Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding

Abstract

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase that targets for degradation cell-cycle regulatory proteins during exit from mitosis and in the G1 phase of the cell cycle. The activity of APC/C in mitosis and in G1 requires interaction with the activator proteins Cdc20 and Cdh1, respectively. Substrates of APC/C-Cdc20 contain a recognition motif called the "destruction box" (D-box). The mode of the action of APC/C activators and their possible role in substrate binding remain poorly understood. Several investigators suggested that Cdc20 and Cdh1 mediate substrate recognition, whereas others proposed that substrates bind to APC/C or to APC/C-activator complexes. All these studies used binding assays, which do not necessarily indicate that substrate binding is functional and leads to product formation. In the present investigation we examined this problem by an "isotope-trapping" approach that directly demonstrates productive substrate binding. With this method we found that the simultaneous presence of both APC/C and Cdc20 is required for functional substrate binding. By contrast, with conventional binding assays we found that either Cdc20 or APC/C can bind substrate by itself, but only at low affinity and relaxed selectivity for D-box. Our results are consistent with models in which interaction of substrate with specific binding sites on both APC/C and Cdc20 is involved in selective and productive substrate binding.

Fig. 1.

The simultaneous presence of mitotic APC/C and Cdc20 is required for productive substrate binding. (A) Outline of isotope-trapping (pulse–chase) procedure. Step 1, binding of labeled substrate to relevant E3 component(s); step 2, dissociation of ³⁵S-substrate–enzyme complex; step 3, product formation from enzyme-bound labeled substrate upon the addition of a mixture of excess unlabeled substrate and all other components necessary for ubiquitylation. (B) Selective inhibition of ubiquitylation of ³⁵S-securin by excess unlabeled N70–2X substrate. Experimental conditions were similar to those described for the pulse–chase assay, except that the indicated concentrations of WT or DM N70–2X proteins were added in the pulse (and not in the chase) incubation, together with APC/C and Cdc20. (C) Productive binding of labeled substrate for conjugate formation in the presence of both APC/C and Cdc20. Experimental conditions were as described in Materials and Methods, with both APC/C and Cdc20 added in the pulse phase. The isotope-trapping incubation (lanes 5 and 5′) was performed in duplicate to test that results were not affected by possible slight changes in mixing conditions upon the addition of the chase mixture. Lanes 1–4 show control incubations, in which the indicated components were added in the indicated phase of the pulse–chase incubation. The position of free ³⁵S-securin and of ³⁵S-securin–MeUb conjugates are indicated on the left. The asterisk indicates a contaminating protein band in the preparation of ³⁵S-securin. Numbers on the right side indicate the positions of molecular mass marker proteins (kDa). The percentage of ³⁵S-securin ligated to MeUb is indicated at the bottom of each lane. (D) The omission of either Cdc20 or APC/C from the pulse mixture abolished the binding of labeled substrate for product formation. Experimental conditions were as described in Materials and Methods, except that the indicated components were added at the indicated phases of the pulse–chase incubation.

Fig. 2.

Cdc20 binds substrate with relaxed selectivity for D-box.(A) The binding of Cdc20, added at the indicated amounts, to WT or DM N70–2X substrate (180 nM) was estimated as described in Materials and Methods. (B) Effect of substrate concentration on the binding of Cdc20. The binding of Cdc20 (0.1 pmol) to WT or DM substrates at the indicated concentrations was estimated by quantitative immunoblotting.

Fig. 3.

Binding of APC/C to substrate in the absence or presence of Cdc20. (A) Influence of substrate concentration. The binding of APC/C (0.015 pmol) to WT N70–2X substrate, added at the concentrations specified, was estimated as described in Materials and Methods. Where indicated, 0.15 pmol Cdc20 was added jointly with APC/C. The immunoblots in lanes 2–5 were done in duplicate for better accuracy of quantitation. (B) Quantitation of the experiment shown in A. There was no significant binding of APC/C to glutathione beads in the absence of substrate, so such correction was not necessary in this case. (C) Influence of Cdc20 (0.05 pmol) on the binding of APC/C (0.015 pmol) to WT or DM substrates. The immunoblots in lanes 1, 2, 5, and 6 were done in duplicate. (D) Quantitation of the experiment shown in C.

Fig. 4.

Possible models to account for the synergistic action of APC/C and Cdc20 in functional and specific binding of substrate. (A) Sequential transfer of substrate from Cdc20 to APC/C. (B) Composite APC/C–Cdc20 substrate binding site. See the text for details. S, substrate.