Fig 1: Substrate‐dependent E2 preferences in APC/C ubiquitination Bottom: Overview of the APC/C–Nek2A structure is shown coloured as in Fig 2. Top: Close up of the Apc2 and Apc4 binding site for Nek2A in the APC/C–Nek2A structure (this study, left), APC/CUbcH10‐Hsl1 structure 27 and APC/CMCC structure 14.Ubiquitination reactions of cyclin A by the APC/CCdc20 in the presence of increasing concentrations of either UbcH10 or UbcH5.Ubiquitination reactions of Nek2A by the APC/CCdc20 in the presence of increasing concentrations of either UbcH10 or UbcH5.Western blot of Hek293 cells showing the siRNA‐mediated depletion of UbcH5, UbcH10 or both E2 enzymes together. Tubulin serves as a loading control.Exemplary still images from time courses between NEBD and anaphase of eGFP‐Nek2A degradation in HEK cells. Cells were either treated with siGL2 as control or depleted of the indicated E2 enzymes. The chromosomes are coloured in cyan and eGFP‐Nek2A in green, with the outline of the cells are indicated with dashed yellow lines. Time is given as hh:mm. Scale bar 10 μm. See also [Link], [Link], [Link], [Link] and Appendix Fig S1.Degradation profiles of eGFP‐cyclin A2 (top) and eGFP‐Nek2A (bottom) in HEK cells during mitosis. The time point of NEBD is marked at 0 min in the graphs. Asterisks indicate values that are significantly different from the same time point of the siGL2 control as determined by a Mann–Whitney U‐test (the statistics are listed in Table EV2). Mean ± SD is shown. The number of cells analysed are N = 66 (Cyclin A2 siGL2), 52 (Cyclin A2 siUbcH5), 28 (Cyclin A2 siUbcH10), 27 (Cyclin A2 siU5/siU10), 80 (Nek2A siGL2), 64 (Nek2A siUbcH5), 21 (Nek2A siUbcH10) and 32 (Nek2A siU5/siU10). All data are from at least two biological replicates. See also [Link], [Link], [Link], [Link]. Source data are available online for this figure.
Fig 2: Nek2A MR tail binding site on Apc2 and Apc4 Left: Cylinder representation of the APC/C–Nek2A complex structure. Apc2 (light blue) and Apc4 (light brown) are highlighted, and the rest of the APC/C is shown in transparency. (i) Two different views of cryo‐EM density for the Apc2 and Apc4 subunits. Secondary structure elements of the Apc2WHB domain are highlighted. (ii) Cryo‐EM density for the second MR tail of Nek2A.Details of the second MR tail binding site formed by the Apc2 (including the repositioned Apc2WHB) and Apc4 subunits. Nek2AMR2 is shown in orange.Nek2A ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants. APC/C2m is the N392A/E395A mutant of Apc2. APC/C2/4m is the N392A/E395A/R48A/H53A/S51A mutant of Apc2 and Apc4.Securin ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants. Source data are available online for this figure.
Fig 3: 3D reconstruction of APC/C–Nek2A complexes A–CWorkflow for focussed 3D classification and multi‐body refinement for 3D reconstruction and refinement of the cryo‐EM density for the MR tail 1 binding site on Apc8A.D–HWorkflow for focussed 3D classification and multi‐body refinement for 3D reconstruction and refinement of the cryo‐EM density for the repositioned APC2WHB domain and for the MR tail 2 binding site on Apc2‐Apc4. Classes 2 and 3 shown in (G) differed in the orientation of the APC2WHB domain, and the occupancy of the MR tail 2 was highest in class 3.
Fig 4: Proteasome-dependent degradation of MCL1 during mitotic arrest.A Stability of MCL1 during mitotic arrest. HeLa cells were synchronized using a double thymidine procedure. G2 samples were harvested at 8 h after release from the second thymidine block (indicated as t = −4 h). Cells were trapped in mitosis (M) using NOC and isolated by shake off (t = 0) before further incubated with NOC. Cells were harvested at different time points. Lysates were prepared and analyzed with immunoblotting. Actin analysis was included to assess protein loading and transfer. Phosphorylated histone H3Ser10 is a marker of mitosis. The intensity of the bands of MCL1, cyclin A, and cyclin B1 was quantified (right-hand panel). B Mitotic arrest stabilizes cyclin B1 and destabilizes MCL1. Cells were synchronized as described in A. The expression of MCL1, cyclin A, and cyclin B1 during G2 and mitosis was quantified from three independent experiments (mean ± SEM). C Mitotic degradation of MCL1 is proteasome-dependent. Cells were synchronized and trapped in mitosis as described above. Mitotic cells were exposed to either buffer or MG132 and harvested after 3 h. The expression of MCL1 was analyzed with immunoblotting. The MCL1 band intensity was quantified (mean ± SEM from three independent experiments). D MCL1 is degraded during mitosis by an APC/C-independent mechanism. APC4KO cells expressing mAIDAPC4 were generated. The cells were synchronized and arrested in mitosis as before. DI were applied to turn off the expression of mAIDAPC4 at the time of second thymidine release. Lysates were prepared and analyzed with immunoblotting. E Disruption of APC4 stabilizes cyclin B1 but not MCL1. Synchronization experiments were performed using mAIDAPC4-expressing APC4KO cells as described in D. The MCL1 and cyclin B1 bands were quantified and shown in the right-hand panels (normalized to G2 expression). Mean ± SEM of three independent experiments.
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