Molecular mechanisms of APC/C release from spindle assembly checkpoint inhibition by APC/C SUMOylation

Summary The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that controls cell cycle transitions. Its regulation by the spindle assembly checkpoint (SAC) is coordinated with the attachment of sister chromatids to the mitotic spindle. APC/C SUMOylation on APC4 ensures timely anaphase onset and chromosome segregation. To understand the structural and functional consequences of APC/C SUMOylation, we reconstituted SUMOylated APC/C for electron cryo-microscopy and biochemical analyses. SUMOylation of the APC/C causes a substantial rearrangement of the WHB domain of APC/C’s cullin subunit (APC2WHB). Although APC/CCdc20 SUMOylation results in a modest impact on normal APC/CCdc20 activity, repositioning APC2WHB reduces the affinity of APC/CCdc20 for the mitotic checkpoint complex (MCC), the effector of the SAC. This attenuates MCC-mediated suppression of APC/CCdc20 activity, allowing for more efficient ubiquitination of APC/CCdc20 substrates in the presence of the MCC. Thus, SUMOylation stimulates the reactivation of APC/CCdc20 when the SAC is silenced, contributing to timely anaphase onset.

(B) Cycling cells were analysed for APC4 SUMOylation. Ubc9 was depleted for 48 h using 20 nM siRNA after which the cells were harvested and analysed by immunoblotting against APC4.
Loading was controlled by immunoblotting against actin and depletion was confirmed by immunoblotting against Ubc9.
(C) Repeats of experiments in Figure 1C. Method is described in Figure 1C and in Methods. Quantifications are shown in a bar graph. Data represent mean with one standard deviation (n=2).  (A) A SUMOylation assay showed that APC/C SUMOylation by SUMO-2 and by a SUMO-2 mutant where all Lys were replaced with Arg (SUMO-2 KR ) both produced two additional SUMOylation bands above APC4. The SUMOylation assay was performed as described in Methods and APC4 was detected by immunoblotting with antibodies against APC4. (D) APC/C SUMOylation by the 6xHis-SUMO-2 isoform is much faster than by 6xHis-SUMO-1 isoform. The SUMOylation assay was performed as described in Methods and APC4 was detected by immunoblotting with antibodies against APC4.
The SUMOylation assay was performed as described in Methods and APC4 and SUMO-2/3 were detected by immunoblotting with antibodies against APC4 and SUMO-2/3, respectively. (H) APC4 phosphorylation state summary at S777 and S779 before and after lambda phosphatase treatment as determined by mass spectrometry peptide analysis.
(I) SDS-PAGE gel of SUMO-2 Δ NTD and SUMO-2 loading controls used for Figure 2D      (E) SUMOylated APC/C map colored by local resolution estimation.
(F) Three-dimensional classification scheme for SUMOylated APC/C structure determination, resulting in classes with APC2 WHB domain density being clearly visible. All particles after 2D classification were refined together to give an initial model (1), which were 3D classified into four classes (2). The two best 3D classes were selected after visual examination, combined and refined together. Bayesian polishing was performed on these best particles with CTF refinement in RELION 3.0 (3). APC2 WHB , APC10, APC3 and APC1 PC repeats were then masked and the signal from the rest of the APC/C was subtracted, and focused 3D classification without alignment was performed to give three classes (4), only one of which had high occupancy of APC2 WHB (5). Full particles with high APC2 WHB occupancy were then refined and the APC/C was split into three bodies for multi body refinement (6), which gave a final map with a well-resolved APC2 WHB .    (E) Three-dimensional classification scheme for SUMOylated APC/C structure determination, resulting in class with clear density at the APC2-APC4 interface, assigned to SUMO-2. The processing was continued after Bayesian polishing described and shown in Figure S3F (1). APC4 and APC2 were masked and the signal was subtracted from the rest of the APC/C and focused 3D classification without alignment was performed to give three classes (2). apoAPC/C (no density), partially activated APC/C were excluded and only class with additional density was taken for subsequent processing. APC/C was refined again (3) and a tight mask around APC4 WD40 domain and APC2 were used for another round of focused 3D classification without alignment (4).
A single class with defined density at APC2-APC4 interface was taken and refined to give a final map (5).  Figure S4E. EMDB 10518 was used to assess the APC/C-Nek2A complex; APC2 WHB is coloured green. SUMO2 is coloured pink.
(F) The SUMOylation reaction proceeded faster when the APC2 WHB was removed. SUMOylation reaction was performed as described in Methods.  Figure 5A, C, E.
(B) Titration of E2 enzymes used in the ubiquitination assay. The assays were performed for 10 min using Hsl1 as a substrate with indicated amounts of UbcH10 and UbcH5 without Ube2S enzymes.
Ubiquitination was detected by immunoblotting against the His-tag of ubiquitin. The assays were performed at least in triplicate.
(D) Comparison of wild-type phosphorylated APC/C activity and phosphorylated APC/C Δ SIM towards securin in the ubiquitination assay. UbcH10 was used as the sole E2 enzyme. Ubiquitination was detected by immunoblotting against securin. The assays were performed at least in triplicate.
(E) Comparison of SUMOylated and non-SUMOylated phosphorylated APC/C Δ SIM ubiquitination activity towards securin using UbcH10 as the sole E2 enzyme. Ubiquitination was detected by immunoblotting against securin. The assays were performed at least in triplicate and quantified as described in Methods. The error bar represents mean with one standard deviation of the ratio of the SUMOylated/non-SUMOylated APC/C activity. One star indicates a significance smaller than 0.05. Significance was calculated using an unpaired Student's t-test. immunoblotting against APC2 shows that equal amounts of APC/C are present in peak fractions, whereas immunoblotting against MCC components, BubR1, Bub3 and Mad2 shows reduced amount of MCC in the SUMOylated APC/C sample. The peak fractions taken for the immunoblotting are shown in Figure S7D.

Data collection
In  Table S1   Table S1. CryoEM data collection, processing information and refinement statistics. Related to Figure 3.