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Address correspondence to Tim Yen, Institute for Cancer Research, The Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: (215) 728-2590. Fax: (215) 728-2412. E-mail: tj_yen{at}fccc.edu
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Abstract |
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Key Words: kinetochore; hBUBR1; anaphase promoting complex; MAD2; mitotic checkpoint
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Introduction |
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Our current understanding of the mechanism by which the APC/C is inhibited by unaligned chromosomes has come primarily from studies of the MAD2 checkpoint protein. Consistent with the behavior of MAD2 mutants in yeast (Hoyt et al., 1991; Li and Murray, 1991; He et al., 1997), disruption of MAD2 function in mammalian cells and Xenopus extracts prevented the establishment of a checkpoint arrest in response to spindle damage (Chen et al., 1998; Gorbsky et al., 1998). The ability of MAD2 to selectively bind unattached kinetochores suggests that one of its functions is to monitor kinetochoremicrotubule interactions. In mammalian cells, the evidence suggests that MAD2 may directly monitor the microtubule occupancy at kinetochores such that kinetochores fully saturated with microtubules exhibit no detectable MAD2, whereas MAD2 is prominently localized to unattached kinetochores (Waters et al., 1998). How this occurs remains unknown, but MAD2 is sensitive to microtubule interactions mediated by the kinetochore motor CENP-E. Depletion of CENP-E from kinetochores in mammalian cells disrupts chromosome alignment, and cells become arrested in mitosis with high levels of MAD2 at unattached kinetochores (Yao et al., 2000). In Xenopus extracts, depletion of CENP-E prevents MAD2 localization to kinetochores, and thus the extracts fail to arrest in mitosis when spindle assembly is inhibited (Abrieu et al., 2000). Despite the differences between how the checkpoint in these two experimental systems responds to loss of CENP-E function, it is clear that CENP-E activity at kinetochores is linked to MAD2 binding to kinetochores.
In addition to its role at kinetochores, MAD2 is thought to have a downstream role in directly blocking APC/C activity. MAD2 was found to bind to the APC/C in cytosol that was obtained from mitotically arrested HeLa cells (Li et al., 1997). Addition of excess MAD2 to mitotic egg extracts blocked exit from mitosis even in the absence of kinetochores (Chen et al., 1998). Furthermore, MAD2 was found to directly inhibit the ubiquitin ligase activity of purified APC/C in vitro (Li et al., 1997; Fang et al., 1998). Interestingly, bacterially expressed MAD2 was found to exist as either monomers or tetramers but only the tetrameric form was found to inhibit the APC/C (Fang et al., 1998). The possibility that there are inactive and active states of MAD2 provided a mechanistic explanation for an existing model in which unattached kinetochores are envisioned to convert MAD2 into a form that can inhibit APC/C activity (Gorbsky et al., 1998; Howell et al., 2000; Shah and Cleveland, 2000). One part of this model, whereby MAD2 is postulated to cycle on and off kinetochores, has been confirmed by FRAP experiments that measured the half-life of kinetochore-bound MAD2 (Howell et al., 2000). These studies estimated that the half-life of MAD2 at unattached kinetochores is 25 s. This rapid turnover rate was predicted to generate sufficient amounts of MAD2 to sustain a prolonged inhibition of the APC/C.
Although the collective studies have shed considerable light on MAD2, whether inhibition of APC/C is specified solely by MAD2 in vivo is unknown. In yeast and mammalian cells, MAD2 has been shown to interact with the APC/C through CDC20, a protein that specifies substrate selectivity by the APC/C (Dawson et al., 1995; Visintin et al., 1997; Fang et al., 1998; Hwang et al., 1998; Kallio et al., 1998; Kim et al., 1998). Furthermore, complexes consisting of MAD3, BUB3, CDC20, and MAD2 were identified in budding (Hardwick et al., 2000) and fission yeasts (Hardwick, K.G., personal communications). In mitotic HeLa cells, APC/C is associated with the hBUBR1 checkpoint kinase (Chan et al., 1999). This observation suggests that inhibition of APC/C in vivo might be achieved through more complex schemes. To obtain some insights into how APC/C is inhibited by the checkpoint in vivo, we set out to identify factors from mitotically arrested HeLa cells that inhibited APC/C. This search yielded a single stable complex named the mitotic checkpoint complex (MCC), consisting of the proteins hBUBR1, hBUB3, CDC20, and MAD2. We report here on the identification and characterization of MCC, and present evidence that this is a physiologically relevant inhibitor of the APC/C.
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Results |
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Next, we sought evidence to directly support our finding that hBUBR1 is part of the inhibitory complex that blocks APC/C activity. Initial attempts to purify MCC with the hBUBR1 antibodies were unsuccessful because the harsh conditions required to elute hBUBR1 from the antibody-linked beads destroyed the complex. An alternative approach was to express a glutathione S-transferase (GST)-tagged hBUBR1 in human embryonic kidney cell line HEK293T and then use glutathione beads to affinity purify it along with the appropriate subunits from extracts of transfected cells (Fig. 4, A and B). Western blot analysis confirmed that the affinity-purified GSThBUBR1 formed the MCC, as it contained endogenous hBUBR1, hBUB3, CDC20, and MAD2. As shown previously, only a small fraction of the total pool of MAD2 was associated with GSThBUBR1:MCC (Fig. 4 A, lane 3). All of these associations are specific to hBUBR1, as these proteins did not associate with GST alone (Fig. 4 A, lane 1). Purified GSThBUBR1:MCC was found to inhibit ubiquitin ligase activity of the APC/C (Fig. 4 B, lane 5) as was seen with conventionally purified MCC (Fig. 4 B, lane 4). GST purified from transfected cells did not exhibit significant inhibitory activity (Fig. 4 B, lane 3).
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It is noteworthy that our radiolabeled immunoprecipitate contained additional prominent bands. As hBUBR1 is known to form a separate complex with CENP-E (which elutes in the void of Superose 6 columns), these complexes were likely to be present in the immunoprecipitates. These additional hBUBR1 complexes explain the higher stoichiometry of hBUBR1 in our calculations. If this is taken into account, we estimate that the MCC subunits exist in near equal stoichiometry. Regardless, the results obtained from the radiolabeling experiment do not support the presence of oligomeric forms of MAD2 in the MCC. Our data show that hBUBR1 forms additional uncharacterized complexes in HeLa cells (Fig. 3 C, fractions 3235); the complexes possessed no inhibitory activity (unpublished data). The identity and functions of these complexes remain to be elucidated. We suggest that the existence of the additional complexes contributed to an increased amount of hBUBR1 in the calculated MCC ratio presented above. Another possibility is that the MCC contains yet unidentified subunits. The complex migrates at 400 kD, whereas its molecular mass estimated by our radiolabeling analysis gives a molecular mass of 300 kD.
MCC is expressed throughout the cell cycle but only targets mitotic APC/C
We showed previously that the 400-kD hBUBR1 complex was present in both interphase and mitotic cells (Chan et al., 1999). Given that hBUBR1 was found to associate with the APC/C only in mitosis (Chan et al., 1999), we expected that only the mitotic form of the MCC would inhibit the APC/C. Surprisingly, hBUBR1 complex isolated from interphase HeLa cells (synchronized in the G1/S boundary) inhibited APC/C activity and contained the same subunits found in mitotic MCC (unpublished data). Furthermore, both interphase and mitotic forms of MCC exhibited similar activities when equivalent amounts of hBUBR1 were tested (Fig. 6 A). Although this MCC preparation was obtained from cells arrested at the G1/S boundary, analysis of synchronized cells obtained by centrifugal elutriation showed that MCC is present in all stages of the cell cycle (unpublished data).
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Sorting out the interactions between chromosomes, APC/C, and MCC
The ability of MCC from interphase cells to block APC/C shows that its activity does not require the presence of unattached kinetochores, as mature kinetochores are not found until mitosis. To clarify the interactions between MCC, APC/C, and kinetochores, we set out to reconstitute the checkpoint inhibition of the APC/C in lysates prepared from HeLa cells. Kinetic studies showed that the APC/C activity in crude mitotic lysates exhibited a reproducible lag of 15 min, compared with the APC/C activity in lysates prepared from asynchronous cells that were >90% interphase (Fig. 7 A, compare
and
). The initial lag seen in the mitotic lysates may have been due to checkpoint inhibition, as lysates were prepared from mitotically arrested cells whose APC/C is associated with MCC. In the absence of chromosomes, the checkpoint inhibition cannot be sustained and the APC/C eventually recovers its activity. This was confirmed when addition of purified chromosomes to the mitotic lysate suppressed the reactivation of ACP/C (Fig. 7 A,
). Chromosomes do not permanently inactivate the APC/C, as APC/C was reactivated when chromosomes were removed from during the incubation (unpublished data). Chromosomes were only effective in mitotic lysates as they modestly reduced APC/C activity in asynchronous lysates (Fig. 7 A, ). This modest reduction can be attributed to the small fraction of mitotic cells that were present in the asynchronous population.
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We next attempted to reconstitute the suppression of APC/C activity observed in crude lysates by using purified components. It was important to verify that the chromosome-induced inhibition of the APC/C activity was not caused by unidentified factors in the crude preparation. As in crude mitotic lysates, we found that chromosomes are able to effectively sustain the APC/C inhibition in a highly purified sample (Fig. 7 C). Given that chromosomes did not exert an effect on MCC or CDC20, its ability to suppress the activation of APC/C maybe mediated through the APC/C.
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Discussion |
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Although oligomerization of recombinant MAD2 was found to enhance its ability to inhibit APC/C activity (Fang et al., 1998), it cannot account for the inhibitory activity of the MCC. Based on the near equal stoichiometry of the MCC subunits, it is unlikely that MAD2 oligomers are present in the MCC despite its ability to oligomerize in bacteria (Fang et al., 1998). It is unclear whether the inhibitory activity of MAD2 is enhanced through its association with the other MCC subunits, or whether the other subunits of MCC also directly block APC/C activity. Taken together, our data strongly suggest that MCC is the physiologically relevant inhibitor of the APC/C in HeLa cells. Interestingly, a similar complex consisting of MAD3, CDC20, and MAD2 has been identified in yeast but its role in regulating APC/C has not been elucidated (Hardwick et al., 2000). It appears that the MCC may be conserved throughout evolution.
It is noteworthy that we did not detect MAD1 in MCC (unpublished data) even though MAD1 and MAD2 form a complex in vivo (Chen et al., 1998, 1999; Jin et al., 1998; Campbell et al., 2001). Studies in Xenopus extracts suggest that MAD1 specifies kinetochore binding by MAD2 but that is not directly involved in inhibiting the APC/C (Chen et al., 1998). We have also not detected the presence of checkpoint proteins such as hBUB1, hZW10, or hROD in the MCC. Therefore, these components are likely to provide functions that are distinct from the MCC.
We believe that MCC inhibits APC/C by directly binding to it rather than via catalytic inactivation. This conclusion is based on the finding that the intracellular concentration of hBUBR1 and APC/C are roughly the same (52 and 65 nM for hBUBR1 and CDC27 in crude lysate, respectively; ratio 1:1 in anti-hBUBR1 immunoprecipitates from whole mitotic lysate; unpublished data). Our unpublished data indicate that there is
400 nM of MAD2 in mitotic HeLa lysates. The gel filtration profiles show that only 510% of the total MAD2 is a part of high molecular mass complexes and comigrates with APC/C and MCC (Fig. 3 A). Given the hBUBR1 and CDC27 concentrations we have a roughly equal ratio between hBUBR1, CDC27, and "active" MAD2 in our extracts (
52, 65, and 2040 nM, respectively). This ratio of hBUBR1 to active MAD2 is similar to our estimate using radiolabeled proteins.
Examination of the MCC and APC/C profiles in a MonoQ column showed that MCC was associated with 75% of the APC/C (Fig. 3 D). However, the remaining 25% of the APC/C most likely lost its association with its MCC, as we found MCC in fractions that did not contain any APC/C (Fig. 3 C). We had relied previously on immunoprecipitation to estimate that
25% of the APC/C was bound to hBUBR1 (Chan et al., 1999). This is likely to be an underestimate, as we now know that the conditions used to wash the immunoprecipitate dissociated much of the APC/C from hBUBR1. The identification of the molecular interactions among the various subunits within APC/C and MCC will be crucial for understanding the mechanism of inhibition.
Roles of MCC and kinetochores in inhibition of APC/C
The finding that MCC is present during interphase and can inhibit the APC/C was unexpected because kinetochores (which are not present during interphase) are thought to generate the inhibitor of the APC/C. However, we found that only APC/C from mitotic cells was sensitive to MCC inhibition. These findings have changed our view of the checkpoint pathway in two important ways (Fig. 8). First, our data suggest that the formation and activity of the APC/C inhibitor can be uncoupled from kinetochores. This may be an important feature considering that APC/C is known to be activated at the onset of mitosis. The existence of a preformed pool of inhibitor would rapidly block precocious ubiquitination activity by the APC/C at the onset of mitosis. It is noteworthy that only the mitotically modified form of the APC/C can be inhibited (Fig. 6, B and C; Fig. 7 A), which can explain why the preformed pool of MCC doesn't inhibit APC before mitosis. By necessity, the inhibition must be reversible so that APC/C can be activated once cells are ready to exit mitosis. We believe that in vivo, the interaction between APC/C and MCC is quite labile in the absence of unattached kinetochores. This would explain why APC/C that is purified from mitotic cells exhibits a lag before it becomes reactivated.
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The role of the kinetochore in generating wait anaphase signal
The biochemical nature of the wait anaphase signal that is generated at kinetochores remains to be elucidated but it is likely to involve many of the checkpoint proteins that reside there. FRAP analysis of MAD2 showed that its half-life at kinetochores is 25 s, and that
1,300 molecules are present on kinetochores at any given time (Howell et al., 2000). The rapid turnover rate supports a model whereby unattached kinetochores catalytically convert MAD2 into a form that inhibits the APC/C upon its release from kinetochores (Chen et al., 1998; Gorbsky et al., 1998; Howell et al., 2000). Our data cannot eliminate this possibility despite the fact that kinetochores are not required for MCC formation or activity. In light of our findings, we propose that MAD2 that is derived from kinetochores may enhance the sensitivity of the APC/C to the inhibitory action of MCC.
As the fate of MAD2 after it has dissociated from kinetochores is not known, other possibilities remain open. An alternative view is that the APC/C inhibition is mediated by checkpoint kinases such as BUBR1 and BUB1 that reside at kinetochores. In this scenario, MAD2 could regulate these kinetochore-associated kinases such that its presence at unattached kinetochores is required to maintain a threshold level of kinase activity. It is noteworthy that hBUBR1 that is associated with kinetochores becomes quantitatively hyperphosphorylated within 15 min after disruption of microtubule attachments (Chan et al., 1999). This may contribute to MAD2 binding, as in vitro studies show that MAD2 will only bind to kinetochores that are phosphorylated (Waters et al., 1998). We speculate that hyperphosphorylation of hBUBR1 stimulates its kinase activity that is required for generating the wait anaphase signal. In vivo, efficient phosphorylation of APC/C by kinetochores may be achieved through a kinase cascade. Our attempt at reconstituting the checkpoint inhibition of APC/C by adding just chromosomes probably does not reflect the in vivo situation. In our experiments, we believe that chromosomes are directly modifying the APC/C while in vivo, an occurrence probably accomplished by a signaling cascade amplifying the signal that originates from a single unattached kinetochore. Whatever the nature of the modification that is introduced to the APC/C by kinetochores, it must be labile in order to relieve MCC inhibitory activity upon chromosome alignment.
MCC interactions with APC/C
Regardless of whether kinetochores modify APC/C to prolonged inhibition by the MCC, our data show that APC/C must undergo mitotic modifications in order for it to be recognized by the MCC. This is supported by the observation that interphase APC/C was insensitive to inhibition by MCC. The insensitivity cannot be due simply to the lack of modifications introduced by kinetochores, as APC/C activity in interphase lysates was unaffected in the presence of chromosomes. We believe that the mitotic modifications that target the MCC to APC/C include phosphorylations of specific APC/C subunits (King et al., 1995; Lahav-Baratz et al., 1995; Charles et al., 1998; Descombes and Nigg, 1998; Kotani et al., 1999; Shirayama et al., 1998) and its association with CDC20 (Fang et al., 1998; Shirayama et al., 1998; Chan et al., 1999; Shteinberg et al., 1999). The importance of CDC20 in checkpoint control is highlighted by the finding that certain CDC20 mutants in fission (Kim et al., 1998) and budding yeast (Hwang et al., 1998; Schott and Hoyt, 1998) exhibit checkpoint defects. Whether this mutation affects formation of an MCC-like complex (Hardwick et al., 2000; Millband, D., and K.G. Hardwick, personal communications) or alters the sensitivity of the APC/C to inhibition by the checkpoint remains to be clarified. How modifications to the APC/C contribute to recognition by MCC remains to be determined, but it has been reported that MAD2, CDC20, and hBUBR1 bind preferentially to mitotically phosphorylated APC/C (Fang et al., 1998; Kallio et al., 1998; Kotani et al., 1999; Wu et al., 2000). We suggest that these previously reported findings reflect the MCCAPC/C association during mitosis. It remains to be seen whether the modifications that are required for APC/C activity are also used by the MCC to inhibit the APC/C.
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Materials and methods |
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Purification of mitotic chromosomes
Chromosomes were purified from mitotically blocked HeLa cells as described (Yen et al., 1991). In brief, HeLa cells were blocked with nocodazole for 18 h, pelleted and hypotonically swollen in RSB (10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM NaCl), and lysed in digitonin containing buffer A (50 mM Tris-HCl, pH 7.5, 80 mM KCl, 2 mM EGTA, 3 mM spermine, 7.5 mM spermidine, 0.1% digitonin supplemented with 1 µM pepstatin, 1 µg/ml leupeptin, 1 mM PMSF, 2 µg/ml aprotinin). Cells were lysed using a dounce (1012 times using a pestle B) and centrifuged at 500 g for 5 min to remove nuclei and cell debris. Chromosomes in the supernatant were pelleted at 1,600 g for 10 min, washed three times in buffer 3 (50 mM Tris-HCl, pH 7.5, 40 mM KCl, 3 mM spermine, 3.75 mM spermidine with protease inhibitors), resuspended in buffer 4 (50 mM Tris-HCl, pH 7.5, 20 mM KCl, 3 mM spermine, 3.75 mM spermidine, 15% glycerol with protease inhibitors), and used immediately or snap frozen. Chromosomes were examined by DAPI staining and kinetochore integrity was monitored by staining for CENP-E and various mitotic checkpoint proteins.
Ubiquitination assay and APC/C isolation
Crude extracts or purified factors were supplemented with purified E1, E2, ATP regenerating system, and iodinated protein Acyclin B substrate (Glotzer et al., 1991), and the APC/C activity was determined as described (Sudakin et al., 1995) in final volume of 10 µl. Standard reactions were performed for 30 min at 30°C but kinetic studies relied on sampling the reaction at various times. Reaction products were separated by SDS-PAGE and the dried gel was analyzed with a phosphorimager (Fujix) to quantify the amount of ubiquitinated substrate. This amount was compared with the amount of input substrate to obtain the percent of substrate that was ubiquitinated in each reaction. In all experiments, the percent of substrate that is conjugated in a positive control reaction that contained APC/C was set at 1 and served as a reference for the activities obtained in other reactions in the same experiment. Most of the figures show normalized activities. APC/C was partially purified from HeLa lysates (15 mg total protein) by fast protein liquid chromatography (FPLC) (Amersham Pharmacia Biotech) using Superose 6 or Superdex 200 gel filtration columns. Gel filtration columns were eluted with buffer A (50 mM Tris-Cl, pH 7.2, 250 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA) and fractions exhibiting APC/C activity were pooled, desalted, and concentrated in buffer B (50 mM Tris-Cl, pH 7.2, 1 mM DTT, 10% glycerol). Further purification of APC/C was achieved by chromatography through a MonoQ column. Proteins were eluted with a salt gradient (0.150.5 M NaCl) in buffer containing 50 mM Hepes, pH 7.4, and 1 mM DTT. BSA was added to the fraction collector tubes (0.2 mg/ml final concentration in collected fractions) and active fractions were washed with buffer B and concentrated. For immunoprecipitation experiments, affinity-purified hBUBR1 or CDC20 antibodies were incubated with APC/C-containing fractions and complexes recovered with 10 µl protein ASepharose beads. The beads were washed five times in ice cold PBS containing 0.1% NP-40 and 10% glycerol, and three times in ice cold buffer containing 50 mM Tris, pH 7.6, 1 mM DTT, 1 mg/ml BSA, and 5% glycerol. After the last wash, the buffer was aspirated completely and the beads were resuspended in 10 µl of the ubiquitination reaction mix as described above. Samples were incubated at 30°C for 30 min and mixed gently every 5 min. The reactions were terminated by SDS-PAGE sample buffer, boiled, and separated by SDS-PAGE. Ubsubstrate conjugates were quantified as described above.
The pET28 plasmid encoding the CDC20 protein was a gift from Dr. M. Brandeis (Hebrew University, Jerusalem, Israel). The CDC20 protein was expressed in vitro in a TNT Quick reticulocyte system (Promega).
Purification of the MCC and of recombinant MAD2
APC/C inhibitory activity was recovered in the 2040% ammonium sulfate cut of an S100 extract. This was subjected to FPLC separation through Superdex 200 gel filtration, MonoQ anion exchange, and Superose 6 gel filtration columns. Gel filtration columns were eluted with buffer A and fractions were desalted and concentrated in buffer B. MonoQ column was eluted with shallow salt gradient (0.20.5 M NaCl) in buffer B without glycerol. To show that MCC can exist independently of APC/C, the Superose 6 fractions of MCC and APC/C were pooled and refractionated on MonoQ anion exchange column by a shallow salt gradient (0.20.4 M NaCl in buffer B). Column fractions were concentrated, desalted into buffer B, and tested for inhibitory activity in the APC/C assay. During the initial purification of the inhibitory factor, equal cell equivalents of column fractions and APC/C were used for the inhibitor assay.
Human MAD2 cDNA was amplified by PCR from HeLa cell cDNA (CLONTECH Laboratories, Inc.) and cloned into pET28A expression vector (Novagen). Recombinant protein was expressed in JM109(DE3) and purified with a nickelagarose column (QIAGEN). Tetrameric recombinant hMAD2 was purified by Superdex 75 gel filtration chromatography as described (Fang, et al. 1998).
Affinity purification of MCC with GSThBUBR1
Full-length hBUBR1 cDNA was obtained by digestion of gfp-hBUBR1 (Chan et al., 1998) with BamH1 and KpnI, and transferred to the mammalian GST vector pEBG (gift from N. Grammatikakis, Lankenau Institute for Medical Research, Wynnewood, PA). Transient transfections were performed by lipofection using Lipofectamine Plus (Life Technologies) as per manufacturer's instruction. Transfected HeLa cells were lysed in 50 mM Tris, pH 7.2, 150 mM NaCl, 1 mM DTT, and protease inhibitors (10 µg/ml AEBSF, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 5 µg/ml chymostatin, 10 µg/ml aprotinin) by repeated freeze and thaw cycles. Lysates were clarified by spinning at 10,000 g at 4°C. GSThBUBR1 was affinity purified from the 10,000 g supernatant by glutathione Sepharose 4B beads (Amersham Pharmacia Biotech), washed five times with wash buffer (50 mM Tris, pH7.2, 250 mM NaCl, 1 mM DTT, and protease inhibitors), and eluted with 10 mM glutathione in lysis buffer. Eluted GSThBUBR1 was dialyzed and concentrated using Centricon10 (Millipore) into 50 mM Tris, pH 7.2, and 1 mM DTT for APC/C assay.
Stoichiometry of MCC
Synchronous populations of HeLa cells released from a G1/S boundary were labeled in 35S-Trans label for 6 h. Mitotic and interphase cells were mechanically separated, lysed in the NP-40 lysis buffer (0.1% NP-40, 10% glycerol in PBS), and the extracts were then incubated with nonimmune IgG or hBUBR1 antibodies. Immunoprecipitates were washed five times in ice cold lysis buffer and separated on SDS-PAGE. Radiolabeled proteins were visualized and quantified with the phosphorimager (Fujix). Cys + Met amounts in the MCC proteins (without initiating Met) have been used to calculate the ratio of the MCC subunits.
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Footnotes |
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Acknowledgments |
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This work was supported by the National Institutes of Health (POI CA 75138; Core Grant CA06927), March of Dimes Foundation, and an appropriation from the Commonwealth of Pennsylvania. V. Sudakin was supported by a fellowship from the Fulbright and Human Frontiers Science Foundations.
Submitted: 16 February 2001
Revised: 11 July 2001
Accepted: 13 July 2001
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