UCSF

The ubiquitin-protein ligase APC/C controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C-Cdc20 substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 minutes later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle-assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1-Cks1 complex and the presence of a Cdc20-binding `ABBA motif' in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C-Cdc20 substrate destruction. We then used a combination of experimental and computational approaches to show that competition among substrates does not contribute significantly to their degradation timing. Instead, the timing and rate of degradation is likely to be primarily determined by the interaction between substrates and APC/C-Cdc20. The mechanisms above could change the binding affinity between the substrate and APC/C-Cdc20, or the catalytic rate once the substrate is bound to APC/C-Cdc20. Depending on parameter region, varying these two properties have different impact on substrate degradation dynamics.