Ste9 (also called Srw1) is the fission yeast member of the Cdh1/Hct1/Fizzy-related subfamily of WD40 beta-propeller coactivators of the anaphase-promoting complex/cyclosome (APC/C), the multisubunit RING E3 ubiquitin ligase that clears mitotic regulators at the end of mitosis and through G1. Ste9 is not itself catalytic: in G1, when Cdc2-cyclin kinase activity is low, the dephosphorylated protein docks onto the APC/C core and switches the ligase on toward its substrates, and purified fission yeast APC/C supplemented with Ste9 ubiquitylates the mitotic cyclin Cdc13 and securin Cut2 in vitro. In vivo, APC/C-Ste9 drives the ubiquitin-dependent destruction of the mitotic B-type cyclins Cdc13 and Cig1 in G1; it is not needed for the wave of cyclin destruction at mitotic exit, which is carried by the Cdc20-type coactivator Slp1, and it does not degrade the S-phase cyclin Cig2, although it restrains Cdc2-Cig2 activity genetically. By keeping Cdc13 low, Ste9 (together with the CDK inhibitor Rum1, with which it is partly redundant) holds cells in a pre-Start G1 state: it lengthens G1 in small cells, is required for G1 arrest upon nitrogen starvation and thus for mating and sexual differentiation, and prevents mitosis from being launched from G1. Cells lacking ste9 are viable and cycle normally in rich medium but are sterile, fail to arrest in G1 when starved, and are synthetically lethal with wee1 mutations because mitotic cyclin control is compromised; overexpression lowers Cdc13-associated kinase activity, blocks mitosis and induces an extra round of DNA replication. Ste9 is switched off outside G1 by Cdc2-Cdc13 phosphorylation on multiple Cdk consensus sites (Ser62, Thr98, Thr177, Ser214), which both dissociates it from the APC/C and targets it for proteolysis; its reactivation in pre-Start G1 requires PP2A-B56 (Par1)-dependent dephosphorylation, and the replication checkpoint kinase Cds1 also phosphorylates and inhibits it during S-phase arrest, protecting the MBF activator Rep2 from APC/C-Ste9-mediated destruction. The protein is nuclear, where the APC/C acts on its substrates, and it is heavily phosphorylated and inactive through most of meiosis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: High-throughput ORFeome localisation (Matsuyama et al.) in which each fission yeast ORF was tagged with YFP and imaged; Ste9/Srw1 was scored as nuclear, and UniProt records this as the sole subcellular-location evidence. This is the compartment in which the APC/C core acts on Cdc13 and where the dephosphorylated Ste9-APC/C interaction occurs in G1. Reason: Direct, if high-throughput, observation of the tagged protein in this species. It is consistent with the nuclear localisation of the orthologous budding yeast Cdh1 during G1 and with the nuclear site of APC/C-dependent cyclin destruction, so it is the location of the core function. The dedicated Ste9 literature does not add cell-cycle-resolved imaging, which is noted as a gap rather than as a reason to doubt this row. 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:10921876 In G 1 , when cdc2โcyclin kinase activity is low, unphosphorylated ste9/srw1 interacts with APC/C. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location keyword mapping (Nucleus), itself derived from the ORFeome YFP localisation of Matsuyama et al. (PMID:16823372). Reason: Electronic transfer of a curated UniProt location that agrees with the experimental HDA row and with the nuclear site of APC/C-Ste9 activity. 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. |
| GO:0010997 anaphase-promoting complex binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro Cdc20/Fizzy family (IPR033010) mapping to APC/C binding. Blanco et al. showed directly in this species that Ste9 co-purifies with the APC/C only in G1, when it is dephosphorylated, and that Cdc2-cyclin phosphorylation in S/G2 dissociates it from the complex; Kimata et al. later reconstituted APC/C activation by in vitro translated Ste9 with purified fission yeast APC/C. Reason: Family-level inference that matches the experimentally established, Cdk-regulated docking of Ste9 onto the APC/C core in fission yeast. This binding is the first step of the core coactivator function and is not a generic protein-binding claim. Supporting Evidence: PMID:10921876 In G 1 , when cdc2โcyclin kinase activity is low, unphosphorylated ste9/srw1 interacts with APC/C. PMID:10921876 APC/Cโste9 interaction occurs only in G 1 when ste9 is in its dephosphorylated form. |
| GO:0031145 anaphase-promoting complex-dependent catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000460086 (Cdc20/Fizzy family) places APC/C-dependent proteolysis at the ancestor of both the Cdc20 and Cdh1 subfamilies; ste9 itself (PomBase:SPAC144.13c) is among the experimental sources, as expected for a gene with its own IDA/IMP for the term. In fission yeast APC/C-Ste9 degrades Cdc13 and Cig1 in G1 and ubiquitylates Cdc13, Cut2 and Mes1 in vitro. Reason: Sound phylogenetic placement that agrees with direct genetic and biochemical evidence in this species. The inherited function is the core function of the gene. Supporting Evidence: PMID:10921876 We show that APC ste9/srw1 specifically promotes the degradation of mitotic cyclins cdc13 and cig1 but not the S-phase cyclin cig2. PMID:21389117 Collectively, these data suggest that Slp1, Ste9, Fzr1/Mfr1, and Fzr2 can all activate APC/C and ubiquitylate cyclin B for destruction, although their activities seem to be regulated differently during the cell cycle. |
| GO:0031145 anaphase-promoting complex-dependent catabolic process | IDA PMID:21389117 Mes1 controls the meiosis I to meiosis II transition by dist... | ACCEPT | Summary: Kimata et al. purified fission yeast APC/C (Lid1-TAP) and reconstituted ubiquitylation of Cut2 and Cdc13 with in vitro translated coactivators: Ste9 gave the strongest stimulation of all five coactivators. In Xenopus egg extracts depleted of endogenous APC/C and Fizzy, added fission yeast APC/C plus Ste9 drove Cdc13 destruction in interphase but not mitotic extracts, and Ste9 also supported Mes1 ubiquitylation and destruction. Reason: Direct biochemical demonstration, with purified components from this species, that Ste9 activates APC/C-dependent ubiquitylation and proteasomal destruction of cyclin B. This is the core process of the gene. The paper's title concerns meiotic coactivators, but the full text (cached) contains the Ste9 assays explicitly. Supporting Evidence: PMID:21389117 Ste9 showed the most robust stimulation of ubiquitylation for both substrates. PMID:21389117 In contrast, in interphase egg extracts, Slp1 was unable to activate Cdc13 destruction whereas Ste9 stimulated Cdc13 destruction efficiently (Figure 5C, bottom). |
| GO:0031145 anaphase-promoting complex-dependent catabolic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning rule assigning APC/C-dependent catabolic process, duplicating the IBA and the experimental IDA/IMP rows for the same term. Reason: Concordant with strong direct evidence in this species that APC/C-Ste9 promotes the ubiquitin-dependent destruction of Cdc13 and Cig1. Supporting Evidence: PMID:10921878 We conclude that the Fizzy-related protein srw1p is a G 1 -specific promoter of mitotic cyclin cdc13p degradation. |
| GO:0031145 anaphase-promoting complex-dependent catabolic process | IMP PMID:21389117 Mes1 controls the meiosis I to meiosis II transition by dist... | ACCEPT | Summary: Mutant-phenotype arm of Kimata et al.: in a Puhp1-HA-ste9 strain in which Ste9 expression is repressed during meiosis, the APC/C substrate Mes1 was significantly stabilised at the end of meiosis II, pointing to a role of Ste9 in Mes1 destruction at meiotic exit. Ste9 was not needed for the meiosis I to meiosis II transition, and it is heavily phosphorylated (inactive) through most of meiosis. Reason: The IMP records a genuine dependence of APC/C substrate destruction on Ste9 in vivo, here at the end of meiosis II, and complements the vegetative G1 evidence for Cdc13 and Cig1. Ste9 is one of several coactivators acting on Mes1 at that stage, so the phenotype is partial, but that does not weaken the process assignment. Supporting Evidence: PMID:21389117 In all the mutants (ste9, fzr2ฮ, and fzr3ฮ), Mes1 levels were significantly increased at the end of meiosis II (5.5โ6 h) rather than in early meiosis, pointing to their roles in Mes1 destruction in meiosis II. PMID:21389117 Because Ste9 is essential for nitrogen starvationโinduced G1 arrest, a prerequisite for meiotic entry, we created Puhp1-HA-ste9 mutants, in which the expression of HA-tagged Ste9 is under the control of the uhp1 promoter and is repressed in meiosis. |
| GO:0031568 mitotic G1 cell size control checkpoint signaling | IGI PMID:9398669 A WD repeat protein controls the cell cycle and differentiat... | ACCEPT | Summary: IGI with cig2 (PomBase:SPCC18B5.03). Yamaguchi et al. isolated srw1 as a multicopy suppressor of hyperactive Cdc2 and showed that srw1-deleted cells fail to arrest in G1 on nitrogen starvation and are sterile, that fertility is restored by deleting the G1/S cyclin Cig2 or by mildly inactivating Cdc2, and that Cdc13 is not degraded in the disruptant on starvation. This is PomBase's term for the pre-Start G1 delay that holds cells below the size threshold for Start, the same term PomBase uses for rum1, cig2, cdc2 and wee1. Reason: Ste9 executes the cyclin-destruction arm of pre-Start G1 control: by removing Cdc13 it keeps Cdc2 activity below the Start threshold so that small or starved cells lengthen or arrest in G1, and Blanco et al. explicitly frame this as lengthening G1 until cells reach the minimum size for DNA replication. The genetic interaction with cig2 identifies the Cdc2-cyclin activity that Ste9 antagonises. The 'signaling' wording is a PomBase convention for this pathway rather than a claim that Ste9 is a signal transducer, and the curator had the full text; the function is the core physiological role of the gene. Supporting Evidence: PMID:9398669 When starved for nitrogen source, they fail to effectively arrest in G1 and die of accelerated mitotic catastrophe if regulation of p34(cdc2)/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. PMID:9398669 Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34(cdc2). PMID:10921876 This is important for small cells that had to lengthen the G 1 -phase until they reach the minimum cell size required to initiate DNA replication or to prevent entry into mitosis from G 1 . |
| GO:0031568 mitotic G1 cell size control checkpoint signaling | IMP PMID:9571240 Fission yeast Ste9, a homolog of Hct1/Cdh1 and Fizzy-related... | ACCEPT | Summary: Kitamura et al. showed that ste9 mutants are sterile because they cannot arrest in G1 on starvation, that ste9 cdc10-ts cells arrested in pre-Start G1 fail to lower Cdc13 and undergo abortive mitosis from G1, that ste9 is synthetically lethal with wee1, and that Ste9 overexpression lowers Cdc13 and H1 kinase activity, blocks mitosis and triggers re-replication. Only the abstract is cached, but it states these findings explicitly. Reason: Loss and gain of function in this species both place Ste9 in the pre-Start G1 control that keeps mitotic CDK activity low until cells pass Start; this is the core physiological output of APC/C-Ste9-dependent Cdc13 destruction. The IMP is retained as a core process alongside the more mechanistic GO:2000134 row from the same paper. Supporting Evidence: PMID:9571240 The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. PMID:9571240 In the double mutants of ste9 cdc10(ts), cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. file:SCHPO/ste9/ste9-deep-research-falcon.md Wild-type fission yeast responds to nitrogen removal by undergoing approximately two divisions and arresting in G1. ste9/srw1-pathway mutants fail to establish this arrest correctly; they can execute an additional S phase and accumulate with G2 DNA content. |
| GO:0097027 ubiquitin-protein transferase activator activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro Cdc20/Fizzy family (IPR033010) mapping to ubiquitin-protein transferase activator activity, the parent of the more specific ubiquitin ligase activator activity (GO:1990757) that ste9 carries by IBA, IDA and IMP. Reason: Broader than the IBA/IDA term but correct: purified fission yeast APC/C ubiquitylates Cut2 and Cdc13 only when an E2 and a coactivator are supplied, and Ste9 gives the strongest stimulation. Handled as in the yeast CDH1 and human FZR1 reviews, where the parent-level InterPro mapping is accepted alongside the specific term. Supporting Evidence: PMID:21389117 Ste9 showed the most robust stimulation of ubiquitylation for both substrates. |
| GO:1905786 positive regulation of anaphase-promoting complex-dependent catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000460086; ste9 itself (PomBase:SPAC144.13c) is among the experimental sources, together with budding yeast CDH1 and CDC20 and Drosophila fizzy. As coactivator, Ste9 activates APC/C-dependent proteolysis: its loss stabilises Cdc13 and Cig1 in G1, its overexpression depletes Cdc13, and non-phosphorylatable Ste9 accelerates Cdc13 turnover. Reason: Sound phylogenetic placement that agrees with direct genetic and biochemical evidence in this species; the positive-regulation term is the conventional process pairing for GO:1990757 across the Cdc20/Cdh1 family. Supporting Evidence: PMID:10921878 Consistent with this view, cdc13p was degraded more quickly in unphosphorylatable srw1 mutant cells (Figure 7 E), indicating that phosphorylation leads to the inhibition of srw1p activity. |
| GO:1905786 positive regulation of anaphase-promoting complex-dependent catabolic process | IMP PMID:10921876 APC(ste9/srw1) promotes degradation of mitotic cyclins in G(... | ACCEPT | Summary: Blanco et al. showed that in ste9-deleted cells Cdc13 and Cig1 are still degraded at the metaphase-anaphase transition but cannot be cleared in G1 (cdc10-arrested or nitrogen-starved cells), that Cig2 is not an APC/C-Ste9 substrate, and that Ste9 is phosphorylated by Cdc2 at multiple sites, which both triggers its proteolysis and dissociates it from the APC/C. Reason: Direct loss-of-function evidence in this species that Ste9 is required for APC/C-mediated destruction of mitotic cyclins specifically in G1, the defining function of the Cdh1-type coactivator. Supporting Evidence: PMID:10921876 APC ste9/srw1 is not necessary for the proteolysis of cdc13 and cig1 that occurs at the metaphaseโanaphase transition but it is absolutely required for their degradation in G 1 . PMID:10921876 ste9 is absolutely required to degrade mitotic cyclins completely when cells need to delay or to stop the cell cycle in G 1 . |
| GO:1905786 positive regulation of anaphase-promoting complex-dependent catabolic process | IMP PMID:10921878 Fission yeast Fizzy-related protein srw1p is a G(1)-specific... | ACCEPT | Summary: Yamaguchi, Okayama and Nurse showed that srw1p is required for Cdc13 degradation during G1 arrest but not at mitotic exit, that it is phosphorylated in vivo and in vitro by Cdc2-Cdc13 on four Cdk consensus sites (S62, T98, T177, S214), that the unphosphorylatable mutant is more stable (half-life >120 min versus 40 min), degrades Cdc13 faster and causes diploidisation, and that this is suppressed by extra Cdc13. Reason: Direct loss- and gain-of-function evidence that Ste9 promotes APC/C-dependent Cdc13 destruction in G1 and that Cdk phosphorylation inhibits this activity; a core process. Supporting Evidence: PMID:10921878 srw1p, a member of the Fizzy-related protein family in fission yeast, is required for the degradation of cdc13p mitotic cyclin B during G 1 arrest. PMID:10921878 Mutant forms of srw1 that could not be phosphorylated on the four Cdk consensus sites in srw1p were more stable than wild-type srw1p: the half-life of the unphosphorylatable srw1p was >120 min, compared with 40 min for wild-type srw1p (Figure 7 B). |
| GO:1990757 ubiquitin ligase activator activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000460086 assigns ubiquitin ligase activator activity to the Cdc20/Cdh1 family; the experimental sources include ste9 itself and all four other fission yeast coactivators (slp1, fzr1/mfr1, fzr2, fzr3) plus budding yeast CDH1 and CDC20. Purified fission yeast APC/C is inactive toward Cut2 and Cdc13 without a coactivator, and Ste9 is the strongest activator of the five in vitro. Reason: Core molecular function, supported by reconstitution with purified components from this species and by the G1-specific cyclin-destruction genetics. The IBA adds that the activity is inherited from the family ancestor rather than lineage-specific. Supporting Evidence: PMID:21389117 Ste9 showed the most robust stimulation of ubiquitylation for both substrates. PMID:21389117 Collectively, these data suggest that Slp1, Ste9, Fzr1/Mfr1, and Fzr2 can all activate APC/C and ubiquitylate cyclin B for destruction, although their activities seem to be regulated differently during the cell cycle. |
| GO:1990757 ubiquitin ligase activator activity | IDA PMID:21389117 Mes1 controls the meiosis I to meiosis II transition by dist... | ACCEPT | Summary: In vitro ubiquitylation with Lid1-TAP-purified fission yeast APC/C, E1, E2 and in vitro translated Ste9: Ste9 gave the strongest, D-box-dependent stimulation of Cut2 and Cdc13 ubiquitylation of the five coactivators, and in APC/C- and Fizzy-depleted Xenopus interphase extracts it drove Cdc13 destruction, whereas in mitotic extracts it was inactive, consistent with Cdk-dependent inhibition of Cdh1 orthologues. Reason: Direct biochemical demonstration of the core molecular function with purified components from this species. The activity is regulated exactly as expected for a Cdh1-type coactivator. Supporting Evidence: PMID:21389117 Ste9 showed the most robust stimulation of ubiquitylation for both substrates. PMID:21389117 As expected, Slp1, but not Ste9, was able to support Cdc13 destruction in mitotic extracts (Figure 5C, top), consistent with the established notion that CDH1 orthologues are inhibited by Cdk1-dependent phosphorylation in mitosis. |
| GO:1990757 ubiquitin ligase activator activity | IMP PMID:10921876 APC(ste9/srw1) promotes degradation of mitotic cyclins in G(... | ACCEPT | Summary: Blanco et al.: ste9 deletion abolishes G1 degradation of Cdc13 and Cig1 while leaving anaphase degradation intact; dephosphorylated Ste9 co-purifies with the APC/C in G1 and Cdc2 phosphorylation dissociates it. The mutant phenotype identifies Ste9 as the factor that switches the APC/C ligase on toward mitotic cyclins in G1. Reason: Loss-of-function evidence for the core activator function, later confirmed biochemically by Kimata et al. (IDA row). The IMP inference from stabilised substrates to activator activity is the standard one for APC/C coactivators. Supporting Evidence: PMID:10921876 APC ste9/srw1 is not necessary for the proteolysis of cdc13 and cig1 that occurs at the metaphaseโanaphase transition but it is absolutely required for their degradation in G 1 . PMID:10921876 In the rest of the cell cycle, phosphorylation of ste9/srw1 by cdc2โcyclin complexes both triggers proteolysis of ste9/srw1 and causes its dissociation from the APC/C. |
| GO:1990757 ubiquitin ligase activator activity | IMP PMID:10921878 Fission yeast Fizzy-related protein srw1p is a G(1)-specific... | ACCEPT | Summary: Yamaguchi et al.: srw1 deletion prevents Cdc13 degradation during G1 arrest but not at mitotic exit, and the unphosphorylatable srw1 mutant degrades Cdc13 faster, showing that Cdc2-Cdc13 phosphorylation inhibits srw1p's activity to promote Cdc13 turnover. Reason: Loss- and gain-of-function evidence for the core activator function in this species, consistent with the IDA reconstitution and the IBA. Supporting Evidence: PMID:10921878 It plays no role in mitotic exit but is necessary to block cell cycle progression in cells arrested in G 1 and has an important role in ensuring proper sexual differentiation that occurs from cells arrested in the G 1 phase of the cycle. PMID:10921878 Consistent with this view, cdc13p was degraded more quickly in unphosphorylatable srw1 mutant cells (Figure 7 E), indicating that phosphorylation leads to the inhibition of srw1p activity. |
| GO:2000134 negative regulation of G1/S transition of mitotic cell cycle | IGI PMID:9571240 Fission yeast Ste9, a homolog of Hct1/Cdh1 and Fizzy-related... | ACCEPT | Summary: IGI with cig2 (PomBase:SPCC18B5.03). Kitamura et al. found that ste9 sterility (failure to arrest in pre-Start G1) is partially suppressed by mutation of the major G1/S cyclin Cig2, and that Ste9 overexpression lowers Cdc13 and the associated H1 kinase activity while blocking mitosis and inducing an extra S phase. The term's definition (a pathway that inhibits cell cycle CDK activity to modulate the G1/S switch) matches this mechanism: Ste9 lowers Cdc2-cyclin B activity by destroying Cdc13 in G1, restraining passage through Start. Reason: The genetic interaction with cig2 and the Cdc13/H1 kinase data identify the CDK activity that Ste9 antagonises, and the mechanism (cyclin destruction lowering Cdc2 activity) is exactly what the term describes. Ste9 does the work of this process rather than merely being required for it, so the row is core. Supporting Evidence: PMID:9571240 Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. PMID:9571240 Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. PMID:9571240 Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase. |
| GO:0005680 anaphase-promoting complex | IDA PMID:10921876 APC(ste9/srw1) promotes degradation of mitotic cyclins in G(... | NEW | Summary: Proposed cellular-component annotation. Blanco et al. showed in fission yeast that dephosphorylated Ste9 associates with the APC/C in G1 (co-purification with the tagged core subunit Cut9) and that Cdc2-cyclin phosphorylation in S/G2 dissociates it, and Kimata et al. reconstituted the active APC/C-Ste9 holoenzyme from purified fission yeast APC/C and in vitro translated Ste9. ComplexPortal lists the srw1 variant of the APC/C (CPX-765). Reason: GO conventionally records Cdc20-family coactivators as part_of the APC/C: budding yeast CDH1 carries GO:0005680 by IPI and IBA, human FZR1 carries it, and fission yeast slp1 carries it by IBA from the same PAINT node (PTN000460086) that supplies ste9's other IBA rows, so the absence on ste9 is a propagation gap rather than a MOD convention. The direct co-association evidence in this species is in PMID:10921876, and the term is needed to express the in_complex of both core functions. This is a component assertion about the G1 holoenzyme, not a claim that Ste9 is a constitutive core subunit. Supporting Evidence: PMID:10921876 In G 1 , when cdc2โcyclin kinase activity is low, unphosphorylated ste9/srw1 interacts with APC/C. PMID:10921876 APC/Cโste9 interaction occurs only in G 1 when ste9 is in its dephosphorylated form. In S-phase and G 2 , ste9 becomes phosphorylated and it does not interact with APC/C. PMID:21389117 To reconstitute APC/C function activated by different coactivators, we immunodepleted endogenous Xenopus APC/C and Fizzy/Cdc20 from Xenopus egg extracts, then added back purified S. pombe APC/C, in vitro translated S. pombe coactivators, and analyzed the destruction of APC/C substrates. |
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Download this section (compressed HTML)Q: ste9 has no cellular-component annotation to GO:0005680 anaphase-promoting complex, whereas yeast CDH1 (IBA, IPI) and human FZR1 carry part_of APC/C and fission yeast slp1 carries it by IBA; Blanco et al. (PMID:10921876) co-purified dephosphorylated Ste9 with the APC/C in G1 and ComplexPortal lists an 'Anaphase-promoting complex, srw1 variant' (CPX-765). This review proposes the term as a NEW part_of annotation on that basis; is the current absence a deliberate PomBase convention for transiently bound coactivators, or a propagation gap that the Blanco co-association evidence (or the PAINT node) should fill?
Q: The two mitotic G1 cell size control checkpoint signaling rows (GO:0031568) rest on nitrogen-starvation G1 arrest, sterility and G1-length phenotypes. Does PomBase intend this term for the cyclin-destruction arm of pre-Start G1 control that Ste9 executes, or for a size-sensing signal upstream of it? If the former, would negative regulation of G1/S transition (GO:2000134, already annotated) be the preferred single term?
Q: Chu et al. (2009, MCB, doi:10.1128/MCB.00562-09) reported the MBF activator Rep2 as an APC/C-Ste9 substrate protected by Cds1 phosphorylation of Ste9 during replication arrest, and Stonyte et al. (2020, iScience) showed PP2A-B56/Par1 is needed for Ste9 dephosphorylation in pre-Start G1. Neither paper is reflected in the current GOA set; should Ste9 receive annotations for these regulatory relationships (e.g. via has_input on the APC/C process, or a replication-checkpoint response term)?
Experiment: Reconstitute ubiquitylation with Lid1-TAP-purified APC/C, E1/E2 and in vitro translated Ste9 (wild-type and WD40 degron-binding-surface mutants modelled on the budding yeast Cdh1 D-box pocket) using Cdc13, Cig1, Cig2 and degron-mutant substrates; complement with in vivo stability of Cig2 chimeras carrying the Cdc13 D-box in G1-arrested wild-type versus ste9 deletion cells.
Hypothesis: Ste9 recognises D-box/KEN-box degrons on Cdc13 and Cig1 through its WD40 propeller, and the exclusion of Cig2 from APC/C-Ste9-mediated destruction reflects the absence or masking of a Ste9-compatible degron rather than a lack of Ste9 activity in S phase.
Type: reconstituted ubiquitylation with degron and receptor-surface mutants
Experiment: Image endogenously tagged Ste9 together with an APC/C core subunit through synchronous cycles and nitrogen starvation in wild-type, par1 deletion and unphosphorylatable ste9-4A backgrounds, quantifying nuclear/cytoplasmic ratio and APC/C co-immunoprecipitation at each stage; test direct dephosphorylation of Cdc2-phosphorylated Ste9 by purified PP2A-B56 in vitro.
Hypothesis: Endogenous Ste9 is nuclear throughout the cycle but its association with the APC/C, not its location, is the cell-cycle-regulated step, with PP2A-B56/Par1 acting directly on the Cdk sites in pre-Start G1.
Type: live-cell imaging with phosphatase genetics and in vitro dephosphorylation
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