mbp tagged apc8 fragments  (New England Biolabs)

Journal: Cell reports

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

doi: 10.1016/j.celrep.2024.114262

Figure Lengend Snippet: 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-EM22 (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 CycBD67 (stable control) were sampled over 1 h and analyzed by SDS-PAGE and autoradiography. Repre- sentative image (G) and analysis (H) of relative CycB levels.

Article Snippet: Purified MBP-tagged Apc8 fragments were incubated with amylose resin (NEB) (400 pmol MBP-tagged proteins on 5 mL packed resin in 400 mL in 1x XBcsf containing 0.1% NP40) at 4 C for 1–2 h with end-over-end rotation (15 rpm).

Techniques: Activation Assay, Comparison, Binding Assay, Incubation, SDS Page, Western Blot, Control, Autoradiography

Journal: Cell reports

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

doi: 10.1016/j.celrep.2024.114262

Figure Lengend Snippet: 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).

Article Snippet: Purified MBP-tagged Apc8 fragments were incubated with amylose resin (NEB) (400 pmol MBP-tagged proteins on 5 mL packed resin in 400 mL in 1x XBcsf containing 0.1% NP40) at 4 C for 1–2 h with end-over-end rotation (15 rpm).

Techniques: Phospho-proteomics, Mass Spectrometry

Journal: Cell reports

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

doi: 10.1016/j.celrep.2024.114262

Figure Lengend Snippet: 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 1F; *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 (CycBD67) 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).

Article Snippet: Purified MBP-tagged Apc8 fragments were incubated with amylose resin (NEB) (400 pmol MBP-tagged proteins on 5 mL packed resin in 400 mL in 1x XBcsf containing 0.1% NP40) at 4 C for 1–2 h with end-over-end rotation (15 rpm).

Techniques: Activation Assay, Binding Assay, Incubation, Immunoprecipitation, SDS Page, Western Blot, Labeling, Autoradiography

Journal: Cell reports

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

doi: 10.1016/j.celrep.2024.114262

Figure Lengend Snippet: 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-a300L 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 (CycBD167) 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.

Article Snippet: Purified MBP-tagged Apc8 fragments were incubated with amylose resin (NEB) (400 pmol MBP-tagged proteins on 5 mL packed resin in 400 mL in 1x XBcsf containing 0.1% NP40) at 4 C for 1–2 h with end-over-end rotation (15 rpm).

Techniques: Phospho-proteomics, In Situ, Mutagenesis, Control, Incubation, SDS Page, Western Blot, In Vitro, Binding Assay, Activation Assay

Journal: Cell reports

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

doi: 10.1016/j.celrep.2024.114262

Figure Lengend Snippet: 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 (DAPC/C) counterparts were prepared. Wild-type (WT) APC/C and its variants shown in (A) were reintroduced into DAPC/C ‘‘cycling’’ extracts and incubated at 23C, 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.

Article Snippet: Purified MBP-tagged Apc8 fragments were incubated with amylose resin (NEB) (400 pmol MBP-tagged proteins on 5 mL packed resin in 400 mL in 1x XBcsf containing 0.1% NP40) at 4 C for 1–2 h with end-over-end rotation (15 rpm).

Techniques: Activation Assay, Incubation, Western Blot, Activity Assay