Different Functionality of Cdc20 Binding Sites within the Mitotic Checkpoint Complex  Katharina Sewart, Silke Hauf  Current Biology  Volume 27, Issue 8, Pages 1213-1220 (April 2017) DOI: 10.1016/j.cub.2017.03.007 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 1213-1220DOI: (10.1016/j.cub.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 The Mad3 C Terminus Binds Slp1Cdc20 and Is Required for Checkpoint Activity (A) Domain structure of H. sapiens BubR1 and S. pombe Mad3 and alignment of yeast Mad3 C-terminal sequences containing Slp1Cdc20-binding motifs. See text for details and Figures S1A and S1B for additional alignments. ABBA2 [7, 8] had not been identified at the time when the APC/C-MCC structure was solved at high resolution [4] but most likely contacts Cdc20M. (B) Cells expressing plo1+-mCherry, the conditional tubulin mutant nda3-KM311, and the indicated mutations in mad3 were analyzed by live-cell imaging at the restrictive temperature of 16°C. The time that each cell spent in prometaphase was determined by localization of Plo1 to spindle pole bodies (circle). Cells that had not yet exited mitosis when filming stopped are indicated by triangles and cells that died during mitosis by filled circles. Pooled data from two independent experiments are shown; n > 55 cells. Control for expression levels and additional data are in Figures S1F and S1G. (C) Anti-GFP immunoprecipitates from mitotic cells of the indicated Mad3-GFP strains were analyzed by immunoblotting using anti-GFP, anti-Slp1, anti-Mad2, and anti-Cdc2 (loading control) antibodies. Results are representative of four independent experiments. (D) Schematic of the association between the “core MCC” and a second Slp1Cdc20 molecule through motifs in the Mad3 C terminus, based on [4]. (E) Anti-GFP immunoprecipitates from mitotic sfGFP-Slp1 cells with the additional genetic modifications indicated on top were analyzed by immunoblotting using anti-GFP, anti-Slp1, anti-Mad3, and anti-Mad2 antibodies. The tagged sfGFP-Slp1Cdc20 was expressed from the endogenous promoter at the endogenous locus; untagged Slp1Cdc20 (Slp1 or Slp1-mr63) was expressed from the endogenous regulatory sequences at the exogenous leu1 locus. (F) Checkpoint function of the indicated strains was analyzed as in (B). Wild-type or C-box mutant Slp1Cdc20 was expressed under the endogenous regulatory sequences (Pslp1-slp1) from the exogenous leu1 locus, and Slp1Cdc20-mr63 was expressed at the endogenous locus either from the endogenous promoter (Pslp1) or from the rad21+ promoter, which reduces the concentration of Slp1Cdc20 (Prad21; Figure S2A) (wt, wild-type). Pooled data from at least two independent experiments are shown; n > 60 cells. See also Figure S1H. (G) Anti-GFP immunoprecipitates from mitotic cells of the indicated strains were analyzed by immunoblotting (long and short exposure for the Slp1 blot). Current Biology 2017 27, 1213-1220DOI: (10.1016/j.cub.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 The Mad3 C Terminus Becomes Dispensable for Checkpoint Activity at Low Slp1Cdc20 Concentrations (A) Checkpoint function of the indicated strains was analyzed as in Figure 1B. Slp1Cdc20 was expressed at the endogenous locus from either its endogenous promoter (Pslp1) or from the rad21+ promoter (Prad21), which lowers the concentration (Figure S2A). Pooled data from two independent experiments are shown; n > 95 cells. (B) Checkpoint function of the indicated strains was analyzed as in Figure 1B. Mad2 and Mad3 were overexpressed to about 200% and 120% of wild-type levels, respectively. Slp1Cdc20 or Slp1Cdc20-mr63 was expressed from the rad21+ promoter at the endogenous locus. Pooled data from two independent experiments are shown; n > 75 cells. (C) Checkpoint function of the indicated strains was analyzed as in Figure 1B, and mitotic arrest was assumed when cells spent more than 5 hr in mitosis. Slp1Cdc20 was expressed from the regulatable nmt41 promoter at the endogenous locus, and expression level was controlled with increasing concentrations of YAM2. Shown is the mean with SEM. Expression levels are in Figure S2B. (D) Representative example of one experiment in (C); n > 120 cells. (E) Schematic for the Mad3 C terminus becoming dispensable at low Slp1Cdc20 concentrations, presumably because all Slp1Cdc20 can be sequestered into the core MCC. Current Biology 2017 27, 1213-1220DOI: (10.1016/j.cub.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 The Mad3 C Terminus and Apc15 Are Required for MCC Disassembly (A) Anti-GFP immunoprecipitates of asynchronously growing Mad3-GFP cells with the indicated mutations were analyzed by immunoblotting. See also Figure S3B. (B) Anti-GFP immunoprecipitates from Mad3-GFP interphase cells with the indicated mutations were analyzed as in (A). Results are representative of three independent experiments. (C) Synchronization for the cells used in (B). Cells were synchronized at the G2/M transition, released, and analyzed for cell-cycle stage using Plo1-mCherry and DNA staining. Cells with Plo1 on spindle pole bodies were considered to be in mitosis; cells with two close nuclei were considered to be in anaphase (n > 150 cells per time point). Example pictures are in Figure S3A. Cells for (B) were harvested at the last time point. (D) Schematic for MCC persistence in different strains. Current Biology 2017 27, 1213-1220DOI: (10.1016/j.cub.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 The Mad3 C Terminus and Apc15 Are Required for MCC Binding to S. pombe APC/C (A) Checkpoint function of the indicated strains was analyzed as in Figure 1B. Slp1Cdc20 was expressed at the endogenous locus from either the endogenous promoter (Pslp1) or from the rad21+ promoter (Prad21). In the 2× Prad21 strains, Slp1Cdc20 was expressed at the endogenous locus and at the exogenous leu1 locus from the rad21+ promoter, which results in an intermediate (int) concentration (Figure S2A). One representative out of two (apc15Δ mad3Δ) or >5 experiments is shown; n > 40 cells. See also Figure S3C. (B) Anti-GFP immunoprecipitates of mitotic cells from the indicated strains were analyzed by immunoblotting using anti-Slp1, anti-Mad3, and anti-Mad2 antibodies. sfGFP-Slp1Cdc20 was expressed from the endogenous promoter at the endogenous locus; Slp1Cdc20-mr63 was expressed from the endogenous regulatory sequences at the exogenous leu1 locus. Results are representative of three independent experiments. (C) Anti-GFP immunoprecipitates from mitotic Mad3-GFP cells, with the additional genetic modifications indicated on top, were analyzed by immunoblotting for binding of myc-tagged Lid1/Apc4 (∗cross-reaction). Results are representative of three independent experiments. (D) Anti-GFP immunoprecipitates from mitotic Cut9/Apc6-GFP cells, with the additional genetic modifications indicated on top, were analyzed for binding of the MCC by immunoblotting. One of two independent experiments is shown. (E) Anti-GFP immunoprecipitates, as well as input and supernatant after precipitation, from mitotic cells of the indicated Mad3-GFP apc15Δ strains were analyzed by immunoblotting. Slp1Cdc20 was expressed as in (A). Results are representative of two independent experiments. (F) In both mad3-ΔCterm and apc15Δ cells, the MCC does not associate with the APC/C, and MCC disassembly is impaired. We hypothesize that reduction of Slp1Cdc20 rescues the checkpoint defect in apc15Δ cells more efficiently, because sequestration within the MCC is more efficient. (G) Schematic depicting the role of Apc15 in binding the MCC to the APC/C. In organisms that contain Bub3 as part of the MCC, Bub3 may redundantly promote MCC-APC/C binding. Current Biology 2017 27, 1213-1220DOI: (10.1016/j.cub.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions