Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2

By V. Sudakin, G. Chan, T. Yen. Journal of Cell Biology, 2001

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The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

Subfields: Cell Biology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/189ab612fab536be37e006258a8f5c49fbc964ad

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Citations in the corpus

• The spindle assembly checkpoint. (2012, background): “Despite discovery of the MCC [18] and the demonstration that BubR1 potentiates Mad2-mediated inhibition of the APC/C in vitro [20], the view that Mad2 was the ultimate downstream APC/C inhibitor remained.”
• Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation (2001, background): “However, this reduction in tension could contribute to checkpoint activation by other mechanisms, such as those that regulate BubR1 inhibition of Cdc20 activation of the APC/C (Skoufias et al., 2001; Sudakin et al., 2001; Tang et al., 2001).”
• The spindle checkpoint: structural insights into dynamic signalling (2002, background): “The other checkpoint protein that has been shown to inhibit Cdc20–APC function directly in vitro is BubR1 (Ref”
• Riding the DUBway: regulation of protein trafficking by deubiquitylating enzymes (2006, neutral): “The seven known human Noxs include Nox1–5 and Duox1–2, with Nox2 (gp91”
• The spindle-assembly checkpoint in space and time (2007, background): “A mitotic checkpoint complex (MCC) that contains three SAC proteins, MAD2, BUBR1/Mad3 and BUB3, as well as CDC20 itsel”
• Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1 (2010, background): “Together with other components, these proteins form an inhibitory complex at the kinetochore that prevents the progression of the cell cycle (32).”