Figure 3. Initiation motifs allow negative feedback regulation of APC/C.

A The N-terminus of Ube2C contains a candidate initiation motif. B. Its initiation motif is required for ubiquitination ³⁵S-Ube2C by APC/C, E1, and ubiquitin, as seen with autoradiography. Asterisks denote ubiquitination of K119 and K121 of Ube2C, which does not require the APC/C. C. The initiation motif is required for rapid degradation of ³⁵S-Ube2C in extracts with active APC/C and low levels of APC/C-substrates. D. The initiation motif in Ube2C is not required for E2-activity, as seen in degradation assays using ³⁵S-cyclin A as reporter. E. The RING-interaction loop is the main APC/C-binding site in Ube2C. Ube2C^C114S, the initiation motif mutant Ube2C^R6A/C114S, or RING-binding deficient Ube2C^C114S/Y91D were tested for their capability to stabilize ³⁵S-securin in extracts. F. The initiation motif is not required for APC/C-binding of Ube2C in vivo. Ube2C^C114S or Ube2C^R6A/C114S were injected into Xenopus tropicalis embryos at the two-cell stage, and cell cycle arrest was scored relative to Ube2C^C114S. Error bars define the standard error derived from three independent experiments. G. APC/C-substrates inhibit Ube2C-ubiquitination through initiation motif competition. Recombinant securin^ΔDΔKEN was added to ubiquitination reactions of ³⁵S-Ube2C by APC/C and/or Ube2S, as indicated.