We have identified a Xenopus
homologue of the budding yeast Mad1 protein and shown that it is a component of the spindle checkpoint. Xmad1 was isolated as a protein that coimmunoprecipitated with Xmad2. When Xmad2 is depleted from extracts, adding back physiological doses does not restore the checkpoint. This observation suggests that other components of the checkpoint bind to Xmad2 and are removed with it from the extract upon immunodepletion. The observation that Xmad1 coimmunoprecipitates with Xmad2 strongly suggests that the additional depleted component is Xmad1, although we cannot exclude the possibility that there are other checkpoint components bound to Xmad2. One protein that has been reported to bind to Mad2 is Cdc20 (Fang et al., 1998
; Hwang et al., 1998
; Kallio et al., 1998
; Kim et al., 1998
), a protein that is necessary for the metaphase to anaphase transition (Dawson et al., 1995
; Visintin et al., 1997
; Kallio et al., 1998
). Since Xmad2-depleted extracts can still exit from mitosis (data not shown), they must still contain functional Cdc20.
As expected, Xmad1 shares several characteristics with Xmad2. First, Xmad1 is important for establishing and maintaining the spindle checkpoint in egg extracts. Second, Xmad1 localizes to the nuclear envelope and the nucleus during interphase, and a fraction of the protein binds to the kinetochores of chromosomes that are not attached to microtubules during prophase and prometaphase. Despite these similarities and the tight interaction between these two proteins, Xmad1 and Xmad2 appear to play distinct roles in the checkpoint. Our studies indicate that Xmad1 likely recruits Xmad2 to unattached kinetochores, where Xmad2 is converted into a form that can prevent the onset of anaphase.
Our results extend previous experiments on the function of Mad1 and Mad2 in the spindle checkpoint. Experiments in budding yeast show that Mad1 and Mad2 bind tightly to each other (Chen, R.-H., K. Hardwick, and A. Murray, unpublished results), and that the presence of Mad2 is required for the hyperphosphorylation of Mad1 that correlates with the activation of the checkpoint (Hardwick and Murray, 1995
). Mad2 has not been localized in yeast cells, and Mad1 shows a punctate localization within the nucleus (Hardwick and Murray, 1995
). It remains a possibility that some of the punctate staining is at kinetochores. We now demonstrate that Xmad1 binds to unattached kinetochores in both tissue culture cells and in frog egg extracts. This is the same pattern of localization for vertebrate homologues of Mad2 (Figs. and ; Chen et al., 1996
; Li and Benezra, 1996
), Bub1 (Taylor and McKeon, 1997
), Bub3 (Taylor et al., 1998
), and Mad3 (Taylor et al., 1998
). The result that anti-Xmad1 antibodies block the association of both Xmad1 and Xmad2 with the kinetochores in egg extracts suggests that the binding of Xmad2 to unattached kinetochores is dependent on Xmad1. Even when Xmad1 and Xmad2 are allowed to bind to unattached kinetochores first, the subsequent addition of anti-Xmad1 antibodies removes both proteins from kinetochores (data not shown), consistent with the idea that both proteins must reside on kinetochores to maintain the checkpoint. Immunodepletion experiments showed that almost all of the Xmad1 is bound with Xmad2, whereas only 40% of Xmad2 is bound with Xmad1 (Fig. A
), suggesting that Xmad1 is the limiting factor in the Xmad1–Xmad2 complex.
Although the spindle checkpoint is normally triggered by unattached kinetochores, we have demonstrated that a high level of Xmad2 in frog egg extracts also inhibits the metaphase to anaphase transition under conditions where the checkpoint is not normally activated. Excess Xmad2 blocks three hallmarks of anaphase: degradation of cyclin B, inactivation of Cdc2, and sister chromatid segregation. Consistent with our result, overexpression of Mad2 homologue in fission yeast also blocks anaphase onset (He et al., 1997
; Kim et al., 1998
), and addition of human Mad2 protein to frog egg extracts also prevents cyclin B degradation (Li et al., 1997
; Fang et al., 1998
). Furthermore, deletion of as few as 10 amino acids from COOH terminus of Xmad2 abolishes the ability of excess protein to induce metaphase arrest (data not shown). Introduction of a high level of the recombinant human Mad2 of similar truncation also fails to induce a mitotic arrest in Xenopus
embryos and to inhibit ubiquitination of cyclin B in vitro (Fang et al., 1998
). Taken together, these results indicate that the phenotypes we observed were not due to some aberrant activity of the recombinant protein. We can rule out several possibilities for how excess Xmad2 induces metaphase arrest. The arrest cannot be dependent on binding of Xmad2 to kinetochores, since it occurs in extracts without any nuclei. In addition, it is unlikely that Xmad2 acts as a competitive inhibitor of proteolysis mediated by the anaphase-promoting complex (APC), because Xmad2 lacks any trace of the destruction boxes found in other APC substrates (Glotzer et al., 1991
; review in Hershko, 1997
and Cohen-Fix and Koshland, 1997
) and Xmad2 levels remain constant during the cell cycle (Fig. ).
Increases in the level or activity of a number of proteins other than Mad2 can also activate the checkpoint in cells that have normal spindles. Adding an active form of Ste11, a budding yeast MAPK kinase family member, arrests frog egg extracts at mitosis (Takenaka et al., 1997
). In budding yeast, overexpression of Mps1, the kinase that is thought to phosphorylate Mad1, arrests cells at mitosis without spindle defects (Hardwick et al., 1996
), and overexpression of Mph1, a S
homologue of Mps1, also induces metaphase arrest (He et al., 1998
). All these observations suggest that the activity or the level of the spindle checkpoint components must be tightly regulated in order for the metaphase to anaphase transition to progress properly.
How is Xmad2 activated by unattached kinetochores and how does it inhibit the onset of anaphase? We speculate that once Xmad1 binds to kinetochores and activates its associated Xmad2, Xmad2 is converted into a form that no longer binds Xmad1 and thus diffuses throughout the cell preventing the onset of anaphase. The free, inactive Xmad2 can then interact with and become activated by kinetochore-bound Xmad1, making the kinetochore a catalytic center for activating the checkpoint (Fig. ). The active molecules of Xmad2 would be slowly inactivated, thus ensuring that the checkpoint would be turned off once all the kinetochores had bound to microtubules. The molecular basis of Xmad2 activation is obscure since we have been unable to detect posttranslational modifications on this protein (unpublished data). One possible form of activation is changes in the state of Xmad2, as indicated by the finding that monomers and tetramers of the recombinant human Mad2 protein differ in their ability to inhibit cyclin degradation in egg extracts (Fang et al., 1998
Figure 11 A model for how Xmad1 and Xmad2 work to activate the spindle checkpoint. Binding of Xmad1 to unattached kinetochores enables its associated Xmad2 to interact with a downstream checkpoint component X. This interaction converts X into a form that is capable (more ...)
The most likely target of Xmad2 is Cdc20. In budding yeast, Cdc20 binds to Mad2 and Mad3 proteins, and mutations in Cdc20 that block this binding act as dominant checkpoint-defective mutants (Hwang et al., 1998
), suggesting that Cdc20 is the target of the checkpoint. Similar results have also been obtained in fission yeast (Kim et al., 1998
). Cdc20 homologues also mediate the association of Mad2 with APC in vertebrates (Gorbsky et al., 1998
; Fang et al., 1998
). A simple model is that interacting with unattached kinetochores converts Xmad2 into a form that can interact and inhibit Cdc20. In the absence of kinetochores, Xmad2 may assume the active conformation at a very slow rate, but this small amount of active Xmad2 cannot efficiently inhibit Cdc20 function. As the level of Xmad2 is increased, the concentration of the active form rises by mass action until it is sufficient to inhibit Cdc20, thus arresting extracts in metaphase (Fig. ). In such a scheme, kinetochore-bound molecules that convert Xmad2 into its active form would be dispensable for excess Xmad2 to arrest the cell cycle, whereas the arrest would require molecules that functioned with Xmad2 to inhibit Cdc20. Our data show that excess Xmad2 can still activate the checkpoint when >95% of Xmad1 is immunodepleted, suggesting that Xmad1 is not involved in signaling events downstream of Xmad2, and that the major role of Xmad1 in the checkpoint is to recruit Xmad2 to unattached kinetochores. Future experiments will be required to determine how kinetochores monitor their interactions with microtubules and control the binding of Xmad1, how Xmad1 binding activates Xmad2, and how the activated Xmad2 inhibits Cdc20 and any other targets of the spindle checkpoint.