MAD2L2 binds to Cdh1 and not Cdc20. (A) ³⁵S-labeled in vitro-translated Cdc20 and Cdh1 prebound to APC on αCdc27 beads. (B) Binding of in vitro-translated ³⁵S-labeled xMAD2L2 to Cdh1–APC on αCdc27 beads but not to Cdc20–APC on beads, iAPC on beads, or αCdc27 beads alone.

xMAD2L2-mediated inhibition of gastrulation is rescued by coinjection with Cdh1. Vegetal views of stage 11 embryos injected (left half of each embryo) at the two-cell stage with (A) 2.5 ng MBP–MAD2L2, (B) 2.5 ng His6–Cdh1, (C) 2.5 ng MBP–MAD2L2 plus 2.5 ng His6–Cdh1, or (D) buffer. Injections of MBD–MAD2L2 result in cells that have enlarged and enhanced nuclear staining properties. One cell of two-cell stage embryos injected with 2.5 ng of MBP–MAD2L2, allowed to develop to stages 8–9, fixed, and stained with propidium iodide (E,F) or Hoechst (G). (E) Uninjected side and (F) injected side of the same embryo. (G) A view showing an injected region (left) next to an uninjected region. (H) Injections of Cdh1 RNA result in enlarged cells that give diffuse nuclear staining. One cell of two-cell embryos were injected with 500 pg of Cdh1 (left side), allowed to develop to stages 10.5, fixed, and stained with Hoechst. (I) Summary of the gastrulation arrest and rescue of the embryos shown in A–D.

MAD2L2 sequence conservation. (A) Comparison of xMAD2L2 with MAD2L2 sequences from human and mouse. Shading indicates residues in common with xMAD2L2; a bold line indicates a putative nuclear localization signal. (B) Sequence comparison of xMAD2L2 with XMAD2. Arrows indicate a conserved C-terminal lysine and valine (A,B). (C) Hoechst staining of eGFP-xMAD2L2 transfected BHK cells (left); eGFP-xMAD2L2 signal (right).

xMAD2L2 inhibits Cdh1 but not Cdc20 activity. (A) Degradation assays in interphase extracts, Cdh1-supplemented interphase extracts, and Cdh1 extracts in the presence of recombinant His–MAD2L2 for mB99, Xkid, securin, and cyc B. Phosphorylation of mB99 in interphase extracts causes a shift; mB99 samples were CIP treated to clarify the difference between phosphorylation and degradation. (B) Gel filtration profiles of recombinant MBP-MAD2L2 on a superdex 200 column using the SMART system (upper panel) and tetramer, trimer, and monomer peaks rerun over the same column (lower panel). (C) t_1/2 of Xkid in Cdh1 extracts (upper panel) or Δ90 extracts (lower panel) with increasing concentrations of MAD2^M, MAD2^O, MBP-xMAD2L2^M or MBP-xMAD2L2^O. Graph reflects typical results from one extract; t_1/2 varies between extracts, but the effect on t_1/2 is consistent. (D) Ubiquitination assays of cyc B using Cdh1-activated APC in the presence of 5 μM MAD2^O or 5 μM MBP–xMAD2L2.

MAD2 and MAD2L2 prevent release of substrate bound to Cdc20 or Cdh1, respectively. Graph of substrate binding assay showing prebound Xkid retained by Cdc20 or Cdh1 beads over time after addition of excess cold Xkid in the presence of 5 μM MAD2^O or MAD2L2, respectively, compared to buffer alone.