Mad3 KEN Boxes Mediate both Cdc20 and Mad3 Turnover, and Are Critical for the Spindle Checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble ‘degron’ signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation

CPF-Associated Phosphatase Activity Opposes Condensin-Mediated Chromosome Condensation

International audienceFunctional links connecting gene transcription and condensin-mediated chromosome condensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3′ end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does not affect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1Dis2 with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologue of vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatases PP1 and Ssu72

Mps1Mph1 kinase phosphorylates Mad3 to inhibit Cdc20Slp1-APC/C and maintain spindle checkpoint arrests

<div><p>The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1^Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1^Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors <i>in vitro</i>, demonstrating that Mad3p modification can directly influence Cdc20^Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1^Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.</p></div

Bub3p facilitates spindle checkpoint silencing in fission yeast

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3 Delta cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest

Swd2.2 facilitates the localization of PP1 phosphatase by interacting with the PNUTS homologue Ppn1.

<p><b>A.</b> The interaction of Ppn1 with PP1^Dis2 requires the three PP1-binding sites of Ppn1. GFP-tagged PP1^Dis2 was immuno-precipitated from cycling cells in the presence of Flag-tagged Ppn1 (Ppn1-3flag) or Flag-tagged Ppn1 lacking the three PP1-binding sites (<i>ppn1ΔPP1(ABC)</i>-3flag). Whole cell extracts (WCE) and the immuno-precipitated material (GFP IP) were analyzed by western blot. <b>B.</b> Asynchronous populations of the indicated strains (Ppn1-3flag or GFP-PP1^Dis2) were grown at 30°C and ChIP-qPCR was performed to analyze their enrichment at various sites along chromosomes. Enrichments were normalized to the values obtained at the RNA Polymerase I-transcribed 18S (mean ± standard deviation from 3 biological replicates). <b>C.</b> Lack of Ppn1 disrupts the nuclear localisation of GFP-tagged PP1^Dis2. GFP-PP1^Dis2 was imaged in dividing cells co-expressing (left panel) the mCherry-tagged nuclear envelope marker Mlp1 or (right panel) the mRFP-tagged nucleolar marker Fib1. <b>DE.</b> Asynchronous populations of the indicated strains were grown at 30°C and ChIP-qPCR was performed (mean ± standard deviation from 6 biological replicates). <b>F.</b> The protein stability of GFP-PP1^Dis2 was assessed by western blot in the various mutant backgrounds used in <b>DE</b>. Tubulin was used as a loading control. <b>G.</b> The interaction between Flag-tagged Swd2.2 and Pk-tagged PP1^Dis2 was analyzed by co-immunoprecipitation in protein extracts prepared from cycling cells in the presence or absence of Ppn1. Protein extracts were treated or not with RNase A prior to immuno-precipitation. Whole cell extracts (WCE) and the immuno-precipitated material (GFP IP) were analyzed by western blot. <b>H.</b> Asynchronous populations of the indicated strains were grown at 30°C and ChIP-qPCR was performed to analyze their enrichment at various sites along chromosomes. <b>I.</b> The protein stability of Flag-tagged Swd2.2 in the presence or absence of Ppn1 was assessed by western blot. Tubulin was used as a loading control. <b>J.</b> Asynchronous populations of the indicated strains were grown at 30°C. Protein extracts were prepared and analyzed by western blot using the indicated antibodies.</p

Swd2.2 antagonizes the association of Condensin with chromatin.

<p><b>A.</b> Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. <b>B.</b> Chromosome segregation in anaphase was visualized after tubulin staining in cells of the indicated genotypes grown for one generation at 34°C. <b>C</b>. Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells was scored. ***p<0,001 Wilcoxon - Mann Whitney. <b>D</b> and <b>E</b>. Same as <b>A</b> and <b>C</b>, except that in <b>E</b>, 3 independent experiments were performed. <b>F.</b> The indicated strains were grown at 34°C for 3 hours and ChIP-qPCR was performed to analyze the amount of Cut14-GFP cross-linked to chromatin (mean ± standard deviation from 6 biological replicates). See text for details for the statistical analysis of the experiments.</p

Lack of Swd2.2 reduces the association of the RNA Pol III transcription machinery with chromatin.

<p><b>A.</b> Asynchronous populations of the indicated strains were grown at 30°C and ChIP-qPCR was performed to analyze the amount of Swd2.2-3flag cross-linked to chromatin (mean ± standard deviation from 4 biological replicates). <b>BC.</b> ChIP qPCR analysis of the indicated strains grown at 30°C. Mean ± standard deviation from 5 biological replicates. *<0,05; **<0,01; Wilcoxon - Mann Whitney. <b>D.</b> Western blot analysis of total protein extracts of the indicated strains. Tubulin (TAT1 antibody) is used as a loading control. <b>E.</b> qRT-PCR analysis of sfc6 expression in swd2.2+ and <i>swd2.2Δ</i> cells (mean ± standard deviation from 3 biological replicates) <b>F.</b> Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures.</p

Swd2.2, Ppn1 and PP1^Dis2 associate as a protein module to the CPF.

<p><b>A.</b> The interaction between Flag-tagged Swd2.2 and GFP-tagged Pfs2 was analyzed by co-immunoprecipitation in protein extracts prepared from cycling cells in the presence or absence of Ppn1. Whole cell extracts (WCE) and the immuno-precipitated material (GFP IP) were analyzed by western blot. <b>BCDE.</b> The proteins indicated at the top were purified by affinity and their associated partners were identified by MS/MS mass-spectrometry analysis (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004415#s4" target="_blank">Methods</a>). The number of unique peptides recovered for each protein is indicated. <b>F.</b> Scheme summarizing the proteomic data. <b>GH.</b> Cells expressing the indicated epitope-tagged proteins were synchronized in early mitosis (mitotic) or not (cycling), using the cold-sensitive <i>nda3KM311</i> mutation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004415#pgen.1004415-Toda1" target="_blank">[33]</a>. <b>G.</b> Flag-tagged Swd2.2 was immuno-precipitated to look at its interaction with the core CPF component Pfs2. <b>H.</b> Flag-tagged PP1^Dis2 was immuno-precipitated to look at its interaction with the DPS component Swd2.2.</p

Ppn1 and Ssu72 oppose condensin-mediated chromosome condensation.

<p><b>A.</b> Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. <b>B.</b> Chromosome segregation in anaphase was visualized after growing cells of the indicated genotypes for one generation at 34°C. Anaphases were scored as defective when lagging chromatin was detected between the two main DNA masses. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells was scored. <b>C.</b> The indicated strains were grown at 34°C for 3 hours and ChIP-qPCR was performed to analyze the amount of Cut14-GFP cross-linked to chromatin (mean ± standard deviation from 8 biological replicates). See text for details for the statistical analysis of the experiments. <b>D.</b> Tetrad dissection was used to show that the double mutant <i>ssu72Δ ppn1ΔPP1(ABC)</i> is dead. <b>E</b>. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures.</p