Immobilized Emi1 can bind to the APC/C^Cdh1 in an “APC/C capture” assay. (A) Recombinant Emi1 protein immobilized on Sepharose beads (see Materials and Methods) was incubated with HeLa cell extracts, and precipitates were analyzed for APC/C binding by immunoblot analysis. The approximate molecular weight at which the indicated subunit migrates is indicated in parentheses. (B) Emi1 binds to the APC/C in the absence of Cdh1. Emi1 beads were incubated with extracts made from HeLa cells transfected with siRNA targeting Cdh1 or APC3 for 48 h.

Emi1 can compete with substrate binding in D-boxand ZBR-dependent manner. (A) The normal N-terminal D-box region of cyclin B1 binds specifically to the APC/C. Recombinant N terminus of cyclin B1 (residues 1–100) or a variant containing a mutated D-box was immobilized on CNBr-activated Sepharose, incubated with cell extracts, and analyzed for APC/C binding with anti-Cdc27 antibody. (B) Emi1 competes with the ability of the cyclin B1 D-box region to bind the APC/ C. Recombinant wild-type or mutant MBP–Emi1 or MBP (0.5, 1, or 2.5 nmol) was added to 1 nmol bead-bound cyclin B1 and incubated with HeLa cell extracts as described in A. The amount of APC/C remaining bound to Cyclin B beads is indicated below, expressed as a percentage of control (no Emi1 addition) sample. Values represent mean of two experiments. (C) Cyclin B1 does not compete efficiently with Emi1 for APC/C binding. N terminus of cyclin B1 or MBP–Emi1 was added at doses of 0.5, 1.0, or 2.5 nmol to capture assay using 1 nmol of bead-bound MBP–Emi1.

Loss of IBR/ZBR function in Emi1 converts the protein from APC/C inhibitor to APC/C substrate. (A) Standard APC/C-dependent ubiquitination assays, using radiolabled, in vitro translated C-terminal Emi1 wild-type or mutant protein as substrate. Reactions were incubated for 60 min, and the formation of ubiquitinated Emi1 was visualized by autoradiography of SDS–polyacrylamide gels. (Db⁻) D-box mutant. (B) Cdh1 over-expression reduces levels of cotransfected Emi1 C401S/ZBR. HA-Cdh1 and Myc-Emi1 variants were cotransfected into HEK293T cells. Lysates were harvested and Emi1 levels detected by anti-Myc immunoblot. A mutant version of Cdh1 lacking the C-terminal IR dipeptide and the C-box (ΔCboxΔIR) was used as a control. (C) A model for evolving APC/C substrates and inhibitors. Emi1 protein binds to the APC/C in a D-box-dependent manner to inhibit APC/C activity through G2 and early mitosis, permitting cyclin accumulation. The ZBR motif of Emi1 contributes to inhibition by blocking substrate access to the APC/C, as well as providing an additional inhibitory function that prevents the ubiquitination of substrates and Emi1 itself. The combination of D-box and ZBR allows Emi1 to compete with APC/C substrates for APC/C binding. Phosphorylation of Emi1 by Cdc2 and Plk1, potentially in conjunction with other events, results in the displacement and proteolytic destruction of Emi1, allowing for APC/C activation and progression through later stages of mitosis.

Emi1 associates with the nuclear form of the APC. (A) Emi1 and the APC/C are coextracted from the high-salt fraction of HeLa cells. HeLa cells were extracted by sequential treatments of nitrogen decompression, 420 mM KCl, 0.1% Triton X-100, and sonication to produce fractions as described in Materials and Methods. Fractions were examined by immunoblot analysis for Emi1 and APC/C subunits (APC3/Cdc27, APC8). (B) Emi1, Cdh1, and APC/C cofractionate during ion exchange and gel filtration chromatography. The nuclear fraction described in A was further fractionated by anion exchange (upper panel) chromatography and subsequently separated by gel filtration (lower panel). (C) Emi1 and APC/C coprecipitate from purified fractions. The fractions indicated in B were pooled and immunoprecipitated with anti-Emi1 antibodies and resolved by SDS-PAGE and silver stained. Excised bands were identified by mass spectrometry. APC/C subunits identified by mass spectromety are indicated by arrows. (D) Anti-Emi1 antibody immunoprecipitates were also examined by immunoblot analysis for Emi1 and APC/C subunits, APC3 (Cdc27), APC5, and APC7. (E) Anti-APC3/Cdc27 efficiently coprecipitates Emi1 from nuclear fractions. Immunoprecipitates were washed with 0.1, 0.25, or 0.5 M KCl; separated by SDS-PAGE; and examined by immunoblot analysis for Cdc27 and Emi1. (F) APC/C subunits associate with expressed epitope-tagged Emi1. HeLa cells were transfected with a construct expressing epitope-tagged Myc-hEmi1 or empty vector. Cell extracts were prepared and anti-Myc precipitates were examined by immunoblot analysis for the presence of Emi1 and APC/C subunits. (G) Immunodepletion of asynchronous nuclear extract by anti-Emi1 antibody results in the removal of the majority of APC/C and Cdh1. Nuclear extracts were incubated with anti-Emi1 or control antibody. Equal amounts of input and flow-through (nonbound) samples were examined by immunoblotting.

The APC/C binds to the C-terminal APC/C inhibitory domain of Emi1 via its conserved D-box. (A) Emi1 protein schematic, key features, and variant proteins. (B) The Emi1 C-terminal domain binds to the APC/C and Cdh1. Capture of the APC/C using recombinant MBP–Emi1 N terminus (1–244) or C terminus (299–447). (C) Efficient Emi1 binding to the APC/C and Cdh1 requires the C-terminal D-box. Capture of APC/C using MBP–Emi1 D-box mutant (Db⁻), MBP–Emi1 ZBR mutant (C401S), or double mutant (Db⁻/C401S).

Emi1’s APC/C inhibitory function requires both the conserved D-box and ZBR functions. (A) APC/ C^Cdh1-dependent in vitro ubiquitination assays, using radiolabeled, in vitro translated cyclin B1 protein as a substrate, were incubated for the indicated times (0, 15, 30 min), and the formation of ubiquitinated cyclin B was visualized by autoradiography of SDS–polyacryl-amide gels. Wild-type, D-box mutant (Db⁻), ZBR mutant (C401S), and D-box, ZBR double-mutant Emi1 was added to the reaction mixture and assayed for the ability to inhibit cyclin B ubiquitination. (B) Quantitation of in vitro APC/C activity showing the amount of cyclin B remaining unconjugated at 30 min for a range of doses of Emi1 variants.