APC/C^Cdc20 binding to cyclin B1 correlates with a substrate retention step, preceding a D box–dependent ubiquitination step. (A) Cells were cotransfected with vectors coding for cyclin B1–Venus and ΔN–cyclin B1–ECFP (left), securin-cerulean and cyclin B1–Venus (middle), or securin-cerulean and cyclin B1–R202A–Venus (right). Cells expressing both proteins were followed through mitosis, and fluorescence levels were plotted. Metaphase (M) and anaphase (A) initiation judged from differential interference contrast images are shown. Graphs are representative of at least two independent experiments. (middle and right) The cells analyzed expressed cyclin B1 or securin at low levels, allowing onset of their destruction at chromosome alignment. With wild-type constructs, sister chromatid separation and cytokinesis were normal. The other fluorescent constructs were expressed to similar levels. At least 20 cells expressing single fusion proteins were analyzed with similar results. (B and C) Wild-type and nondegradable mutants of cyclin B1 bind equally well to APC/C^Cdc20 in mitosis, whereas non-Cdk–binding mutants of cyclin B1 do not bind. (B) Cells expressing Cdk-binding mutant cyclin B1–R202A–Venus (lane 1), nondegradable ΔN–cyclin B1–ECFP (lane 2), or control cyclin B1–Venus (lane 3) were synchronized in the spindle checkpoint and collected by mitotic shake off. Successful expression of the constructs was validated by fluorescence microscopy (not depicted). (C) Cells expressing control cyclin B1–Venus (lane 1), Cdk-binding mutant cyclin B1–R202A–Venus (lane 2), the non-Cdk–binding N-terminal destruction region of cyclin B1 fused to GFP (lane 3), or nondegradable R42A D box mutants (lanes 4 and 5) were synchronized in the spindle checkpoint and collected by mitotic shake off. (B and C) Fusion proteins were immunoprecipitated by anti-GFP antibodies, equally recognizing GFP or spectral variants. IPs were blotted and probed with antibodies against the indicated proteins. (D) Cells were treated with nocodazole, and cyclin B1 IPs were performed on extracts of the collected mitotic cells (lane 1) or on extracts from an asynchronous population (lane 2). Cells expressing the first 97 amino acids of cyclin B1 fused to GFP either containing an intact D box (lane 3) or an R42A-mutated D box (lane 4) were collected in mitosis. Anti-GFP antibodies were used for IP of the fusion proteins from extracts (lanes 3 and 4). IPs were probed for cyclin B1 and APC3. (lane 1) Binding of APC/C to endogenous cyclin B1 in mitosis as reference is shown. In prometaphase, binding is not detectable to the N-terminal fragments of cyclin B1. (E and F) Mitotic cells were collected by shake off after taxol treatment and released from the spindle checkpoint by addition of aurora B inhibitor ZM447439 but kept in mitosis by the addition of proteasome inhibitor MG132. After 1 h, cells were lysed and extracts used for IP of fusion proteins of GFP and the cyclin B1 N terminus (1–86), (cycB1-N (1X)-GFP), or tandem or sixfold repeats of the same region (cycB1-N (2X)-GFP and cycB1-N (6X)-GFP, respectively). IPs were blotted for APC3, Cdc20, or Cdk1 (as a negative control [ctrl]; E) or the cyclin B1 fragments (F). APC3 long exposure (APC3 le) is an overexposed blot to reveal minor APC/C binding to the single- or tandem-repeat cyclin B1 fragment. Markers are given in kilodaltons. WCE, whole cell extract.