Cell Cycle-Regulated Recognition of the Destruction Box of Cyclin B by the APC/C in Xenopus Egg Extracts  Hiroyuki Yamano, Julian Gannon, Hiro Mahbubani, Tim Hunt  Molecular Cell  Volume 13, Issue 1, Pages 137-147 (January 2004) DOI: 10.1016/S1097-2765(03)00480-5

Figure 1 Affinity Chromatography with Immobilized D-Boxes (A) Schematic diagram of the D-box affinity columns; wild-type D-boxes are indicated by open and mutant D-boxes by filled rectangles. (B) Flow-through of the various D-box columns were used for testing cyclin proteolysis in frog egg extracts. Full-length fission yeast cyclin B (Cdc13) and a version lacking 67 N-terminal residues (Δ67, stable control) were used as substrates. CaCl2 was added to initiate proteolysis. Molecular Cell 2004 13, 137-147DOI: (10.1016/S1097-2765(03)00480-5)

Figure 2 The APC/C, but Not Fizzy, Is Retained by a D-Box Affinity Matrix (A) CSF-arrested frog egg extracts (input, I) were applied to the indicated affinity matrices, and separated into flow-through (F) and bound (B) fractions followed by immunoblotting with anti-Apc3, anti-Fizzy, or anti-PSTAIRE antibodies. (B) Same as (A) using N70-2X column, but CSF or APC/C depleted CSF extracts were used. (C) Titration of N70. Aliquots of biotinylated N70: 30, 10, 5, 1, 0.5, 0.25, or 0 μg, were attached to Streptavidin-magnetic beads. Next, 20 μl of CSF extract were applied onto the beads. After incubation at 23°C for 20 min, the beads were isolated by a magnetic stand. The supernatants were analyzed by immunoblotting with anti-Apc3, anti-Fizzy, anti-PSTAIRE, or anti-UbcP4 (Ubcx homolog in fission yeast) antibodies. (D) Cyclin destruction assay using the supernatants from (C). CaCl2 was added to trigger cyclin proteolysis. (E) Purified APC/C with or without additional ubiquitin was added to the supernatant of the N70-2X column showed in (A), followed by cyclin destruction assays. (F) Same as (A), but interphase extracts were used as input. Molecular Cell 2004 13, 137-147DOI: (10.1016/S1097-2765(03)00480-5)

Figure 3 Interaction between the D-Box and the APC/C in Egg Extracts (A) Large volumes (2 ml) of anaphase extracts (Δ90-arrested egg extracts) were applied to 100 μl N70-GST or Dm-GST columns, and the appearance of proteins in successive drops (∼30 μl) of the flow-through was monitored by immunoblotting with anti-Apc3, anti-Fizzy, and anti-PSTAIRE antibodies. Lane E, bound proteins eluted by reduced glutathione. (B) Quantitation of (A). For each protein, intensities are plotted relative to the level after saturation of the column. (C) Sucrose gradient centrifugation of anaphase egg extracts. Anaphase HSS was overlaid on a 15%–40% sucrose density gradient and spun at 40,000 rpm for 12 hr. Fractions were analyzed by immunoblotting with anti-Apc3, anti-Fizzy, and anti-PSTAIRE antibodies. Molecular Cell 2004 13, 137-147DOI: (10.1016/S1097-2765(03)00480-5)

Figure 4 Fizzy-free APC/C Binds to the D-box, but Not Mutant D-Box Affinity Matrix (A) Purified APC/C or “Fizzy-free” APC/C from CSF extracts was analyzed by immunoblotting with anti-Apc3 or anti-Fizzy antibodies. (B) Top, Fizzy-free APC (input, I) was applied onto biotinylated N70 or Dm columns, and after incubation at 23°C for 20 min, flow-through (F) and bound (B) fractions were separated. The fractions analyzed by immunoblotting with anti-Apc3 antibody. Bottom, quantitation of the immunoblots with relative value against input (I). (C) Same as (B), but using securin-GST or securin-D-box/KEN-box double mutant-GST matrices. Molecular Cell 2004 13, 137-147DOI: (10.1016/S1097-2765(03)00480-5)

Figure 5 Interaction with the D-Box and the APC/C Is Cell Cycle Regulated (A) Purification of the APC/C from anaphase or interphase extracts. Using AF3.1-immunoaffinity chromatography and specific elution peptides, the soluble APC/C was purified, and visualized by silver staining. The numbers in figure refer to subunit numbers of the APC/C. The bands marked with asterisks are unknown. (B) Parallel samples from (A) were analyzed by immunoblotting with anti-Apc3 and anti-Fizzy antibodies. (C) APC/C purified from either anaphase or interphase extracts was applied onto streptavidin-coated sensor chips of a Biacore apparatus, loaded with biotinylated N70 or Dm in surface plasmon resonance (SPR). The APC/C was injected at time 0, and injection continued for 100 s. (D) Metaphase (CSF-arrested; Meta), anaphase (Δ90 arrested; Ana), or interphase (Int) extracts were subjected to biotinylated N70 or Dm affinity chromatography. After incubation at 23°C for 20 min, the bound fractions were isolated by a magnetic stand, washed several times, and analyzed by immunoblotting with anti-Apc3, anti-Fizzy, anti-PSTAIRE, or anti-UbcP4 (Ubcx homolog in fission yeast) antibodies. Molecular Cell 2004 13, 137-147DOI: (10.1016/S1097-2765(03)00480-5)

Figure 6 Qualitative Difference of the APC/C Isolated from Different Cell Cycle Stages in Egg Extracts (A) The APC/C was depleted from CSF extracts using AF3.1 coupled beads. The APC/C depletion process was repeated; first depletion for single depletion (lane 2) and second depletion for sequential double depletion (lane 3). Untreated extract (lane 1) and both depleted extracts were analyzed by immunoblotting with anti-Apc3, anti-Fizzy, anti-PSTAIRE, or anti-UbcP4 (Ubcx homolog in fission yeast) antibodies. (B) The APC/C-depleted CSF extracts were assayed for cyclin destruction with or without added CaCl2 as indicated and the following additions: lanes 1–8, buffer; lanes 9–16, APC/C purified from CSF extracts; lanes 17–24, APC/C from anaphase extract; and lanes 25–32, APC/C from interphase extract. Molecular Cell 2004 13, 137-147DOI: (10.1016/S1097-2765(03)00480-5)

Figure 7 Model for Activation of the D-Box Receptor and the APC/C-Dependent Proteolysis The APC/C purified from interphase, metaphase (CSF-arrested frog egg extract), or anaphase has different activity in terms of recognition and proteolysis of cyclin B. In interphase, cyclin B is neither recognized or ubiquitylated. During mitosis, mitotic kinase(s) phosphorylate the APC/C, priming it for activation by Fizzy and turning on recognition of the D-box. This form of the APC/C is not fully active, however, owing to inhibitory signals emanating from the spindle assembly checkpoint (perhaps acting to sequester Fizzy), and cyclin B is consequently stable. In anaphase, the APC/C is fully activated by as yet unknown mechanisms (here indicated by a further conformational change), and cyclin B is rapidly ubiquitylated, leading to its proteolysis. Note that Fizzy is not a permanent member of the complex, and that tight interactions with E2 ubiquitin-conjugating enzymes have never been observed. The phosphorylation sites are indicated in purely cartoon form; there is no direct evidence that polo kinase must phosphorylate the APC/C to activate it fully, although depletion of polo precludes such activation. Molecular Cell 2004 13, 137-147DOI: (10.1016/S1097-2765(03)00480-5)