, UV-visible and Mössbauer spectroscopy, in vivo 55Fe radiolabeling, genetic epistasis","journal":"Nature Chemical Biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution + spectroscopic characterization + in vivo transfer assay, highly cited foundational paper","pmids":["17401378"],"is_preprint":false},{"year":2005,"finding":"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.","method":"Genetic depletion, enzyme activity assays, subcellular fractionation, genetic interaction analysis","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, highly cited, replicated in subsequent studies","pmids":["15728363"],"is_preprint":false},{"year":2008,"finding":"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.","method":"RNA interference, enzyme activity assays, co-immunoprecipitation, iron metabolism assays in HeLa cells","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus functional KD with defined biochemical phenotypes in human cells","pmids":["18573874"],"is_preprint":false},{"year":2012,"finding":"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.","method":"Site-directed mutagenesis, Mössbauer spectroscopy, EPR, genetic complementation, yeast two-hybrid","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with spectroscopic validation and genetic readouts","pmids":["22362766"],"is_preprint":false},{"year":2013,"finding":"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.","method":"55Fe radiolabeling in yeast, mutant analysis of heterocomplex stability, iron release kinetics","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple mutant alleles with quantitative in vivo iron-binding and release measurements","pmids":["23798678"],"is_preprint":false},{"year":2015,"finding":"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.","method":"In vitro ATPase assay, site-directed mutagenesis, fluorescent nucleotide analog binding","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro enzymatic assay with mutagenesis controls","pmids":["26195633"],"is_preprint":false},{"year":2019,"finding":"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.","method":"Site-directed mutagenesis, in vivo and in vitro Fe-S cluster assembly/transfer assays, nucleotide binding measurements","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple ATPase site mutants linked to defined in vivo and in vitro phenotypes","pmids":["30865432"],"is_preprint":false},{"year":2019,"finding":"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.","method":"Fluorescent nucleotide titration, site-directed mutagenesis, ATPase assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — rigorous in vitro dissection of subunit-specific roles with mutagenesis","pmids":["30785732"],"is_preprint":false},{"year":2020,"finding":"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.","method":"In vitro cluster transfer assays, NMR spectroscopy, UV-visible and Mössbauer spectroscopy, analytical ultracentrifugation","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 — reconstituted human protein cluster transfer with multiple spectroscopic and biophysical methods","pmids":["32429669"],"is_preprint":false},{"year":2023,"finding":"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.","method":"In vitro reconstitution, NMR, UV-visible spectroscopy, Mössbauer spectroscopy","journal":"Protein Science","confidence":"High","confidence_rationale":"Tier 1 — reconstituted multi-protein in vitro assembly with spectroscopic characterization","pmids":["36916754"],"is_preprint":false},{"year":2006,"finding":"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.","method":"Co-immunoprecipitation, RNAi knockdown, immunofluorescence microscopy, in vitro motor assays","journal":"Journal of Cell Science","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus RNAi phenotype with genetic epistasis (double knockdown)","pmids":["16638812"],"is_preprint":false},{"year":2013,"finding":"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.","method":"RNAi, immunofluorescence, live imaging, Co-immunoprecipitation, C. elegans genetics","journal":"Cellular and Molecular Life Sciences","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal organisms and methods, defined subcellular localization with functional consequence","pmids":["23807208"],"is_preprint":false},{"year":2015,"finding":"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.","method":"Co-immunoprecipitation, shRNAi, immunofluorescence, cell cycle analysis","journal":"Cellular and Molecular Life Sciences","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus RNAi phenotype; single lab, functional consequences inferred from KATNAL2 KD","pmids":["26153462"],"is_preprint":false},{"year":2012,"finding":"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.","method":"Forward genetic screen, mouse mutant analysis, immunofluorescence, siRNA knockdown in lung epithelial cells","journal":"PLoS One","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo mouse mutant with defined cellular phenotypes plus in vitro knockdown confirmation","pmids":["23028652"],"is_preprint":false},{"year":2009,"finding":"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.","method":"GFP fusion live-cell imaging in HeLa cells","journal":"Molecular Biology Reports","confidence":"Low","confidence_rationale":"Tier 3 — single method (fluorescent protein tagging), single lab, no functional consequence tested","pmids":["19263241"],"is_preprint":false}],"current_model":"NUBP1 (human Nbp35) is a cytosolic P-loop ATPase that, together with NUBP2 (Cfd1), forms a heterotetrameric scaffold that receives [2Fe-2S] clusters from the chaperone GLRX3 (with electrons from anamorsin), reductively couples them into labile [4Fe-4S] clusters at both its N-terminal (CX13CX2CX5C) and C-terminal (CPXC) cysteine motifs, and then transfers these clusters to cytosolic and nuclear apo-Fe-S proteins in a CIA pathway-dependent manner; independently, NUBP1 localizes to centrioles and basal bodies where it acts—together with NUBP2 and the motor KIFC5A—as a negative regulator of ciliogenesis and a positive regulator of centrosome duplication, with ATPase activity allosterically coupled to both cluster assembly and inter-protein transfer steps."},"narrative":{"teleology":[{"year":2005,"claim":"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","pmids":["15728363"],"confidence":"High","gaps":["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"]},{"year":2006,"claim":"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","pmids":["16638812"],"confidence":"High","gaps":["Whether the centrosome function requires ATPase or Fe-S cluster activity was unknown","Mechanism of centrosome duplication control was not defined"]},{"year":2007,"claim":"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","pmids":["17401378"],"confidence":"High","gaps":["Source of electrons and [2Fe-2S] precursors feeding the scaffold was unknown","Role of ATP hydrolysis in cluster assembly was not tested"]},{"year":2008,"claim":"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","pmids":["18573874"],"confidence":"High","gaps":["In vitro reconstitution with human proteins had not been performed","Whether the human system uses the same cluster transfer mechanism was unproven"]},{"year":2012,"claim":"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","pmids":["22362766"],"confidence":"High","gaps":["Structural basis of bridging coordination was inferred, not crystallographically resolved","How ATP hydrolysis mechanistically couples to cluster loading remained unclear"]},{"year":2012,"claim":"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","pmids":["23028652"],"confidence":"Medium","gaps":["Whether the lung phenotype reflects Fe-S or centrosome dysfunction was not distinguished","Single genetic screen without independent allelic confirmation"]},{"year":2013,"claim":"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","pmids":["23807208"],"confidence":"High","gaps":["Mechanism by which NUBP1 suppresses ciliogenesis was not defined","Whether CCT/TRiC interaction is functionally relevant to ciliogenesis was not tested"]},{"year":2013,"claim":"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","pmids":["23798678"],"confidence":"High","gaps":["Structural basis for increased lability upon heterocomplex formation was unknown","Whether the same asymmetry applies to the human complex was not shown"]},{"year":2015,"claim":"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","pmids":["26195633"],"confidence":"High","gaps":["Connection between ATPase cycle and cluster assembly/transfer was not yet established","No structural information on the ATP-bound state"]},{"year":2019,"claim":"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","pmids":["30865432","30785732"],"confidence":"High","gaps":["No structural snapshot of the allosteric conformational change","Whether ATP hydrolysis drives cluster release or cluster loading (or both) could not be kinetically separated"]},{"year":2020,"claim":"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","pmids":["32429669"],"confidence":"High","gaps":["Role of NUBP2 in the transfer from GLRX3 was not addressed","Electron source for reductive coupling in this system was glutathione, not anamorsin"]},{"year":2023,"claim":"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","pmids":["36916754"],"confidence":"High","gaps":["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"]},{"year":null,"claim":"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.","evidence":"","pmids":[],"confidence":"Low","gaps":["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":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[5,6,7]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[5,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,2,8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,14]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[10,11]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,2,8,9]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[10,11]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[10,11]}],"complexes":["NUBP1-NUBP2 (Nbp35-Cfd1) heterotetramer"],"partners":["NUBP2","GLRX3","KIFC5A","KATNAL2","CYBC1"],"other_free_text":[]},"mechanistic_narrative":"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]."},"prefetch_data":{"uniprot":{"accession":"P53384","full_name":"Cytosolic Fe-S cluster assembly factor NUBP1","aliases":["Nucleotide-binding protein 1","NBP 1"],"length_aa":320,"mass_kda":34.5,"function":"Component of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery (PubMed:18573874). Required for maturation of extramitochondrial Fe-S proteins (PubMed:18573874). The NUBP1-NUBP2 heterotetramer forms a Fe-S scaffold complex, mediating the de novo assembly of an Fe-S cluster and its transfer to target apoproteins (PubMed:18573874). Implicated in the regulation of centrosome duplication (By similarity). Negatively regulates cilium formation and structure (By similarity)","subcellular_location":"Cytoplasm; Nucleus; Cell projection; Cytoplasm, cytoskeleton, cilium axoneme; Cytoplasm, cytoskeleton, cilium basal body; Cytoplasm, cytoskeleton, microtubule organizing center; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriole; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome","url":"https://www.uniprot.org/uniprotkb/P53384/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NUBP1","classification":"Common Essential","n_dependent_lines":1165,"n_total_lines":1208,"dependency_fraction":0.9644039735099338},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NUBP1","total_profiled":1310},"omim":[{"mim_id":"614778","title":"CYTOSOLIC IRON-SULFUR ASSEMBLY COMPONENT 2B; CIAO2B","url":"https://www.omim.org/entry/614778"},{"mim_id":"614777","title":"MMS19 HOMOLOG, CYTOSOLIC IRON-SULFUR ASSEMBLY COMPONENT; MMS19","url":"https://www.omim.org/entry/614777"},{"mim_id":"613622","title":"FAD-DEPENDENT OXIDOREDUCTASE DOMAIN-CONTAINING PROTEIN 1; FOXRED1","url":"https://www.omim.org/entry/613622"},{"mim_id":"610779","title":"NUCLEOTIDE-BINDING PROTEIN 2; NUBP2","url":"https://www.omim.org/entry/610779"},{"mim_id":"604333","title":"WD40 REPEAT-CONTAINING PROTEIN CIAO1; CIAO1","url":"https://www.omim.org/entry/604333"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Mid piece","reliability":"Supported"},{"location":"Principal piece","reliability":"Supported"},{"location":"End piece","reliability":"Supported"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NUBP1"},"hgnc":{"alias_symbol":["NBP35","CIAO5"],"prev_symbol":["NBP1"]},"alphafold":{"accession":"P53384","domains":[{"cath_id":"-","chopping":"2-39","consensus_level":"medium","plddt":74.0234,"start":2,"end":39},{"cath_id":"3.40.50.300","chopping":"44-320","consensus_level":"high","plddt":92.1869,"start":44,"end":320}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P53384","model_url":"https://alphafold.ebi.ac.uk/files/AF-P53384-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P53384-F1-predicted_aligned_error_v6.png","plddt_mean":89.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NUBP1","jax_strain_url":"https://www.jax.org/strain/search?query=NUBP1"},"sequence":{"accession":"P53384","fasta_url":"https://rest.uniprot.org/uniprotkb/P53384.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P53384/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P53384"}},"corpus_meta":[{"pmid":"17401378","id":"PMC_17401378","title":"The Cfd1-Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol.","date":"2007","source":"Nature chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/17401378","citation_count":143,"is_preprint":false},{"pmid":"15728363","id":"PMC_15728363","title":"The eukaryotic P loop NTPase Nbp35: an essential component of the cytosolic and nuclear iron-sulfur protein assembly machinery.","date":"2005","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15728363","citation_count":129,"is_preprint":false},{"pmid":"18573874","id":"PMC_18573874","title":"Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis.","date":"2008","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18573874","citation_count":87,"is_preprint":false},{"pmid":"22362766","id":"PMC_22362766","title":"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.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22362766","citation_count":86,"is_preprint":false},{"pmid":"18957412","id":"PMC_18957412","title":"The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18957412","citation_count":54,"is_preprint":false},{"pmid":"19114487","id":"PMC_19114487","title":"Archaeal ApbC/Nbp35 homologs function as iron-sulfur cluster carrier proteins.","date":"2008","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/19114487","citation_count":37,"is_preprint":false},{"pmid":"8921898","id":"PMC_8921898","title":"NBP35 encodes an essential and evolutionary conserved protein in Saccharomyces cerevisiae with homology to a superfamily of bacterial ATPases.","date":"1996","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/8921898","citation_count":33,"is_preprint":false},{"pmid":"26153462","id":"PMC_26153462","title":"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.","date":"2015","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/26153462","citation_count":30,"is_preprint":false},{"pmid":"21785410","id":"PMC_21785410","title":"Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication.","date":"2011","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/21785410","citation_count":30,"is_preprint":false},{"pmid":"23798678","id":"PMC_23798678","title":"Interaction with Cfd1 increases the kinetic lability of FeS on the Nbp35 scaffold.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23798678","citation_count":29,"is_preprint":false},{"pmid":"16638812","id":"PMC_16638812","title":"Motor protein KIFC5A interacts with Nubp1 and Nubp2, and is implicated in the regulation of centrosome duplication.","date":"2006","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16638812","citation_count":29,"is_preprint":false},{"pmid":"23807208","id":"PMC_23807208","title":"The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis.","date":"2013","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/23807208","citation_count":24,"is_preprint":false},{"pmid":"19084504","id":"PMC_19084504","title":"Arabidopsis cytosolic Nbp35 homodimer can assemble both [2Fe-2S] and [4Fe-4S] clusters in two distinct domains.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/19084504","citation_count":21,"is_preprint":false},{"pmid":"10767562","id":"PMC_10767562","title":"NBP1 (Nap1 binding protein 1), an essential gene for G2/M transition of Saccharomyces cerevisiae, encodes a protein of distinct sub-nuclear localization.","date":"2000","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/10767562","citation_count":20,"is_preprint":false},{"pmid":"26195633","id":"PMC_26195633","title":"The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26195633","citation_count":19,"is_preprint":false},{"pmid":"32429669","id":"PMC_32429669","title":"GLRX3 Acts as a [2Fe-2S] Cluster Chaperone in the Cytosolic Iron-Sulfur Assembly Machinery Transferring [2Fe-2S] Clusters to NUBP1.","date":"2020","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/32429669","citation_count":18,"is_preprint":false},{"pmid":"27801963","id":"PMC_27801963","title":"NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana.","date":"2017","source":"The Plant journal : for cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/27801963","citation_count":18,"is_preprint":false},{"pmid":"23028652","id":"PMC_23028652","title":"Nubp1 is required for lung branching morphogenesis and distal progenitor cell survival in mice.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23028652","citation_count":17,"is_preprint":false},{"pmid":"10486206","id":"PMC_10486206","title":"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.","date":"1999","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/10486206","citation_count":17,"is_preprint":false},{"pmid":"25271645","id":"PMC_25271645","title":"Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25271645","citation_count":14,"is_preprint":false},{"pmid":"30865432","id":"PMC_30865432","title":"Coupling Nucleotide Binding and Hydrolysis to Iron-Sulfur Cluster Acquisition and Transfer Revealed through Genetic Dissection of the Nbp35 ATPase Site.","date":"2019","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30865432","citation_count":12,"is_preprint":false},{"pmid":"19263241","id":"PMC_19263241","title":"Comparison of intracellular localization of Nubp1 and Nubp2 using GFP fusion proteins.","date":"2009","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/19263241","citation_count":11,"is_preprint":false},{"pmid":"30679587","id":"PMC_30679587","title":"The bacterial MrpORP is a novel Mrp/NBP35 protein involved in iron-sulfur biogenesis.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30679587","citation_count":7,"is_preprint":false},{"pmid":"36916754","id":"PMC_36916754","title":"Unraveling the mechanism of [4Fe-4S] cluster assembly on the N-terminal cluster binding site of NUBP1.","date":"2023","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/36916754","citation_count":5,"is_preprint":false},{"pmid":"31709520","id":"PMC_31709520","title":"The Nbp35/ApbC homolog acts as a nonessential [4Fe-4S] transfer protein in methanogenic archaea.","date":"2019","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/31709520","citation_count":3,"is_preprint":false},{"pmid":"30785732","id":"PMC_30785732","title":"The Cfd1 Subunit of the Nbp35-Cfd1 Iron Sulfur Cluster Scaffolding Complex Controls Nucleotide Binding.","date":"2019","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30785732","citation_count":3,"is_preprint":false},{"pmid":"37774824","id":"PMC_37774824","title":"Interaction between Cfd1 and Nbp35 proteins involved in cytosolic FeS cluster assembly machinery deciphers a stable complexation in Leishmania donovani.","date":"2023","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/37774824","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14266,"output_tokens":4002,"usd":0.051414},"stage2":{"model":"claude-opus-4-6","input_tokens":7435,"output_tokens":3263,"usd":0.178125},"total_usd":0.229539,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro reconstitution, UV-visible and Mössbauer spectroscopy, in vivo 55Fe radiolabeling, genetic epistasis\",\n      \"journal\": \"Nature Chemical Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution + spectroscopic characterization + in vivo transfer assay, highly cited foundational paper\",\n      \"pmids\": [\"17401378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"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.\",\n      \"method\": \"Genetic depletion, enzyme activity assays, subcellular fractionation, genetic interaction analysis\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, highly cited, replicated in subsequent studies\",\n      \"pmids\": [\"15728363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"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.\",\n      \"method\": \"RNA interference, enzyme activity assays, co-immunoprecipitation, iron metabolism assays in HeLa cells\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus functional KD with defined biochemical phenotypes in human cells\",\n      \"pmids\": [\"18573874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"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.\",\n      \"method\": \"Site-directed mutagenesis, Mössbauer spectroscopy, EPR, genetic complementation, yeast two-hybrid\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with spectroscopic validation and genetic readouts\",\n      \"pmids\": [\"22362766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"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.\",\n      \"method\": \"55Fe radiolabeling in yeast, mutant analysis of heterocomplex stability, iron release kinetics\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple mutant alleles with quantitative in vivo iron-binding and release measurements\",\n      \"pmids\": [\"23798678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro ATPase assay, site-directed mutagenesis, fluorescent nucleotide analog binding\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro enzymatic assay with mutagenesis controls\",\n      \"pmids\": [\"26195633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"Site-directed mutagenesis, in vivo and in vitro Fe-S cluster assembly/transfer assays, nucleotide binding measurements\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple ATPase site mutants linked to defined in vivo and in vitro phenotypes\",\n      \"pmids\": [\"30865432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"Fluorescent nucleotide titration, site-directed mutagenesis, ATPase assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — rigorous in vitro dissection of subunit-specific roles with mutagenesis\",\n      \"pmids\": [\"30785732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro cluster transfer assays, NMR spectroscopy, UV-visible and Mössbauer spectroscopy, analytical ultracentrifugation\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted human protein cluster transfer with multiple spectroscopic and biophysical methods\",\n      \"pmids\": [\"32429669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro reconstitution, NMR, UV-visible spectroscopy, Mössbauer spectroscopy\",\n      \"journal\": \"Protein Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted multi-protein in vitro assembly with spectroscopic characterization\",\n      \"pmids\": [\"36916754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, immunofluorescence microscopy, in vitro motor assays\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus RNAi phenotype with genetic epistasis (double knockdown)\",\n      \"pmids\": [\"16638812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"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.\",\n      \"method\": \"RNAi, immunofluorescence, live imaging, Co-immunoprecipitation, C. elegans genetics\",\n      \"journal\": \"Cellular and Molecular Life Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal organisms and methods, defined subcellular localization with functional consequence\",\n      \"pmids\": [\"23807208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, shRNAi, immunofluorescence, cell cycle analysis\",\n      \"journal\": \"Cellular and Molecular Life Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus RNAi phenotype; single lab, functional consequences inferred from KATNAL2 KD\",\n      \"pmids\": [\"26153462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"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.\",\n      \"method\": \"Forward genetic screen, mouse mutant analysis, immunofluorescence, siRNA knockdown in lung epithelial cells\",\n      \"journal\": \"PLoS One\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo mouse mutant with defined cellular phenotypes plus in vitro knockdown confirmation\",\n      \"pmids\": [\"23028652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"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.\",\n      \"method\": \"GFP fusion live-cell imaging in HeLa cells\",\n      \"journal\": \"Molecular Biology Reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single method (fluorescent protein tagging), single lab, no functional consequence tested\",\n      \"pmids\": [\"19263241\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NUBP1 (human Nbp35) is a cytosolic P-loop ATPase that, together with NUBP2 (Cfd1), forms a heterotetrameric scaffold that receives [2Fe-2S] clusters from the chaperone GLRX3 (with electrons from anamorsin), reductively couples them into labile [4Fe-4S] clusters at both its N-terminal (CX13CX2CX5C) and C-terminal (CPXC) cysteine motifs, and then transfers these clusters to cytosolic and nuclear apo-Fe-S proteins in a CIA pathway-dependent manner; independently, NUBP1 localizes to centrioles and basal bodies where it acts—together with NUBP2 and the motor KIFC5A—as a negative regulator of ciliogenesis and a positive regulator of centrosome duplication, with ATPase activity allosterically coupled to both cluster assembly and inter-protein transfer steps.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"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].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"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.\",\n      \"evidence\": \"Genetic depletion of Nbp35 in yeast with enzyme activity assays and subcellular fractionation\",\n      \"pmids\": [\"15728363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The biochemical nature of the Fe-S cluster on Nbp35 was undefined\",\n        \"How Nbp35 cooperates with Cfd1 was unclear\",\n        \"No reconstituted in vitro system existed\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"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.\",\n      \"evidence\": \"Co-immunoprecipitation, RNAi knockdown, and immunofluorescence in mouse cells\",\n      \"pmids\": [\"16638812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the centrosome function requires ATPase or Fe-S cluster activity was unknown\",\n        \"Mechanism of centrosome duplication control was not defined\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"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.\",\n      \"evidence\": \"In vitro reconstitution with UV-visible and Mössbauer spectroscopy, 55Fe radiolabeling, and genetic epistasis in yeast\",\n      \"pmids\": [\"17401378\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Source of electrons and [2Fe-2S] precursors feeding the scaffold was unknown\",\n        \"Role of ATP hydrolysis in cluster assembly was not tested\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"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.\",\n      \"evidence\": \"RNAi in HeLa cells with enzyme activity assays, co-immunoprecipitation, and iron metabolism readouts\",\n      \"pmids\": [\"18573874\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In vitro reconstitution with human proteins had not been performed\",\n        \"Whether the human system uses the same cluster transfer mechanism was unproven\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"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.\",\n      \"evidence\": \"Site-directed mutagenesis with Mössbauer/EPR spectroscopy and genetic complementation in yeast\",\n      \"pmids\": [\"22362766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of bridging coordination was inferred, not crystallographically resolved\",\n        \"How ATP hydrolysis mechanistically couples to cluster loading remained unclear\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"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.\",\n      \"evidence\": \"Forward genetic screen and mouse mutant analysis with immunofluorescence\",\n      \"pmids\": [\"23028652\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether the lung phenotype reflects Fe-S or centrosome dysfunction was not distinguished\",\n        \"Single genetic screen without independent allelic confirmation\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"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.\",\n      \"evidence\": \"RNAi in NIH 3T3 cells and C. elegans, immunofluorescence, live imaging, co-immunoprecipitation\",\n      \"pmids\": [\"23807208\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which NUBP1 suppresses ciliogenesis was not defined\",\n        \"Whether CCT/TRiC interaction is functionally relevant to ciliogenesis was not tested\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"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.\",\n      \"evidence\": \"55Fe radiolabeling and mutant analysis of heterocomplex stability in yeast\",\n      \"pmids\": [\"23798678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for increased lability upon heterocomplex formation was unknown\",\n        \"Whether the same asymmetry applies to the human complex was not shown\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"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.\",\n      \"evidence\": \"In vitro ATPase assays with purified proteins and fluorescent nucleotide binding\",\n      \"pmids\": [\"26195633\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Connection between ATPase cycle and cluster assembly/transfer was not yet established\",\n        \"No structural information on the ATP-bound state\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"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.\",\n      \"evidence\": \"Site-directed mutagenesis of ATPase motifs combined with in vivo/in vitro cluster assembly, transfer assays, and nucleotide binding measurements in yeast\",\n      \"pmids\": [\"30865432\", \"30785732\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structural snapshot of the allosteric conformational change\",\n        \"Whether ATP hydrolysis drives cluster release or cluster loading (or both) could not be kinetically separated\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"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.\",\n      \"evidence\": \"In vitro cluster transfer with NMR, UV-visible, Mössbauer spectroscopy, and analytical ultracentrifugation using purified human proteins\",\n      \"pmids\": [\"32429669\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Role of NUBP2 in the transfer from GLRX3 was not addressed\",\n        \"Electron source for reductive coupling in this system was glutathione, not anamorsin\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"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.\",\n      \"evidence\": \"In vitro reconstitution with NMR, UV-visible, and Mössbauer spectroscopy using purified human proteins\",\n      \"pmids\": [\"36916754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How the C-terminal CPXC site acquires its cluster from GLRX3/anamorsin was not resolved\",\n        \"Full reconstitution including NUBP2, downstream CIA factors, and apo-target proteins has not been achieved\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"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.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No separation-of-function mutations distinguishing Fe-S and centrosome roles\",\n        \"No structural model of full-length NUBP1 or the NUBP1-NUBP2 heterotetramer\",\n        \"Mechanism by which NUBP1 negatively regulates ciliogenesis is undefined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [5, 6, 7]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2, 8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 14]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 2, 8, 9]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [10, 11]}\n    ],\n    \"complexes\": [\n      \"NUBP1-NUBP2 (Nbp35-Cfd1) heterotetramer\"\n    ],\n    \"partners\": [\n      \"NUBP2\",\n      \"GLRX3\",\n      \"KIFC5A\",\n      \"KATNAL2\",\n      \"CYBC1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}