Figure 4.

A Free Pool of Cdc20^M-Mad3-Mad2-Cdc20^A Accumulates in apc15Δ Mutants

(A) The MCC (Cdc20^M-Mad3-Mad2)-Cdc20^A complex can be found in the APC/C-depleted supernatant, and the complex accumulates in apc15Δ. Mitotic lysates were prepared from cdc25-22 cdc20-HA cdc20-FLAG Apc6^Cut9-GFP (60 min after cdc25 block and release) and then immunodepleted for APC/C complexes through four rounds of cut9^apc6-GFP immunodepletion. Cdc20-FLAG was then immunoprecipitated from the resulting supernatant and immunoblotted to look for associated Cdc20-HA and checkpoint proteins. The MCC (Cdc20^M-Mad3-Mad2)-Cdc20^A is immunoprecipitated without Apc6^Cut9-GFP or Apc15. This experiment was repeated twice.

(B) Models of MCC binding and Cdc20 ubiquitination, in wild-type cells and apc15 mutants. (i) When the checkpoint is on, the MCC binds to Cdc20-APC/C and in fission yeast this interaction is stabilized by Apc15. The C terminus of Mad3 (KEN2 and associated ABBA motifs) is critical for this stable interaction with the second molecule of Cdc20 (Cdc20^A). (iia) In apc15Δ cells, the MCC could preferentially bind free Cdc20^A. (iib) In the absence of Apc15, the MCC complex is weakly bound and Cdc20^M is inefficiently ubiquitinated. It is released with short ubiquitin chains in the form of Cdc20^M-Mad3-Mad2-Cdc20^A. Note that both molecules of Cdc20 are released from the APC/C. (iii) In wild-type cells Cdc20^M is efficiently poly-ubiquitinated, leading to its degradation. Apc14 function is required for efficient release of the MCC. (iv) The checkpoint is off; APC/C is now free to be bound by the Cdc20^A activator, which can catalyze the poly-ubiquitination of securin and cyclin, leading to anaphase onset.

See also Figure S4.