Mad3 KEN Boxes Mediate both Cdc20 and Mad3 Turnover, and Are Critical for the Spindle Checkpoint

Emma M.J King,Sjaak J.A Van Der Sar,Kevin G Hardwick,Sue Biggins

doi:10.1371/journal.pone.0000342

Emma M.J King, Sjaak J.A Van Der Sar + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0000342

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Abstract

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/CCdc20 is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C Cdc20 inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble ‘degron’ signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.

Highlights

During mitosis and meiosis cells segregate their replicated genomes to daughter nuclei [1]
Are these proteins required for normal spindle checkpoint function [28,29,30], but in fission yeast we have shown that Mad3p is even required for the overexpression of Mad2p to induce a metaphase arrest [31]
Mutation of the N-terminal Mad3p KEN box completely prevents Cdc20p binding. This mutation blocks MCC formation, dramatically reduces Cdc20p turnover in mitosis, and has a checkpoint null phenotype. We propose that this N-terminal KEN box, which is conserved from yeast Mad3p to human BubR1, is a critical link required for MCC formation and spindle checkpoint inhibition of Cdc20p

Summary

INTRODUCTION

During mitosis and meiosis cells segregate their replicated genomes to daughter nuclei [1]. One thing lacking from these models is a clear role for the Mad3p/BubR1 checkpoint component Are these proteins required for normal spindle checkpoint function [28,29,30], but in fission yeast we have shown that Mad3p is even required for the overexpression of Mad2p to induce a metaphase arrest [31]. Mutation of the N-terminal Mad3p KEN box (mad3-KEN30AAA) completely prevents Cdc20p binding This mutation blocks MCC formation, dramatically reduces Cdc20p turnover in mitosis, and has a checkpoint null phenotype. We propose that this N-terminal KEN box, which is conserved from yeast Mad3p to human BubR1, is a critical link required for MCC formation and spindle checkpoint inhibition of Cdc20p. We analyse the effects of Mad3p overproduction and observe chromosome bi-orientation defects and significant chromosome loss during the recovery from checkpoint arrest

RESULTS

DISCUSSION

MATERIALS AND METHODS