title: Accumulation of Mad2-Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Nezi
authors.forename: Giulia
authors.initials: G
authors.lastname: Rancati
authors.forename: Anna
authors.initials: A
authors.lastname: De Antoni
authors.forename: Sebastiano
authors.initials: S
authors.lastname: Pasqualato
authors.forename: Simonetta
authors.initials: S
authors.lastname: Piatti
authors.forename: Andrea
authors.initials: A
authors.lastname: Musacchio
mesh_terms: D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D064246:Mad2 Proteins; D008938:Mitosis / Q000502:physiology*; D008954:Models, Biological; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: C577495:CDC20 protein, S cerevisiae; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C501382:MAD1 protein, S cerevisiae; C577695:MAD2 protein, S cerevisiae; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins