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PMID: 17406666 Published · epublish English Journal Article Research Support, Non-U.S. Gov't

Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint.

PloS one ·Vol. 2 ·No. 4 ·2007-04-04 ·Pages e342

King EM, van der Sar SJ, Hardwick KG

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/C(Cdc20) 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.

MeSH Terms
Amino Acid Sequence Binding Sites Cdc20 Proteins Cell Cycle Proteins/chemistry,metabolism,physiology Conserved Sequence Molecular Sequence Data Nuclear Proteins/chemistry,metabolism,physiology Saccharomyces cerevisiae/cytology Saccharomyces cerevisiae Proteins/chemistry,metabolism,physiology Sequence Homology, Amino Acid Spindle Apparatus
Chemicals
CDC20 protein, S cerevisiae Cdc20 Proteins Cell Cycle Proteins MAD3 protein, S cerevisiae Nuclear Proteins Saccharomyces cerevisiae Proteins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
King Emma M J
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
van der Sar Sjaak J A
Hardwick Kevin G
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Article Info
Journal
PloS one
Abbr.
PLoS One
ISSN
1932-6203
DOI
10.1371/journal.pone.0000342
Published
2007-04-04
Epub
2007-00-04
Pages
e342
Language
English
Region
United States
NLM ID
101285081
PMCID
PMC1829190
Subset
IM
Grants
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1371/journal.pone.0000342 PMC Full Text View journal details
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