Figure 6.
Regulation of the MCC. (A) MCC is present and active in interphase HeLa cells. hBUBR1 complex was purified from HeLa cells that were arrested at the G1/S boundary by a double thymidine block. The hBUBR1 and APC/C inhibitor profiles (tested by APC/C partially purified on gel filtration column) from the final Superose 6 column are shown. (B) Only APC/C from mitotic cells is sensitive to inhibition by MCC. APC/C purified from mitotic (M) and interphase (I) cells were incubated in the absence and presence of the MCC that was purified from interphase HeLa cells. Relative APC/C activity denotes the ratio of the ubiquitin ligase activity in reactions performed in the presence and absence of MCC. (C) hBUBR1 binds preferentially APC/C that contains hyperphosphorylated CDC27. Mitotic HeLa lysates were fractionated through a Superose 6 column as described and the portion containing intact APC was immunoprecipitated with hBUBR1 antibodies. The immunoprecipitate (IP) and remaining supernatant (Sup) were separated on SDS-PAGE and probed with hBUBR1, CDC27, CDC16, and APC7 antibodies.