Darling S et al. (2024), The C-terminal disordered loop domain of Apc8 u...

XB-ART-60852

Cell Rep 2024 May 25;436:114262. doi: 10.1016/j.celrep.2024.114262.

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The C-terminal disordered loop domain of Apc8 unlocks APC/C mitotic activation.

Darling S , Fujimitsu K , Chia KH , Zou J , Rappsilber J , Yamano H .

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The anaphase-promoting complex/cyclosome (APC/C) is a critical and tightly regulated E3 ligase that orchestrates the cellular life cycle by controlling the degradation of cell cycle regulators. An intriguing feature of this complex is an autoinhibition mechanism: an intrinsically disordered loop domain, Apc1-300L, blocks Cdc20 coactivator binding, yet phosphorylation of Apc1-300L counteracts this autoinhibition. Many such disordered loops within APC/C remain unexplored. Our systematic analysis of loop-deficient APC/C mutants uncovered a pivotal role for Apc8's C-terminal loop (Apc8-L) in mitotic activation. Apc8-L directly recruits the CDK adaptor protein, Xe-p9/Cks2, positioning the Xe-p9-CDK-CycB complex near Apc1-300L. This stimulates the phosphorylation and removal of Apc1-300L, prompting the formation of active APC/CCdc20. Strikingly, without both Apc8-L and Apc3-L, the APC/C is rendered inactive during mitosis, highlighting Apc8-L's synergistic role with other loops and kinases. This study broadens our understanding of the intricate dynamics in APC/C regulation and provides insights on the regulation of macromolecular complexes.

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Species referenced: Xenopus laevis
Genes referenced: anapc1 apc2 cdc20 cdh1 cdk1 ensa mastl mbp plk1 tpr
GO keywords: mitotic cell cycle [+]
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	Figure S1. Expression and quality assessment of loop-deleted rAPC/C mutants relative to wild type (WT), related to Figures 1, 2, and 4. (A) Relative levels of recombinant APC/C (rAPC/C) in coactivator binding assays as shown in Figure 1E. Endogenous APC/C was depleted from anaphase-arrested extracts, which were then supplemented with either WT or mutant rAPC/Cs. Samples were collected immediately after rAPC/C addition to accurately reflect the initial, unphosphorylated state of rAPC/Cs, and to confirm similar amounts of WT or mutant rAPC/Cs were used for each experiment. Levels of individual APC/C subunits were analysed using SDS-PAGE and immunoblotting. (B) Stability of Apc3 and Apc3-L deficient mutants during anaphase (Figure 1E). Extract preparation was followed analogously to (A). Samples were taken immediately after rAPC/C addition (T0) and after a 45-minute incubation in anaphasic conditions (T45). Anaphase extracts containing rAPC/Cs were treated with calf intestinal phosphatase (CIP) and analysed through SDS-PAGE and immunoblotting. CIP-treatment collapsed highly phosphorylated Apc3 bands permitting direct comparison of Apc3 levels and stability. The relative levels of Apc3 within the rAPC/Cs remained consistent, regardless of the presence or absence of the Apc3-L. (C and D) Analysis of rAPC/Cs used in Figures 2B and 4B, conducted similarly to (A), assessing the relative levels and quality of rAPC/Cs in coactivator binding assays.
	Figure S1. Expression and quality assessment of loop-deleted rAPC/C mutants relative to wild type (WT), related to Figures 1, 2, and 4. (A) Relative levels of recombinant APC/C (rAPC/C) in coactivator binding assays as shown in Figure 1E. Endogenous APC/C was depleted from anaphase-arrested extracts, which were then supplemented with either WT or mutant rAPC/Cs. Samples were collected immediately after rAPC/C addition to accurately reflect the initial, unphosphorylated state of rAPC/Cs, and to confirm similar amounts of WT or mutant rAPC/Cs were used for each experiment. Levels of individual APC/C subunits were analysed using SDS-PAGE and immunoblotting. (B) Stability of Apc3 and Apc3-L deficient mutants during anaphase (Figure 1E). Extract preparation was followed analogously to (A). Samples were taken immediately after rAPC/C addition (T0) and after a 45-minute incubation in anaphasic conditions (T45). Anaphase extracts containing rAPC/Cs were treated with calf intestinal phosphatase (CIP) and analysed through SDS-PAGE and immunoblotting. CIP-treatment collapsed highly phosphorylated Apc3 bands permitting direct comparison of Apc3 levels and stability. The relative levels of Apc3 within the rAPC/Cs remained consistent, regardless of the presence or absence of the Apc3-L. (C and D) Analysis of rAPC/Cs used in Figures 2B and 4B, conducted similarly to (A), assessing the relative levels and quality of rAPC/Cs in coactivator binding assays.
	Figure S2. Apc8-loop conservation and mitotic coactivator binding assay using non-phosphorylatable Cdc20-5A, related to Figure 1 and Figure 2. (A) Alignment of predicted C-terminal loop domains of vertebrate Apc8 (Hs; Homo sapiens human, Pt; Pan troglodytes chimpanzee, Mm; Mus musculus mouse, Gg; Gallus gallus chicken, Xl; Xenopus laevis frog, Dr; Danio rerio zebrafish). Conserved residues are highlighted with an asterisk (*), physiochemically similar residues are highlighted with a period (.). Conserved CDK-targeted residues are highlighted in yellow. Bold lines indicate the favoured CDK consensus sequence (pS/T-P-x-K/R). (B) Cdc20-5A binding assay. Endogenous APC/Cs and Cdc20 were both depleted from anaphase-arrested Xenopus egg extracts; equal amounts of extracts were first supplemented with in vitro-translated Cdc20-5A, then the indicated loop-deficient rAPC/Cs. Immobilised rAPC/Cs were recovered by immunoprecipitation, washed harshly and Cdc20-5A loading was analysed by SDS-PAGE and immunoblotting. (C) Quantitative analysis of the proportion of Cdc20-5A bound to rAPC/C after 45 minutes (n=3). Quantification and statistical analysis was performed analogous to Figure 1F.
	Figure S3. CLMS analysis of APC/C complex interactions, related to Figure 3. (A) Circular plots (xiVIEW) of APC/C intra- and inter-subunit crosslinks identified by crosslinking mass spectrometry (CLMS). Crosslinks in (A) represent stable proximities that were consistently identified in both unphosphorylated and phosphorylated APC/C complexes. Grey lines represent all the observed intra- and inter-subunit crosslinks between APC/C subunits; intra-subunit linkages are drawn exteriorly; inter-subunit crosslinks are interiorly. Apc11 and Apc15 were excluded from the plot as no crosslinks were observed. A subset of stable, phosphorylation-unreactive, linkages within APC/C’s three sub-complexes have been highlighted for comparative analysis in (B): Platform: XeApc1-K29—XeApc5-K241 (teal), XeApc4-K408—XeApc5-K415 (orange); Catalytic: XeApc2-K596/K674—XeApc4-K282 (green) and TPR substrate recognition lobe: XeApc7-K345—XeApc16-K68 (blue).(B) Structural comparison of stable crosslinks identified in (A) to published cryo-EM structures [S1]. Sequence alignments of XeAPC/C and HsAPC/C subunits were performed to identify corresponding lysine residues between Xenopus CLMS and human cryo-EM datasets. Panels represent expanded views of stable crosslinks extrapolated to human APC/C: HsApc1-K29—HsApc5-K238 (teal box, bottom left), HsApc4-K415—HsApc5-K411 (orange box, top right), HsApc2-K608/K686—HsApc4-K289 (green box, bottom right) and HsApc7-K379—HsApc16-K68 (blue box, top left). The pink dashed lines and numbers represent the measured distances from cryo-EM structure between the corresponding Cα atoms of the lysine residues at the linkage site. All measured distances fall within the distance constraint of BS3 (26-30 Å) [S2]. (C and D) Circular plots (xiVIEW) of dynamic, phosphorylation-responsive, crosslinks identified by CLMS. Crosslinks in (C) represent linkages that were lost upon APC/C phosphorylation. Crosslinks predicted to dissociate between lysines in Apc1-300L and lysines adjacent to coactivator binding site (C-box) in Apc8 have been highlighted with purple dashed lines [S1]. Crosslinks in (D) represent crosslinks that were formed only in hyper-phosphorylated APC/C complexes.
	Figure S3. CLMS analysis of APC/C complex interactions, related to Figure 3. (A) Circular plots (xiVIEW) of APC/C intra- and inter-subunit crosslinks identified by crosslinking mass spectrometry (CLMS). Crosslinks in (A) represent stable proximities that were consistently identified in both unphosphorylated and phosphorylated APC/C complexes. Grey lines represent all the observed intra- and inter-subunit crosslinks between APC/C subunits; intra-subunit linkages are drawn exteriorly; inter-subunit crosslinks are interiorly. Apc11 and Apc15 were excluded from the plot as no crosslinks were observed. A subset of stable, phosphorylation-unreactive, linkages within APC/C’s three sub-complexes have been highlighted for comparative analysis in (B): Platform: XeApc1-K29—XeApc5-K241 (teal), XeApc4-K408—XeApc5-K415 (orange); Catalytic: XeApc2-K596/K674—XeApc4-K282 (green) and TPR substrate recognition lobe: XeApc7-K345—XeApc16-K68 (blue).(B) Structural comparison of stable crosslinks identified in (A) to published cryo-EM structures [S1]. Sequence alignments of XeAPC/C and HsAPC/C subunits were performed to identify corresponding lysine residues between Xenopus CLMS and human cryo-EM datasets. Panels represent expanded views of stable crosslinks extrapolated to human APC/C: HsApc1-K29—HsApc5-K238 (teal box, bottom left), HsApc4-K415—HsApc5-K411 (orange box, top right), HsApc2-K608/K686—HsApc4-K289 (green box, bottom right) and HsApc7-K379—HsApc16-K68 (blue box, top left). The pink dashed lines and numbers represent the measured distances from cryo-EM structure between the corresponding Cα atoms of the lysine residues at the linkage site. All measured distances fall within the distance constraint of BS3 (26-30 Å) [S2]. (C and D) Circular plots (xiVIEW) of dynamic, phosphorylation-responsive, crosslinks identified by CLMS. Crosslinks in (C) represent linkages that were lost upon APC/C phosphorylation. Crosslinks predicted to dissociate between lysines in Apc1-300L and lysines adjacent to coactivator binding site (C-box) in Apc8 have been highlighted with purple dashed lines [S1]. Crosslinks in (D) represent crosslinks that were formed only in hyper-phosphorylated APC/C complexes.
	Figure S4. Phosphorylation of Apc8-L and its impact on Cdc20 binding, related to Figure 4. (A-C) Cyclin-B destruction assay in interphase Xenopus egg extracts. Equal amounts of mock-depleted or Xe-APC/C-depleted extracts were supplemented with mutant rAPC/Cs. In vitro-translated APC/C substrates (CycB and a non-degradable control CycB∆67) were added to each reaction prior to the initiation of the destruction assay by spiking in recombinant Cdh1, the interphase APC/C coactivator. Levels of 35S-labelled APC/C substrate (CycB) and its stable counterpart (CycB∆67) were sampled over 1 hour and analysed by SDS-PAGE and autoradiography. Representative images (A and B) and analysis (C) of the relative CycB levels, normalised to CycB∆67, over destruction time course. (D) Alignment of Xenopus Apc8-L (540-580) and human Apc8-L (558-597). Conserved residues are marked with an asterisk (*). All predicted phospho-residues are circled in light blue. Bold lines highlight the favoured CDK ([pS/T]-P-x-[K/R]) and PLK1 ([D/E/N/Q]-x(1,2)-[pS/T]) consensus sequences respectively. (E) Phosphorylation of Apc8-L by mitotic kinases. Recombinant wild type Apc8-L or alanine mutants (4A and 8A) were phosphorylated in vitro by CDK2/cyclin A (lanes 1-3), Plx1 (lanes 5-7) or Gwl (Hs: MASTL) (lanes 9-10) and analysed by SDS–PAGE and autoradiography (32P). Bottom: Coomassie blue staining image of the MBP-fused proteins used (labelled as CBB). As positive controls, MBP-tagged ENSA-6His protein was used with CDK and Gwl, while casein was used with Plx1 kinase. (F and G) Coactivator binding assay in mitotic Xenopus egg extracts. The endogenous APC/C was depleted from anaphase-arrested Xenopus egg extracts, and equal quantities of these extracts were incubated with a selection of mutated Apc8 rAPC/Cs. Immobilised rAPC/Cs were then recovered, subjected to thorough washing, and Cdc20 binding was analysed by SDS-PAGE and immunoblotting (C). Panel D shows a quantitative analysis of the fraction of Cdc20 associated with rAPC/C after 45 minutes, based on three independent experiments. Error bars represent the standard deviation (SD). Statistical analysis was performed using a one-way ANOVA followed by Tukey’s post-hoc test; NS denotes not significant.
	Figure S4. Phosphorylation of Apc8-L and its impact on Cdc20 binding, related to Figure 4. (A-C) Cyclin-B destruction assay in interphase Xenopus egg extracts. Equal amounts of mock-depleted or Xe-APC/C-depleted extracts were supplemented with mutant rAPC/Cs. In vitro-translated APC/C substrates (CycB and a non-degradable control CycB∆67) were added to each reaction prior to the initiation of the destruction assay by spiking in recombinant Cdh1, the interphase APC/C coactivator. Levels of 35S-labelled APC/C substrate (CycB) and its stable counterpart (CycB∆67) were sampled over 1 hour and analysed by SDS-PAGE and autoradiography. Representative images (A and B) and analysis (C) of the relative CycB levels, normalised to CycB∆67, over destruction time course. (D) Alignment of Xenopus Apc8-L (540-580) and human Apc8-L (558-597). Conserved residues are marked with an asterisk (*). All predicted phospho-residues are circled in light blue. Bold lines highlight the favoured CDK ([pS/T]-P-x-[K/R]) and PLK1 ([D/E/N/Q]-x(1,2)-[pS/T]) consensus sequences respectively. (E) Phosphorylation of Apc8-L by mitotic kinases. Recombinant wild type Apc8-L or alanine mutants (4A and 8A) were phosphorylated in vitro by CDK2/cyclin A (lanes 1-3), Plx1 (lanes 5-7) or Gwl (Hs: MASTL) (lanes 9-10) and analysed by SDS–PAGE and autoradiography (32P). Bottom: Coomassie blue staining image of the MBP-fused proteins used (labelled as CBB). As positive controls, MBP-tagged ENSA-6His protein was used with CDK and Gwl, while casein was used with Plx1 kinase. (F and G) Coactivator binding assay in mitotic Xenopus egg extracts. The endogenous APC/C was depleted from anaphase-arrested Xenopus egg extracts, and equal quantities of these extracts were incubated with a selection of mutated Apc8 rAPC/Cs. Immobilised rAPC/Cs were then recovered, subjected to thorough washing, and Cdc20 binding was analysed by SDS-PAGE and immunoblotting (C). Panel D shows a quantitative analysis of the fraction of Cdc20 associated with rAPC/C after 45 minutes, based on three independent experiments. Error bars represent the standard deviation (SD). Statistical analysis was performed using a one-way ANOVA followed by Tukey’s post-hoc test; NS denotes not significant.
	Figure S5. Developing and validating the Apc1-300L cleavable assay for phosphorylation analysis, related to Figure 5. (A) Schematic representing the architecture of apoAPC/C (left panel) and Apc1 subunit (right panel) which have been engineered with cleavable Apc1-300L for a loop release assay. HRV-3C cleavage sites are represented in yellow, the physiologically inert ALFA tag is situated N-terminally of wild-type Apc1-300L sequence and is represented by orange hexagon. (B) Anaphase phosphorylation assay comparing the phospho-activation profiles of the endogenous APC/C (Mock), wild-type rAPC/C (WT) and rAPC/C with a cleavable, ALFA-tagged Apc1-300L (1-α3C). Interphase Xenopus extracts were prepared and endogenous APC/Cs were depleted (unless otherwise stated, Mock). Non-degradable cyclin B (CycB∆167) was added to trigger anaphase entry (time point T0) and reactions were sampled routinely over a 75-minute time course. Phosphorylation status was analysed by SDS-PAGE and immunoblotting for pan Apc1, Apc3 and Apc8 subunits. (C and D) Cdc20 binding assay in anaphase Xenopus egg extracts to compare coactivator loading between wild-type and loop-cleavable apoAPC/C. Endogenous APC/C was depleted from anaphase-arrested Xenopus egg extracts. Equal amounts of these extracts were incubated with wild-type, Apc3∆L or Apc8∆L rAPC/Cs. Labels for rAPC/Cs with cleavable, ALFA-tagged, Apc1-300L (rAPC/C_1-3C) are highlighted in teal. Immobilised rAPC/Cs were recovered by immunoprecipitation, washed harshly and Cdc20 loading was analysed by SDS-PAGE and immunoblotting (C). (D) Quantitative analysis of the proportion of Cdc20 bound to rAPC/C after 60 minutes. (E) Wild-type rAPC/Cs harbouring a cleavable, ALFA-tagged, Apc1-300L (rAPC/C_1-3C) were compared to equivalent rAPC/Cs, which expressed a loop-deficient Apc3 (rAPC/C_1-3C/3∆L). These rAPC/Cs were phosphorylated in anaphasic egg extracts for 1 hour. rAPC/Cs were isolated then washed harshly to strip all interacting proteins. Immobilised rAPC/Cs were incubated with equal volumes of HRV-3C protease to cleave Apc1-300L for subsequent analysis. Cleaved Apc1-300L was then visualised by SDS-PAGE and immunoblotting for its N-terminal ALFA tag. 3C-x-Apc1 (visualised by α-Apc1 immunoblotting) and 3C-x-α300L (visualised by α-ALFA immunoblotting) denote the Apc1 HRV-3C protease cleavage products.
	Figure S6. Anaphase-specific Apc8-L binding assay, related to Figure 5. (A and B) Bacterially purified MBP-tagged Apc8-L fragments were immobilised on amylose resin and equilibrated prior to their addition into egg extract. Fusion proteins were incubated in anaphase-arrested extract for 60 minutes. Subsequent to immunoprecipitation, rigorous washing, and elution of associated proteins, loop-dependent interactions were analysed by SDS-PAGE and immunoblotting for the CDK complex components (Xe-p9/Cks2, CycB, CDK1) and Plx1, another mitotic kinase. Representative image (A) and quantitative analysis (B) of the proportion of Xe-p9/Cks2 bound to wild-type and non phosphorylatable Apc8-loop fragments (n=2). Error bars represent SD.
	Figure S7. Delayed phosphorylation of Apc1-300L in Apc8-L deficient APC/C, related to Figure 5. (A-C) Quantification of Apc1 phosphorylation in anaphase phosphorylation assay assessing APC/C phospho-activation upon Apc8-L deletion (from Figure 5G). Interphase Xenopus extracts were prepared, and endogenous APC/Cs were depleted. Non-degradable cyclin B (CycB∆167) was added to trigger anaphase entry, marked as time point T0. Samples were taken at the indicated time points. Phosphorylation status of Apc1 was analysed by SDS-PAGE and immunoblotting for three Apc1 phospho-specific antibodies targeting CDK-favoured phospho-residues within Apc1-300L: (A) pS314/S318, (B) pS358 and (C) pT378/S380. Phosphorylation of Apc1 in wild-type APC/C (black circles), phosphorylation of Apc1 in Apc8∆L APC/C complexes (white circles). Intensities for phospho-residues were normalised to Apc2 (depicted in Figure 5G).
	Graphical abstract
	Figure 1. Unveiling the role of Apc8-L in APC/CCdc20 activation via systematic IDR deletion (A) Schematic representing the intrinsically disordered regions (IDRs) or “loops” present in the APC/C complex: loops chosen for analysis are highlighted as bold black lines, and gray lines represent loops excluded from this screen. (B and C) Comparison of Apc1 N-terminal WD40 domain structures using Cryo-EM 22 (Human 1–612) (B) and AlphaFold2 (Xenopus 1–615) with its three IDRs: Apc1-35L (35–70), Apc1-300L (298–399), and Apc1-500L (515–585) (C) models. (D) Panel of increasingly loop-deficient rAPC/C mutants. Mutations are listed underneath. (E and F) Coactivator binding assay in mitotic Xenopus egg extracts. Endogenous Xe-APC/C was depleted from anaphase-arrested extracts prior to loop-deficient rAPC/C incubation. Immobilized rAPC/Cs were recovered and washed harshly, and Cdc20 binding was analyzed by SDS-PAGE and immunoblotting (E). (F) Quantitative analysis of the proportion of Cdc20 bound to rAPC/C after 60 min (based on data from three independent experiments, n = 3). Cdc20 levels were first normalized to Apc2 levels, and then wild-type intensities were arbitrarily set to 1.0. Error bars, SD, one-way ANOVA with Tukey’s post-hoc test; NS: not significant, ∗∗p ≤ 0.01. (G and H) Anaphase cyclin B destruction assay. Mock-depleted or Xe-APC/C-depleted extracts supplemented with loop-deficient rAPC/Cs were anaphase-arrested. Levels of APC/C substrates CycB and CycBΔ67 (stable control) were sampled over 1 h and analyzed by SDS-PAGE and autoradiography. Representative image (G) and analysis (H) of relative CycB levels.
	Figure 2. Apc8-L regulates mitotic APC/C activation in a 300L-dependent mechanism (A) Panel of loop-deficient rAPC/C mutants. (B and C) Mitotic coactivator binding assay. APC/C was depleted from anaphase-arrested Xenopus egg extracts; equal amounts of extracts were incubated with the panel of loop-deficient rAPC/Cs in (A). Immobilized rAPC/Cs were recovered by immunoprecipitation and washed harshly, and Cdc20 loading was analyzed by SDS-PAGE and immunoblotting (B). (C) Quantitative analysis of the proportion of Cdc20 bound to rAPC/C after 45 min (n = 3). Quantification and statistical analysis was performed analogous to Figure 1 F, ∗p ≤ 0.02, ∗∗∗p ≤ 0.001. (D and E) Cyclin B destruction assay in anaphase extracts. Equal amounts of Xe-APC/C-depleted extracts were supplemented with loop-deficient rAPC/Cs prior to anaphase arrest. Levels of 35S-labeled APC/C substrate (CycB) and its stable counterpart (CycBΔ67) were sampled over 75 min and analyzed by SDS-PAGE and autoradiography. (D) shows a representative autoradiograph, and (E) presents the quantitative analysis of relative CycB levels over time.
	Figure 3. Phosphorylation regulates Apc8-L and Apc1-300L proximity (A) Workflow for crosslinking mass spectrometry (CLMS) analysis of phosphorylation-modulated Xenopus rAPC/C complex interactions. (B and C) Circular plots (xiVIEW) of intra- and inter-protein crosslinks within unphosphorylated (B) and hyper-phosphorylated (C) APC/C complexes. Gray lines represent all the observed intra- and inter-subunit crosslinks between APC/C subunits; intra-subunit linkages are drawn exteriorly; inter-subunit crosslinks are drawn interiorly. Apc11 and Apc15 were excluded from the plot as no crosslinks were observed. Apc1-300L crosslinks are highlighted in teal, and C-terminal Apc8 linkages are highlighted in pink. Dashed boxes refer to expanded sections in (D). (D) Expanded view of Apc8-Apc1 phosphorylation-responsive crosslinks. C-terminal Apc8-K529 is in proximity to Apc1-K311 (left panel). This linkage is lost upon hyper-phosphorylation of APC/C (right panel). (E) AlphaFold2 modeling of APC/C subcomplex orients Apc8-L in proximity to Apc1-300L. A subset of Xenopus APC/C subunits (Apc1, N-terminal WD40 domain only [1–615] in teal, Apc5 [1–759] in orange, and an Apc8A/Apc8B homodimer [1–580] in pink) were modeled using AlphaFold2 (i). The subcomplex was successfully predicted, compared to published cryo-EM structures. 22 (ii). The right panel represents an expanded view of a predicted interconnection between Apc8-L (red) and Apc1-300L (green).
	Figure 4. CDK-dependence and synergistic activation of APC/C by Apc8-L and Apc3-L (A) Panel of loop-deficient or non-phosphorylatable rAPC/C mutants. (B and C) Mitotic Cdc20 binding assay. Endogenous APC/C was depleted from anaphase-arrested Xenopus egg extracts; equal amounts of extracts were incubated with the panel of loop-deficient rAPC/Cs in (A). Immobilized rAPC/Cs were recovered by immunoprecipitation and washed harshly, and Cdc20 loading was analyzed by SDS-PAGE and immunoblotting (B). (C) Quantitative analysis of the proportion of Cdc20 bound to rAPC/C after 45 min (n = 3). Quantification and statistical analysis was performed analogous to Figure 1 F; ∗p ≤ 0.05, ∗∗∗p ≤ 0.001. (D–G) Anaphase cyclin B destruction assay. Equal amounts of APC/C-depleted extracts were supplemented with loop-deficient rAPC/Cs and then arrested after anaphase entry. Levels of 35S-labeled APC/C substrate (CycB) and its stable counterpart (CycBΔ67) were sampled over 75 min and analyzed by SDS-PAGE and autoradiography with representative images (D and E) and relative CycB level analysis (F and G).
	Figure 5. Apc8-L influences Apc1-300L phosphorylation via direct Xe-p9/Cks2 recruitment (A and B) Workflow of the in situ phosphorylation assay (A). (B) Wild-type rAPC/Cs and rAPC/C with mutant Apc8, each with cleavable ALFA-Apc1-300L, were phosphorylated using anaphase extracts, or control interphase extracts, for 1 h. For an unmodified control, rAPC/C not incubated with extract is shown as “negative.” Immobilized rAPC/Cs were washed harshly before ALFA-Apc1-300Ls were excised by HRV-3C protease. Reactions were analyzed by SDS-PAGE and immunoblotting: 3C-x-Apc1 and 3C-x-α300L denote the Apc1 HRV-3C cleavage products. (C–F) In vitro Xe-p9/Cks2 binding assay with bacterially purified MBP-tagged Apc8-L fragments. Equal amounts of pre-phosphorylated Apc8-loop proteins were incubated with purified Xe-p9/Cks2 for 30 min. Phosphorylation-dependent interactions were analyzed by SDS-PAGE and immunoblotting. Representative images (C and E) and quantitative analysis (D and F) (n = 3). His-Xe-p9 levels were first normalized to MBP eluate levels, and then intensities of MBP-Apc8-L-WT were arbitrarily set to 1.0. Error bars, SD, one-way ANOVA with Tukey’s post-hoc test; ∗∗∗p ≤ 0.001. (G) Anaphase phosphorylation assay assessing APC/C phospho-activation upon Apc8-L deletion. Interphase Xenopus extracts were prepared, and endogenous APC/Cs were depleted. Non-degradable cyclin B (CycBΔ167) was added to trigger anaphase entry, marked as time point T0. Samples were taken at the indicated time points. Phosphorylation status was analyzed by SDS-PAGE and immunoblotting for Apc1 and Apc8 subunits, as well as three phospho-specific antibodies targeting CDK-favored phospho-residues within Apc1-300L.
	Figure 6. Cooperative activation of the APC/C by Apc3-L and Apc8-L through Apc1-300L displacement in Xenopus egg extracts (A) Panel of APC/Cs used in “cycling” egg extract experiment. (B) Xenopus “cycling” egg extracts and their APC/C-depleted (ΔAPC/C) counterparts were prepared. Wild-type (WT) APC/C and its variants shown in (A) were reintroduced into ΔAPC/C “cycling” extracts and incubated at 23°C, with samples collected every 10 min for analysis. The dynamic behavior of the APC/C complex and its regulation of the cell cycle were monitored through immunoblotting of cyclin B, phosphorylated Plx1 (pPlx1), Cdk1, and Apc5 to assess the restoration of cell cycle progression and APC/C activity in the depleted extracts.
	Figure 7. Model representing Apc8-L’s cooperative role in APC/C mitotic activation (A) Proposed model for Apc8-loop-dependent APC/C activation via Apc1-300L. Apc8-L directly recruits the CDK adaptor protein, Xe-p9/Cks2, stimulating Apc1-300L phosphorylation, which in turn triggers Apc1-300L displacement, thereby facilitating the formation of an active APC/CCdc20 complex. (B) Dual pathways mediated by Apc3-L and Apc8-L synergistically activate the APC/C. In interphase, the Cdc20 C-box binding site is blocked by Apc1-300L, preventing Cdc20 from associating with the APC/C and resulting in its inactivity. During mitosis, Apc8-L and Apc3-L work together to phosphorylate Apc1-300L, enabling APC/C activation upon Cdc20 association. If either Apc3-L or Apc8-L is restricted, Apc1-300L phosphorylation decreases due to one defective pathway, resulting in only partial activation. When both Apc8-L and Apc3-L are restricted, Apc1-300L phosphorylation is significantly impaired, leading to near-total APC/C inactivation.