Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint.
Inappropriate attachment/tension between chromosomal kinetochores and the kinetochore microtubules activates the spindle assembly checkpoint, which delays anaphase by blocking the ubiquitin-mediated degradation of securin/Pds1p by APCCdc20. The checkpoint proteins Mad2 and Mad3/BubR1 bind to Cdc20, although how they inhibit APCCdc20 is unclear. We investigated the roles of two evolutionarily conserved KEN boxes and a D box within Mad3/BubR1. Although such motifs usually mediate APC-substrate recognition and ubiquitination, they have no apparent role in Mad3p turnover in Saccharomyces cerevisiae. Instead, these motifs are important for Mad3p function in the checkpoint and for binding to Cdc20p. We show that the Mad3p D box and KEN boxes function together to mediate Cdc20p-Mad3p interaction and that Mad3p and an anaphase-promoting complex (APC) substrate, Hsl1p, compete for Cdc20p binding in a D-box- and KEN-box-dependent manner. In vivo, we observed an increased binding of Cdc20p to Mad3p and decreased binding to Hsl1p upon checkpoint activation. Furthermore, we demonstrate that Mad2p stimulates the association between Mad3p and Cdc20p and that this stimulated binding requires KEN box 1 within Mad3p. These findings implicate Mad3p as a pseudosubstrate inhibitor of APCCdc20, competing with APC substrates for Cdc20p binding. We present a model aimed at unifying previous analyses of checkpoint function by focusing on the Mad3-Cdc20 interaction.
Burton
Janet L
JL
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Solomon
Mark J
MJ
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D001665:Binding Sites; D001667:Binding, Competitive; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D064246:Mad2 Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
C577495:CDC20 protein, S cerevisiae; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577695:MAD2 protein, S cerevisiae; C406764:MAD3 protein, S cerevisiae; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011494:Protein Kinases; C106494:HSL1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases