Figure 6

The E3 responsible for Myf5 ubiquitylation is not the APC/C. (A) In vitro translated Xkid (upper panel) or Myf5 (lower panel) were incubated in 15 μl Xenopus interphase egg extract in which Cdh1 has been translated [39]. After the times indicated, 2.5 μl were analyzed by SDS-PAGE and fluorography. (B) 1 ml of mitotic extract was activated with 1 μM microcystin LR (in order to fully activate APC/C), then fractionated on a DEAE column (see Methods). Bound proteins were eluted in two steps with buffer A containing 0.25 M and 0.5 M NaCl, respectively. Each fraction (lanes 2 & 3) was compared to a control reaction containing buffer (lane 1) for its ability to mediate ubiquitylation of either cyclin B (upper panel) or Myf5 (lower panel). Lanes 4–7: the 0.25 M NaCl eluate that mediates both cyclin B and Myf5 ubiquitylation was then subjected to immunoprecipitations using either anti-Cdc27 or control antibodies. For each immunoprecipitation, both the material bound to the beads or the supernatant were analyzed for ubiquitylation activity using cyclin B (upper panel) or Myf5 (lower panel) as a substrate. (Ub)_n-cycB and (Ub)_n-Myf5 indicate poly-ubiquitylated forms of cyclin B and Myf5, respectively.