Cell cycle-regulated proteolysis of mitotic target proteins

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugat ing enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase t ransition and continues through G1 phase of the next cell cycle. We have us ed cell-free systems from mammalian somatic tells collected at different ce ll cycle stages (GO, G1, S, G2, and M) to investigate the regulated degrada tion of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an i nhibitor of anaphase onset identified in fission yeast). All four are degra ded by G1 extracts but not by extracts of S phase cells. Maintenance of des truction during G1 requires the activity of a PP2A-like phosphatase. Destru ction of each target is dependent on the presence of an N-terminal destruct ion box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a domina nt activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is a ctivated after chromosome alignment at the metaphase plate. In support of t his, we show that addition of dominant negative UbcH10 to GI extracts block s destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we rep ort that injection of dominant negative Ubc3/Cdc34, whose role in G1-S cont rol is well established and has been implicated in kinetochore function dur ing mitosis in yeast, dramatically interferes with congression of chromosom es to the metaphase plate. These results demonstrate that the regulated ubi quitination and destruction of critical mitotic proteins is highly conserve d from yeast to humans.