Fission Yeast Apc15 Stabilizes MCC-Cdc20-APC/C Complexes, Ensuring Efficient Cdc20 Ubiquitination and Checkpoint Arrest

During mitosis, cells must segregate the replicated copies of their genome to their daughter cells with extremely high fidelity. Segregation errors lead to an abnormal chromosome number (aneuploidy), which typically results in disease or cell death [1]. Chromosome segregation and anaphase onset are initiated through the action of the multi-subunit E3 ubiquitin ligase known as the anaphase-promoting complex or cyclosome (APC/C [2]). The APC/C is inhibited by the spindle checkpoint in the presence of kinetochore attachment defects [3, 4]. Here we demonstrate that two non-essential APC/C subunits (Apc14 and Apc15) regulate association of spindle checkpoint proteins, in the form of the mitotic checkpoint complex (MCC), with the APC/C. apc14Δ mutants display increased MCC association with the APC/C and are unable to silence the checkpoint efficiently. Conversely, apc15Δ mutants display reduced association between the MCC and APC/C, are defective in poly-ubiquitination of Cdc20, and are checkpoint defective. In vitro reconstitution studies have shown that human MCC-APC/C can contain two molecules of Cdc20 [5–7]. Using a yeast strain expressing two Cdc20 genes with different epitope tags, we show by co-immunoprecipitation that this is true in vivo. MCC binding to the second molecule of Cdc20 is mediated via the C-terminal KEN box in Mad3. Somewhat surprisingly, complexes containing both molecules of Cdc20 accumulate in apc15Δ cells, and the implications of this observation are discussed. [Display omitted] •Two small fission yeast APC/C subunits regulate MCC binding•Apc14 is required for checkpoint silencing and Apc15 for checkpoint arrest•Apc15 enhances Cdc20 ubiquitination and turnover•Two Cdc20 molecules are found in MCC-APC/C and free MCC-Cdc20A accumulates in apc15Δ May et al. dissect the roles of fission yeast Apc14 and Apc15 in spindle checkpoint regulation of APC/C activity. Apc15 is needed for efficient Cdc20 turnover and checkpoint arrest in fission yeast. Apc15 and the C terminus of Mad3 are shown to co-ordinate the interactions of two molecules of Cdc20 within inhibited APC/C complexes.