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Published 3 July 2006. doi:10.1083/jcb.200602109
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 174, Number 1, 39-51
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Article

Accumulation of Mad2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae



Luigi Nezi1, Giulia Rancati2, Anna De Antoni1, Sebastiano Pasqualato1,3, Simonetta Piatti2, and Andrea Musacchio1,3

1 Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy
2 Department of Biotechnology and Bioscience, University of Milan-Bicocca, 20126 Milan, Italy
3 The Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology Foundation, 20139 Milan, Italy

Correspondence to Andrea Musacchio: andrea.musacchio{at}ifom-ieo-campus.it; or Simonetta Piatti: simonetta.piatti{at}unimib.it

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.

L. Nezi and G. Rancati contributed equally to this paper.

G. Rancati's present address is Stowers Institute for Medical Research, Kansas City, MO 64110.

Abbreviations used in this paper: GSH, glutathione–Sepharose; IP, immunoprecipitation; MAD, mitotic arrest deficient; SAC, spindle assembly checkpoint; SEC, size-exclusion chromatography.


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