Conformation-specific binding of p31comet antagonizes the function of Mad2 in the spindle checkpoint
2004; Springer Nature; Volume: 23; Issue: 15 Linguagem: Inglês
10.1038/sj.emboj.7600322
ISSN1460-2075
AutoresGuo-Hong Xia, Xuelian Luo, Toshiyuki Habu, Josep Rizo, Tomohiro Matsumoto, Hongtao Yu,
Tópico(s)Chromosomal and Genetic Variations
ResumoArticle15 July 2004free access Conformation-specific binding of p31comet antagonizes the function of Mad2 in the spindle checkpoint Guohong Xia Guohong Xia Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Xuelian Luo Xuelian Luo Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Department of Biochemistry, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Toshiyuki Habu Toshiyuki Habu Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan Search for more papers by this author Josep Rizo Josep Rizo Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Department of Biochemistry, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Tomohiro Matsumoto Tomohiro Matsumoto Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan Search for more papers by this author Hongtao Yu Corresponding Author Hongtao Yu Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Guohong Xia Guohong Xia Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Xuelian Luo Xuelian Luo Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Department of Biochemistry, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Toshiyuki Habu Toshiyuki Habu Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan Search for more papers by this author Josep Rizo Josep Rizo Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Department of Biochemistry, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Tomohiro Matsumoto Tomohiro Matsumoto Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan Search for more papers by this author Hongtao Yu Corresponding Author Hongtao Yu Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Author Information Guohong Xia1, Xuelian Luo1,2, Toshiyuki Habu3, Josep Rizo1,2, Tomohiro Matsumoto3 and Hongtao Yu 1 1Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA 2Department of Biochemistry, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA 3Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan *Corresponding author. Department of Pharmacology, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA. Tel.: +1 214 648 9697; Fax: +1 214 648 2971; E-mail: [email protected] The EMBO Journal (2004)23:3133-3143https://doi.org/10.1038/sj.emboj.7600322 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase in response to misaligned sister chromatids during mitosis. Upon checkpoint activation, Mad2 binds directly to Cdc20 and inhibits the anaphase-promoting complex or cyclosome (APC/C). Cdc20 binding triggers a dramatic conformational change of Mad2. Consistent with an earlier report, we show herein that depletion of p31comet (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31comet protein antagonizes the ability of Mad2 to inhibit APC/CCdc20 in vitro and in Xenopus egg extracts. Interestingly, p31comet binds selectively to the Cdc20-bound conformation of Mad2. Binding of p31comet to Mad2 does not prevent the interaction between Mad2 and Cdc20 in vitro. During checkpoint inactivation in HeLa cells, p31comet forms a transient complex with APC/CCdc20-bound Mad2. Purified p31comet enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2–Cdc20 interaction. Therefore, our results suggest that p31comet counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint. Introduction During the cell division cycle, cells duplicate their chromosomes once and only once and segregate the sister chromatids evenly in mitosis, thus faithfully transmitting their genetic information to the daughter cells. The replicated chromosomes are held together by the cohesin protein complex (Nasmyth, 2002). At the metaphase–anaphase transition, a large ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C) mediates the degradation of securin, an inhibitor of separase (Peters, 2002). The active separase then cleaves a subunit of the cohesin complex, Scc1, leading to the dissolution of chromosome cohesion and the onset of sister-chromatid separation (Nasmyth, 2002). To maintain genetic stability, the spindle checkpoint delays the onset of chromosome segregation in response to sister chromatids that have not attached to microtubules emanating from the two opposite poles of the mitotic spindle (biorientation) (Rudner and Murray, 1996; Gorbsky, 2001; Wassmann and Benezra, 2001; Millband et al, 2002; Musacchio and Hardwick, 2002; Yu, 2002; Bharadwaj and Yu, 2004). A single kinetochore that is not captured by microtubules and not under tension activates this checkpoint (Li and Nicklas, 1995; Rieder et al, 1995; Nicklas, 1997). APC/C is a critical target of the spindle checkpoint (Peters, 2002; Yu, 2002). Inhibition of APC/C by the checkpoint causes the stabilization of securin, inhibition of separase, and a delay in sister-chromatid separation (Peters, 2002). Progress has been made toward understanding the mechanism by which the spindle checkpoint inhibits APC/C in response to spindle defects (Millband et al, 2002; Yu, 2002). In vertebrates, Mad2 and BubR1 act synergistically to inhibit APC/C in vitro through their direct binding to Cdc20 (Li et al, 1997; Fang et al, 1998a; Hwang et al, 1998; Kim et al, 1998; Wassmann and Benezra, 1998; Sudakin et al, 2001; Tang et al, 2001; Fang, 2002). Furthermore, BubR1 and Mad2 can assemble into a single mitotic checkpoint complex (MCC) containing BubR1, Bub3, Mad2, and Cdc20 in vivo (Hardwick et al, 2000; Fraschini et al, 2001; Sudakin et al, 2001; Millband and Hardwick, 2002). It is unclear whether Mad2 and BubR1 act exclusively in the context of MCC or they also form independent BubR1–Bub3–Cdc20 and Mad2–Cdc20 complexes to inhibit APC/C in living cells. Nevertheless, it has been firmly established that the Mad2–Cdc20 interaction is absolutely required for the proper execution of the spindle checkpoint (Fang et al, 1998a; Hwang et al, 1998; Kim et al, 1998; Dobles et al, 2000; Michel et al, 2001). Recent structural data have provided insights into the Mad2–Cdc20 interaction (Luo et al, 2000, 2002; Sironi et al, 2002). Mad2 recognizes a short conserved peptide motif within Cdc20 (Luo et al, 2000, 2002). Binding of the Cdc20 peptide to Mad2 triggers a dramatic structural rearrangement of Mad2, which involves the shuffling of several β strands in the main β sheet of Mad2 (Luo et al, 2000, 2002). Interestingly, Mad1 contains a small Mad2-binding motif similar to that of Cdc20, and Mad1 binding triggers the same large conformational change of Mad2 as does Cdc20 (Luo et al, 2000, 2002; Sironi et al, 2002). Mad1 is also required for the kinetochore localization of Mad2 (Chen et al, 1998; Chung and Chen, 2002; Luo et al, 2002). Very recently, we have shown that Mad2 possesses two natively folded conformations at equilibrium, referred to as N1 and N2 (Luo et al, 2004). N2-Mad2 resembles the structure of the Mad1- or Cdc20-bound Mad2 and is more active in APC/C inhibition. The Mad2-binding domain of Mad1 facilitates the N1–N2 conformational switch of Mad2 in vitro. Taken together, these results suggest that, upon checkpoint activation, Mad1 recruits Mad2 to unattached kinetochores and facilitates the formation of N2-Mad2 and thus the establishment of the Mad2–Cdc20 interaction (Chen et al, 1998; Chung and Chen, 2002; Luo et al, 2002). The structural studies on the Mad1–Mad2 and Mad2–Cdc20 complexes have revealed another intriguing feature of these interactions. The dissociation of Mad1–Mad2 and Mad2–Cdc20 complexes requires the partial unfolding of the C-terminal region of Mad2, which imposes a significant energetic barrier on these processes (Luo et al, 2000, 2002; Sironi et al, 2002). This suggests the existence of other factors that may facilitate the disassembly of the Mad2–Cdc20-containing checkpoint complexes when the checkpoint is satisfied. A novel human Mad2-binding protein called Cmt2 has recently been identified in a yeast two-hybrid screen (Habu et al, 2002). As Cmt2 has been in use for the Charcot–Marie–Tooth disease type 2 (CMT2) gene, we propose to rename this Mad2-binding protein, p31comet, for its comet tail-like cellular localization pattern in mitosis (unpublished results, TH and TM). In HeLa cells, the association of the endogenous p31comet protein with Mad2 coincides with the dissociation of the Mad2–Cdc20 interaction, suggesting that p31comet might play a role in silencing the spindle checkpoint (Habu et al, 2002). Consistent with this notion, we report herein that HeLa cells depleted of p31comet by RNA interference (RNAi) escape from the checkpoint-mediated mitotic arrest with significantly slower kinetics. P31comet blocks the biochemical function of Mad2 in vitro and in Xenopus egg extracts. Strikingly, p31comet only interacts with the Cdc20-bound conformation of Mad2. P31comet binding and Cdc20 binding to Mad2 are not mutually exclusive, indicating that p31comet and Cdc20 bind to different sites on Mad2. Moreover, p31comet, Mad2, and Cdc20 form a ternary complex in vitro and in vivo, and this complex is transiently enriched as cells exit from mitosis. Purified recombinant p31comet protein moderately activates APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2–Cdc20 interaction. Our results indicate that p31comet counteracts the function of Mad2 and is required for spindle checkpoint silencing. Results P31comet is required for the inactivation of the spindle checkpoint To test the hypothesis that p31comet is required for checkpoint inactivation, we examined the effect of RNAi-mediated depletion of p31comet on the mitotic arrest of HeLa cells caused by spindle-damaging agents. Transfection of p31comet-specific siRNA into HeLa cells reduced the protein level of p31comet to 15% of that of the cells that received a control siRNA (Figure 1A). Cells depleted of p31comet did not exhibit any discernable cell cycle phenotype, as judged by FACS (Figure 1C) and cell morphology (data not shown). This indicates that p31comet at 15% of its endogenous concentration is sufficient for cell cycle progression under normal conditions. If p31comet is required for the inactivation of the spindle checkpoint, downregulation of p31comet is expected to cause a delay in the activation of APC/CCdc20 and consequently a delay in mitotic exit after the removal of spindle-damaging agents. HeLa cells transfected with control or p31comet siRNA were arrested in mitosis with nocodazole. After 18 h of nocodazole treatment, cells were then grown in fresh medium to allow them to exit from mitosis. As shown in Figure 1B, two key APC/C substrates, securin and cyclin B1, were degraded in control cells at around 2 h after the removal of nocodazole. In contrast, securin and cyclin B1 were inefficiently degraded in p31comet RNAi cells, resulting in higher levels of both proteins after the removal of nocodazole as compared to control cells. Consistent with the biochemical readout, FACS analysis revealed that p31comet RNAi cells exited from mitosis with a much slower kinetics as compared to the control cells (Figure 1C). For example, at 1.5 h after the release from nocodazole-mediated mitotic arrest, 62% of the control cells had returned to the G1 phase with a 2N DNA content, while only 35% of the p31comet RNAi cells exited from mitosis. These data suggest that p31comet is required for the efficient inactivation of the spindle checkpoint established by extensive spindle damage. The phenotypes of p31comet RNAi cells are consistent with those described for cells transfected with antisense oligonucleotides against p31comet (Habu et al, 2002), with one minor exception. In the latter case, cells also experience a delay in mitotic exit following the release from nocodazole-mediated mitotic arrest. However, a significant portion of cells treated with antisense p31comet underwent cell death. We do not know the exact reason for the lower incidence of cell death in p31comet RNAi cells following the release from nocodazole-mediated mitotic arrest. It is possible that RNAi and antisense technologies knock down the protein level of p31comet with different kinetics and to different extents, thus resulting in slightly different phenotypes. Figure 1.P31comet is required for the inactivation of the spindle checkpoint. (A) HeLa cells transfected with the control or p31comet siRNA duplexes were dissolved in SDS sample buffer, separated on SDS–PAGE, and blotted with anti-APC2 and anti-p31comet antibodies. (B) HeLa cells transfected with control or p31comet siRNA were treated with 100 ng/ml nocodazole for 18 h and released into fresh medium. The total cell lysates of log-phase cells and cell samples taken at the indicated time points were separated on SDS–PAGE and blotted with the indicated antibodies. (C) FACS analysis of some of the cell samples described in (B). The peaks corresponding to 2N and 4N DNA contents are labeled. (D) FACS analysis of HeLa cells that were transfected with control or p31comet siRNA and treated with the indicated concentrations of nocodazole. The peaks corresponding to 2N and 4N DNA contents are labeled. (E) HeLa Tet-on cells transfected with the control or p31comet siRNA were treated with varying concentrations of nocodazole for 16 h and stained with Hoechst 33342. The mitotic indices were determined by directly observing the cells with an inverted fluorescence microscope. The mitotic cells were round and contained condensed DNA, while the interphase cells were flat with decondensed DNA. This experiment was repeated three times and standard deviations are included as error bars. Download figure Download PowerPoint We next checked the behavior of p31comet RNAi cells in the presence of lower concentrations of nocodazole. As shown in Figure 1D and E, p31comet RNAi cells underwent mitotic arrest more efficiently at lower concentrations of nocodazole (e.g. 12.5 ng/ml). This again is consistent with the notion that p31comet counteracts the spindle checkpoint function of Mad2. Higher levels of p31comet inactivate Mad2 and the spindle checkpoint in living cells (Habu et al, 2002), whereas lower levels of p31comet lead to checkpoint activation at a lower threshold of spindle damage. P31comet blocks the ability of Mad2 to inhibit APC/CCdc20 in vitro and in Xenopus extracts Binding of Mad2 to Cdc20 inhibits the ubiquitin ligase activity of APC/C in vitro (Fang et al, 1998a). We next checked whether p31comet blocked the ability of Mad2 to inhibit APC/CCdc20 in a ubiquitination assay reconstituted with purified components (Figure 2A). As shown previously, addition of purified Cdc20 protein to interphase Xenopus APC/C activated its ligase activity using a fragment of human cyclin B1 (residues 1–102) as the substrate (Figure 2B, compare lanes 1 and 2) (Fang et al, 1998b; Kramer et al, 1998). Preincubation of Cdc20 with the dimeric wild-type Mad2 greatly reduced the activity of APC/CCdc20 (Figure 2B, lanes 2 and 3). Addition of increasing amounts of p31comet together with Mad2 partially restored the activity of APC/CCdc20 (Figure 2B, lanes 4–8). As a control, addition of increasing amounts of recombinant human Mob1, a protein that does not interact with Mad2, did not affect the inhibition of APC/CCdc20 by Mad2 (Figure 2B, lanes 9–13). We also performed the APC/C assay in the presence of ΔC-Mad2 and p31comet (Figure 2C). As the C-terminal region of Mad2 is required for the Mad2–Cdc20 interaction, a Mad2 mutant with its C-terminal 10 residues deleted (ΔC-Mad2) was inactive in APC/C inhibition (Figure 2C). Addition of p31comet or Mob1 along with ΔC-Mad2 did not affect the activity of APC/C (Figure 2C). This indicates that p31comet does not have a general APC/C-stimulatory effect. Our data clearly demonstrate that p31comet partially reverses the APC/C-inhibitory activity of Mad2 in vitro. Figure 2.P31comet antagonizes the ability of Mad2 to inhibit the activity of APC/CCdc20. (A) The purified His6-tagged p31comet, Mad2, ΔC-Mad2, Mob1, and Cdc20 proteins were separated on SDS–PAGE and stained with Coomassie blue. (B) The ubiquitination activity of APC/C was assayed with a Myc-tagged N-terminal fragment of human cyclin B1. The reaction mixtures were separated on SDS–PAGE and blotted with the anti-Myc antibody. The positions of the cyclin B1 substrate and the cyclin B1–ubiquitin conjugates are labeled. Incubation of APC/C isolated from interphase Xenopus egg extracts (I-APC/C) with human Cdc20 greatly stimulated the activity of APC/C (compare lanes 1 and 2). The purified Mad2 protein (4 μM) inhibited the activity of APC/CCdc20 using cyclin B1 as a substrate (compare lanes 2 and 3). Addition of increasing amounts of p31comet (250 nM–4 μM) together with Mad2 restored the activity of APC/CCdc20 (compare lane 3 with lanes 4–8). Addition of a control protein Mob1 (250 nM–4 μM) did not have any effect on Mad2-mediated inhibition of APC/CCdc20 (lanes 9–13). Addition of p31comet (4 μM) alone in the absence of Mad2 did not stimulate the activity of APC/CCdc20 (lanes 14–18). (C) Same as in panel B (lanes 1–13) except that the ΔC-Mad2 protein (4 μM) was used in the assays. (D) P31comet reverses Mad2-mediated inhibition of APC/CCdc20 in Xenopus egg extracts. In vitro-translated 35S-labeled full-length human cyclin B1, a known APC/C substrate, was added to mitotic Xenopus egg extracts in the presence of XB buffer, recombinant purified wild-type Mad2, p31comet, or both Mad2 and p31comet. The final concentrations of Mad2 and p31comet were 20 μM. Samples were taken at the indicated time points and analyzed by SDS–PAGE followed by autoradiography. The half-life of cyclin B in each experiment is indicated. Download figure Download PowerPoint We next tested the effect of p31comet on Mad2-mediated APC/C inhibition in Xenopus egg extracts. Consistent with the earlier reports (Fang et al, 1998a), cyclin B1 was rapidly degraded in mitotic Xenopus egg extracts that contained active APC/CCdc20 (Figure 2D). Addition of dimeric wild-type Mad2 inhibited APC/CCdc20 in these extracts and stabilized cyclin B1 (Figure 2D) (Fang et al, 1998a). Addition of p31comet together with Mad2 to these extracts again partially restored degradation of cyclin B1 (Figure 2D). As a control, p31comet alone did not perturb cyclin B1 degradation in these extracts (Figure 2D). Therefore, p31comet also blocked the APC/C-inhibitory activity of Mad2 in Xenopus egg extracts. P31comet selectively binds to the Cdc20-bound conformation of Mad2 It has been previously shown that bacterially expressed wild-type Mad2 protein exists as two species: a monomer and a dimer (Fang et al, 1998a). Interestingly, only the dimeric form of Mad2 is active in inhibiting APC/C in Xenopus egg extracts (Fang et al, 1998a). A point mutant of Mad2, Mad2R133A, exists exclusively as a monomer (Sironi et al, 2001). Surprisingly, we have recently shown that Mad2R133A also exists as two distinct monomeric conformers, referred to as N1 and N2, which can be separated by anion exchange chromatography (Luo et al, 2004). N2-Mad2R133A is much more potent in APC/C inhibition in Xenopus extracts (Luo et al, 2004). The structures of both conformers have been determined by NMR spectroscopy (Figure 3A). The structure of N2-Mad2 resembles that of the Cdc20-bound form of Mad2, thus explaining why N2-Mad2 is more active in APC/C inhibition. Our NMR studies also indicate that the wild-type Mad2 monomer exhibits the N1 conformation, whereas the Mad2 dimer is in the N2 conformation. More interestingly, the Mad2-binding domain of Mad1 facilitates the structural conversion from N1-Mad2 to N2-Mad2 in vitro. These data suggest that the N1–N2 conformational change of Mad2 is required for its APC/C-inhibitory activity, and this structural transition might be regulated by other spindle checkpoint proteins, such as Mad1. Figure 3.P31comet selectively binds to the N2 conformation of Mad2. (A) Ribbon drawing of the structures of the N1 and N2 forms of Mad2. The structural elements of Mad2 that undergo major changes between the N1 and N2 conformers are colored yellow, while the rest of the structural elements are in blue. The strands are numbered 1–8, while the helices are labeled A–C in the N1-Mad2 structure. The secondary structure elements of the N2-Mad2 are labeled in a similar manner with the exception of β9, which is unstructured in N1-Mad2. The structures were generated with Molscript and Raster3D. (B) Various forms of His6-Mad2 were bound to Ni2+-NTA beads and incubated with 35S-labeled Mad1C, Cdc20N, and p31comet proteins. Empty beads were used as a control, and 50% of the input protein used in each binding reaction was loaded for comparison. After washing, the proteins retained on beads were analyzed by SDS–PAGE followed by autoradiography. The Mad2 proteins bound to the beads were analyzed by SDS–PAGE followed by Coomassie blue staining. (C) ITC analysis of the binding between p31comet and N2-Mad2R133A. The dissociation constant (Kd) and the stoichiometry (N) of binding are indicated. Download figure Download PowerPoint As the interaction between Mad2 and p31comet is regulated during the cell cycle (Habu et al, 2002), we tested whether the conformational change of Mad2 regulates the Mad2–p31comet interaction. Various purified His6-tagged Mad2 proteins were immobilized on Ni2+-NTA beads and incubated with 35S-labeled p31comet, Cdc20, or Mad1 proteins translated in reticulocyte lysate. After washing, the proteins bound to beads were analyzed by SDS–PAGE followed by autoradiography. As shown in Figure 3B, p31comet was selectively retained on beads containing N2-Mad2wt dimer or N2-Mad2R133A monomer. There was no detectable binding between p31comet and N1-Mad2 (Figure 3B). In contrast, N1-Mad2wt and N1-Mad2R133A bound more tightly to the C-terminal domain of Mad1 (Mad1C) as compared to N2-Mad2, while both N1 and N2 forms of Mad2 bound equally well to the N-terminal fragment (residues 1–174) of Cdc20 (Figure 3B). Therefore, these Mad2-binding proteins exhibit different binding preferences toward the N1 and N2 forms of Mad2. P31comet preferably binds to N2-Mad2. We do not yet fully understand why Mad1 prefers N1-Mad2 because the binding of N1-Mad2 to Mad1 is a complicated process (Luo et al, 2004). However, this is very likely due to a difference in binding kinetics between Mad1 and the two forms of Mad2. To confirm the findings of these qualitative binding assays, we used isothermal calorimetry titration (ITC) to measure the binding affinity between p31comet and Mad2 quantitatively (Figure 3C). The dissociation constant (Kd) of the p31comet–N2-Mad2R133A complex was determined to be 0.24 μM (Figure 3C), while there was no detectable binding between p31comet and N1-Mad2 (data not shown). Thus, the ITC experiment demonstrates that p31comet only binds to the N2 form of Mad2. Furthermore, the stoichiometry of binding between p31comet and Mad2 was very close to 1:1. The native molecular weight of the Mad2wt–p31comet or the Mad2R133A–p31comet complexes was estimated to be around 60 kDa by gel filtration chromatography (data not shown). These data indicate that p31comet selectively forms a simple 1:1 heterodimer with N2-Mad2 with high affinity. This finding was further confirmed by NMR studies (Figure 4). Addition of p31comet caused dramatic line broadening and disappearance of most HSQC signals of N2-Mad2R133A due to the formation of the 60 kDa p31comet–Mad2 complex (Figure 4A). In contrast, at a final concentration of 80 μM, addition of p31comet did not alter the pattern of the 1H–15N HSQC spectrum of N1-Mad2R133A, demonstrating that there is no binding even at high concentrations (Figure 4B). Thus, the Kd between p31comet and N1-Mad2 is greater than 80 μM. This in turn indicates that p31comet binds to N2-Mad2 preferably with a selectivity greater than 330-fold. Figure 4.P31comet selectively binds to N2-Mad2 but not to N1-Mad2. (A) Overlay of the 1H–15N HSQC spectra of the free 15N-labeled N2-Mad2R133A (in black) and the mixture of 60 μM 15N-labeled N2-Mad2R133A and 80 μM unlabeled p31comet (in red). (B) Overlay of the 1H–15N HSQC spectra of the free 15N-labeled N1-Mad2R133A (in black) and the mixture of 60 μM 15N-labeled N1-Mad2R133A and 80 μM unlabeled p31comet (in red). Download figure Download PowerPoint We next tested whether p31comet only interacts with N2-Mad2 in living cells. As the C-terminal region of Mad2 is also required for the N1–N2 structural transition, ΔC-Mad2 exclusively adopts the N1 conformation and cannot undergo the N1–N2 conversion (data not shown). We cotransfected plasmids encoding Myc-tagged Mad2 or ΔC-Mad2 and HA-tagged p31comet into HeLa cells and performed co-immunoprecipitation experiments. As shown in Figure 5A, a significant portion of HA-p31comet was precipitated with anti-Myc beads when the Myc-tagged Mad2 was coexpressed. The Myc-tagged Mad2 protein was also detected in the anti-HA immunoprecipitates when coexpressed with HA-p31comet (Figure 5A). As expected, p31comet did not interact with ΔC-Mad2 in this assay (Figure 5A). The expression levels of Mad2, ΔC-Mad2, and p31comet in the lysates were similar in these samples (Figure 5B). Therefore, p31comet does not interact with ΔC-Mad2 in living cells, presumably due to the inability of ΔC-Mad2 to adopt the N2 conformation. Figure 5.P31comet does not bind to ΔC-Mad2 in living cells. (A) HeLa Tet-on cells were transfected with the indicated plasmids and lysed. The resulting lysates were immunoprecipicated with anti-Myc or anti-HA beads and the immunoprecipitates were then blotted with anti-Myc or anti-HA. (B) The cell lysates in (A) were blotted with anti-Myc or anti-HA. Download figure Download PowerPoint Cdc20 and p31comet bind to distinct sites on Mad2 As described above, p31comet blocks the ability of Mad2 to inhibit APC/CCdc20 in vitro and in Xenopus egg extracts. P31comet also selectively binds to the N2 conformation of Mad2 with high affinity. The most straightforward model to explain these findings is that p31comet competes with Cdc20 for binding to the N2 conformer of Mad2. We tested this possibility directly. As shown in Figure 6A, the full-length Cdc20 was selectively retained on glutathione–agarose beads bound to GST-Mad2. Surprisingly, addition of His6-tagged p31comet did not affect the binding of Cdc20 (Figure 6A). This suggested that Mad2, Cdc20, and p31comet might transiently form a ternary complex. We also performed the binding experiment in the reverse order. The preformed Mad2–Cdc20 complex also associated with 35S-labeled p31comet (Figure 6B). We next measured the binding affinities among Mad2, p31comet, and Cdc20 using ITC (Figure 6). A Cdc20 peptide (Cdc20P1) containing the Mad2-binding motif of Cdc20 bound to N2-Mad2R133A with a dissociation constant (Kd) of 0.65 μM (Figure 6C and D). Consistent with the qualitative beads pull-down experiments, Cdc20P1 bound to the Mad2–p31comet complex with a similar affinity (Kd=0.69 μM) (Figure 6D). Thus, these data indicate that binding of p31comet to Mad2 does not prevent Mad2 from binding to Cdc20. Mad2, Cdc20, and p31comet can indeed form a ternary complex. This also indicates that p31comet and Cdc20 use distinct binding mode to interact with Mad2, and they bind to different sites on Mad2. It should be mentioned that the Mad2–Cdc20P1 affinity measured in this study was somewhat lower than, but similar to, the affinity between Mad2R133A and a longer Cdc20 peptide (Kd=0.1 μM) reported by Sironi et al (2002). This difference is very likely due to the use of a longer Cdc20 peptide in their experiment (Sironi et al, 2002). Figure 6.Mad2, Cdc20, and p31comet form a ternary complex in vitro. (A) GST-Mad2 was bound to glutathione–agarose beads and incubated with 35S-labeled full-length human Cdc20 in the presence of 20 μg of His6-tagged Mob1 or p31comet proteins. GST, His6-Mob1, or His6-p31comet alone was used as negative controls and 20% of the input 35S-labeled Cdc20 protein used in the binding reaction was loaded for comparison. After washing, the proteins retained on beads were analyzed by SDS–PAGE followed by autoradiography. The samples were also blotted with α-p31comet. (B) GST-Mad2 or the GST-Mad2–Cdc20 complex was bound to glutathione–agarose beads and incubated with 35S-labeled p31comet. GST was used as a negative control and 20% of the input 35S-labeled p31comet prot
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