p31comet Promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process

Abstract

Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31(comet) in this process. It is known that p31(comet) prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31(comet) blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31(comet) causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31(comet) binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC.

Fig. 1.

p31^comet stimulates the release of APC/C from checkpoint inhibition and accelerates the disassembly of MCC. (A) Effect of p31^comet on APC/C activation in checkpoint extracts. Extracts were incubated with ATP as described in Methods, in the absence (“Control”) or presence of bacterially expressed p31^comet, as indicated. Samples taken at the times indicated were immunoprecipitated with anti-Cdc27 beads and APC/C activity in the ligation of ¹²⁵I-cyclin to ubiquitin was assayed. (B) Effect of p31^comet on the disassembly of MCC in checkpoint extracts incubated with ATP. Extracts were incubated with p31^comet or without it (“Control”), as described in A. Samples withdrawn at the indicated times were subjected to sequential immunoprecipitations with anti-Cdc27 and anti-BubR1 antibodies (see Methods). The amounts of MCC components associated with anti-BubR1 immunoprecipitates were estimated with quantitative immunoblotting. (C) Binding of ³⁵S-labeled p31^comet to components in anti-BubR1 immunoprecipitates. Extracts were incubated with ATP as described in A, (“Control”), and samples were subjected to immunoprecipitation with anti-BubR1 as described in B. The binding of ³⁵S- p31^comet to anti-BubR1 immunoprecipitates was estimated as described in Methods. (D) Relationship of bound ³⁵S- p31^comet to MCC components in anti-BubR1 immunoprecipitates. Binding of ³⁵S- p31^comet to immunoprecipitates was estimated as in C, and the amounts of Mad2, Cdc20, and BubR1 in the same immunoprecipitates were measured as in B. Ratios were normalized to 1.0 at time zero of incubation.

Fig. 2.

MCC is dissociated by the joint action p31^comet and ATP. (A) p31^comet abrogates the inhibition of purified APC/C by MCC. Reaction mixture was similar to that described in ref.  for the assay of APC/C activity, except that purified mitotic APC/C and recombinant Cdc20 were supplemented as in ref. . Where indicated, 250 nM p31^comet was added. MCC and MCF2 were purified from salt eluate of APC/C immunoprecipitate by chromatography on MonoQ as described (14). Residual MCC was removed from the preparation of MCF2 by immunodepletion with anti-BubR1 (14). The amounts of MCC and MCF2 used were adjusted to produce an inhibition of approximately 40–50% of APC/C activity. The ligation of ¹²⁵I-cyclin to ubiquitin was determined following an incubation of 60 min at 30 °C. Results were expressed relative to APC/C activity in the control incubation (without inhibitor and without p31^comet). (B) p31^comet and β,γ-hydrolyzable ATP synergistically promote the release of Mad2 and Cdc20 from anti-BubR1 immunoprecipitates. The release of MCC components from anti-BubR1 immunoprecipitates to supernatants was determined as described in Methods. Where indicated, ATP or AMP–PNP were added at 5 mM, and p31^comet at 250 nM. “Input” was the amount of MCC components associated with anti-BubR1 immunoprecipitate prior to release. (C) Time course of the release of Mad2 and Cdc20 from anti-BubR1 immunoprecipitates incubated with p31^comet and ATP. Reaction conditions were similar to those in B. Samples of released material were analyzed by quantitative immunoblotting. (D) Nucleotide specificity of MCC dissociation. BubR1 immunoprecipitates were incubated with 2 mM of the indicated nucleotides, in the absence (“Control”) or presence of 250 nM p31^comet. GMP–PNP, guanosine-5′-(β,γ-imido)triphosphate. The release of MCC components was determined as described in Methods. “No nucl.,” without nucleotide added. Shown is the release of Mad2; similar results were obtained with the release of Cdc20.

Fig. 3.

Dissociation of soluble MCC by incubation with p31^comet and ATP. (A) Analysis of MCC dissociation by gel filtration chromatography. MCC was partially purified from salt eluate of APC/C immunoprecipitate by gel filtration chromatography on Superose-6, as described in Methods. Incubation mixtures contained in a volume of 100 μl: 40 mM Tris-HCl. (pH 7.6), 5 mM MgCl₂, 1 mM DTT and 80 μl MCC. Where indicated, 5 mM ATP and 250 nM p31^comet were added. Following incubation at 23 °C for 60 minutes, samples were separated on a 2.4-ml Superdex 20 PC3.2/30 column (GE Healthcare) equilibrated with Buffer B. Fractions of 100 μl were collected at a flow rate of 50 μl/ min. Samples of column fractions of 10 μl were analyzed by immunoblotting for the indicated proteins. (B) Quantitation of results from A. The amount of each protein in each fraction was expressed as the percentage of total eluted protein. The elution position of the following marker proteins is indicated (kDa): ferritin, 440; aldolase, 160; BSA, 66; cytochrome C, 12. (C) Coimmunoprecipitation of Cdc20 and Mad2 in material released from MCC. Anti-BubR1 immunoprecipitates were incubated with p31^comet and ATP and material released to the supernatant was collected as described under Methods. Samples of 40 μl of released material were subjected to immunoprecipitation with 5 μg of affinity-purified rabbit antibodies against Cdc20 or Mad2 (12) adsorbed to 10 μl Affi-prep Protein A beads (Bio-Rad), or with a similar amount of nonimmune rabbit IgG (Pierce), as indicated. Equal samples of supernatants (S) and precipitates (P) were subjected to immunoblotting with the indicated monoclonal antibodies.