Featured Article - May 2009
Short description: The molecular basis of inhibition of the anaphase-promoting complex/cyclosome revealed.Science 323, 1477-1481 (2009)
Chromosome segregation is carefully regulated to reduce defects arising from abnormal numbers of chromosomes. Cell division is halted by the spindle checkpoint until all chromosomes are connected to the mitotic or meiotic spindle, and only once they are correctly attached does the next step of the cell cycle, anaphase, begin. The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin-protein ligase, and its coactivator Cdc20 are crucial for cell-cycle progression, and now a new study in Science by Herzog et al. 1 reveals how they are inhibited by spindle checkpoint proteins.
APC/C was purified in two different states. Checkpoint-inhibited APC/C was isolated from HeLa cells trapped in an early phase of mitosis, prometaphase, by addition of the chemical taxol. An inactive form of APC/C was isolated from cells in which the checkpoint had been inactivated by the kinase inhibitor Hesperadin. Four times the amount of checkpoint proteins were associated with APC/C in cells in which the checkpoint was active than in Hesperadin-treated cells.
Further examination of the active checkpoint revealed two forms of active APC/C. One contains the mitotic checkpoint proteins BubR1, Bub3, Cdc20 and Mad2 (together known as APC/CMCC); the other lacks these additional proteins (and is known as apo-ACP/C).
The structures of APC/CMCC and apo-APC/C were examined by electron microscopy, along with that of apo-APC/C with Cdc20 bound (APC/CCdc20). The team used angular reconstitution techniques to build three-dimensional models and showed that apo-APC/C consists of two domains: the 'platform' and the 'arc lamp'. These domains exist in three different relative conformations, which probably reflects flexibility in the complex.
By analyzing APC/CMCC, the authors established that a large additional density at the 'front' of the platform domain corresponds to the proteins of the mitotic checkpoint complex and that addition of this complex locks APC/C in a closed conformation. A further three-dimensional model of apo-APC with Cdc20 bound showed a 50 kDa mass on the front of APC/C that partly overlaps the site where MCC binds to the APC/C.
These studies show that upon activation, the mitotic checkpoint proteins BubR1, Bub3 and Mad2 associate with APC/CCdc20. In biochemical experiments the authors observed that the binding of checkpoint proteins to APC/C coincides with loss of substrate binding. These findings indicate that the checkpoint proteins inhibit APC/C by preventing the recruitment of substrates. Because previous work has revealed that Cdc20 functions as a substrate adaptor, the authors speculate that binding of checkpoint proteins to Cdc20 may prevent substrates binding to the APC/CCdc20 active site.
F. Herzog et al. Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex.
Science 323, 1477-1481 (2009).