Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

Proteins and assays

Proteins used in this study are human except for yeast Hsl1. Recombinant APC/C and its variants contain 100 phosphomimetic mutations ( Qiao et al., 2016 ; Weissmann et al., 2016 ). CDC20, MCC and variants were expressed in Hi5 insect cells and then purified by nickel affinity, cation exchange and size exclusion chromatography. Substrate ubiquitination assays were performed as described, with MCC titrated from 15–250 nM ( Brown et al., 2016 ; Qiao et al., 2016 ). CDC20 ubiquitination assays were adapted from ( Foe et al., 2011 ; Foster and Morgan, 2012 ) to use methyl UB, and extended to include UBE2S with WT UB. To distinguish CDC20 ubiquitination targets, CDC20 A and CDC20 M were N-terminally Myc- and Flag-tagged, respectively, for 2-color western detection by secondary antibodies conjugated with DyLight 488 and Alexa 633, respectively, during a single scan with Typhoon FLA 9500. Hydrolysis of oxyester-linked UBE2C~UB ( Brown et al., 2015 ) was performed three independent times, with 1 μM APC/C or APC/CΔ15, 1 μM CDC20, 5 μM UBE2C~UB, ± 1 μM MCC at 30°C. Reaction products were visualized in Coomassie blue-stained 4–12% NuPAGE gels (Life Technologies). Complex preparation for EM Complexes were purified by sequential affinity pulldowns, followed by specific polishing steps. APC/C CDC20 , and APC/C CDC20 -MCC, or versions lacking APC15, were prepared by either coexpressing APC/C, CDC20, and/or MCC, or by mixing lysates from Hi5 insect cell cultures expressing components independently. Subcomplexes crosslinked to E2 active sites [UBE2C-substrate-UB (FLAG-tagged donor UB mimic), UBE2C-MCC (His 6 -FLAG-tagged BUBR1), UBE2S-UBv-UB (untagged)] were generated largely as described previously ( Brown et al., 2015 ; Brown et al., 2016 ). 2- or 3-way crosslinking between cysteine side-chains was performed through use of BMOE or TMEA (Pierce), respectively. Complexes were initially purified based on affinity tag(s) on APC/C, either a C-terminal Twin-Strep tag on APC4 or sequentially via an N-terminal Twin-Strep tag on APC2 and N-terminal GST-tag on APC16. Complexes were enriched by FLAG-affinity for BUBR1 or a donor UB mimic in some crosslinked complexes, with detailed purification procedures described in Supplemental Information .

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