Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint - PubMed (original) (raw)

Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint

Emma M J King et al. PLoS One. 2007.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Mad3p contains two conserved KEN boxes.

A) Mad3/BubR1 domain structure–schematic diagrams of the Mad3 and Bub proteins indicate the organisation of their functional domains. B) Clustal×alignment of the two conserved KEN boxes found in Mad3/BubR1. Species indicated are Saccharomyces cerevisiae (Scer), Saccharomyces paradoxus (Spar), Saccharomyces mikatae (Smik), Saccharomyces kudriavzevii (Skud), Saccharomyces bayanus (Sbay), Saccharomyces castellii (Scas), Schizosaccharomyces pombe (Spom), Candida albicans (Calb), Arabidopsis thaliana (Atha), Drosophila melanogaster (Dmel) and Homo sapiens (Hsap). Numbers indicate residue position within protein sequence.

Figure 2. Mad3p is an APC/C substrate.

A) Mad3p is unstable in G1. Cells expressing GAL-MAD3 were first synchronised in G1 (with alpha factor) or mitosis (with nocodazole) in raffinose media, and then a pulse of Mad3p expression was induced by 30 minutes of growth in media containing 2% galactose. Cells were then washed with glucose media (YPD) and cycloheximide was added to inhibit new protein synthesis. The G1 and mitotic arrests were maintained, samples taken at the times indicated, and whole cell extracts immunoblotted with anti-Mad3p antibodies to determine the remaining level of Mad3p. B) Mad3p turnover in G1 is APC/C dependent. Cells expressing GAL-MAD3 were synchronised in G1 (with α-factor) in raffinose media, and then a pulse of Mad3p expression was induced by 30 minutes of growth in media containing 2% galactose. Cells were then shifted to 36°C (restrictive temperature for _cdc16_-ts) for 30 minutes, washed with glucose media (YPD) and cycloheximide was added to inhibit new protein synthesis. The G1 arrest was maintained, samples taken at the times indicated, and whole cell extracts immunoblotted with α-Mad3p antibodies to determine the remaining level of Mad3p. C) SCF mutants do not stabilise Mad3p in G1. cdc4 and cdc34 strains expressing GAL-MAD3 were analysed for Mad3p turnover as in B. D) mad3KEN30AAA stabilises Mad3p in G1. mad3Δ cells containing GAL driven wild-type or mad3-ken mutants were arrested in alpha factor, and then Mad3p was induced for 30 minutes with galactose media. Cells were then washed in glucose media and cycloheximide was added to prevent new protein synthesis. Samples were taken at the times indicated, and whole cell extracts immunoblotted with anti-Mad3p antibodies for the level of Mad3p.

Figure 3. mad3-KEN-AAA mutants are spindle checkpoint defective.

A) The mad3-ken mutants failed to complement the benomyl sensitivity of mad3Δ. The constructs indicated were transformed into the mad3Δ strain, and then diluted and plated onto YPD plates containing the indicated concentration of benomyl (µg/ml). Photos were taken after three days growth at 24°C. B) The mad3-ken mutants died rapidly. The strains from above were grown to log phase, synchronised in G1 with α-factor, released and then nocodazole was added to 20 µg/ml. At the times indicated, cells were taken, diluted, plated out and scored for their ability to form colonies on rich media. C) The mad3-ken mutants failed to maintain sister chromatid cohesion. In parallel these strains, which contain GFP-marked chromosomes, were scored for sister chromatid cohesion at 0 (α-factor arrested) and four hours after release into nocodazole-containing media. D) The mad3-ken mutants are unable to prevent securin and cyclin from being degraded. Similar strains to those above, but containing Pds1-Myc13 were synchronised in α-factor, then released into nocodazole media (20 µg/ml). Time points were taken, whole cell extracts made and immunoblotted for Pds1 (anti-myc) and cyclin levels (anti-Clb2p).

Figure 4. mad3-KEN30AAA fails to form a mitotic checkpoint complex (MCC).

A) mad3-KEN-AAA mutants can bind Bub3p. Native extracts were made from the indicated strains, containing Bub3-myc13, and Mad3p complexes were immunoprecipitated then immunoblotted for Mad3p and Bub3p (anti-myc). B) mad3-KEN30AAA mutants don't bind to Mad2p or Cdc20p. Cells were arrested in mitosis (with hydroxyurea and nocodazole) and native extracts were made from the indicated strains, containing Cdc20p-myc13. Mad3p was immunoprecipitated then immunoblotted for Mad3p, Mad2p and Cdc20p (anti-myc).

Figure 5. mad3-KEN30AAA stabilises Cdc20p in mitosis.

A) Strains were arrested in mitosis (with nocodazole and hydroxyurea), and cycloheximide was added (time 0) to prevent new protein synthesis. Time points were taken and immunoblotted for Cdc20p levels (anti-myc) and Mad3p levels. B) Quantitation of the Cdc20p and Mad3p levels.

Figure 6. Over-expression of Mad3p induces micro-tubule drug sensitivity, but only subtly perturbs the spindle checkpoint.

A) Strains expressing extra integrated copies of wild-type MAD3 or ken mutant mad3 were immunoblotted to quantitate their expression levels. B) Overexpression of Mad3p induces benomyl sensitivity. The over-expression strains were diluted and plated onto rich YPD media with or without the addition of 15 µg/ml of benomyl. Plates were photographed after 3 days growth at 24°C. C) Strains overexpressing Mad3p die quickly in the presence of nocodazole. The indicated strains were pre-synchronised in G1 with alpha factor, released into media containing 20 µg/ml nocodazole, and cells were plated out at indicated times. Viability was scored as the percentage of colonies formed relative to the zero time point. D) Mad3p overexpression has a mild effect on sister-chromatid cohesion. The overexpression strains, which containing GFP marked chromosome V, were synchronised in G1 with alpha factor, then released into media containing nocodazole. Sister-chromatid separation was scored as the percentage of cells containing two clearly separated GFP spots.

Figure 7. Over-expression of Mad3p induces bi-orientation and chromosome segregation defects.

A) Mad3p overexpression induces chromosome mis-segregation following checkpoint “challenge”. Strains were pre-synchronised with α-factor, released and then arrested with nocodazole. The nocodazole was then washed out and cells arrested in the next G1. Cells were fixed and analysed for the presence of GFP-marked chromosomes. Chromosome V was labelled with GFP and spindle poles with SPC42-mCherry. Arrowheads mark G1 cells containing two copies of chromosome V. B) Mad3p overexpression induces chromosome bi-orientation defects. Cells were pre-synchronised with α-factor, then depleted for Cdc20p through the addition of Methionine. Cells were then released from G1 into media containing nocodazole and benomyl. The microtubule drugs were then washed out to allow soindle assembly, but Cdc20p remained represed to arrest cells in metaphase. Cells were briefly fixed and scored for bi-orientation (breathing centromeres). Centromere IV was labelled with GFP and spindle poles with _SPC42_-tomato. Scale bars are 5 microns.

Figure 8. Models of Mad3p KEN box interactions.

a) Mad3 KEN box interactions. Mad2p and Mad3-KEN30 are both required for stable Mad3p-Cdc20p binding. b) Cdc20p turnover in mitosis: this is dependent on Mad3-KEN30, and Mad2p, suggesting that this Mad3 KEN box acts “in trans” as a Cdc20p degron. c) Mad3p turnover in G1. Mad3p is degraded in a Mad3-KEN30, Cdh1, and APC/C dependent manner.

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Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint - PubMed (original) (raw)