Contribution of the “ABBA motif” to Clb5 degradation. (A) Sequence alignment of Clb5 orthologues from closely related yeast species, showing the putative ABBA motif in Clb5 in alignment with other known ABBA motifs. Different colors represent chemical properties of the residues. (B) Degradation profiles of Clb5-2A-GFP and wild-type Clb5-GFP, as in Fig. 3 A; n > 70 cells per strain. In the inset, n > 170 cells per strain. (C) Degradation profiles of Clb5-2A3D-GFP and securin-2A-GFP, as in Fig. 3 A; n > 60 cells per strain. (D) Sequence alignment of budding yeast Cdc20 and Cdh1; purple dots mark the potential ABBA motif interacting residues that are different between Cdc20 and Cdh1. Below is the degradation profile of Clb5-2A-GFP or wild-type Clb5-GFP in a CDC20-GAG background, compared with the wild-type CDC20 background; n > 57 cells per strain. In the inset, n > 75 cells per strain. (E) Analysis of Clb5 and Clb5-2A ubiquitination by APC/C^Cdh1 or APC/C^Cdc20-GAG in vitro, as described in the Materials and methods. Reactions with securin were included as controls. (F) Degradation of Clb5-GFP and Clb5-2A-GFP in nocodazole-treated cells. Asynchronous cells were plated on an agarose pad with 15 µg/ml nocodazole 10 min before the start of imaging. Clb5-GFP dynamics before spindle reformation (SAC inactivation) were analyzed. Representative traces were selected to minimize overlap and omit cells that were not in mitosis. The right panel shows the rates of degradation calculated by fitting single-cell GFP traces to a linear decay; n > 55 cells per strain.