Faithful segregation of genetic material during cell division is essential for the viability of all organisms. For each chromosome, DNA replication creates two identical copies, which are segregated from each other at mitosis. Segregation is directed by the kinetochore, a specialized multi-protein structure that assembles on centromeric DNA and binds to and moves along microtubules. Normal segregation depends on the two sister kinetochores attaching to microtubules from opposite spindle poles during mitosis. Eukaryotes use a control circuit called the spindle checkpoint to ensure accurate segregation. During unperturbed mitosis, an E3 ubiquitin ligase known as the anaphase-promoting complex (APC) and its co-activator Cdc20 triggers anaphase and chromosome segregation by catalyzing the ubiquitination and destruction of securin (Pds1 in budding yeast) (). The absence of microtubule attachment [1
] or the lack of tension at the kinetochore (because of chromosome failing to attach to opposite spindle poles) [3
] activates the checkpoint, which arrests cells at the metaphase-to-anaphase transition by targeting APC and Cdc20 for inhibition (for reviews see [6
]). In the budding yeast, Saccharomyces cerevisiae
, the key players of the spindle checkpoint include Mad1, Mad2, Mad3, Bub1, Bub3, Mps1, and Ipl1, all of which are highly conserved among eukaryotes [1
Figure 1 A model for spindle checkpoint activation (adapted from [6, 7]). (A) During mitosis, when all chromosomes are properly attached to microtubules, the anaphase-promoting complex (APC) and its co-activator Cdc20 polyubiquitinate different substrates such (more ...)
Although the checkpoint proteins have been studied extensively, we lack a molecular description of how events at the kinetochore are converted into inhibition of the APC. Several models have been described including the conformational change (Mad2-template) model [6
], which proposes that Mad1-Mad2 complexes associate with kinetochores that lack microtubule attachments and recruit an “open” Mad2 conformer (O-Mad2), facilitating the formation of the “closed” Mad2 (C-Mad2)-Cdc20 complex (). Besides the recruitment of Mad1 and Mad2 to unattached kinetochores, experiments such as fluorescent protein localization and coimmunoprecipitation (co-IP) have shown that in budding yeast both Bub1 and Bub3 can associate with kinetochore [10
] and Mad1 [11
], while Mad3 can interact with both Mad2 and Bub3 [12
]. This complicated network of interactions can potentially bring different checkpoint proteins together at the kinetochores in response to attachment errors and lead to formation of additional inhibitory complexes. One example is the mitotic checkpoint complex (MCC), which is proposed to consist of Mad2, Mad3, Bub3 and Cdc20 and has been shown to be a potent inhibitor of APCCdc20
] (). Inhibition of APC activity arrests cells in metaphase and provides the cells a chance to correct the attachment errors at the kinetochores. The spindle checkpoint hence ensures that cells only progress through mitosis when all chromosomes are properly attached.
The initial studies that identified Cdc20 as the target of the spindle checkpoint showed that both Mad2 and Mad3 bind to Cdc20 [15
]. We have investigated the consequences of this binding by manipulating the linkage between Mad2, Mad3, and Cdc20. Expressing physically-linked Mad2 and Mad3 induces a metaphase arrest that does not require functional kinetochores or other checkpoint proteins, indicating the Mad2-Mad3 fusion alone is sufficient to inhibit APC activity. We also show that tethering Mad2 directly to Cdc20 can lead to similar arrest that does not require Mad3 or other checkpoint components, supporting the idea that the Mad2-Mad3 fusion induces metaphase arrest by promoting an intimate association between Mad2 and Cdc20. Our results suggest that the most downstream event in spindle checkpoint activation is the cooperative binding of Mad2 and Mad3 to Cdc20.