Extended Data Figure 6 : Stabilizing the interaction between MAD2 and CDC20.

From: The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C

Extended Data Figure 6

a, Schematic of how a stabilized MCC might block cells in metaphase. At prometaphase, when the SAC is ‘ON’, CDC20 is inhibited both by incorporation into the MCC and through binding to the MCC. At metaphase when the SAC is ‘OFF’, CDC20 is released from the MCC and activates the APC/C. We postulate that stabilizing an exogenous MCC to prevent its disassembly should also prevent endogenous CDC20 from activating the APC/C, which results in an anaphase delay. b, Schematic of a stabilized MCC. To stabilize the MCC we took advantage of the binding between yellow fluorescent protein (Venus) and GFP-binding domain (GBP), which is a 13kDa domain from a camelid antibody that binds strongly and specifically to GFP and YFP18. MAD2 and BUBR1 were tagged with Venus and the GBP domain was tagged to CDC20. We refer to the MCC containing a stabilized MAD2–CDC20 interaction as MCCM2, and that with stabilized BUBR1–CDC20 as MCCR1. c, GBP- and Venus-fusion proteins bind stably to each other in vivo. HeLa cell lines expressing siRNA-resistant myc–CDC20 or myc–GBP–CDC20 were transfected with plasmids encoding either Venus alone or Venus–MAD2, followed by siRNA treatment against CDC20. After a single thymidine block and release, the cells were arrested at prometaphase by treating with nocodazole, and harvested by mitotic shake-off 48 h after the siRNA treatment. Proteins were immunoprecipitated with anti-myc epitope antibodies before analysis by quantitative immunoblotting with the indicated antibodies. WT, myc–CDC20; GBP, myc–GBP–CDC20. Results in panel c are representative of three independent biological replicates.