MAD2B directly inhibits activation of APC by CDC20 and CDH1. (A) Interphase APC (iAPC) was immunopurified from Xenopus extracts by anti-CDC27 antibody/protein A beads and then incubated with purified recombinant CDH1, CDH1 plus MAD2B or CDH1 plus MAD2. The ability of treated APC to ubiquitinate radioactive N-terminal fragment of cyclin B was assayed. (B) Interphase APC was immunopurified by anti-CDC27 antibody/protein A beads and then incubated with purified recombinant CDC20, CDC20 plus MAD2B or CDC20 plus MAD2. The ability of treated APC to ubiquitinate radioactive N-terminal fragment of cyclin B was assayed.

Inhibition of APC by MAD2B is not specific to cyclin B. Interphase APC (lanes B) was immunopurified by anti-CDC27 antibody/protein A beads and then incubated with recombinant CDH1 (lanes C) or with CDH1 plus MAD2B (lanes D). The ability of treated APC to ubiquitinate [³⁵S]Met-labeled Plk1, ARK2, CDC20, and full-length cyclin B was assayed.

MAD2B inhibits APC activity in Xenopus extracts. (A) Purified recombinant MAD2B was analyzed by 12% SDS-PAGE. (B) Degradation of cyclin B in Xenopus extracts. CDH1 was incubated with Xenopus interphase extracts in the presence of various concentrations of MAD2B (II–V). Similarly, MAD2B (VII–IX) and MAD2 (X–XII) were incubated with Xenopus mitotic extracts. The stability of radioactive N-terminal fragment (amino acids 1–102) of cyclin B was assayed in these extracts. As controls, interphase (I) and mitotic (VI) extracts were analyzed in parallel. The MAD2B and MAD2 concentrations indicated are final concentration in extracts. The recombinant MAD2 protein exists in two different forms (oligomer and monomer) and only the oligomer is an active inhibitor of APC (Fang et al. 1998b). Thus, MAD2 oligomer was used in experiments described in this paper. (C) APC activity assayed in reconstituted ubiquitination reactions. APC from extracts prepared in B was immunopurified by anti-CDC27 antibody/protein A beads and its ability to ubiquitinate N-terminal fragment of cyclin B analyzed. (I ext), interphase extracts; (M ext), mitotic extracts.

Inhibition by MAD2B is targeted to CDC20 and CDH1. (A) Interphase APC beads were incubated with purified recombinant CDC20 (I), with MAD2B (II), with MAD2 (III), with CDC20 plus MAD2B (IV), or with CDC20 plus MAD2 (V). Recombinant proteins were then removed and APC beads incubated with a buffer (I, IV, and V) or with recombinant CDC20 (II and III). The ability of treated APC to ubiquitinate radioactive N-terminal fragment of cyclin B was assayed. (B) Interphase APC beads were incubated with purified recombinant CDH1 (I), with MAD2B (II), or with CDH1 plus MAD2B (III). Recombinant proteins were then removed and APC beads incubated with a buffer (I and III) or with recombinant CDH1 (II). The ability of treated APC to ubiquitinate radioactive N-terminal fragment of cyclin B was assayed. (C) Interphase APC beads were first incubated with recombinant CDH1. MAD2B was then added and incubated with the mixture of CDH1 and APC. The ability of treated APC to ubiquitinate radioactive N-terminal fragment of cyclin B was assayed.

MAD2B interacts with CDC20 and CDH1, but not with MAD1. (A) His–MAD2B (lanes 2,4) or BSA (lanes 3,6) were incubated with ³⁵S-labeled CDC20 (lanes 2,3) and CDH1 (lanes 5,6) and MAD2B complexes were purified by Ni⁺; beads and analyzed by SDS-PAGE. (Lanes 1,3) 1/10 of input CDC20 and CDH1. (B–D) HA–MAD2 was incubated with Myc-MAD1 (B). HA–MAD2B was incubated with Myc–MAD1 in the presence (D) or absence (C) of Myc–MAD2. HA-tagged proteins were then immunopurified by an anti-HA antibody (lane 2) or by control IgG (lane 3). The association of HA-tagged proteins with Myc-tagged proteins was analyzed by SDS-PAGE. (Lane 1) 1/10 of input proteins in the binding assay. In this experiment, six copies of the Myc tag were fused to the amino termini of MAD1 and MAD2 and two copies of the HA tag were fused to MAD2B. The large difference in the migration rate between Myc–MAD2 and HA–MAD2B was due to the difference in the copy number of the tags.