Zeng X , King RW .

GM66492 NIGMS NIH HHS , R01 GM066492-09 NIGMS NIH HHS , R01 GM066492 NIGMS NIH HHS , R56 GM066492 NIGMS NIH HHS

	Figure 2. TAME induces Cdc20 dissociation from the APC by promoting Cdc20 ubiquitination(a) TAME-induced Cdc20 dissociation can be reconstituted in vitro with immunopurified mitotic APCCdc20, E1 (250 nM), UbcH10 (2 μM) and ubiquitin (150 μM). Immunoprecipitated APCCdc20 was incubated with various components as shown for 10 min. Cdc20 was analyzed by Western blot separately in the bead-bound (B) and the supernatant (S) fractions. Numbers represent the relative band intensity of the unmodified Cdc20. (b) TAME-induced Cdc20 dissociation can be reconstituted in vitro with immunopurified mitotic APC and in vitro-translated human Cdc20. The same assay was performed as in a, except that the APC was first washed with high salt buffer to remove Cdc20 and then re-loaded with in vitro-translated human Cdc20.
	Figure 3. Ubiquitination upstream of the CRY-box of Cdc20 reduces its binding affinity for the APC(a) Schematic illustration of human Cdc20. Asterisks indicate lysine residues. (b) Ubiquitination/dissociation of Cdc20 requires lysines in the N-terminal region. In vitro ubiquitination assays were performed with APC re-loaded with double HA-tagged human WT or Cdc20 mutants as described in Fig. 2b. (c) Deubiquitination of ubiquitinated Cdc20 increases its affinity for the APC. Supernatant from an in vitro Cdc20 ubiquitination assay containing ubiquitinated Cdc20 was divided into two aliquots and one aliquot was incubated with the catalytic domain of Usp2 (Usp2-CD). Both aliquots were then re-incubated with a fresh batch of high salt-washed APC to assess the binding of Cdc20. (d) Unmodified Cdc20 outcompetes ubiquitinated Cdc20 for binding to the APC. Ubiquitinated Cdc20 (Ub-Cdc20) was generated as in (c) and mixed with reticulocyte lysate containing unmodified Cdc20 at different ratios. The mixture was then incubated with high salt-washed APC and Cdc20 binding was assessed by Western blot. (e) Cdc201-164R is more resistant to TAME-induced dissociation than WT Cdc20. APC beads loaded with WT or Cdc201-164R were incubated in mitotic extract +/- TAME for 10 min. A fraction of the beads were then treated with Usp2-CD.
	Figure 4. CycB-NT promotes Cdc20 binding to the APC and suppresses Cdc20 ubiquitination(a) Cyclin B-NT increases the steady-state level of Cdc20 bound to the APC in non-treated or TAME-treated mitotic Xenopus extract. A cyclin B1 N-terminal fragment (CycB-NT, 6 μM) or the same fragment without the D-box (CycB-NT ΔD-box, 6 μM) and TAME (200 μM) were added to mitotic extract as indicated. APC was immunoprecipitated from the extract and protein levels were analyzed by Western blot. (b) CycB-NT promotes Cdc20 binding to the APC in the absence of the IR-tail. APC cleared of Cdc20 by TAME-treatment was incubated with in vitro-translated WT Cdc20 or Cdc20ΔIR for 10 minutes. CycB-NT (2 μM) and TAME (200 μM) were added as indicated. (c) CycB-NT suppresses Cdc20 ubiquitination. The same assay was performed as in Fig. 2b in the presence or absence of 500 nM cycB-NT.
	Figure 5. TAME causes premature termination of cycB-NT ubiquitination(a) TAME reduces cycBNT ubiquitination level in mitotic extract. CycB-NT was isolated from mitotic extract treated with ubiquitin vinyl sulfone (20 μM) and ubiquitin (20 μM) (+UbVS) or buffer (-UbVS) +/- TAME (200 μM) as indicated and detected by anti-HA blot. (b) TAME reduces cycB-NT ubiquitination level in a reconstituted ubiquitination assay, which is accompanied by loss of Cdc20 from the APC. A reconstituted ubiquitination assay was performed with immunopurified APCCdc20 and 500 nM cycB-NT +/- TAME. Quantification of Cdc20 and Apc10 levels bound to the beads is shown. (c) TAME's effect on cycB-NT ubiquitination can be recapitulated with Cdc20ΔIR. The same assay as in b was performed with APC reloaded with WT Cdc20 or Cdc20ΔIR. (d) Ubiquitinated cycB-NT has reduced binding affinity for Cdc20. Unmodified or ubiquitinated cycB-NT was immobilized on an HA-antibody resin and then incubated with in vitro-translated Flag-Cdc20. Resin bound cycB-NT and Cdc20 were analyzed by anti-HA and anti-Flag Western blot, respectively. (e) Ubiquitinated cycB-NT cannot promote Cdc20 binding to the APC in the presence of TAME. Unmodified or ubiquitinated cycB-NT was mixed with in vitro-translated Flag-Cdc20 and then incubated with APC +/- TAME. The binding of Cdc20 was assessed by anti-Flag Western blot. (f) TAME prevents further ubiquitination of partially ubiquitinated cycB-NT. Unmodified or ubiquitinated cycB-NT, along with ubiquitination reaction components, were mixed with in vitro-translated Flag-Cdc20 and then incubated with APC +/- TAME.
	Figure 6. UBE2S extends ubiquitin chains on cycB-NT in the presence of TAME in a single substrate binding cycle and promotes substrate degradation in TAME-treated extract(a) UBE2S extends ubiquitin chains on cycB-NT in the presence of TAME in a single substrate binding cycle. APC beads were pre-incubated with HA-tagged cycB-NT and then mixed with an excess of untagged competitor and the ubiquitination reaction mixture for 2 min. UbcH10/UBE2S (2 μM) was added as indicated. The fraction of ubiquitinated cycB-NT carrying at least 4 ubiquitin molecules was quantitated. (b) UBE2S promotes substrate degradation in TAME-treated extract. Mitotic extract was incubated with UbcH10 (2 μM), UBE2S (2 μM), ubiquitin (20 μM) and TAME (200 μM) for 10 min as indicated and then the luciferase reporter was added. Samples were collected at indicated time points and the reporter level was measured by luminometer. Error bar: SEM from 3 independent experiments. (c) UBE2S and UbcH10 rescue substrate degradation much less effectively in APC-depleted extract than in TAME-treated extract. APC-depleted or TAME-treated extracts were supplemented with UbcH10 (2 μM), UBE2S (2 μM), ubiquitin (20 μM) and the luciferase reporter, as indicated. Samples were collected at 60 min and the reporter level was measured by luminometer. Results were normalized to the value obtained under the +TAME/-E2 condition, which was set arbitrarily at 100%. Error bar: SEM from 3 independent experiments.
	Figure 7. Schematic illustration of TAME's mechanism(a) In the absence of substrate, TAME promotes ubiquitination and dissociation of pre-bound Cdc20 and blocks the binding of free Cdc20 to the APC. (b) Substrate (cyclin B1) requires multiple APC binding cycles to become sufficiently ubiquitinated for proteolysis in a UbcH10-mediated reaction. (c) TAME reduces the kcat of the APCCdc20/substrate complex. In a UbcH10-mediated reaction, the partially ubiquitinated substrate cannot undergo further ubiquitination because it fails to promote Cdc20 binding to the APC in the presence of TAME. Instead, it is reversed to the unmodified state by deubiquitination. In a UBE2S-mediated reaction, substrate becomes sufficiently ubiquitinated for proteolysis in a single binding cycle in the presence of TAME.

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