{"gene":"MAD1L1","run_date":"2026-04-28T18:30:27","timeline":{"discoveries":[{"year":1998,"finding":"Human MAD1 (TXBP181/HsMAD1) is the human homolog of yeast mitotic checkpoint MAD1; it functions as a homodimer, is hyperphosphorylated during S/G2/M phases and upon nocodazole treatment, binds HsMAD2, localizes to the centrosome during metaphase and to the spindle midzone/midbody during anaphase/telophase, and is targeted by HTLV-I oncoprotein Tax, whose expression causes multinucleated cells consistent with loss of MAD1 function.","method":"Co-immunoprecipitation, immunofluorescence, gel filtration, transfection/overexpression with phenotypic readout","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (co-IP, localization, functional assay), foundational paper with 433 citations","pmids":["9546394"],"is_preprint":false},{"year":2001,"finding":"HsMAD1 and HsMAD2 localize to nuclear pore complexes (NPCs) during interphase, as demonstrated by co-labeling with NPC antibodies and co-purification with enriched nuclear envelope fractions; MAD1 associates with MAD2 but not p55CDC in this context.","method":"Immunofluorescence co-localization, nuclear envelope fractionation, co-immunoprecipitation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-localization and biochemical fractionation in same study","pmids":["11181178"],"is_preprint":false},{"year":2002,"finding":"Crystal structure of the tetrameric Mad1-Mad2 core complex reveals an asymmetric tetramer with elongated Mad1 monomers forming a coiled-coil and two connected sub-complexes with Mad2; Mad2 C-terminal tails wrap around Mad1 as 'safety belts'. Mad1 acts as a competitive inhibitor of the Mad2-Cdc20 complex, and unlocking the Mad2 C-terminal tail is required for ligand release.","method":"X-ray crystallography, in vitro competition assay, mutagenesis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation (competition assay, mutagenesis)","pmids":["12006501"],"is_preprint":false},{"year":2002,"finding":"RNAi-mediated suppression of MAD1 in mammalian cells causes loss of Mad2 kinetochore localization and spindle checkpoint impairment; Mad1 and Cdc20 share a conserved Mad2-binding motif consensus; binding of a Mad1 or Cdc20 motif peptide triggers extensive rearrangement of Mad2 tertiary structure.","method":"RNAi knockdown, in vitro binding assay, structural analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — RNAi loss-of-function with defined phenotype plus in vitro structural analysis, replicated across labs","pmids":["11804586"],"is_preprint":false},{"year":2001,"finding":"Mad2 forms incompatible complexes with Mad1 and Cdc20 (neither requiring Mad2 oligomerization); interaction of Mad2 with Mad1 is crucial for kinetochore localization of Mad2, where Mad2 then interacts with Cdc20.","method":"Co-immunoprecipitation, mutagenesis, cell microinjection, kinetochore localization assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (co-IP, mutagenesis, localization), replicated","pmids":["11707408"],"is_preprint":false},{"year":2005,"finding":"A closed conformer of Mad2 constitutively bound to Mad1 (C-Mad2) serves as the kinetochore receptor for cytosolic open Mad2 (O-Mad2); interaction of open and closed Mad2 conformers is essential for the spindle assembly checkpoint. This 'Mad2 template' model proposes that C-Mad2 bound to Mad1 acts as a template for conversion of O-Mad2 into C-Mad2 bound to Cdc20.","method":"Conformer-specific binding assays, fluorescence imaging (FRAP), mutagenesis, in vitro reconstitution","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods, foundational model paper widely replicated","pmids":["15694304"],"is_preprint":false},{"year":2002,"finding":"Hec1 is required for recruitment of Mps1 kinase and Mad1/Mad2 complexes to kinetochores; depletion of Hec1 by RNAi impairs chromosome congression and causes persistent spindle checkpoint activation.","method":"RNAi depletion, immunofluorescence kinetochore localization","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — clean RNAi knockdown with defined kinetochore localization phenotype, highly cited","pmids":["12351790"],"is_preprint":false},{"year":2001,"finding":"Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E in Xenopus egg extracts; immunodepletion of Bub1 abolishes checkpoint and kinetochore binding of these proteins; reintroduction of kinase-deficient Bub1 restores checkpoint and localization, demonstrating kinase-independent scaffolding.","method":"Immunodepletion, add-back reconstitution, kinase-dead mutant, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — reconstitution with mutant analysis in Xenopus extract system","pmids":["11402067"],"is_preprint":false},{"year":2002,"finding":"BubR1 immunodepletion in Xenopus egg extracts greatly reduces kinetochore binding of Mad1, Mad2, Bub1, Bub3, and CENP-E; localization and hyperphosphorylation of BubR1 at kinetochores depends on Bub1 and Mad1 (but not Mad2), placing Mad1 upstream of BubR1 phosphorylation.","method":"Immunodepletion, kinetochore localization assay, phosphorylation analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — epistasis via immunodepletion with defined localization and phosphorylation readouts","pmids":["12163471"],"is_preprint":false},{"year":1999,"finding":"Mad2 forms a tight complex with Mad1 in budding yeast, independent of cell cycle stage and other checkpoint proteins; this association is critical for checkpoint function and for hyperphosphorylation of Mad1. Deletion/mutation analysis confirms that Mad1-Mad2 association is required for spindle checkpoint.","method":"Gel filtration, co-immunoprecipitation, deletion/mutagenesis analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — multiple biochemical methods with functional validation by mutagenesis","pmids":["10436016"],"is_preprint":false},{"year":2008,"finding":"Tpr (a nuclear pore complex component) directly binds Mad1 and Mad2; Tpr depletion disrupts NPC localization of Mad1 and Mad2 during interphase, decreases levels of Mad1-bound Mad2, and decreases Mad1 at kinetochores during prometaphase, correlating with inability of Mad1 to activate Mad2 and inhibit APC/Cdc20.","method":"Mass spectrometry of affinity-purified Mad2 complexes, direct binding assay, RNAi depletion, immunofluorescence","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — MS identification plus direct binding plus RNAi functional validation","pmids":["18981471"],"is_preprint":false},{"year":2010,"finding":"Mps1 kinase activity is required for recruitment of O-Mad2 to the kinetochore-bound Mad1-C-Mad2 core complex; inhibiting Mps1 before mitosis abolishes Mad1 and Mad2 kinetochore recruitment, whereas inhibiting it after mitotic entry leaves the Mad1-C-Mad2 core bound but prevents O-Mad2 recruitment. Mps1 can dimerize and transphosphorylate.","method":"Small-molecule Mps1 inhibitor (AZ3146), immunofluorescence, biochemical fractionation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — chemical genetic approach with timed inhibition, multiple orthogonal readouts","pmids":["20624899"],"is_preprint":false},{"year":2014,"finding":"Mad1 kinetochore localization in budding yeast is mediated by Mps1-dependent phosphorylation of a region within Bub1; tethering this Bub1 region to kinetochores bypasses Mps1-mediated upstream checkpoint requirements; Mad1-Bub1 interaction and kinetochore association can be reconstituted in vitro with Mps1 and Mad2.","method":"In vitro reconstitution, mutagenesis, epistasis (bypass tethering), kinetochore localization assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus bypass epistasis and mutagenesis","pmids":["24402315"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of the C-terminal domain (CTD) of human Mad1 reveals a homodimer with fold similar to kinetochore-binding domains of Spc25 and Csm1; mutagenesis of the CTD interface diminishes kinetochore targeting and implicates Bub1 as the Mad1 CTD receptor.","method":"X-ray crystallography, mutagenesis, kinetochore localization assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis validation of kinetochore targeting","pmids":["22493223"],"is_preprint":false},{"year":2021,"finding":"Crystal structure of the Mad1 CTD bound to two phosphorylated Bub1 CD1 peptides at 1.75 Å resolution shows that phosphorylated Bub1 Thr461 directly contacts Arg617 of the Mad1 RLK motif and caps the CD1 α-helix dipole; in solution, only one Bub1 CD1 peptide binds the Mad1 homodimer, reflecting inherent coiled-coil asymmetry.","method":"X-ray crystallography, solution binding assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with biochemical validation","pmids":["34013668"],"is_preprint":false},{"year":2011,"finding":"Mad2 requires association with Mad1 to adopt the closed conformation (C-Mad2) in vivo; p31comet-dependent 'capping' regulates the activity of the Mad1:C-Mad2 complex; microinjection of C-Mad2-specific antibody or Mad1-neutralizing antibody abruptly terminates the spindle assembly checkpoint and accelerates mitotic progression.","method":"Conformation-specific monoclonal antibody, antibody microinjection, live-cell imaging","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 — conformer-specific antibody with in vivo functional validation by microinjection","pmids":["21772247"],"is_preprint":false},{"year":2011,"finding":"Constitutive targeting of Mad1 to bioriented kinetochores (via engineered construct) is sufficient to cause metaphase arrest dependent on Mad1-Mad2 binding; Aurora B, Mps1, and BubR1 kinases (but not Polo-like kinase) are needed to maintain checkpoint arrest when Mad1 is present at kinetochores, placing them downstream of Mad1-Mad2 recruitment.","method":"Kinetochore-targeted Mad1 construct, chemical kinase inhibitors, live-cell imaging, mutagenesis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — engineered gain-of-function with pharmacological epistasis and mutagenesis","pmids":["21394085"],"is_preprint":false},{"year":2014,"finding":"Recruiting Mad1 to metaphase kinetochores via chemically induced dimerization (CID) is sufficient to reactivate the spindle assembly checkpoint without increasing Mps1 or BubR1 at kinetochores; Mad2 binding and a conserved C-terminal motif of Mad1 are both required for checkpoint reactivation, suggesting Mad1 scaffolds formation of a higher-order mitotic checkpoint complex.","method":"Chemically induced dimerization, live-cell imaging, mutagenesis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — temporally controlled CID with mutagenesis validation","pmids":["24637323"],"is_preprint":false},{"year":2019,"finding":"MAD1 directly interacts with the N-terminal 100 amino acids of CDK1-CCNB1 (Cyclin B1); CCNB1 is recruited to unattached kinetochores through this interaction in an MPS1-dependent manner; this MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop for timely MPS1 kinetochore recruitment and sustained checkpoint arrest.","method":"Proteomics/co-immunoprecipitation, in vitro binding with truncation mutants, live-cell imaging, RNAi","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — proteomic identification plus co-IP plus functional RNAi with multiple readouts","pmids":["30674583"],"is_preprint":false},{"year":2020,"finding":"Cyclin B1 directly binds to an acidic face in the N-terminal region of MAD1 (mapped by in vitro reconstitution) and scaffolds MAD1 to the kinetochore corona; point mutations abolishing this interaction eliminate MAD1 corona localization and weaken the spindle assembly checkpoint; corona-localized MAD1 loses dependence on MPS1 kinase after corona establishment, ensuring robust SAC.","method":"In vitro reconstitution, mutagenesis, live-cell imaging, RNAi","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro reconstitution plus mutagenesis plus functional imaging","pmids":["32202322"],"is_preprint":false},{"year":2020,"finding":"Cyclin B1-Cdk1 is targeted to the nuclear pore complex by binding an acidic face of MAD1; localized Cyclin B1-Cdk1 is required for proper release of MAD1 from Tpr at the NPC, allowing MAD1 recruitment to kinetochores before nuclear envelope breakdown.","method":"Co-immunoprecipitation, live-cell imaging, phosphorylation assays, RNAi","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — co-IP with functional imaging and RNAi, multiple orthogonal methods","pmids":["32236513"],"is_preprint":false},{"year":2014,"finding":"MAD-1 (C. elegans) interacts with BUB-1 through a specific segment of the MAD-1 coiled coil; mutations selectively disrupting BUB-1 interaction eliminate MAD-1 localization to unattached kinetochores and meiotic chromosomes and abrogate checkpoint signaling; this BUB-1 C-terminal/kinase domain region mediates MAD-1 kinetochore targeting independently of kinase activity.","method":"Mutagenesis, co-immunoprecipitation, kinetochore localization assay, C. elegans genetics","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — mutagenesis with reciprocal co-IP and in vivo functional validation","pmids":["24567362"],"is_preprint":false},{"year":2003,"finding":"Tpr is required for Mad1-c-Mad2 recruitment to nuclear pores during interphase and stabilizes Mad1 and Mad2 protein levels; Tpr depletion reduces Mad2 (but not Mad1) at kinetochores, and the SAC robustness depends on Mad2 kinetochore levels maintained by Tpr.","method":"Co-immunoprecipitation, RNAi, immunofluorescence, protein half-life measurements","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — co-IP plus RNAi with defined protein stability and localization readouts","pmids":["24344181"],"is_preprint":false},{"year":2010,"finding":"Nup153 directly binds Mad1 via Nup153's N-terminal domain; Nup153 overexpression causes hypophosphorylation of Mad1 and spindle checkpoint inactivation; Nup153 depletion reduces Mad1 at nuclear pores and delays Mad1 dissociation from kinetochores at metaphase. Nup153 binding to Mad1 affects Mad1 phosphorylation status but not its interaction with Mad2.","method":"In vitro direct binding assay, overexpression, RNAi, immunofluorescence, phosphorylation analysis","journal":"Nucleus","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro direct binding with RNAi functional analysis, single lab","pmids":["21327106"],"is_preprint":false},{"year":2003,"finding":"Human CENP-I is required for kinetochore localization of MAD1, MAD2, and CENP-F; CENP-I depletion prevents mitotic arrest despite unattached kinetochores, and the delay that does occur is MAD2-dependent, indicating that collective signal from many unattached kinetochores is needed to sustain arrest.","method":"RNAi depletion, immunofluorescence, cell cycle analysis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — RNAi with defined checkpoint and localization phenotypes, replicated","pmids":["12640463"],"is_preprint":false},{"year":2003,"finding":"Depletion of either Nuf2 or Hec1 by RNAi leads to 5-fold or greater reduction of Mad1 and Mad2 at kinetochores during a prometaphase block; Mad1/Mad2 reduction is reversible upon spindle depolymerization, indicating Nuf2/Hec1 prevent microtubule-dependent stripping of Mad1/Mad2.","method":"RNAi, quantitative immunofluorescence","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi with quantitative kinetochore localization, single lab","pmids":["14654001"],"is_preprint":false},{"year":2004,"finding":"NEK2A interacts with MAD1 in vitro and in vivo via a leucine zipper-containing C-terminal domain of MAD1; NEK2A localizes to kinetochores; NEK2A depletion by siRNA abolishes MAD2 (but not MAD1, BUB1, or HEC1) kinetochore association and impairs spindle checkpoint signaling.","method":"Co-immunoprecipitation, in vitro binding, siRNA, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP plus in vitro binding plus siRNA functional analysis, single lab","pmids":["14978040"],"is_preprint":false},{"year":2007,"finding":"PRP4 kinase is required for recruitment or maintenance of MPS1, MAD1, and MAD2 at kinetochores; PRP4 depletion by RNAi induces mitotic acceleration, lagging chromatids, aneuploidy, and failure to arrest after nocodazole treatment.","method":"RNAi, immunofluorescence, mitotic index assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi with defined checkpoint localization and functional phenotype, single lab","pmids":["17998396"],"is_preprint":false},{"year":2002,"finding":"Two leucine zipper domains (amino acids 501-522 and 557-571) in human MAD1 are required for binding MAD2; a polymorphism at codon 558 (Arg→His) reduces MAD2 binding and mitotic checkpoint enforcement, representing loss-of-heterozygosity at the MAD1 locus in a human breast cancer.","method":"Mutagenesis, co-immunoprecipitation, mitotic index assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — mutagenesis with functional validation and human cancer evidence","pmids":["12042300"],"is_preprint":false},{"year":2003,"finding":"Human Mad2 is phosphorylated on multiple serine residues in a cell-cycle-dependent manner; only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo; a phosphomimetic Mad2 mutant fails to interact with Mad1 or APC/C and acts as dominant-negative.","method":"In vivo phosphorylation analysis, co-immunoprecipitation, dominant-negative mutagenesis","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo PTM with co-IP and mutagenesis, single lab","pmids":["12574116"],"is_preprint":false},{"year":1999,"finding":"BUB1 phosphorylates MAD1 in vitro; BUB1 and BUB3 form a complex and interact with MAD1; BUB1 autophosphorylation and MAD1 phosphorylation require Lys821 in the BUB1 kinase motif.","method":"In vitro kinase assay, co-immunoprecipitation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro kinase assay with mutagenesis, single paper","pmids":["10198256"],"is_preprint":false},{"year":2014,"finding":"ATM kinase phosphorylates Mad1 at Serine 214; this phosphorylation promotes Mad1 homodimerization and heterodimerization with Mad2, contributes to spindle assembly checkpoint activation, and is required for chromosomal stability.","method":"In vitro kinase assay, phospho-specific antibody, co-immunoprecipitation, flow cytometry","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro kinase assay with co-IP and functional validation, single lab","pmids":["24728176"],"is_preprint":false},{"year":2019,"finding":"ULK1 phosphorylates Mad1 at Ser546, promoting Mad1 kinetochore recruitment; phosphorylated Ser546-Mad1 shows enhanced interaction with the RZZ (Rod/ZW10/Zwilch) complex, which may serve as its kinetochore receptor; ULK1 deletion increases chromosomal instability.","method":"In vitro kinase assay, co-immunoprecipitation, RNAi, immunofluorescence","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro kinase assay with co-IP and localization readout, single lab","pmids":["31291454"],"is_preprint":false},{"year":2017,"finding":"MAD1 N-terminal domain (NTD) and C-terminal domain (CTD) both bind O-Mad2 and C-Mad2 conformers (unlike the MIM which binds only C-Mad2); NTD and CTD interact with each other and with MPS1 kinase; MPS1 phosphorylates both NTD and CTD, decreasing their mutual interaction and CTD's interaction with MPS1; phosphorylation of CTD residue Thr-716 compromises Mad2 binding and checkpoint responses.","method":"Co-immunoprecipitation, in vitro kinase assay, mutagenesis, conformer-specific binding assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple binding and kinase assays with mutagenesis, single lab","pmids":["29162720"],"is_preprint":false},{"year":2014,"finding":"A Golgi-localized pool of Mad1 (independent of Mad2) controls secretion of α5 integrin; Mad1 depletion impairs integrin secretion, cell attachment, adhesion, FAK activation, and cell motility; Mad1 overexpression accelerates directed cell migration.","method":"Immunofluorescence co-localization with Golgi markers, subcellular fractionation, RNAi, integrin secretion assay, cell motility assay","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 — localization tied to functional consequence via RNAi with multiple readouts, single lab","pmids":["25447996"],"is_preprint":false},{"year":2019,"finding":"Upregulated Mad1 localizes to PML nuclear bodies; the C-terminus of Mad1 directly interacts with PML (interaction enhanced by sumoylation); upregulated Mad1 displaces MDM2 from PML, freeing MDM2 to ubiquitinate and destabilize p53, thereby promoting tumor growth.","method":"Co-immunoprecipitation, direct binding assay, immunofluorescence, sumoylation assay, orthotopic tumor assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding plus localization plus in vivo tumor assay, single lab","pmids":["30948704"],"is_preprint":false},{"year":2015,"finding":"Fission yeast Mad1 binds the kinesin-5 motor Cut7 (Eg5 homolog); Mad1 recruits Cut7 to kinetochores of misaligned chromosomes to promote chromosome congression; human Mad1 similarly recruits CENP-E motor to kinetochores, revealing a conserved dual function of Mad1 in checkpoint signaling and chromosome gliding.","method":"Protein-protein interaction screen, co-immunoprecipitation, live-cell imaging, genetic analysis","journal":"Nature cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with live imaging, conserved in two organisms, single study","pmids":["26258632"],"is_preprint":false},{"year":2014,"finding":"Mad1 has a direct role in the spindle assembly checkpoint beyond Mad2 kinetochore recruitment: even when C-Mad2 is artificially recruited to kinetochores, Mad1 is still required for mitotic arrest; the C-terminal globular domain of Mad1 and conserved residues within it are required for this additional checkpoint function.","method":"Artificial C-Mad2 kinetochore tethering, mutagenesis, mitotic index assay","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 — bypass experiment with mutagenesis, single lab","pmids":["24477933"],"is_preprint":false},{"year":2016,"finding":"Cep57 localizes to kinetochores, binds Mis12 (KMN network), and interacts with Mad1; Cep57 depletion decreases Mad1-Mad2 kinetochore localization and reduces spindle checkpoint signaling; Cep57 microtubule-binding activity is involved in timely Mad1 removal from kinetochores.","method":"Co-immunoprecipitation, RNAi, immunofluorescence, spindle checkpoint assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with RNAi functional validation, single lab","pmids":["26743940"],"is_preprint":false},{"year":2014,"finding":"CENP-I is required for stable association of RZZ complex and Mad1 with kinetochores and inhibits their dynein-mediated removal; Aurora B regulates RZZ/Mad1 association; CENP-I and Aurora B act as a molecular switch controlling Mad1 kinetochore dynamics.","method":"RNAi, Aurora B inhibitor, immunofluorescence, kinetochore localization quantification","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi epistasis with pharmacological inhibition and localization readout, single lab","pmids":["24862574"],"is_preprint":false},{"year":2015,"finding":"The RZZ complex localizes to the N-terminus of KNL1 downstream of Bub1, and mediates robust Mad1/Mad2 kinetochore localization in human cells; both Bub1/KNL1-dependent and KNL1/Bub1-independent mechanisms exist for RZZ and Mad1/Mad2 kinetochore recruitment.","method":"RNAi, kinetochore localization quantification, rescue experiments","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 — systematic RNAi epistasis with multiple constructs, single lab","pmids":["26581576"],"is_preprint":false},{"year":1996,"finding":"Mad1 heterodimerizes with Max and represses transcription from E-box-containing promoters (competing with Myc:Max); the SIN3 corepressor interaction domain at the N-terminus of Mad1 and the PAH2 domain of Sin3 are required for transcriptional repression; Mad1 inhibits cell cycle progression from G1 to S phase, and this inhibitory activity requires its transcriptional repression function.","method":"Transcription reporter assay, cell cycle analysis (FACS), mutagenesis, co-immunoprecipitation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — mutagenesis linking repressor domain to cell cycle function, widely replicated","pmids":["8649388"],"is_preprint":false},{"year":1999,"finding":"Amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction; these residues form an amphipathic alpha-helix, and hydrophobic residues on the helix face are required for mSin3A PAH2 binding; this minimal SID can function as an autonomous portable repression domain.","method":"Mutagenesis, NMR/structural analysis, in vitro binding assay, transcription reporter assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — structural analysis with mutagenesis and functional validation","pmids":["10551834"],"is_preprint":false},{"year":2000,"finding":"NMR solution structure of the Sin3B PAH2 domain in complex with the N-terminal Mad1 peptide reveals a 'wedged helical bundle'; four α-helices of PAH2 form a hydrophobic cleft accommodating an amphipathic Mad1 α-helix; Mad1 binding stabilizes PAH2 secondary structure elements.","method":"NMR structure determination","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 — NMR structure of protein complex","pmids":["11101889"],"is_preprint":false},{"year":2008,"finding":"c-IAP1 (a RING finger E3 ubiquitin ligase) catalyzes ubiquitination of Mad1 (the Myc/Max/Mad transcriptional repressor), accelerating its degradation via the 26S proteasome, thereby reducing Mad1 levels and cooperating with Myc to promote cell proliferation.","method":"In vitro ubiquitination assay, co-immunoprecipitation, proteasome inhibitor experiments, cell proliferation assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — in vitro ubiquitination reconstitution with functional validation","pmids":["18082613"],"is_preprint":false},{"year":2008,"finding":"RSK (p90 ribosomal kinase) and S6K (p70 S6 kinase) phosphorylate Serine 145 of Mad1 (Myc/Max/Mad family member) upon serum or insulin stimulation; Ser-145 phosphorylation accelerates Mad1 ubiquitination and degradation via the 26S proteasome, promoting Myc transcriptional activity.","method":"In vitro kinase assay, mutagenesis, ubiquitination assay, pulse-chase degradation analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with mutagenesis and degradation analysis","pmids":["18451027"],"is_preprint":false},{"year":2009,"finding":"Mad1 (Myc/Max/Mad family) recruits the histone demethylase RBP2 to the hTERT gene promoter; Mad1-RBP2 interaction and co-occupancy at the hTERT promoter correlates with H3-K4 demethylation and transcriptional repression during differentiation; RBP2 depletion derepresses hTERT expression.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), RNAi, histone methylation assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus ChIP plus RNAi with functional readout, single lab","pmids":["19762557"],"is_preprint":false},{"year":2004,"finding":"Mad1 (Myc/Max/Mad) interacts directly with the UBF promoter and represses rDNA transcription; granulocytic cells lacking Mad1 display increased rDNA transcription, protein synthesis, and cell volume; siRNA knockdown shows UBF is required for c-MYC-induced rDNA transcription.","method":"Nuclear run-on assay, chromatin immunoprecipitation, siRNA, cell size measurement","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (run-on, ChIP, siRNA), single lab","pmids":["15282543"],"is_preprint":false},{"year":2001,"finding":"During differentiation of HL60 cells, c-Myc occupancy at hTERT E-boxes is replaced by Mad1 occupancy in vivo, correlating with reduced histone acetylation at the promoter; histone deacetylase inhibitor trichostatin A attenuates hTERT repression during differentiation.","method":"Chromatin immunoprecipitation (ChIP), reporter assay, HDAC inhibitor treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP demonstrating in vivo promoter occupancy switch with histone modification readout","pmids":["11274400"],"is_preprint":false},{"year":2003,"finding":"p53 represses MAD1 transcription through a novel 38-bp element in the MAD1 promoter distinct from canonical p53 binding sites; p53, HDAC1, and mSin3A associate with the MAD1 promoter in vivo (by ChIP); HDAC inhibitor trichostatin A relieves p53-mediated MAD1 repression.","method":"Promoter reporter assay, ChIP, HDAC inhibitor treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — reporter assay with ChIP validation, single lab","pmids":["12876282"],"is_preprint":false},{"year":2012,"finding":"miR-125b represses Mad1 (spindle checkpoint) expression post-transcriptionally; exogenous miR-125b overexpression downregulates Mad1, delays cells at metaphase, and promotes apoptotic death with elevated chromosomal abnormalities.","method":"miRNA overexpression, immunoblot, cell cycle analysis, chromosome aberration assay","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2-3 — miRNA overexpression with functional phenotype, single lab","pmids":["23099851"],"is_preprint":false},{"year":2012,"finding":"Overexpression of Mad1 (spindle checkpoint) causes aneuploidy and chromosomal instability through mislocalization of Mad2 away from kinetochores, weakening the mitotic checkpoint; cells overexpressing Mad1 are resistant to microtubule poisons.","method":"Mad1 overexpression, immunofluorescence quantification of Mad2 kinetochore levels, chromosome counting, drug sensitivity assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — overexpression with mechanistic localization readout and functional consequence, single lab","pmids":["22778409"],"is_preprint":false},{"year":2017,"finding":"RARS-MAD1L1 fusion protein interacts with AIMP2 (by co-immunoprecipitation), resulting in activation of the FUBP1/c-Myc pathway; silencing of FUBP1 or c-Myc inhibitor abrogates cancer stem cell-like characteristics induced by the fusion.","method":"Co-immunoprecipitation, ChIP, siRNA, colony/sphere formation assay","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP with functional validation for a fusion protein context, single lab","pmids":["29133573"],"is_preprint":false}],"current_model":"MAD1L1 (Mad1) encodes a conserved homodimeric scaffold protein with two distinct molecular functions: (1) as a core spindle assembly checkpoint (SAC) component, Mad1 constitutively associates with closed-conformation Mad2 (C-Mad2) at kinetochores—recruited via Mps1-phosphorylated Bub1, the RZZ complex, and Cyclin B1 scaffold at the corona—where the Mad1:C-Mad2 tetramer acts as a catalytic template converting cytosolic open Mad2 into the closed form that sequesters Cdc20 to inhibit APC/C; and (2) as a bHLH-LZ transcriptional repressor (the Myc/Max/Mad-family Mad1, gene MXD1/MAD1 in some contexts), Mad1 heterodimerizes with Max to occupy E-box promoters and recruits the mSin3A-HDAC corepressor complex through an amphipathic N-terminal SID helix, antagonizing Myc-driven transcription; additionally, Mad1 localizes to nuclear pores (via Tpr) during interphase to maintain Mad2 proteostasis, to the Golgi to regulate integrin secretion and cell migration, and to PML bodies where it displaces MDM2 to destabilize p53."},"narrative":{"teleology":[{"year":1998,"claim":"Identification of human MAD1 as a checkpoint homodimer established that the yeast SAC architecture is conserved in mammals and that Mad1 functions through direct Mad2 binding and cell-cycle-dependent hyperphosphorylation.","evidence":"Co-IP, gel filtration, immunofluorescence, and HTLV-I Tax overexpression phenotyping in human cells","pmids":["9546394"],"confidence":"High","gaps":["Kinase(s) responsible for hyperphosphorylation unidentified","Mechanism of Tax-mediated checkpoint disruption not resolved"]},{"year":1999,"claim":"Demonstrating that Mad1–Mad2 association is constitutive and independent of cell cycle stage resolved that the complex is pre-formed rather than assembled de novo during mitosis, shifting focus to how the stable complex is activated.","evidence":"Gel filtration and co-IP with deletion/mutagenesis in budding yeast","pmids":["10436016"],"confidence":"High","gaps":["How Mad1–Mad2 complex converts cytosolic Mad2 remained unknown","Structural basis of constitutive binding unresolved"]},{"year":2001,"claim":"Discovery that Mad1 and Mad2 reside at nuclear pore complexes during interphase revealed an unexpected non-mitotic reservoir, raising the question of whether NPC localization functionally contributes to checkpoint competence.","evidence":"Immunofluorescence co-localization with NPC markers and nuclear envelope fractionation","pmids":["11181178"],"confidence":"High","gaps":["NPC-binding partner of Mad1 not identified","Functional significance of interphase NPC pool unclear"]},{"year":2001,"claim":"Bub1 was established as an upstream scaffold required for Mad1 kinetochore targeting, and kinase-dead Bub1 rescued both localization and checkpoint, indicating a kinase-independent scaffolding mechanism.","evidence":"Immunodepletion and kinase-dead add-back reconstitution in Xenopus egg extracts","pmids":["11402067"],"confidence":"High","gaps":["Direct binding interface between Bub1 and Mad1 not mapped","Whether Bub1 kinase activity has additional roles at kinetochore not excluded"]},{"year":2002,"claim":"The crystal structure of the Mad1–Mad2 tetramer revealed the 'safety belt' mechanism by which Mad2 C-terminal tails wrap around Mad1, explaining how Mad1 acts as a competitive inhibitor of the Mad2–Cdc20 interaction and establishing the structural foundation of the template model.","evidence":"X-ray crystallography with in vitro competition assays and mutagenesis","pmids":["12006501"],"confidence":"High","gaps":["How O-Mad2 is recruited to the Mad1:C-Mad2 complex remained unknown","Full-length Mad1 structure unavailable"]},{"year":2002,"claim":"RNAi depletion of Mad1 abolished Mad2 kinetochore localization and checkpoint function, confirming Mad1 is essential for Mad2 activation in mammalian cells; identification of a shared Mad2-binding motif in Mad1 and Cdc20 explained substrate mimicry.","evidence":"RNAi knockdown, in vitro peptide binding, structural analysis of Mad2 conformational change","pmids":["11804586"],"confidence":"High","gaps":["Conformer-specific mechanism of Mad2 conversion not yet demonstrated in vivo"]},{"year":2005,"claim":"The 'Mad2 template model' was established: C-Mad2 constitutively bound to Mad1 at kinetochores serves as a catalytic template that recruits and converts O-Mad2 to C-Mad2–Cdc20, answering how a single unattached kinetochore amplifies the checkpoint signal.","evidence":"Conformer-specific binding assays, FRAP, mutagenesis, and in vitro reconstitution","pmids":["15694304"],"confidence":"High","gaps":["Rate-limiting step in the catalytic cycle undefined","Role of p31comet in capping the template not fully characterized"]},{"year":2008,"claim":"Tpr was identified as the direct NPC anchor for Mad1–Mad2, resolving the interphase binding partner; Tpr depletion reduced Mad1 at both NPCs and kinetochores and diminished Mad2 protein levels, establishing a proteostatic function for the NPC-localized pool.","evidence":"Affinity purification–mass spectrometry, direct binding assay, RNAi with localization and protein level readouts","pmids":["18981471"],"confidence":"High","gaps":["Whether Tpr directly hands off Mad1 to kinetochores or indirectly supports it via protein stabilization not resolved"]},{"year":2010,"claim":"Mps1 kinase was shown to be required not just for Mad1 kinetochore recruitment but specifically for O-Mad2 recruitment to the already-bound Mad1:C-Mad2 core, separating two temporally distinct steps in checkpoint activation.","evidence":"Timed chemical inhibition of Mps1 (AZ3146) with immunofluorescence in human cells","pmids":["20624899"],"confidence":"High","gaps":["Direct Mps1 substrate on Mad1 for O-Mad2 recruitment step not identified at this point"]},{"year":2011,"claim":"Constitutive Mad1 kinetochore tethering was sufficient for metaphase arrest dependent on Mad1–Mad2 binding, establishing that Mad1 presence at kinetochores is the rate-limiting determinant of checkpoint activation and that downstream kinases (Aurora B, Mps1, BubR1) are needed to maintain but not initiate this arrest.","evidence":"Engineered kinetochore-targeted Mad1, chemical kinase inhibitors, live-cell imaging","pmids":["21394085"],"confidence":"High","gaps":["What additional checkpoint function the Mad1 CTD provides beyond Mad2 recruitment remained unclear"]},{"year":2012,"claim":"Crystal structure of the Mad1 CTD revealed a homodimeric fold resembling Spc25/Csm1 kinetochore-binding domains, and mutagenesis implicated Bub1 as the direct CTD receptor, providing the first structural basis for Mad1 kinetochore docking.","evidence":"X-ray crystallography with mutagenesis and kinetochore localization assay","pmids":["22493223"],"confidence":"High","gaps":["Atomic-resolution structure of Mad1 CTD–Bub1 complex not yet solved"]},{"year":2014,"claim":"Mps1-dependent phosphorylation of Bub1 was reconstituted in vitro as the direct mechanism for Mad1 kinetochore recruitment, closing the kinase–scaffold–effector pathway; artificial Bub1 tethering bypassed Mps1 upstream requirements, confirming Bub1 phosphorylation as the key regulated step.","evidence":"In vitro reconstitution, bypass tethering, mutagenesis in budding yeast","pmids":["24402315"],"confidence":"High","gaps":["Phosphorylation sites on Bub1 not fully mapped in human system at this point"]},{"year":2014,"claim":"A checkpoint function for Mad1 beyond Mad2 kinetochore recruitment was established: even when C-Mad2 is artificially tethered to kinetochores, Mad1 and specifically its CTD are still required for mitotic arrest, indicating Mad1 scaffolds additional checkpoint signaling.","evidence":"Artificial C-Mad2 tethering, mutagenesis, mitotic index assay","pmids":["24477933"],"confidence":"Medium","gaps":["Identity of the additional Mad1 CTD interactor(s) required for this function unknown","Mechanism not reconstituted in vitro"]},{"year":2014,"claim":"A Mad2-independent Golgi-localized pool of Mad1 was discovered that regulates α5-integrin secretion, cell adhesion, and directed cell migration, revealing a non-mitotic function entirely separate from the SAC.","evidence":"Golgi co-localization, subcellular fractionation, RNAi with integrin secretion and cell motility assays","pmids":["25447996"],"confidence":"Medium","gaps":["Molecular mechanism of Mad1 action at the Golgi unknown","Whether this pool is regulated during cell cycle not addressed","Not independently replicated"]},{"year":2019,"claim":"MAD1 was shown to directly recruit Cyclin B1–CDK1 to unattached kinetochores through its N-terminal acidic face, establishing a positive-feedback loop: kinetochore-localized CDK1 sustains Mps1 recruitment, which in turn maintains Mad1 at kinetochores.","evidence":"Proteomics/co-IP, in vitro binding with truncation mutants, live-cell imaging, RNAi","pmids":["30948704"],"confidence":"High","gaps":["CDK1 substrates in this feedback loop not comprehensively identified"]},{"year":2019,"claim":"Mad1 localization to PML nuclear bodies and direct binding to PML was shown to displace MDM2, leading to p53 destabilization and tumor promotion, establishing a SAC-independent oncogenic mechanism for Mad1 overexpression.","evidence":"Co-IP, direct binding assay, immunofluorescence, sumoylation assay, orthotopic tumor assay","pmids":["30948704"],"confidence":"Medium","gaps":["Whether PML-body function operates in non-tumor contexts unknown","Stoichiometry and regulation of the Mad1–PML interaction not defined","Single-lab finding"]},{"year":2020,"claim":"Cyclin B1 was identified as the scaffold that anchors Mad1 at the kinetochore corona through a direct interaction with Mad1's N-terminal acidic surface; this corona pool becomes Mps1-independent after establishment, explaining how robust SAC signaling persists even as Mps1 activity fluctuates.","evidence":"In vitro reconstitution, point mutagenesis abolishing interaction, live-cell imaging","pmids":["32202322"],"confidence":"High","gaps":["Structure of the Mad1–Cyclin B1 interface not resolved","Whether corona and Bub1-dependent pools function additively or redundantly not fully established"]},{"year":2020,"claim":"Cyclin B1–Cdk1 was shown to localize to NPCs via Mad1 binding, where it phosphorylates Tpr to release Mad1 before nuclear envelope breakdown, connecting the interphase NPC pool to rapid kinetochore loading at mitotic entry.","evidence":"Co-IP, live-cell imaging, phosphorylation assays, RNAi","pmids":["32236513"],"confidence":"High","gaps":["Specific Tpr phosphorylation sites mediating Mad1 release not mapped","Whether other kinases contribute to NPC release not excluded"]},{"year":2021,"claim":"A 1.75 Å crystal structure of Mad1 CTD bound to phospho-Bub1 CD1 peptides defined the atomic contacts: pThr461 of Bub1 contacts Arg617 of the Mad1 RLK motif; asymmetric binding of only one Bub1 peptide per Mad1 homodimer in solution revealed functional consequences of coiled-coil asymmetry.","evidence":"X-ray crystallography, solution binding assay","pmids":["34013668"],"confidence":"High","gaps":["Functional consequence of asymmetric Bub1 binding for checkpoint signaling not determined","Full-length Mad1–Bub1 complex structure unavailable"]},{"year":null,"claim":"Key open questions include the structural basis of the Mad1–Cyclin B1 corona interface, how the Golgi-localized Mad1 pool regulates integrin trafficking at the molecular level, how the dual NPC and kinetochore pools are coordinately regulated across the cell cycle, and the full-length structure of the Mad1 homodimer.","evidence":"","pmids":[],"confidence":"High","gaps":["Full-length Mad1 structure unavailable","Molecular mechanism of Mad1's Golgi function unknown","Quantitative contribution of NPC versus corona versus Bub1 kinetochore pools to checkpoint strength not measured"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,5,16,17]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2,4,13,14]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,3,6,13,14]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[1,10,22]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[34]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[35]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,3,5,11,16,17]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,10,20,22]}],"complexes":["Mad1:C-Mad2 tetramer","MCC (mitotic checkpoint complex)"],"partners":["MAD2L1","BUB1","TPR","CCNB1","MPS1","CEP57","NUP153","PML"],"other_free_text":[]},"mechanistic_narrative":"MAD1L1 encodes a coiled-coil homodimeric protein that functions as the central scaffold of the spindle assembly checkpoint (SAC), constitutively binding closed-conformation Mad2 (C-Mad2) at kinetochores to catalyze conversion of cytosolic open-Mad2 (O-Mad2) into C-Mad2–Cdc20 complexes that inhibit the APC/C and prevent premature anaphase onset [PMID:15694304, PMID:12006501, PMID:11804586]. Mad1 is recruited to unattached kinetochores through Mps1-phosphorylated Bub1 via its C-terminal domain and is additionally scaffolded at the kinetochore corona by direct binding of Cyclin B1 to an acidic N-terminal surface, creating a positive-feedback loop that sustains Mps1 recruitment and checkpoint robustness [PMID:24402315, PMID:34013668, PMID:30948704, PMID:32202322]. During interphase, Mad1 localizes to nuclear pore complexes through Tpr binding, which stabilizes Mad2 protein levels and primes the Mad1–C-Mad2 complex for rapid kinetochore deployment upon mitotic entry; Cyclin B1–Cdk1 at the NPC promotes timely Mad1 release from Tpr before nuclear envelope breakdown [PMID:11181178, PMID:18981471, PMID:32236513]. Beyond its SAC role, a Golgi-localized pool of Mad1 regulates α5-integrin secretion and cell migration independently of Mad2, and overexpression of Mad1 at PML nuclear bodies displaces MDM2 to destabilize p53 [PMID:25447996, PMID:30948704]."},"prefetch_data":{"uniprot":{"accession":"Q9Y6D9","full_name":"Mitotic spindle assembly checkpoint protein MAD1","aliases":["Mitotic arrest deficient 1-like protein 1","MAD1-like protein 1","Mitotic checkpoint MAD1 protein homolog","HsMAD1","hMAD1","Tax-binding protein 181"],"length_aa":718,"mass_kda":83.1,"function":"Component of the spindle-assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at the metaphase plate (PubMed:10049595, PubMed:20133940, PubMed:29162720). Forms a heterotetrameric complex with the closed conformation form of MAD2L1 (C-MAD2) at unattached kinetochores during prometaphase, recruits an open conformation of MAD2L1 (O-MAD2) and promotes the conversion of O-MAD2 to C-MAD2, which ensures mitotic checkpoint signaling (PubMed:29162720) Sequesters MAD2L1 in the cytoplasm preventing its function as an activator of the mitotic spindle assembly checkpoint (SAC) resulting in SAC impairment and chromosomal instability in hepatocellular carcinomas","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9Y6D9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAD1L1","classification":"Not Classified","n_dependent_lines":54,"n_total_lines":1208,"dependency_fraction":0.04470198675496689},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAD1L1","total_profiled":1310},"omim":[{"mim_id":"620189","title":"MOSAIC VARIEGATED ANEUPLOIDY SYNDROME 7 WITH INFLAMMATION AND TUMOR PREDISPOSITION; MVA7","url":"https://www.omim.org/entry/620189"},{"mim_id":"618136","title":"MAD2L1-BINDING PROTEIN; MAD2L1BP","url":"https://www.omim.org/entry/618136"},{"mim_id":"615890","title":"DYNEIN, CYTOPLASMIC 1, LIGHT INTERMEDIATE CHAIN 1; DYNC1LI1","url":"https://www.omim.org/entry/615890"},{"mim_id":"613713","title":"PCI DOMAIN-CONTAINING PROTEIN 2; PCID2","url":"https://www.omim.org/entry/613713"},{"mim_id":"610266","title":"TAO KINASE 1; TAOK1","url":"https://www.omim.org/entry/610266"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MAD1L1"},"hgnc":{"alias_symbol":["HsMAD1","TXBP181","MAD1","PIG9","TP53I9"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y6D9","domains":[{"cath_id":"-","chopping":"334-491","consensus_level":"medium","plddt":81.7897,"start":334,"end":491},{"cath_id":"3.30.457.60","chopping":"639-718","consensus_level":"high","plddt":81.658,"start":639,"end":718},{"cath_id":"1.20.5","chopping":"59-242","consensus_level":"medium","plddt":93.0331,"start":59,"end":242}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6D9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6D9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6D9-F1-predicted_aligned_error_v6.png","plddt_mean":81.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAD1L1","jax_strain_url":"https://www.jax.org/strain/search?query=MAD1L1"},"sequence":{"accession":"Q9Y6D9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y6D9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y6D9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6D9"}},"corpus_meta":[{"pmid":"9546394","id":"PMC_9546394","title":"Human 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consistent with loss of MAD1 function.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, gel filtration, transfection/overexpression with phenotypic readout\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (co-IP, localization, functional assay), foundational paper with 433 citations\",\n      \"pmids\": [\"9546394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HsMAD1 and HsMAD2 localize to nuclear pore complexes (NPCs) during interphase, as demonstrated by co-labeling with NPC antibodies and co-purification with enriched nuclear envelope fractions; MAD1 associates with MAD2 but not p55CDC in this context.\",\n      \"method\": \"Immunofluorescence co-localization, nuclear envelope fractionation, co-immunoprecipitation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-localization and biochemical fractionation in same study\",\n      \"pmids\": [\"11181178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Crystal structure of the tetrameric Mad1-Mad2 core complex reveals an asymmetric tetramer with elongated Mad1 monomers forming a coiled-coil and two connected sub-complexes with Mad2; Mad2 C-terminal tails wrap around Mad1 as 'safety belts'. Mad1 acts as a competitive inhibitor of the Mad2-Cdc20 complex, and unlocking the Mad2 C-terminal tail is required for ligand release.\",\n      \"method\": \"X-ray crystallography, in vitro competition assay, mutagenesis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation (competition assay, mutagenesis)\",\n      \"pmids\": [\"12006501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"RNAi-mediated suppression of MAD1 in mammalian cells causes loss of Mad2 kinetochore localization and spindle checkpoint impairment; Mad1 and Cdc20 share a conserved Mad2-binding motif consensus; binding of a Mad1 or Cdc20 motif peptide triggers extensive rearrangement of Mad2 tertiary structure.\",\n      \"method\": \"RNAi knockdown, in vitro binding assay, structural analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — RNAi loss-of-function with defined phenotype plus in vitro structural analysis, replicated across labs\",\n      \"pmids\": [\"11804586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mad2 forms incompatible complexes with Mad1 and Cdc20 (neither requiring Mad2 oligomerization); interaction of Mad2 with Mad1 is crucial for kinetochore localization of Mad2, where Mad2 then interacts with Cdc20.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis, cell microinjection, kinetochore localization assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (co-IP, mutagenesis, localization), replicated\",\n      \"pmids\": [\"11707408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"A closed conformer of Mad2 constitutively bound to Mad1 (C-Mad2) serves as the kinetochore receptor for cytosolic open Mad2 (O-Mad2); interaction of open and closed Mad2 conformers is essential for the spindle assembly checkpoint. This 'Mad2 template' model proposes that C-Mad2 bound to Mad1 acts as a template for conversion of O-Mad2 into C-Mad2 bound to Cdc20.\",\n      \"method\": \"Conformer-specific binding assays, fluorescence imaging (FRAP), mutagenesis, in vitro reconstitution\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods, foundational model paper widely replicated\",\n      \"pmids\": [\"15694304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Hec1 is required for recruitment of Mps1 kinase and Mad1/Mad2 complexes to kinetochores; depletion of Hec1 by RNAi impairs chromosome congression and causes persistent spindle checkpoint activation.\",\n      \"method\": \"RNAi depletion, immunofluorescence kinetochore localization\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean RNAi knockdown with defined kinetochore localization phenotype, highly cited\",\n      \"pmids\": [\"12351790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E in Xenopus egg extracts; immunodepletion of Bub1 abolishes checkpoint and kinetochore binding of these proteins; reintroduction of kinase-deficient Bub1 restores checkpoint and localization, demonstrating kinase-independent scaffolding.\",\n      \"method\": \"Immunodepletion, add-back reconstitution, kinase-dead mutant, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstitution with mutant analysis in Xenopus extract system\",\n      \"pmids\": [\"11402067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"BubR1 immunodepletion in Xenopus egg extracts greatly reduces kinetochore binding of Mad1, Mad2, Bub1, Bub3, and CENP-E; localization and hyperphosphorylation of BubR1 at kinetochores depends on Bub1 and Mad1 (but not Mad2), placing Mad1 upstream of BubR1 phosphorylation.\",\n      \"method\": \"Immunodepletion, kinetochore localization assay, phosphorylation analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis via immunodepletion with defined localization and phosphorylation readouts\",\n      \"pmids\": [\"12163471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Mad2 forms a tight complex with Mad1 in budding yeast, independent of cell cycle stage and other checkpoint proteins; this association is critical for checkpoint function and for hyperphosphorylation of Mad1. Deletion/mutation analysis confirms that Mad1-Mad2 association is required for spindle checkpoint.\",\n      \"method\": \"Gel filtration, co-immunoprecipitation, deletion/mutagenesis analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple biochemical methods with functional validation by mutagenesis\",\n      \"pmids\": [\"10436016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Tpr (a nuclear pore complex component) directly binds Mad1 and Mad2; Tpr depletion disrupts NPC localization of Mad1 and Mad2 during interphase, decreases levels of Mad1-bound Mad2, and decreases Mad1 at kinetochores during prometaphase, correlating with inability of Mad1 to activate Mad2 and inhibit APC/Cdc20.\",\n      \"method\": \"Mass spectrometry of affinity-purified Mad2 complexes, direct binding assay, RNAi depletion, immunofluorescence\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS identification plus direct binding plus RNAi functional validation\",\n      \"pmids\": [\"18981471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mps1 kinase activity is required for recruitment of O-Mad2 to the kinetochore-bound Mad1-C-Mad2 core complex; inhibiting Mps1 before mitosis abolishes Mad1 and Mad2 kinetochore recruitment, whereas inhibiting it after mitotic entry leaves the Mad1-C-Mad2 core bound but prevents O-Mad2 recruitment. Mps1 can dimerize and transphosphorylate.\",\n      \"method\": \"Small-molecule Mps1 inhibitor (AZ3146), immunofluorescence, biochemical fractionation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — chemical genetic approach with timed inhibition, multiple orthogonal readouts\",\n      \"pmids\": [\"20624899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mad1 kinetochore localization in budding yeast is mediated by Mps1-dependent phosphorylation of a region within Bub1; tethering this Bub1 region to kinetochores bypasses Mps1-mediated upstream checkpoint requirements; Mad1-Bub1 interaction and kinetochore association can be reconstituted in vitro with Mps1 and Mad2.\",\n      \"method\": \"In vitro reconstitution, mutagenesis, epistasis (bypass tethering), kinetochore localization assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus bypass epistasis and mutagenesis\",\n      \"pmids\": [\"24402315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of the C-terminal domain (CTD) of human Mad1 reveals a homodimer with fold similar to kinetochore-binding domains of Spc25 and Csm1; mutagenesis of the CTD interface diminishes kinetochore targeting and implicates Bub1 as the Mad1 CTD receptor.\",\n      \"method\": \"X-ray crystallography, mutagenesis, kinetochore localization assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis validation of kinetochore targeting\",\n      \"pmids\": [\"22493223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structure of the Mad1 CTD bound to two phosphorylated Bub1 CD1 peptides at 1.75 Å resolution shows that phosphorylated Bub1 Thr461 directly contacts Arg617 of the Mad1 RLK motif and caps the CD1 α-helix dipole; in solution, only one Bub1 CD1 peptide binds the Mad1 homodimer, reflecting inherent coiled-coil asymmetry.\",\n      \"method\": \"X-ray crystallography, solution binding assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with biochemical validation\",\n      \"pmids\": [\"34013668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Mad2 requires association with Mad1 to adopt the closed conformation (C-Mad2) in vivo; p31comet-dependent 'capping' regulates the activity of the Mad1:C-Mad2 complex; microinjection of C-Mad2-specific antibody or Mad1-neutralizing antibody abruptly terminates the spindle assembly checkpoint and accelerates mitotic progression.\",\n      \"method\": \"Conformation-specific monoclonal antibody, antibody microinjection, live-cell imaging\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — conformer-specific antibody with in vivo functional validation by microinjection\",\n      \"pmids\": [\"21772247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Constitutive targeting of Mad1 to bioriented kinetochores (via engineered construct) is sufficient to cause metaphase arrest dependent on Mad1-Mad2 binding; Aurora B, Mps1, and BubR1 kinases (but not Polo-like kinase) are needed to maintain checkpoint arrest when Mad1 is present at kinetochores, placing them downstream of Mad1-Mad2 recruitment.\",\n      \"method\": \"Kinetochore-targeted Mad1 construct, chemical kinase inhibitors, live-cell imaging, mutagenesis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — engineered gain-of-function with pharmacological epistasis and mutagenesis\",\n      \"pmids\": [\"21394085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Recruiting Mad1 to metaphase kinetochores via chemically induced dimerization (CID) is sufficient to reactivate the spindle assembly checkpoint without increasing Mps1 or BubR1 at kinetochores; Mad2 binding and a conserved C-terminal motif of Mad1 are both required for checkpoint reactivation, suggesting Mad1 scaffolds formation of a higher-order mitotic checkpoint complex.\",\n      \"method\": \"Chemically induced dimerization, live-cell imaging, mutagenesis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — temporally controlled CID with mutagenesis validation\",\n      \"pmids\": [\"24637323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MAD1 directly interacts with the N-terminal 100 amino acids of CDK1-CCNB1 (Cyclin B1); CCNB1 is recruited to unattached kinetochores through this interaction in an MPS1-dependent manner; this MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop for timely MPS1 kinetochore recruitment and sustained checkpoint arrest.\",\n      \"method\": \"Proteomics/co-immunoprecipitation, in vitro binding with truncation mutants, live-cell imaging, RNAi\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — proteomic identification plus co-IP plus functional RNAi with multiple readouts\",\n      \"pmids\": [\"30674583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cyclin B1 directly binds to an acidic face in the N-terminal region of MAD1 (mapped by in vitro reconstitution) and scaffolds MAD1 to the kinetochore corona; point mutations abolishing this interaction eliminate MAD1 corona localization and weaken the spindle assembly checkpoint; corona-localized MAD1 loses dependence on MPS1 kinase after corona establishment, ensuring robust SAC.\",\n      \"method\": \"In vitro reconstitution, mutagenesis, live-cell imaging, RNAi\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution plus mutagenesis plus functional imaging\",\n      \"pmids\": [\"32202322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cyclin B1-Cdk1 is targeted to the nuclear pore complex by binding an acidic face of MAD1; localized Cyclin B1-Cdk1 is required for proper release of MAD1 from Tpr at the NPC, allowing MAD1 recruitment to kinetochores before nuclear envelope breakdown.\",\n      \"method\": \"Co-immunoprecipitation, live-cell imaging, phosphorylation assays, RNAi\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with functional imaging and RNAi, multiple orthogonal methods\",\n      \"pmids\": [\"32236513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MAD-1 (C. elegans) interacts with BUB-1 through a specific segment of the MAD-1 coiled coil; mutations selectively disrupting BUB-1 interaction eliminate MAD-1 localization to unattached kinetochores and meiotic chromosomes and abrogate checkpoint signaling; this BUB-1 C-terminal/kinase domain region mediates MAD-1 kinetochore targeting independently of kinase activity.\",\n      \"method\": \"Mutagenesis, co-immunoprecipitation, kinetochore localization assay, C. elegans genetics\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with reciprocal co-IP and in vivo functional validation\",\n      \"pmids\": [\"24567362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Tpr is required for Mad1-c-Mad2 recruitment to nuclear pores during interphase and stabilizes Mad1 and Mad2 protein levels; Tpr depletion reduces Mad2 (but not Mad1) at kinetochores, and the SAC robustness depends on Mad2 kinetochore levels maintained by Tpr.\",\n      \"method\": \"Co-immunoprecipitation, RNAi, immunofluorescence, protein half-life measurements\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus RNAi with defined protein stability and localization readouts\",\n      \"pmids\": [\"24344181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Nup153 directly binds Mad1 via Nup153's N-terminal domain; Nup153 overexpression causes hypophosphorylation of Mad1 and spindle checkpoint inactivation; Nup153 depletion reduces Mad1 at nuclear pores and delays Mad1 dissociation from kinetochores at metaphase. Nup153 binding to Mad1 affects Mad1 phosphorylation status but not its interaction with Mad2.\",\n      \"method\": \"In vitro direct binding assay, overexpression, RNAi, immunofluorescence, phosphorylation analysis\",\n      \"journal\": \"Nucleus\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro direct binding with RNAi functional analysis, single lab\",\n      \"pmids\": [\"21327106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human CENP-I is required for kinetochore localization of MAD1, MAD2, and CENP-F; CENP-I depletion prevents mitotic arrest despite unattached kinetochores, and the delay that does occur is MAD2-dependent, indicating that collective signal from many unattached kinetochores is needed to sustain arrest.\",\n      \"method\": \"RNAi depletion, immunofluorescence, cell cycle analysis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — RNAi with defined checkpoint and localization phenotypes, replicated\",\n      \"pmids\": [\"12640463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Depletion of either Nuf2 or Hec1 by RNAi leads to 5-fold or greater reduction of Mad1 and Mad2 at kinetochores during a prometaphase block; Mad1/Mad2 reduction is reversible upon spindle depolymerization, indicating Nuf2/Hec1 prevent microtubule-dependent stripping of Mad1/Mad2.\",\n      \"method\": \"RNAi, quantitative immunofluorescence\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi with quantitative kinetochore localization, single lab\",\n      \"pmids\": [\"14654001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"NEK2A interacts with MAD1 in vitro and in vivo via a leucine zipper-containing C-terminal domain of MAD1; NEK2A localizes to kinetochores; NEK2A depletion by siRNA abolishes MAD2 (but not MAD1, BUB1, or HEC1) kinetochore association and impairs spindle checkpoint signaling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding, siRNA, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP plus in vitro binding plus siRNA functional analysis, single lab\",\n      \"pmids\": [\"14978040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PRP4 kinase is required for recruitment or maintenance of MPS1, MAD1, and MAD2 at kinetochores; PRP4 depletion by RNAi induces mitotic acceleration, lagging chromatids, aneuploidy, and failure to arrest after nocodazole treatment.\",\n      \"method\": \"RNAi, immunofluorescence, mitotic index assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi with defined checkpoint localization and functional phenotype, single lab\",\n      \"pmids\": [\"17998396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Two leucine zipper domains (amino acids 501-522 and 557-571) in human MAD1 are required for binding MAD2; a polymorphism at codon 558 (Arg→His) reduces MAD2 binding and mitotic checkpoint enforcement, representing loss-of-heterozygosity at the MAD1 locus in a human breast cancer.\",\n      \"method\": \"Mutagenesis, co-immunoprecipitation, mitotic index assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with functional validation and human cancer evidence\",\n      \"pmids\": [\"12042300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human Mad2 is phosphorylated on multiple serine residues in a cell-cycle-dependent manner; only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo; a phosphomimetic Mad2 mutant fails to interact with Mad1 or APC/C and acts as dominant-negative.\",\n      \"method\": \"In vivo phosphorylation analysis, co-immunoprecipitation, dominant-negative mutagenesis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo PTM with co-IP and mutagenesis, single lab\",\n      \"pmids\": [\"12574116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"BUB1 phosphorylates MAD1 in vitro; BUB1 and BUB3 form a complex and interact with MAD1; BUB1 autophosphorylation and MAD1 phosphorylation require Lys821 in the BUB1 kinase motif.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro kinase assay with mutagenesis, single paper\",\n      \"pmids\": [\"10198256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ATM kinase phosphorylates Mad1 at Serine 214; this phosphorylation promotes Mad1 homodimerization and heterodimerization with Mad2, contributes to spindle assembly checkpoint activation, and is required for chromosomal stability.\",\n      \"method\": \"In vitro kinase assay, phospho-specific antibody, co-immunoprecipitation, flow cytometry\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro kinase assay with co-IP and functional validation, single lab\",\n      \"pmids\": [\"24728176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ULK1 phosphorylates Mad1 at Ser546, promoting Mad1 kinetochore recruitment; phosphorylated Ser546-Mad1 shows enhanced interaction with the RZZ (Rod/ZW10/Zwilch) complex, which may serve as its kinetochore receptor; ULK1 deletion increases chromosomal instability.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, RNAi, immunofluorescence\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro kinase assay with co-IP and localization readout, single lab\",\n      \"pmids\": [\"31291454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MAD1 N-terminal domain (NTD) and C-terminal domain (CTD) both bind O-Mad2 and C-Mad2 conformers (unlike the MIM which binds only C-Mad2); NTD and CTD interact with each other and with MPS1 kinase; MPS1 phosphorylates both NTD and CTD, decreasing their mutual interaction and CTD's interaction with MPS1; phosphorylation of CTD residue Thr-716 compromises Mad2 binding and checkpoint responses.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, mutagenesis, conformer-specific binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple binding and kinase assays with mutagenesis, single lab\",\n      \"pmids\": [\"29162720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A Golgi-localized pool of Mad1 (independent of Mad2) controls secretion of α5 integrin; Mad1 depletion impairs integrin secretion, cell attachment, adhesion, FAK activation, and cell motility; Mad1 overexpression accelerates directed cell migration.\",\n      \"method\": \"Immunofluorescence co-localization with Golgi markers, subcellular fractionation, RNAi, integrin secretion assay, cell motility assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — localization tied to functional consequence via RNAi with multiple readouts, single lab\",\n      \"pmids\": [\"25447996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Upregulated Mad1 localizes to PML nuclear bodies; the C-terminus of Mad1 directly interacts with PML (interaction enhanced by sumoylation); upregulated Mad1 displaces MDM2 from PML, freeing MDM2 to ubiquitinate and destabilize p53, thereby promoting tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, direct binding assay, immunofluorescence, sumoylation assay, orthotopic tumor assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding plus localization plus in vivo tumor assay, single lab\",\n      \"pmids\": [\"30948704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Fission yeast Mad1 binds the kinesin-5 motor Cut7 (Eg5 homolog); Mad1 recruits Cut7 to kinetochores of misaligned chromosomes to promote chromosome congression; human Mad1 similarly recruits CENP-E motor to kinetochores, revealing a conserved dual function of Mad1 in checkpoint signaling and chromosome gliding.\",\n      \"method\": \"Protein-protein interaction screen, co-immunoprecipitation, live-cell imaging, genetic analysis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with live imaging, conserved in two organisms, single study\",\n      \"pmids\": [\"26258632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mad1 has a direct role in the spindle assembly checkpoint beyond Mad2 kinetochore recruitment: even when C-Mad2 is artificially recruited to kinetochores, Mad1 is still required for mitotic arrest; the C-terminal globular domain of Mad1 and conserved residues within it are required for this additional checkpoint function.\",\n      \"method\": \"Artificial C-Mad2 kinetochore tethering, mutagenesis, mitotic index assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — bypass experiment with mutagenesis, single lab\",\n      \"pmids\": [\"24477933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cep57 localizes to kinetochores, binds Mis12 (KMN network), and interacts with Mad1; Cep57 depletion decreases Mad1-Mad2 kinetochore localization and reduces spindle checkpoint signaling; Cep57 microtubule-binding activity is involved in timely Mad1 removal from kinetochores.\",\n      \"method\": \"Co-immunoprecipitation, RNAi, immunofluorescence, spindle checkpoint assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with RNAi functional validation, single lab\",\n      \"pmids\": [\"26743940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CENP-I is required for stable association of RZZ complex and Mad1 with kinetochores and inhibits their dynein-mediated removal; Aurora B regulates RZZ/Mad1 association; CENP-I and Aurora B act as a molecular switch controlling Mad1 kinetochore dynamics.\",\n      \"method\": \"RNAi, Aurora B inhibitor, immunofluorescence, kinetochore localization quantification\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi epistasis with pharmacological inhibition and localization readout, single lab\",\n      \"pmids\": [\"24862574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The RZZ complex localizes to the N-terminus of KNL1 downstream of Bub1, and mediates robust Mad1/Mad2 kinetochore localization in human cells; both Bub1/KNL1-dependent and KNL1/Bub1-independent mechanisms exist for RZZ and Mad1/Mad2 kinetochore recruitment.\",\n      \"method\": \"RNAi, kinetochore localization quantification, rescue experiments\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic RNAi epistasis with multiple constructs, single lab\",\n      \"pmids\": [\"26581576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Mad1 heterodimerizes with Max and represses transcription from E-box-containing promoters (competing with Myc:Max); the SIN3 corepressor interaction domain at the N-terminus of Mad1 and the PAH2 domain of Sin3 are required for transcriptional repression; Mad1 inhibits cell cycle progression from G1 to S phase, and this inhibitory activity requires its transcriptional repression function.\",\n      \"method\": \"Transcription reporter assay, cell cycle analysis (FACS), mutagenesis, co-immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis linking repressor domain to cell cycle function, widely replicated\",\n      \"pmids\": [\"8649388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction; these residues form an amphipathic alpha-helix, and hydrophobic residues on the helix face are required for mSin3A PAH2 binding; this minimal SID can function as an autonomous portable repression domain.\",\n      \"method\": \"Mutagenesis, NMR/structural analysis, in vitro binding assay, transcription reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural analysis with mutagenesis and functional validation\",\n      \"pmids\": [\"10551834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"NMR solution structure of the Sin3B PAH2 domain in complex with the N-terminal Mad1 peptide reveals a 'wedged helical bundle'; four α-helices of PAH2 form a hydrophobic cleft accommodating an amphipathic Mad1 α-helix; Mad1 binding stabilizes PAH2 secondary structure elements.\",\n      \"method\": \"NMR structure determination\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure of protein complex\",\n      \"pmids\": [\"11101889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"c-IAP1 (a RING finger E3 ubiquitin ligase) catalyzes ubiquitination of Mad1 (the Myc/Max/Mad transcriptional repressor), accelerating its degradation via the 26S proteasome, thereby reducing Mad1 levels and cooperating with Myc to promote cell proliferation.\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation, proteasome inhibitor experiments, cell proliferation assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro ubiquitination reconstitution with functional validation\",\n      \"pmids\": [\"18082613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RSK (p90 ribosomal kinase) and S6K (p70 S6 kinase) phosphorylate Serine 145 of Mad1 (Myc/Max/Mad family member) upon serum or insulin stimulation; Ser-145 phosphorylation accelerates Mad1 ubiquitination and degradation via the 26S proteasome, promoting Myc transcriptional activity.\",\n      \"method\": \"In vitro kinase assay, mutagenesis, ubiquitination assay, pulse-chase degradation analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with mutagenesis and degradation analysis\",\n      \"pmids\": [\"18451027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mad1 (Myc/Max/Mad family) recruits the histone demethylase RBP2 to the hTERT gene promoter; Mad1-RBP2 interaction and co-occupancy at the hTERT promoter correlates with H3-K4 demethylation and transcriptional repression during differentiation; RBP2 depletion derepresses hTERT expression.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), RNAi, histone methylation assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus ChIP plus RNAi with functional readout, single lab\",\n      \"pmids\": [\"19762557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Mad1 (Myc/Max/Mad) interacts directly with the UBF promoter and represses rDNA transcription; granulocytic cells lacking Mad1 display increased rDNA transcription, protein synthesis, and cell volume; siRNA knockdown shows UBF is required for c-MYC-induced rDNA transcription.\",\n      \"method\": \"Nuclear run-on assay, chromatin immunoprecipitation, siRNA, cell size measurement\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (run-on, ChIP, siRNA), single lab\",\n      \"pmids\": [\"15282543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"During differentiation of HL60 cells, c-Myc occupancy at hTERT E-boxes is replaced by Mad1 occupancy in vivo, correlating with reduced histone acetylation at the promoter; histone deacetylase inhibitor trichostatin A attenuates hTERT repression during differentiation.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), reporter assay, HDAC inhibitor treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP demonstrating in vivo promoter occupancy switch with histone modification readout\",\n      \"pmids\": [\"11274400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"p53 represses MAD1 transcription through a novel 38-bp element in the MAD1 promoter distinct from canonical p53 binding sites; p53, HDAC1, and mSin3A associate with the MAD1 promoter in vivo (by ChIP); HDAC inhibitor trichostatin A relieves p53-mediated MAD1 repression.\",\n      \"method\": \"Promoter reporter assay, ChIP, HDAC inhibitor treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reporter assay with ChIP validation, single lab\",\n      \"pmids\": [\"12876282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"miR-125b represses Mad1 (spindle checkpoint) expression post-transcriptionally; exogenous miR-125b overexpression downregulates Mad1, delays cells at metaphase, and promotes apoptotic death with elevated chromosomal abnormalities.\",\n      \"method\": \"miRNA overexpression, immunoblot, cell cycle analysis, chromosome aberration assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — miRNA overexpression with functional phenotype, single lab\",\n      \"pmids\": [\"23099851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Overexpression of Mad1 (spindle checkpoint) causes aneuploidy and chromosomal instability through mislocalization of Mad2 away from kinetochores, weakening the mitotic checkpoint; cells overexpressing Mad1 are resistant to microtubule poisons.\",\n      \"method\": \"Mad1 overexpression, immunofluorescence quantification of Mad2 kinetochore levels, chromosome counting, drug sensitivity assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — overexpression with mechanistic localization readout and functional consequence, single lab\",\n      \"pmids\": [\"22778409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RARS-MAD1L1 fusion protein interacts with AIMP2 (by co-immunoprecipitation), resulting in activation of the FUBP1/c-Myc pathway; silencing of FUBP1 or c-Myc inhibitor abrogates cancer stem cell-like characteristics induced by the fusion.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, siRNA, colony/sphere formation assay\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP with functional validation for a fusion protein context, single lab\",\n      \"pmids\": [\"29133573\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAD1L1 (Mad1) encodes a conserved homodimeric scaffold protein with two distinct molecular functions: (1) as a core spindle assembly checkpoint (SAC) component, Mad1 constitutively associates with closed-conformation Mad2 (C-Mad2) at kinetochores—recruited via Mps1-phosphorylated Bub1, the RZZ complex, and Cyclin B1 scaffold at the corona—where the Mad1:C-Mad2 tetramer acts as a catalytic template converting cytosolic open Mad2 into the closed form that sequesters Cdc20 to inhibit APC/C; and (2) as a bHLH-LZ transcriptional repressor (the Myc/Max/Mad-family Mad1, gene MXD1/MAD1 in some contexts), Mad1 heterodimerizes with Max to occupy E-box promoters and recruits the mSin3A-HDAC corepressor complex through an amphipathic N-terminal SID helix, antagonizing Myc-driven transcription; additionally, Mad1 localizes to nuclear pores (via Tpr) during interphase to maintain Mad2 proteostasis, to the Golgi to regulate integrin secretion and cell migration, and to PML bodies where it displaces MDM2 to destabilize p53.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MAD1L1 encodes a coiled-coil homodimeric protein that functions as the central scaffold of the spindle assembly checkpoint (SAC), constitutively binding closed-conformation Mad2 (C-Mad2) at kinetochores to catalyze conversion of cytosolic open-Mad2 (O-Mad2) into C-Mad2–Cdc20 complexes that inhibit the APC/C and prevent premature anaphase onset [PMID:15694304, PMID:12006501, PMID:11804586]. Mad1 is recruited to unattached kinetochores through Mps1-phosphorylated Bub1 via its C-terminal domain and is additionally scaffolded at the kinetochore corona by direct binding of Cyclin B1 to an acidic N-terminal surface, creating a positive-feedback loop that sustains Mps1 recruitment and checkpoint robustness [PMID:24402315, PMID:34013668, PMID:30948704, PMID:32202322]. During interphase, Mad1 localizes to nuclear pore complexes through Tpr binding, which stabilizes Mad2 protein levels and primes the Mad1–C-Mad2 complex for rapid kinetochore deployment upon mitotic entry; Cyclin B1–Cdk1 at the NPC promotes timely Mad1 release from Tpr before nuclear envelope breakdown [PMID:11181178, PMID:18981471, PMID:32236513]. Beyond its SAC role, a Golgi-localized pool of Mad1 regulates α5-integrin secretion and cell migration independently of Mad2, and overexpression of Mad1 at PML nuclear bodies displaces MDM2 to destabilize p53 [PMID:25447996, PMID:30948704].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of human MAD1 as a checkpoint homodimer established that the yeast SAC architecture is conserved in mammals and that Mad1 functions through direct Mad2 binding and cell-cycle-dependent hyperphosphorylation.\",\n      \"evidence\": \"Co-IP, gel filtration, immunofluorescence, and HTLV-I Tax overexpression phenotyping in human cells\",\n      \"pmids\": [\"9546394\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase(s) responsible for hyperphosphorylation unidentified\", \"Mechanism of Tax-mediated checkpoint disruption not resolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrating that Mad1–Mad2 association is constitutive and independent of cell cycle stage resolved that the complex is pre-formed rather than assembled de novo during mitosis, shifting focus to how the stable complex is activated.\",\n      \"evidence\": \"Gel filtration and co-IP with deletion/mutagenesis in budding yeast\",\n      \"pmids\": [\"10436016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Mad1–Mad2 complex converts cytosolic Mad2 remained unknown\", \"Structural basis of constitutive binding unresolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Discovery that Mad1 and Mad2 reside at nuclear pore complexes during interphase revealed an unexpected non-mitotic reservoir, raising the question of whether NPC localization functionally contributes to checkpoint competence.\",\n      \"evidence\": \"Immunofluorescence co-localization with NPC markers and nuclear envelope fractionation\",\n      \"pmids\": [\"11181178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NPC-binding partner of Mad1 not identified\", \"Functional significance of interphase NPC pool unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Bub1 was established as an upstream scaffold required for Mad1 kinetochore targeting, and kinase-dead Bub1 rescued both localization and checkpoint, indicating a kinase-independent scaffolding mechanism.\",\n      \"evidence\": \"Immunodepletion and kinase-dead add-back reconstitution in Xenopus egg extracts\",\n      \"pmids\": [\"11402067\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding interface between Bub1 and Mad1 not mapped\", \"Whether Bub1 kinase activity has additional roles at kinetochore not excluded\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"The crystal structure of the Mad1–Mad2 tetramer revealed the 'safety belt' mechanism by which Mad2 C-terminal tails wrap around Mad1, explaining how Mad1 acts as a competitive inhibitor of the Mad2–Cdc20 interaction and establishing the structural foundation of the template model.\",\n      \"evidence\": \"X-ray crystallography with in vitro competition assays and mutagenesis\",\n      \"pmids\": [\"12006501\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How O-Mad2 is recruited to the Mad1:C-Mad2 complex remained unknown\", \"Full-length Mad1 structure unavailable\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"RNAi depletion of Mad1 abolished Mad2 kinetochore localization and checkpoint function, confirming Mad1 is essential for Mad2 activation in mammalian cells; identification of a shared Mad2-binding motif in Mad1 and Cdc20 explained substrate mimicry.\",\n      \"evidence\": \"RNAi knockdown, in vitro peptide binding, structural analysis of Mad2 conformational change\",\n      \"pmids\": [\"11804586\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformer-specific mechanism of Mad2 conversion not yet demonstrated in vivo\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The 'Mad2 template model' was established: C-Mad2 constitutively bound to Mad1 at kinetochores serves as a catalytic template that recruits and converts O-Mad2 to C-Mad2–Cdc20, answering how a single unattached kinetochore amplifies the checkpoint signal.\",\n      \"evidence\": \"Conformer-specific binding assays, FRAP, mutagenesis, and in vitro reconstitution\",\n      \"pmids\": [\"15694304\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Rate-limiting step in the catalytic cycle undefined\", \"Role of p31comet in capping the template not fully characterized\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Tpr was identified as the direct NPC anchor for Mad1–Mad2, resolving the interphase binding partner; Tpr depletion reduced Mad1 at both NPCs and kinetochores and diminished Mad2 protein levels, establishing a proteostatic function for the NPC-localized pool.\",\n      \"evidence\": \"Affinity purification–mass spectrometry, direct binding assay, RNAi with localization and protein level readouts\",\n      \"pmids\": [\"18981471\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Tpr directly hands off Mad1 to kinetochores or indirectly supports it via protein stabilization not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mps1 kinase was shown to be required not just for Mad1 kinetochore recruitment but specifically for O-Mad2 recruitment to the already-bound Mad1:C-Mad2 core, separating two temporally distinct steps in checkpoint activation.\",\n      \"evidence\": \"Timed chemical inhibition of Mps1 (AZ3146) with immunofluorescence in human cells\",\n      \"pmids\": [\"20624899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Mps1 substrate on Mad1 for O-Mad2 recruitment step not identified at this point\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Constitutive Mad1 kinetochore tethering was sufficient for metaphase arrest dependent on Mad1–Mad2 binding, establishing that Mad1 presence at kinetochores is the rate-limiting determinant of checkpoint activation and that downstream kinases (Aurora B, Mps1, BubR1) are needed to maintain but not initiate this arrest.\",\n      \"evidence\": \"Engineered kinetochore-targeted Mad1, chemical kinase inhibitors, live-cell imaging\",\n      \"pmids\": [\"21394085\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What additional checkpoint function the Mad1 CTD provides beyond Mad2 recruitment remained unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Crystal structure of the Mad1 CTD revealed a homodimeric fold resembling Spc25/Csm1 kinetochore-binding domains, and mutagenesis implicated Bub1 as the direct CTD receptor, providing the first structural basis for Mad1 kinetochore docking.\",\n      \"evidence\": \"X-ray crystallography with mutagenesis and kinetochore localization assay\",\n      \"pmids\": [\"22493223\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of Mad1 CTD–Bub1 complex not yet solved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mps1-dependent phosphorylation of Bub1 was reconstituted in vitro as the direct mechanism for Mad1 kinetochore recruitment, closing the kinase–scaffold–effector pathway; artificial Bub1 tethering bypassed Mps1 upstream requirements, confirming Bub1 phosphorylation as the key regulated step.\",\n      \"evidence\": \"In vitro reconstitution, bypass tethering, mutagenesis in budding yeast\",\n      \"pmids\": [\"24402315\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphorylation sites on Bub1 not fully mapped in human system at this point\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"A checkpoint function for Mad1 beyond Mad2 kinetochore recruitment was established: even when C-Mad2 is artificially tethered to kinetochores, Mad1 and specifically its CTD are still required for mitotic arrest, indicating Mad1 scaffolds additional checkpoint signaling.\",\n      \"evidence\": \"Artificial C-Mad2 tethering, mutagenesis, mitotic index assay\",\n      \"pmids\": [\"24477933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the additional Mad1 CTD interactor(s) required for this function unknown\", \"Mechanism not reconstituted in vitro\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"A Mad2-independent Golgi-localized pool of Mad1 was discovered that regulates α5-integrin secretion, cell adhesion, and directed cell migration, revealing a non-mitotic function entirely separate from the SAC.\",\n      \"evidence\": \"Golgi co-localization, subcellular fractionation, RNAi with integrin secretion and cell motility assays\",\n      \"pmids\": [\"25447996\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of Mad1 action at the Golgi unknown\", \"Whether this pool is regulated during cell cycle not addressed\", \"Not independently replicated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"MAD1 was shown to directly recruit Cyclin B1–CDK1 to unattached kinetochores through its N-terminal acidic face, establishing a positive-feedback loop: kinetochore-localized CDK1 sustains Mps1 recruitment, which in turn maintains Mad1 at kinetochores.\",\n      \"evidence\": \"Proteomics/co-IP, in vitro binding with truncation mutants, live-cell imaging, RNAi\",\n      \"pmids\": [\"30948704\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CDK1 substrates in this feedback loop not comprehensively identified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mad1 localization to PML nuclear bodies and direct binding to PML was shown to displace MDM2, leading to p53 destabilization and tumor promotion, establishing a SAC-independent oncogenic mechanism for Mad1 overexpression.\",\n      \"evidence\": \"Co-IP, direct binding assay, immunofluorescence, sumoylation assay, orthotopic tumor assay\",\n      \"pmids\": [\"30948704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PML-body function operates in non-tumor contexts unknown\", \"Stoichiometry and regulation of the Mad1–PML interaction not defined\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Cyclin B1 was identified as the scaffold that anchors Mad1 at the kinetochore corona through a direct interaction with Mad1's N-terminal acidic surface; this corona pool becomes Mps1-independent after establishment, explaining how robust SAC signaling persists even as Mps1 activity fluctuates.\",\n      \"evidence\": \"In vitro reconstitution, point mutagenesis abolishing interaction, live-cell imaging\",\n      \"pmids\": [\"32202322\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the Mad1–Cyclin B1 interface not resolved\", \"Whether corona and Bub1-dependent pools function additively or redundantly not fully established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Cyclin B1–Cdk1 was shown to localize to NPCs via Mad1 binding, where it phosphorylates Tpr to release Mad1 before nuclear envelope breakdown, connecting the interphase NPC pool to rapid kinetochore loading at mitotic entry.\",\n      \"evidence\": \"Co-IP, live-cell imaging, phosphorylation assays, RNAi\",\n      \"pmids\": [\"32236513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific Tpr phosphorylation sites mediating Mad1 release not mapped\", \"Whether other kinases contribute to NPC release not excluded\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A 1.75 Å crystal structure of Mad1 CTD bound to phospho-Bub1 CD1 peptides defined the atomic contacts: pThr461 of Bub1 contacts Arg617 of the Mad1 RLK motif; asymmetric binding of only one Bub1 peptide per Mad1 homodimer in solution revealed functional consequences of coiled-coil asymmetry.\",\n      \"evidence\": \"X-ray crystallography, solution binding assay\",\n      \"pmids\": [\"34013668\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of asymmetric Bub1 binding for checkpoint signaling not determined\", \"Full-length Mad1–Bub1 complex structure unavailable\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the structural basis of the Mad1–Cyclin B1 corona interface, how the Golgi-localized Mad1 pool regulates integrin trafficking at the molecular level, how the dual NPC and kinetochore pools are coordinately regulated across the cell cycle, and the full-length structure of the Mad1 homodimer.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length Mad1 structure unavailable\", \"Molecular mechanism of Mad1's Golgi function unknown\", \"Quantitative contribution of NPC versus corona versus Bub1 kinetochore pools to checkpoint strength not measured\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 5, 16, 17]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2, 4, 13, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 3, 6, 13, 14]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [1, 10, 22]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [34]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 3, 5, 11, 16, 17]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 10, 20, 22]}\n    ],\n    \"complexes\": [\n      \"Mad1:C-Mad2 tetramer\",\n      \"MCC (mitotic checkpoint complex)\"\n    ],\n    \"partners\": [\n      \"MAD2L1\",\n      \"BUB1\",\n      \"TPR\",\n      \"CCNB1\",\n      \"MPS1\",\n      \"CEP57\",\n      \"NUP153\",\n      \"PML\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}