Fig. 5. Both fzy and fzr can activate the APC/C to ubiquitinate securin. (A) An in vitro ubiquitination assay was performed on ³⁵S-labeled securin expressed in reticulocyte lysate (lane 1) with a partially purified APC/C from mitotic HeLa cells in the presence of bacterially expressed E1, E2-C, an energy-regenerating system and ubiquitin aldehyde, which inhibits de-ubiquitination. This reaction was supplemented with either fzy (lane 2), fzr (lane 3) or both (lane 4). (B) Some of the ubiquitinated securin must have been degraded by the proteasome in the reticulocyte lysate. We therefore repeated the ubiquitination reaction in the presence (lane 6) or absence (lane 5) of ATP-γ-S, which enables ubiquitination but not proteasome-specific degradation.