S. pombe APC/C Stably Interacts with Both Mad2 and Mad3
—Our previous TAP of the APC/C from asynchronously growing S. pombe
identified 13 core subunits (31
). Using the TAP-tagged Lid1(Apc4) subunit, we purified APC/C from cells arrested in prometaphase using the nda3-KM311
cold-sensitive tubulin allele. The nda3-KM311
mutant has no microtubules at 18 °C and arrests with hypercondensed chromosomes due to spindle checkpoint activation (32
). All other APC/C subunits identified previously (31
) co-purified with Lid1, along with the Cdc20 activator (). Strikingly, this mitotic APC/C also contained the Mad2 and Mad3 spindle checkpoint proteins. In a reverse experiment, Mad2, Cdc20, and all 13 core APC/C subunits were detected in a Mad3-TAP eluate purified from nda3
arrested cells. No Mad1 or Bub (budding uninhibited by benzimidazole) proteins were detected in these complexes. These results, which were confirmed by immunoprecipitation and blotting (), indicate that both Mad2 and Mad3 can stably interact with the APC/C in S. pombe
. The stable associations we detected by TAP and mass spectrometry allowed us to address the mechanism by which Mad2 and Mad3 interact with the core APC/C in S. pombe
FIGURE 1. S. pombe Mad3 and Mad2 stably interact with mitotic APC/C. A, Lid1-TAP and Mad3-TAP purifications were performed from mitotically arrested S. pombe strains. The TAP eluates were subjected to trypsin digestion and MudPIT mass spectrometric analysis. The (more ...) Mad3 and Mad2 Interact with the APC/C Every Mitosis
—We also found that Mad3 and Mad2 are stably associated with the APC/C in an mts3-1
mutant arrest (33
arrests in a metaphase-like state due to decreased proteasome activity and the stabilization of securin and cyclin B (34
). These cells are not sensitive to microtubule destabilizing agents, and components of the SAC do not accumulate on kinetochores in mts3-1
cells (data not shown). This shows that Mad3 and Mad2 associate with the APC/C in a mitotic arrest that is independent of spindle checkpoint signaling. This was confirmed by accumulating cells in mitosis through overexpression of the N-terminal 70 amino acids of S. pombe
cyclin B that lacks lysines (N70-K0: see “Experimental Procedures”). N70-K0 competes with endogenous APC/C substrates (35
), and cells accumulate in metaphase independently of spindle checkpoint action. We could reproducibly detect both Mad2 and Mad3 bound to APC/C in N70-K0-induced mitotic delays (see Figs. and ).
FIGURE 3. The mad3-KEN20AAA mutation abolishes stable APC/C association of both Mad3 and Mad2. A, wild-type and slp1-362 strains were arrested at metaphase. APC/C was pulled down via Lid1-TAP using IgG Dynabeads and immunoblotted for Mad3-GFP and Mad2. B, the Mad3 (more ...)
FIGURE 4. The mad3-KEN20AAA mutation abolishes MCC formation, whereas upstream checkpoint signaling components are not necessary. A, strains were arrested at metaphase through overexpression of the N terminus of cyclin (N70-K0), and arrests were scored by mitotic (more ...)
To determine whether the Mad protein-APC/C complex assembles and disassembles during every cell cycle, we used the cdc25 mutant to presynchronize cultures in G2. Cells were then released from the block point, and samples were taken at 15-min intervals. Mad2 was immunoprecipitated, and we looked for associated Cdc20 and APC/C proteins by immunoblotting. Mad2 was found to associate with the APC/C specifically during mitosis (). Similarly, during an nda3 release time course, Lid1-Myc was detectable in a Mad2 immunoprecipitate up until the point of septation ().
FIGURE 2. The Mad-APC/C complex is formed every mitosis. A, cdc25 cultures were synchronized in G2 and then released at 25 °C. Samples were taken at 15-min time points throughout mitosis, Mad2 was immunoprecipitated, immunoprecipitates were separated by (more ...)
Sucrose gradient fractionation was carried out of extracts made from nda3 arrested cells. Pools of Mad2, Mad3-GFP, and Cdc20/Slp1-HA were seen to co-fractionate with Lid1-TAP ().
To determine what fraction of the total APC/C pool associates with checkpoint proteins, we made extracts from nda3-KM311 and mts3-1 arrested cells, immunodepleted Mad2 tagged at its endogenous locus with HTB using magnetic streptavidin beads (, left panels), and quantitatively analyzed the amount of Lid1 associated with the beads and in the supernatant (see “Experimental Procedures”). We found that up to 8% of the APC/C was co-depleted with Mad2 (, right panel). Thus, the levels of checkpoint proteins associated with APC/C increase specifically in mitosis and are significant, with at least 5% of the core APC/C bound by Mad2.
Mad3-KEN20, Cdc20, and Mad2 Mediate the Mad3-APC/C Interaction
—How is the Mad3-APC/C interaction mediated? First, Mad3 interaction with the APC/C was observed to be dependent on Cdc20. The conditional cdc20
, arrests in a metaphase-like state because the mutant Slp1-362 protein is unable to bind the APC/C at restrictive temperature (36
). In a Lid1-TAP from this mutant, Slp1-362, Mad3, and Mad2 were all undetectable by mass spectrometry (33
). Immunoblotting confirmed these mass spectrometry results; Lid1-TAP pull downs from N70-K0-overproducing cells contained both Mad3 and Mad2, but the pull down from mitotic slp1-362
cells did not (). We conclude that stable checkpoint protein interactions with the mitotic APC/C are Cdc20-dependent.
Cdc20 recognizes destruction (D) boxes and KEN boxes of APC/C substrates. Sequence analysis of Mad3 from different organisms revealed two conserved KEN boxes (). To study their potential role in mediating an interaction with Cdc20-APC/C, we made three mutants in which the KEN boxes were replaced by a triple alanine (AAA) substitution: mad3-KEN20AAA, mad3-KEN271AAA, and mad3-KEN20/271AAA. The mutant proteins are present at wild-type levels and can be recruited to kinetochores (Figs. and ), so mutation of these residues does not lead to gross changes in stability of the Mad3 protein or its localization. The mad3-KEN mutants were enriched in mitosis with N70-K0, Lid1-TAP was pulled down, and the associated Mad proteins were analyzed by immunoblotting. The mad3-KEN20AAA mutant abolished Mad3, and importantly also Mad2, association with the APC/C, whereas the mad3-KEN271AAA mutant had little effect ( and supplemental Fig. S1). As expected, the double KEN box mutant abolished APC/C binding. Very similar results were obtained using mad3-KEN mts3-1 mutants (supplemental Fig. S2). Thus, Mad3- and Mad2-APC/C association is dependent on the highly conserved N-terminal KEN box that is found in Mad3 and BubR1.
FIGURE 5. Mutation of Mad3 KEN boxes abolishes spindle checkpoint arrests. A, nda3-KM311 cultures were shifted to their restrictive temperature (18 °C), and cells were analyzed microscopically for chromosome condensation and mis-segregation. Images of the (more ...)
Mad2 interaction with the APC/C is most likely bridged by direct interaction with Cdc20 (9
). To determine whether Mad3 requires Mad2 to stably bind the APC/C, we overproduced N70-K0 in mad2
Δ cells so that they accumulate in mitosis, pulled down Lid1-TAP, and immunoblotted for Mad3-GFP. Mad3-APC/C binding was Mad2-dependent (). Thus, Mad3 is dependent on its own N-terminal KEN box, Mad2, and Cdc20 for APC/C association. Unexpectedly, Mad2 is also dependent on the N-terminal Mad3 KEN box for stable APC/C association (). Thus, Mad2 and Mad3 are inter-dependent for stable APC/C binding.
KEN20 Mediates Cdc20 Binding and MCC Formation
—Mad3 is part of the MCC, which in fission yeast is comprised exclusively of Mad3, Mad2, and Cdc20. In other organisms, Bub3 is also a component of the MCC. However, S. pombe
Mad3 lacks the GLEBS motif that is necessary and sufficient for Saccharomyces cerevisiae
Mad3 to bind Bub3 (37
). Thus, although Bub3 is important for the kinetochore targeting of Mad3 (28
), Bub3 is not stably associated with either MCC or APC/C in fission yeast ( and data not shown).
Due to the co-dependence of Mad2 and Mad3 for stable APC/C association, we considered the possibility that this stable association requires formation of the MCC. To test the importance of the Mad3 KEN boxes for MCC formation, we enriched for mitotic wild-type and KEN mutant cells in mitosis using N70. We then carried out immunoprecipitations of Mad3-GFP and Cdc20/Slp1-HA from these mitotic extracts and found that mutation of the N-terminal KEN box (mad3-KEN20AAA) abolished the interaction between Mad3 and both Cdc20 and Mad2, demonstrating that it is necessary for MCC formation (). Mutation of the C-terminal KEN box (mad3-KEN271AAA) did not have this effect on Cdc20 or Mad2 binding. It is important to note here that neither the mad3-KEN20AAA mutation () nor deletion of mad3 (data not shown) interfered with Mad2-Cdc20 binding. This contrasts with its effect on Mad2-APC/C binding, which was abolished in mad3-KEN20AAA (), and supports our proposal that checkpoint protein-APC/C association requires the integrity of the MCC in vivo ().
FIGURE 6. Models of Mad3 interactions. A, model of stepwise assembly of the checkpoint protein APC/C complex. Note that this model is based on genetic dependencies and does not necessarily imply that this order of assembly would be observed during biochemical reconstitution. (more ...)
MCC Formation Is Independent of Upstream Checkpoint Signaling and Kinetochore Targeting
—It has been demonstrated that budding yeast MCC forms independently of kinetochores. For example, in ndc10-1
mutants, where kinetochores are completely absent at restrictive temperatures, the MCC assembles with normal kinetics and to normal levels (39
). We attempted similar experiments with the fission yeast nuf2-1
), but these cells were too sick for detailed biochemistry. Instead, we analyzed the MCC in fission yeast strains lacking Bub3, Bub1, Mph1, and Mad1. These proteins are critical for kinetochore targeting of other checkpoint components; Bub3 and Bub1 are both required for Mad3 localization (38
), and Mad1 is necessary for Mad2 recruitment to fission yeast kinetochores (43
). In all of these mutants, which were enriched in mitotic cells due to N70 overexpression, we could readily detect an interaction between Mad2, Mad3, and Cdc20 (). The only mutant demonstrating significant quantitative reduction in MCC levels was mad1
, and in this case, the effect was typically only 2–3-fold (). This experiment demonstrates that fission yeast MCC can be formed independently of upstream checkpoint signaling components and that kinetochore targeting of the MCC components is not necessary for its assembly. Similar experiments, pulling down Lid1-TAP, showed that the Mad2 and Mad3 interaction with the APC/C was also independent of these upstream factors (, and data not shown). It is important to note that although MCC and MCC-APC/C are still being assembled in these fission yeast mutants, the checkpoint is not functional and Cdc20-APC/C is not efficiently inhibited.
Mad3 KEN Boxes Are Required for Spindle Checkpoint Function—Three assays were employed to test the functional requirement for Mad3-KEN boxes in spindle checkpoint signaling. First, we made nda3-KM311 mad3-KEN-AAA double mutants and assayed their ability to arrest and maintain viability at 18 °C. Both KEN mutants behaved like the mad3Δ strain in this assay; cells failed to arrest with condensed chromosomes, mis-segregated their DNA, displayed the cut (cells untimely torn) phenotype (), and lost viability rapidly after 3 h at 18 °C (). During this experiment, we also analyzed the recruitment of GFP-tagged mad3 KEN mutant proteins to kinetochores. The Mad3 KEN mutant proteins were recruited to kinetochores. However, quantitation of the numbers of cells with bright Mad3-GFP foci during the nda3-KM311 time course confirmed that the KEN mutants were unable to arrest in mitosis as the mutant cultures failed to accumulate cells with bright Mad3-GFP foci ().
Mad2 or Mph1 overexpression arrests wild-type S. pombe
cells at metaphase (44
), and this was scored by staining cells with anti-tubulin antibodies and counting mitotic spindles in cultures overproducing these proteins. When Mad2 or Mph1 was overexpressed in the mad3 KEN
mutants, they showed a greatly decreased ability to arrest at metaphase. This arrest was scored by staining cells with anti-tubulin antibodies and counting the frequency of mitotic spindles (). Thus, using three independent assays, we find that mutation of Mad3 KEN boxes abrogates spindle checkpoint arrests.