Mad2 and Mad3 Cooperate to Arrest Budding Yeast in Mitosis

Abstract

Summary Background The spindle checkpoint ensures accurate chromosome transmission by delaying chromosome segregation until all chromosomes are correctly aligned on the mitotic spindle. The checkpoint is activated by kinetochores that are not attached to microtubules or are attached but not under tension and arrests cells at metaphase by inhibiting the anaphase-promoting complex (APC) and its co-activator Cdc20. Despite numerous studies, we still do not understand how the checkpoint proteins coordinate with each other to inhibit APCCdc20 activity. Results To ask how the checkpoint components induce metaphase arrest, we constructed fusions of checkpoint proteins and expressed them in the budding yeast, Saccharomyces cerevisiae, to mimic possible protein interactions during checkpoint activation. We found that expression of a Mad2-Mad3 protein fusion or non-covalently linked Mad2 and Mad3, but not the overexpression of the two separate proteins, induces metaphase arrest that is independent of functional kinetochores or other checkpoint proteins. We further showed that artificially tethering Mad2 to Cdc20 also arrests cells in metaphase independently of other checkpoint components. Conclusion Our results suggest that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, which is sufficient to inhibit APC activity and is the most downstream event in spindle checkpoint activation.

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…While various subcomplexes between Cdc20 and checkpoint proteins exist, the most potent inhibitor of the APC is a soluble mitotic checkpoint complex (MCC) containing Mad2, Mad3, Bub3, and Cdc20 (Brady and Hardwick 2000;Hardwick et al 2000;Fraschini et al 2001b;Sudakin et al 2001;Tang et al 2001;Fang 2002;Chao et al 2012). A variety of data suggest that the key inhibition comes from the binding of Mad2 and Mad3 to Cdc20 (Chao et al 2012;Lau and Murray 2012).…”

Bimodal activation of BubR1 by Bub3 sustains mitotic checkpoint signaling

“…However, the identity of the ultimate mitotic checkpoint inhibitor remains controversial, with some investigators arguing that Mad2 plays the predominant role (16) and others arguing that the inhibitory activity of APC/C Cdc20 is provided by BubR1 (13,17), or both BubR1 and Mad2 (14,15). We recently demonstrated that the N-terminal Mad3 homology domain of BubR1 (including one of its two Cdc20 binding sites) (18), along with its associated Bub3, but not Mad2, accounts for the inhibition of APC/C Cdc20 both in vitro using purified components and in vivo after induced degradation of either BubR1 or Mad2 (13).…”

The Mad1–Mad2 balancing act – a damaged spindle checkpoint in chromosome instability and cancer

“…In most experimental systems, this effect requires Mad1, but this is not the case in Xenopus extracts or when Mad2 is artificially tethered to Cdc20 (Fig. 3B, dashed arrow) Fang et al, 1998;Howell et al, 2000;Lau and Murray, 2012). The imbalance in the 'flow' of Cdc20 into the checkpoint pathway, which leads to its destruction by the 26S proteasome, can cause an extended mitotic arrest (Fig.…”

“…Mad2 binding to Mad1 also occurs through the 'closed' Mad2 conformation (Luo et al, 2002). The conversion of Mad2 from its 'open' into its 'closed' form involves an extensive conformational change and has been shown to be the rate-limiting step in spindle checkpoint signaling (De Antoni et al, 2005;Vink et al, 2006;Hewitt et al, 2010;Maldonado and Kapoor, 2011;Lau and Murray, 2012 (Sironi et al, 2002). At the kinetochore, or after being released from the kinetochore, the Mad2-Cdc20 heterodimer binds to a pseudo-substrate that comprises the checkpoint proteins Mad3 and Bub3 (budding uninhibited by benzimidazoles 3) (Fig.…”

“…In budding yeast, C. elegans and mammalian tissue culture cells, the cell cycle delay that is induced by an excess of Mad2 depends on the presence of Mad1 (Sironi et al, 2001;Essex et al, 2009;Rossio et al, 2010a). However, in budding yeast, the requirement for Mad1 can be bypassed by tethering Mad2 to Cdc20 (Lau and Murray, 2012). In tissue culture cells, the stabilization of kinetochore-microtubule attachments induced by excess Mad2 does not require Mad1, although the effect appears to be reduced when Mad1 is absent (Kabeche and Compton, 2012).…”