mad3 encodes the fission-yeast orthologue of BubR1/Mad3, a 310-residue, non-catalytic spindle assembly checkpoint (SAC) protein. Like budding-yeast Mad3, and unlike vertebrate BubR1, it has no C-terminal kinase domain: it consists of an N-terminal KEN box (KEN20, KEN1) followed by the Bub1/Mad3 TPR-like helical domain (residues ~56-218, crystallised in PDB 4AEZ), and a disordered C-terminus carrying a second KEN box (KEN271, KEN2) flanked by ABBA and D-box-like motifs and a cluster of Mph1 (Mps1) phosphorylation sites. Fission-yeast Mad3 lacks the GLEBS motif through which other Mad3/BubR1 proteins bind Bub3, so Bub3 is not a stable subunit of the S. pombe mitotic checkpoint complex (MCC), which consists of Mad3, closed Mad2 and the APC/C activator Slp1 (Cdc20). Mad3 is a nuclear protein in this closed-mitosis organism. When kinetochores are unattached, Mad3-GFP accumulates at them in a Bub1-, Bub3- and Mph1-dependent (but Mad1- and Mad2-independent) manner; recruitment is mediated by a direct interaction between the Bub1 TPR domain and the Mad3 TPR domain, downstream of Mph1 phosphorylation of Spc7 (KNL1) MELT motifs. Within the MCC the Mad3 N-terminal KEN box occupies the degron-binding site of Slp1 and, together with Mad2, obstructs substrate recognition by APC/C-Slp1, so that securin (Cut2) and cyclin B (Cdc13) are not ubiquitinated and anaphase is delayed. Mad3 and Mad2 associate stably with the APC/C every mitosis in a Slp1-, Mad2- and KEN1-dependent manner; MCC formation and APC/C binding take place in the nucleoplasm independently of kinetochore targeting, but kinetochore-generated modifications, in particular Mph1 phosphorylation of the Mad3 C-terminus near KEN2, are needed to stabilise MCC-APC/C binding and to maintain checkpoint arrests, and Mad3 phosphomimetics are more potent APC/C inhibitors in vitro. Mad3 is the only checkpoint protein required for overexpressed Mad2 to arrest cells in metaphase. mad3 is not essential for viability, but mad3 delta cells are hypersensitive to benomyl and carbendazim, cannot arrest when microtubules are absent (nda3-KM311), precociously separate sister chromatids, lose minichromosomes at elevated rates and display the cut phenotype.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000776 kinetochore | NAS PMID:15930132 Spindle checkpoint signaling requires the mis6 kinetochore s... | ACCEPT | Summary: NAS row (PomBase) citing Saitoh et al. 2005, a study of Mis6-complex and Nuf2-complex requirements for Mad2 and Bub1 kinetochore loading; the cached full text does not mention Mad3, so the statement was presumably taken from the authors' general framing of checkpoint proteins accumulating at unattached kinetochores. Mad3 kinetochore localisation is directly documented elsewhere for this gene. Reason: The term is correct and is the site where Mad3 acts in checkpoint signal generation. Millband and Hardwick 2002 show that functional Mad3-GFP is recruited to unattached kinetochores early in mitosis, accumulates there on prolonged checkpoint activation, and depends on Bub1, Bub3 and Mph1 but not Mad1 or Mad2 (PMID:11909965), and Leontiou et al. 2019 show that the Bub1 TPR domain directly recruits Mad3 (PMID:31257143). The NAS evidence from PMID:15930132 is weak for Mad3 specifically, but the annotation is supported by direct evidence and is retained as core. Supporting Evidence: PMID:11909965 Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. PMID:11909965 We find Mad3-GFP kinetochore localization to be dependent upon Bub1p, Bub3p, and the Mph1p kinase, but not upon Mad1p or Mad2p. PMID:15930132 During early mitosis, the spindle checkpoint proteins, such as Mad2 and Bub1, accumulate at kinetochores that do not associate with the spindle. |
| GO:0000776 kinetochore | NAS PMID:15930132 Spindle checkpoint signaling requires the mis6 kinetochore s... | ACCEPT | Summary: ComplexPortal NAS row for kinetochore localisation citing Saitoh et al. 2005, whose cached text does not mention Mad3; the localisation itself is directly established by Mad3-GFP imaging in Millband and Hardwick 2002. Reason: Correct localisation. Mad3-GFP forms punctate foci at unattached kinetochores during early mitosis and on checkpoint activation, colocalising with centromeric markers, in a Bub1/Bub3/Mph1- dependent manner (PMID:11909965); the Bub1 TPR domain is necessary and sufficient to recruit Mad3 (PMID:31257143). Retained; the direct-evidence references are added. Supporting Evidence: PMID:11909965 Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. PMID:31257143 We conclude that an important fission yeast function of the highly conserved Bub1-TPR domain is to recruit and interact with Mad3. |
| GO:0005515 protein binding | IPI PMID:11909965 Fission yeast Mad3p is required for Mad2p to inhibit the ana... | MODIFY | Summary: IntAct IPI recording that Mad3p coimmunoprecipitates Mad2p (O14417) in Millband and Hardwick 2002, alongside Bub3p and the checkpoint effector Slp1/Cdc20p; the same paper shows Mad3 is required for overexpressed Mad2 to arrest cells in metaphase. Reason: Bare protein binding is uninformative. The Mad3-Mad2 association reported here is the Mad3-Mad2-Slp1 mitotic checkpoint complex, the fission-yeast MCC later defined biochemically (PMID:18556659) and structurally (PMID:22437499); the informative resolution is membership of the mitotic checkpoint complex (GO:0033597), the same resolution used for the Mad3-Mad2 rows in the budding-yeast MAD3 and S. pombe mad2 reviews in this repository. Proposed replacements: mitotic checkpoint complex Supporting Evidence: PMID:11909965 Mad3p coimmunoprecipitates Bub3p, Mad2p, and the spindle checkpoint effector Slp1/Cdc20p. PMID:11909965 We demonstrate that Mad3p function is required for the overexpression of Mad2p to result in a metaphase arrest. PMID:18556659 Mad3 is part of the MCC, which in fission yeast is comprised exclusively of Mad3, Mad2, and Cdc20. |
| GO:0005515 protein binding | IPI PMID:12676091 S. pombe aurora kinase/survivin is required for chromosome c... | MODIFY | Summary: IntAct IPI from Petersen and Hagan 2003 (abstract-only cache), who show that Ark1 (Aurora)/ survivin function is dispensable for Mad3 kinetochore association but is required for Mad2 to form a complex with Mad3 (O14417) on checkpoint activation. Reason: Bare protein binding is uninformative. The Mad2-Mad3 complex whose Ark1-dependence is reported here is the checkpoint-induced mitotic checkpoint complex (Mad3-Mad2-Slp1), so the informative resolution is GO:0033597. The Ark1 dependence of MCC assembly is a useful regulatory observation but does not by itself define a distinct molecular function for Mad3. Proposed replacements: mitotic checkpoint complex Supporting Evidence: PMID:12676091 Ark1/survivin function was not required for the association of Bub1 or Mad3 with the kinetochores. PMID:12676091 However, it was required for two aspects of Mad2 function that accompany checkpoint activation: full-scale association with kinetochores and formation of a complex with Mad3. |
| GO:0005515 protein binding | IPI PMID:22437499 Structure of the mitotic checkpoint complex. | MODIFY | Summary: IntAct IPI from the 2.3 A crystal structure of the S. pombe MCC (Chao et al. 2012, PDB 4AEZ, Mad3 residues 1-223), in which closed Mad2 (O14417) stabilises the complex by positioning the Mad3 KEN-box degron to bind Cdc20/Slp1. Reason: Bare protein binding is uninformative. This paper defines the Mad2-Mad3-Cdc20 mitotic checkpoint complex at atomic resolution, so the informative resolution of the Mad3-Mad2 contact is complex membership (GO:0033597), which PomBase and ComplexPortal already annotate from this paper; the inhibitory output of the complex is captured by the GO:1990948 rows. Proposed replacements: mitotic checkpoint complex Supporting Evidence: PMID:22437499 Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. PMID:22437499 Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. |
| GO:0005515 protein binding | IPI PMID:31257143 The Bub1-TPR Domain Interacts Directly with Mad3 to Generate... | REMOVE | Summary: PomBase IPI from Leontiou et al. 2019 showing that the Bub1 (SPCC1322.12c) TPR domain is necessary and sufficient to recruit Mad3 to a tetO array in vivo and that purified Bub1-TPR and GFP-Mad3-TPR form a stable complex by size-exclusion chromatography without phosphorylation. Reason: Bare protein binding is uninformative about what Mad3 does. The interaction is genuine, direct and functionally important (it is how Mad3 reaches Bub1/kinetochores in an organism whose Mad3 lacks a Bub3-binding GLEBS motif), but on the Mad3 side it is a recruitment event rather than a distinct molecular function; the adaptor/scaffold activity belongs to Bub1 and is captured on the bub1 review as protein-macromolecule adaptor activity, while Mad3's own function is ubiquitin ligase inhibitor activity within the MCC. Removal does not dispute the interaction, which is recorded in the notes and in the kinetochore rows. Supporting Evidence: PMID:31257143 Size exclusion chromatography profiles demonstrate that simply mixing the two proteins together in vitro was sufficient to produce a stable Bub1-Mad3 TPR complex. PMID:31257143 SpMad3 interacts with kinetochores in a Bub1-dependent fashion, but SpMad3 lacks a Bub3 binding domain of its own [36, 42]. file:SCHPO/mad3/mad3-deep-research-falcon.md The Bub1 TPR domain is therefore necessary and sufficient for recruitment and can bind Mad3 directly. |
| GO:0005634 nucleus | EXP PMID:22660415 MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 t... | ACCEPT | Summary: UniProt EXP nucleus annotation from Yamagishi et al. 2012 (abstract-only cache), who imaged SAC components including Mad3 at kinetochores in spc7 MELT mutants; UniProt records Nucleus and Chromosome, centromere, kinetochore from this paper and PMID:11909965. Reason: Correct. Fission yeast undergoes a closed mitosis and Mad3 acts on nuclear kinetochores and on the nucleoplasmic MCC-APC/C; Mad3-GFP gives diffuse nuclear fluorescence with kinetochore foci (PMID:11909965), and Sczaniecka et al. 2008 place MCC formation and APC/C binding in the nucleoplasm (PMID:18556659). The abstract of the cited paper states that the phospho-Spc7-dependent Bub1-Bub3 platform is required for Mad1-Mad2-Mad3 localisation, consistent with the annotation. Supporting Evidence: PMID:22660415 This phosphorylation promotes the in vitro binding to the Bub1-Bub3 complex, which is required for kinetochore-based SAC activation (Mad1-Mad2-Mad3 localization) and chromosome alignment. PMID:18556659 We propose that MCC formation and APC/C binding take place in the nucleoplasm every mitosis, independently of kinetochore-based SAC signaling. |
| GO:0005634 nucleus | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: High-throughput ORFeome YFP-localisation screen (Matsuyama et al. 2006) scoring Mad3 as nuclear. Reason: Consistent with the gene-specific imaging of endogenous Mad3-GFP (nuclear, with kinetochore foci; PMID:11909965) and with the nuclear site of MCC-APC/C action in this closed-mitosis organism (PMID:18556659). Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. PMID:18556659 We propose that MCC formation and APC/C binding take place in the nucleoplasm every mitosis, independently of kinetochore-based SAC signaling. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000361608 places nuclear localisation on the Bub1/Mad3 family; descendant evidence includes S. pombe mad3 itself (SPCC1795.01c), S. pombe bub1, budding-yeast BUB1/MAD3, human BUB1/BUB1B and the worm orthologues. Reason: The target is inside the clade that inherited the function and has its own experimental nuclear localisation (PMID:22660415, PMID:16823372), which is why it appears in its own WITH/FROM; this is the expected marker that experimental grounding exists on the target. Nuclear localisation is where Mad3 functions. Supporting Evidence: PMID:11909965 Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Automated nucleus assignment from the UniProt subcellular-location line (Nucleus) and an ARBA rule. Reason: Consistent with the experimental nucleus annotations (EXP, HDA) and with the nuclear site of MCC-APC/C action (PMID:18556659). Supporting Evidence: PMID:18556659 We propose that MCC formation and APC/C binding take place in the nucleoplasm every mitosis, independently of kinetochore-based SAC signaling. |
| GO:0005829 cytosol | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | KEEP AS NON CORE | Summary: High-throughput ORFeome YFP-localisation screen (Matsuyama et al. 2006) in which Mad3 was also scored in the cytosol, presumably reflecting a diffuse cytoplasmic signal from the plasmid-expressed fusion in interphase cells. Reason: Not wrong, but not where Mad3 functions. Gene-specific imaging of endogenous Mad3-GFP reports nuclear signal with kinetochore foci during mitosis (PMID:11909965), MCC assembly and APC/C binding occur in the nucleoplasm of this closed-mitosis organism (PMID:18556659), and no cytosolic activity of Mad3 has been described. A soluble cytoplasmic pool is plausible (Mad3 has no described NLS and is 36 kDa), so the row is kept as a non-core localisation rather than removed. Supporting Evidence: PMID:16823372 Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. PMID:18556659 We propose that MCC formation and APC/C binding take place in the nucleoplasm every mitosis, independently of kinetochore-based SAC signaling. |
| GO:0007094 mitotic spindle assembly checkpoint signaling | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000361607 places mitotic spindle assembly checkpoint signaling on the Bub1/Mad3 family, seeded by experimental annotations from plant, fly, fission and budding yeast, human and worm Bub1 and Mad3/BubR1 proteins, including S. pombe mad3 itself. Reason: This is the core function of Mad3 and is directly demonstrated for the target. mad3 delta cells cannot arrest when microtubules are depolymerised or absent, precociously separate sister chromatids, and are required for Mad2- or Mph1-overexpression arrests (PMID:11909965, PMID:18556659, PMID:22521786). The appearance of SPCC1795.01c in its own WITH/FROM is the expected marker that this experimental evidence contributed to the IBD placement. Supporting Evidence: PMID:11909965 We have identified the fission yeast mad3 + gene and found it to be nonessential for normal cell division, but required for spindle checkpoint function. PMID:22521786 By analysing the area under this curve it can be seen that, during the 6 hour time course, almost all of the Ξmad3 cells (>90%) have exited mitosis, as expected for a checkpoint mutant (Figure 4D). |
| GO:0007094 mitotic spindle assembly checkpoint signaling | IEA GO_REF:0000120 | ACCEPT | Summary: Automated assignment from the InterPro Bub1/Mad3 family signature (IPR015661) and an ARBA rule. Reason: The InterPro2GO mapping is correct for this protein; the term is experimentally established for the target (PMID:11909965, PMID:18556659, PMID:26882497). Supporting Evidence: PMID:11909965 We have identified the fission yeast mad3 + gene and found it to be nonessential for normal cell division, but required for spindle checkpoint function. |
| GO:0007094 mitotic spindle assembly checkpoint signaling | IMP PMID:11909965 Fission yeast Mad3p is required for Mad2p to inhibit the ana... | ACCEPT | Summary: Founding characterisation of fission-yeast mad3 (Millband and Hardwick 2002). mad3 delta cells are benomyl-hypersensitive, fail to arrest with microtubule defects, rereplicate DNA in a cut7-24 background, lose minichromosomes at elevated rates and precociously separate sister chromatids; Mad3 is required for overexpressed Mad2 to arrest cells in metaphase whereas Mad1, Bub1 and Bub3 are not. Reason: Direct loss-of-function evidence that Mad3 is a core, downstream component of mitotic spindle assembly checkpoint signalling in S. pombe, acting between Mad2 and the APC/C. Core function. Supporting Evidence: PMID:11909965 Cells deleted for mad3 + are hypersensitive to microtubule poisons such as benomyl, have elevated levels of minichromosome loss rates, and rereplicate their DNA when spindle function is compromised. PMID:11909965 Furthermore, direct visualization of sister separation events revealed that mad3 Ξ cells precociously separate their sister chromatids when challenged with microtubule poisons. PMID:11909965 We demonstrate that Mad3p function is required for the overexpression of Mad2p to result in a metaphase arrest. file:SCHPO/mad3/mad3-deep-research-falcon.md Only 8% of *mad3Ξ* cells underwent the short-spindle arrest caused by Mad2 overexpression, versus approximately 70% in checkpoint-competent comparison cultures. |
| GO:0007094 mitotic spindle assembly checkpoint signaling | IMP PMID:22521786 Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 b... | ACCEPT | Summary: Shepperd et al. 2012 (Mph1 phosphorylation of Spc7 MELT motifs recruits Bub1-Bub3) use mad3 delta as the checkpoint-null control in nda3-KM311 arrests, where >90% of mad3 delta cells exit mitosis, and show that the anaphase delay of spc7-MELT mutants is Mad3-dependent. Reason: Direct IMP evidence in the target organism that Mad3 is required for checkpoint arrest and for the SAC-dependent mitotic delay caused by kinetochore attachment defects. Core function. Supporting Evidence: PMID:22521786 Using Ξmad3 as a checkpoint null mutant control we saw a wave of cells with Nsk1-GFP at spindle poles. PMID:22521786 Consistent with this hypothesis, we found that the delay in anaphase onset observed in these mutants is dependent on Mad3 (Figure 4C). |
| GO:0007094 mitotic spindle assembly checkpoint signaling | IMP PMID:26882497 Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC... | ACCEPT | Summary: Zich et al. 2016 identify Mad3 as an Mph1 (Mps1) substrate; non-phosphorylatable mad3-C9A (C-terminal sites flanking KEN2) is targeted to kinetochores and assembled into MCC but shows reduced APC/C binding and cannot maintain checkpoint arrests, synergistically with mad2-S92A. Reason: Direct IMP evidence that the Mph1-phosphorylated form of Mad3 is required to maintain mitotic spindle assembly checkpoint arrests in S. pombe. Core function. Supporting Evidence: PMID:26882497 We conclude that reduced phosphorylation of either Mad3p or Mad2p impairs maintenance of a checkpoint arrest, and that reduced phosphorylation of both proteins further reduces their ability to maintain a robust checkpoint response. PMID:26882497 These data strongly support a model in which Mps1Mph1-dependent phosphorylation of both Mad2p and Mad3p are required to stabilise their APC/C binding and thus to maintain the MCC-APC/C complex and spindle checkpoint arrest. |
| GO:0007094 mitotic spindle assembly checkpoint signaling | NAS PMID:18556659 The spindle checkpoint functions of Mad3 and Mad2 depend on ... | ACCEPT | Summary: ComplexPortal NAS row citing Sczaniecka et al. 2008, which in fact provides direct evidence (KEN-box mutants behave like mad3 delta in nda3-KM311, Mad2-overexpression and Mph1-overexpression arrest assays). Reason: Correct core-function term; the cited paper supports it with genetic data stronger than the NAS code implies. Supporting Evidence: PMID:18556659 Both KEN mutants behaved like the mad3Ξ strain in this assay; cells failed to arrest with condensed chromosomes, mis-segregated their DNA, displayed the cut (cells untimely torn) phenotype (Fig. 5A), and lost viability rapidly after 3 h at 18 Β°C (Fig. 5B). |
| GO:0010997 anaphase-promoting complex binding | IPI PMID:18556659 The spindle checkpoint functions of Mad3 and Mad2 depend on ... | ACCEPT | Summary: Sczaniecka et al. 2008 purify APC/C via Lid1(Apc4)-TAP from nda3-arrested cells and recover Mad3, Mad2 and Slp1/Cdc20 (SPAC19G12.01c) by mass spectrometry; Mad3-GFP, Mad2 and Slp1-HA co-fractionate with Lid1-TAP on sucrose gradients, and the Mad3-APC/C association is abolished in slp1-362, in mad2 delta and by the mad3-KEN20AAA mutation. Reason: A specific, well-supported binding term describing the stable Slp1- and KEN1-dependent association of Mad3 with the core APC/C that underlies its inhibitory function; the interaction partner Slp1 is the APC/C subunit through which Mad3 engages the complex. Complementary to the ubiquitin ligase inhibitor activity rows and retained as core. Supporting Evidence: PMID:18556659 Pools of Mad2, Mad3-GFP, and Cdc20/Slp1-HA were seen to co-fractionate with Lid1-TAP (Fig. 2C). PMID:18556659 Thus, Mad3 is dependent on its own N-terminal KEN box, Mad2, and Cdc20 for APC/C association. |
| GO:0032991 protein-containing complex | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning assignment of the root complex term. Reason: The root term is uninformative. The complex Mad3 belongs to is the mitotic checkpoint complex (Mad3-Mad2-Slp1), established by co-immunoprecipitation, APC/C co-purification and a crystal structure (PMID:11909965, PMID:18556659, PMID:22437499) and already annotated as GO:0033597 by IDA/IPI; the same resolution was applied to the budding-yeast MAD3 review. Proposed replacements: mitotic checkpoint complex Supporting Evidence: PMID:18556659 Mad3 is part of the MCC, which in fission yeast is comprised exclusively of Mad3, Mad2, and Cdc20. |
| GO:0033597 mitotic checkpoint complex | IDA PMID:18556659 The spindle checkpoint functions of Mad3 and Mad2 depend on ... | ACCEPT | Summary: Sczaniecka et al. 2008 immunoprecipitate Mad3-GFP and Slp1-HA from mitotic extracts and show Mad3, Mad2 and Slp1 form the fission-yeast MCC; the N-terminal KEN box (KEN20) is required for MCC formation, whereas Bub3 (which S. pombe Mad3 cannot bind, lacking a GLEBS motif) is not a stable MCC subunit. Reason: Direct evidence for MCC membership in the target organism; the GO definition lists the budding-yeast and mammalian compositions, and the fission-yeast complex lacking Bub3 is a legitimate variant of the same complex. Core. Supporting Evidence: PMID:18556659 Mad3 is part of the MCC, which in fission yeast is comprised exclusively of Mad3, Mad2, and Cdc20. PMID:18556659 Thus, although Bub3 is important for the kinetochore targeting of Mad3 (28, 38), Bub3 is not stably associated with either MCC or APC/C in fission yeast (Fig. 1A and data not shown). |
| GO:0033597 mitotic checkpoint complex | IDA PMID:22437499 Structure of the mitotic checkpoint complex. | ACCEPT | Summary: Crystal structure of the S. pombe MCC (Mad2, Mad3 and Cdc20/Slp1; PDB 4AEZ), showing how C-Mad2 positions the Mad3 KEN box to occupy the Cdc20 degron-binding site and how the complex displaces Cdc20 from the Apc10 D-box co-receptor. Reason: Definitive structural evidence of MCC membership for the target protein. Core. Supporting Evidence: PMID:22437499 Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. |
| GO:0033597 mitotic checkpoint complex | IPI PMID:22437499 Structure of the mitotic checkpoint complex. | ACCEPT | Summary: ComplexPortal (CPX-3924) IPI row for MCC membership derived from the Chao et al. 2012 crystal structure. Reason: Same evidence as the IDA row above; duplicate rows with different codes are acceptable. Core. Supporting Evidence: PMID:22437499 Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. |
| GO:0045841 negative regulation of mitotic metaphase/anaphase transition | IPI PMID:18556659 The spindle checkpoint functions of Mad3 and Mad2 depend on ... | ACCEPT | Summary: Sczaniecka et al. 2008 show that Mad3, through its KEN20 box, binds Slp1/Cdc20 (SPAC19G12.01c) and the APC/C and that KEN-box mutants cannot delay anaphase; cells fail to arrest, mis-segregate DNA and cut. Reason: Mad3 does the work of this process directly, by occupying the substrate-recognition site of Slp1 within the MCC so that securin and cyclin are not ubiquitinated. The term is an ancestor of GO:0007094 in the current ontology, so it adds no coverage beyond the checkpoint rows, but it is correct and specifically supported; retained as core. Supporting Evidence: PMID:18556659 Mutation of an N-terminal Mad3 KEN box prevents stable Mad3-Cdc20 binding, as well as Mad3 and Mad2 binding to APC/C, and abrogates the spindle checkpoint. PMID:18556659 Our findings demonstrate the importance of Mad3, and in particular, its conserved N-terminal KEN box, for MCC formation, for APC/C binding, and for inhibition of anaphase by the SAC. |
| GO:0140678 molecular function inhibitor activity | EXP PMID:22437499 Structure of the mitotic checkpoint complex. | MODIFY | Summary: PomBase contributes_to annotation from the MCC crystal structure, recording that Mad3, as part of the MCC, inhibits APC/C by obstructing degron-recognition sites on Cdc20 and displacing Cdc20 from the Apc10 D-box co-receptor. Reason: The term is correct but unnecessarily general. The inhibited target is the APC/C, an E3 ubiquitin ligase, and the paper's own framing is MCC-mediated APC/C inhibition; the child term ubiquitin ligase inhibitor activity (GO:1990948, a descendant of GO:0140678) is already annotated to Mad3 by IDA from the in vitro securin-ubiquitination assays of Zich et al. 2016 (PMID:26882497) and is the term used for budding-yeast Mad3 and human BUB1B in this repository. The contributes_to qualifier remains appropriate, since inhibition is a property of the Mad3-Mad2-Slp1 complex. Proposed replacements: ubiquitin ligase inhibitor activity Supporting Evidence: PMID:22437499 The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. PMID:26882497 We conclude that mimicking phosphorylation of Mad3p near its C-terminal KEN2 box can directly improve its ability to inhibit Cdc20Slp1-APC/C activity. |
| GO:1990948 ubiquitin ligase inhibitor activity | IDA PMID:26882497 Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC... | ACCEPT | Summary: Zich et al. 2016 reconstitute fission-yeast APC/C ubiquitination of securin (Cut2) with purified E1, E2s, APC/C and Slp1, and show that recombinant Mad3 inhibits the reaction and that Mad3 C-terminal phosphomimetic mutants are progressively more potent inhibitors (0, 3, 4 and 7 D/E residues). Reason: Direct in vitro demonstration that Mad3 inhibits the E3 ubiquitin ligase activity of APC/C-Slp1, which is the molecular function underlying its checkpoint role. Core function. Supporting Evidence: PMID:26882497 These experiments demonstrate that the Mad3 phosphomimics are better in vitro APC/C inhibitors than the recombinant wild-type Mad3 protein and that they display a graded increase in potency (c.f. 0,3,4 and 7 phospho-mimicking D/E residues). PMID:26882497 We conclude that mimicking phosphorylation of Mad3p near its C-terminal KEN2 box can directly improve its ability to inhibit Cdc20Slp1-APC/C activity. |
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Download this section (compressed HTML)Q: Fission-yeast Mad3 lacks a GLEBS motif and Bub3 is not a stable MCC subunit, yet Bub3 is required for Mad3 kinetochore recruitment. Should the GO definition of mitotic checkpoint complex (GO:0033597), which lists Bub3 as a subunit in yeast and mammals, be amended to accommodate the three-subunit S. pombe complex, or should a taxon-specific note be added?
Q: Is the C-terminal ABBA/KEN2 region of Mad3 engaging a second Slp1 molecule in vivo (as proposed from motif mutagenesis by Sewart and Hauf 2017), and does that second Cdc20-binding event represent a distinct inhibitory mechanism from KEN1-mediated MCC formation?
Q: The 2024 reports linking Pmk1 MAPK signalling to stress-induced Slp1 turnover implicate Mad3; is this a separable function of Mad3 (e.g. promoting Slp1 autoubiquitination, as budding-yeast Mad3 does) or simply a manifestation of canonical MCC-mediated APC/C inhibition?
Experiment: Map the Bub1-TPR-binding surface on the Mad3 TPR domain by mutagenesis guided by the 4AEZ structure, confirm loss of binding by size-exclusion chromatography with purified TPR domains, then test whether combining the binding-defective allele with the mad3-7D/E phosphomimetic restores maintenance of nda3-KM311 and carbendazim checkpoint arrests and MCC-APC/C co-purification.
Hypothesis: Mad3 C-terminal Mph1 phosphorylation and the Bub1-TPR interaction act in the same kinetochore-based activation step, so that a Bub1-TPR-binding-defective Mad3 is rescued by phosphomimetic C-terminal mutations.
Type: genetic
Experiment: Use the reconstituted S. pombe APC/C assay (purified E1, Ubc1/4/11, APC/C, Slp1) with wild-type, KEN20AAA, KEN271AAA and phosphomimetic Mad3 in the presence and absence of closed Mad2, and measure Slp1 autoubiquitination versus Cut2 ubiquitination, to determine whether Mad3 promotes Slp1 turnover independently of substrate inhibition.
Hypothesis: Fission-yeast Mad3, like budding-yeast Mad3-Bub3, stimulates Slp1 autoubiquitination by APC/C, which would provide the mechanism for Mad3-dependent stress-induced Slp1 turnover.
Type: biochemical
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