Emi2 inhibition of the anaphase-promoting complex/cyclosome absolutely requires Emi2 binding via the C-terminal RL tail

Abstract

Emi2 (also called Erp1) inhibits the anaphase-promoting complex/cyclosome (APC/C) and thereby causes metaphase II arrest in unfertilized vertebrate eggs. Both the D-box and the zinc-binding region (ZBR) of Emi2 have been implicated in APC/C inhibition. However, it is not well known how Emi2 interacts with and hence inhibits the APC/C. Here we show that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. When expressed in Xenopus oocytes and egg extracts, Emi2 lacking the RL tail fails to interact with and inhibit the APC/C. The RL tail itself can directly bind to the APC/C, and, when added to egg extracts, either an excess of RL tail peptides or anti-RL tail peptide antibody can dissociate endogenous Emi2 from the APC/C, thus allowing APC/C activation. Furthermore, and importantly, the RL tail-mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C. Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail. We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.

Figure 1.

Requirement of the C-terminal tail for Emi2 activity during the MI/MII transition and Meta-II arrest. (A) Domain organization of Xenopus Emi2 protein. Plk1, CaMKII, and p90rsk phosphorylate Ser or Thr residues (dotted) in the N-terminal region of Emi2 and regulate Emi2 stability and activity. The D-box (DB) and the ZBR in the C-terminal region serve to inhibit the APC/C, whereas the function of the C-terminal (CT) tail is not known. (B) Conservation of the C-terminal amino acid sequence in Emi2 proteins from various vertebrate species. (C) Immature oocytes were injected with Emi2 MO together with or without 300 pg of full-length 3′UTR-containing and MO-resistant mRNA encoding the indicated (Myc-)Emi2 constructs, cultured overnight, treated with progesterone, and subjected after GVBD to immunoblotting (IB) for the indicated proteins (for Emi2, oocyte extracts were treated with λ phosphatase before immunoblotting). Oocytes were also photographed 3 h after GVBD. Asterisk, background protein; exo, exogenous (Myc-)Emi2; endo, endogenous Emi2. (D) CSF extracts were incubated with or without 20 ng/μl mRNA encoding the indicated (Myc-)Emi2 constructs for 1 h, treated with cycloheximide for 5 min, further treated with calcium (CaCl₂), and then subjected to immunoblotting for the indicated proteins. Four independent experiments were performed for both C and D, and, for each, a typical result is shown.

Figure 2.

Direct binding of Emi2 to the APC/C via the C-terminal RL tail. (A) CSF extracts were incubated with 20 ng/μl mRNA encoding the indicated (Myc-)Emi2 constructs for 2 h, subjected to immunoprecipitation (IP) either with anti-Myc antibody (left) or anti-Cdc27 antibody (right), and then immunoblotted for the indicated proteins. (B) CSF extracts were incubated with 30 ng/μl mRNA encoding the indicated (Myc-)Emi2 constructs for 1 h and subjected to Myc immunoprecipitation followed by Cdc27 immunoblotting. (C) CSF extracts were incubated with uncoupled control beads or beads coupled with Emi2 C-terminal tail peptides (residues 638-651 with or without an RL→AA mutation); the beads were then pulled down (PD) and subjected to immunoblotting for Cdc27 and Cdc23. (D) APC/C complexes immunopurified from CSF extracts were incubated with beads coupled with Emi2 C-terminal tail peptides and processed as in C. α-Tubulin served as a control of APC/C purification. (E) CSF extracts were added with buffer (as control) or an excess (2 mM) of C-terminal tail peptides and subjected either to immunoblotting for the indicated proteins at the indicated times (left) or to Emi2 immunoprecipitation followed by Cdc27 immunoblotting 5 min after the peptide addition (right). (F) CSF extracts were incubated with buffer, control rabbit IgG, or antibody against the C-terminal tail peptide (α-Emi2-RL; 40 ng/μl), and subjected to immunoblotting for the indicated proteins (left). CSF extracts were also incubated with control IgG, α-Emi2-RL, or anti-Emi2 (N-terminus) antibody (α-Emi2(N)) for 30 min and subjected to immunoprecipitation (with the respective antibodies) followed by Emi2 or Cdc27 immunoblotting (right). At least three independent experiments were performed for A–F, and for each a typical result is shown.

Figure 3.

Binding of the Emi2 RL motif to the APC/C at a site(s) distinct from that occupied by the Cdc20 IR domain. (A) CSF extracts were incubated with an excess (2 mM) of the indicated C-terminal RL motif peptides for 5 min and subjected to Cdc27 immunoprecipitation followed by Emi2 or Cdc20 immunoblotting. (B) CSF extracts were treated with calcium and then subjected to either control or Cdc27 immunoprecipitation followed by immunoblotting for the indicated proteins. On calcium treatment, Emi2 underwent hyperphosphorylation and degradation, as previously shown (Rauh et al., 2005). (C) CSF extracts were subjected to either control or Emi2 immunoprecipitation followed by immunoblotting for Cdc27 and Cdc20. The anti-Emi2 antibody used was anti-Emi2 (N-terminus) antibody, not anti-RL motif peptide antibody (which could not precipitate Cdc27 or Cdc20; data not shown, but see Figure 2F). Four, three, and four independent experiments were performed for A–C, respectively, and, for each a typical result is shown.

Figure 4.

Comparison of the binding affinities of the D-box, the ZBR, and the RL tail to the APC/C. (A) Schematic representation of various Emi2-C constructs. Each Emi2-C construct is N-terminally tagged with three Myc-epitopes. Asterisk, Ala mutation of the essential residue(s) in the indicated motifs (see text for details). (B and C) CSF extracts were incubated with 35 ng/μl mRNA encoding the indicated (Myc-)Emi2-C constructs for 1 h and subjected to Myc immunoprecipitation followed by Cdc27 immunoblotting (top). The levels of coprecipitated Cdc27 were quantitated and normalized to (Myc-)Emi2-C proteins; the value obtained for Cdc27 coprecipitated with WT Emi2-C was set at 100, and all values are means ± SD of five independent experiments (bottom).

Figure 5.

Binding of Emi1 to the APC/C via the RL tail. (A) Conservation of the C-terminal amino acid sequence in Emi1 proteins from various species. Emi2 (Xenopus) also has conserved amino acids (in red) at the corresponding sites of Emi1 (dotted residues are those essential for APC/C binding). (B) CSF extracts were incubated with 35 ng/μl mRNA encoding the indicated Xenopus (Myc-)Emi1 constructs (proteolysis-resistant forms; Ohsumi et al., 2004) for 2 h and subjected to Myc immunoprecipitation followed by Cdc27 immunoblotting. (C) CSF extracts were incubated with uncoupled control beads or beads coupled with the indicated Xenopus Emi1 C-terminal tail peptides (residues 379-392); the beads were then pulled down and subjected to immunoblotting for Cdc27. (D) CSF extracts were added with buffer (as control) or an excess (2 mM) of the indicated Xenopus Emi1 C-terminal peptides and subjected either to immunoblotting for the indicated proteins at the indicated times (top) or to Emi2 immunoprecipitation followed by Cdc27 immunoblotting 5 min after the peptide addition (bottom). (E) HEK293 cells were transfected with cDNA encoding the indicated human (Myc-)Emi1 constructs, cultured for 48 h, and subjected to Myc immunoprecipitation followed by immunoblotting for the indicated proteins (α-tubulin being a loading and IP control). (F) HEK293 cells transfected as in E were subjected to immunoblotting for the indicated proteins (left). The levels of geminin and cyclin A were quantitated and normalized to the level of α-tubulin; the value obtained for nontransfected cells was set at 1, and all values are means ± SD of five independent experiments (middle and right). At least three independent experiments were performed for B–E, and, for each a typical result is shown.

Figure 6.

A two-step model for the inhibition of the APC/C by Emi1/Emi2. Emi1/Emi2 inhibit the APC/C by two steps: (1) docking onto the APC/C via the RL tail, and (2) interaction and inhibition of the APC/C by the D-box and the ZBR.