THE CYCLOSOME, A LARGE COMPLEX CONTAINING CYCLIN-SELECTIVE UBIQUITIN LIGASE ACTIVITY, TARGETS CYCLINS FOR DESTRUCTION AT THE END OF MITOSIS

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has r evealed four components required for cyclin-ubiquitin ligation and pro teolysis: a nonspecific ubiquitin-activating enzyme E(1), a soluble fr action containing a ubiquitin carrier protein activity called E(2)-C, a crude particulate fraction containing a ubiquitin ligase (E(3)) acti vity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel similar to 1500-kDa complex, termed the cyclosome, which contain s a cyclin-selective ubiquitin Ligase activity, E(3)-C. E(3)-C is pres ent but inactive during interphase; it can be activated in vitro by th e addition of cdc2, enabling the transfer of ubiquitin from E(2)-C to cyclin. The kinetics of E(3)-C activation suggest the existence of one or more intermediates between cdc2 and E(3)-C. Cyclosome-associated E (3)-C acts on both cyclin A and B, and requires the presence of wild-t ype N-terminal destruction box motifs in each cyclin. Ubiquitinated cy clins are then rapidly recognized and degraded by the proteasome. Thes e results identify the cyclosome-associated E(3)-C as the component of the cyclin destruction machinery whose activity is ultimately regulat ed by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.