Cell cycle–regulated proteolysis of mitotic target proteins

H Bastians, LM Topper, GL Gorbsky… - Molecular biology of …, 1999 - Am Soc Cell Biol
H Bastians, LM Topper, GL Gorbsky, JV Ruderman
Molecular biology of the cell, 1999Am Soc Cell Biol
The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-
promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10),
begins around the time of the metaphase–anaphase transition and continues through G1
phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells
collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated
degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H …
The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase–anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation 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 inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C–dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1–S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.
Am Soc Cell Biol