Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset. Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset. It is critical that cells segregate their chromosomes perfectly in every mitosis. Segregation errors result in aneuploidy, which typically leads to disease or cell death (1Hassold T. Hunt P. Nat. Rev. Genet. 2001; 2: 280-291Crossref PubMed Scopus (1744) Google Scholar). To ensure that their genomes are replicated, repaired, and segregated with high fidelity, cells have evolved checkpoint controls. The spindle assembly checkpoint (SAC) 5The abbreviations used are:SACspindle assembly checkpointAPC/Canaphase-promoting complex/cyclosomeMCCmitotic checkpoint complexMadmitotic arrest-defectiveTAPtandem affinity purificationGFPgreen fluorescent proteinHTBHis6-TEV-biotinTEVtobacco etch virusPIPES1,4-piperazinediethanesulfonic acidHAhemagglutininDAPI4′,6-diamidino-2-phenylindole. 5The abbreviations used are:SACspindle assembly checkpointAPC/Canaphase-promoting complex/cyclosomeMCCmitotic checkpoint complexMadmitotic arrest-defectiveTAPtandem affinity purificationGFPgreen fluorescent proteinHTBHis6-TEV-biotinTEVtobacco etch virusPIPES1,4-piperazinediethanesulfonic acidHAhemagglutininDAPI4′,6-diamidino-2-phenylindole. delays anaphase onset until all sister chromatid pairs are bioriented on the mitotic spindle (2Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell. Biol. 2007; 8: 379-393Crossref PubMed Scopus (1712) Google Scholar, 3Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 4Taylor S.S. Scott M.I. Holland A.J. Chromosome Res. 2004; 12: 599-616Crossref PubMed Scopus (124) Google Scholar). The SAC works by preventing activation of the ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C (5Peters J.M. Nat. Rev. Mol. Cell. Biol. 2006; 7: 644-656Crossref PubMed Scopus (1003) Google Scholar)). In order for the APC/C to become active, it must interact with a transiently available activator; early in mitosis, this is Slp1/Cdc20, and later in mitosis and in G1, it switches to Ste9/Cdh1 (6Visintin R. Prinz S. Amon A. Science. 1997; 278: 460-463Crossref PubMed Scopus (713) Google Scholar, 7Kitamura K. Maekawa H. Shimoda C. Mol. Biol. Cell. 1998; 9: 1065-1080Crossref PubMed Scopus (94) Google Scholar, 8Kominami K. Seth-Smith H. Toda T. EMBO J. 1998; 17: 5388-5399Crossref PubMed Scopus (87) Google Scholar). spindle assembly checkpoint anaphase-promoting complex/cyclosome mitotic checkpoint complex mitotic arrest-defective tandem affinity purification green fluorescent protein His6-TEV-biotin tobacco etch virus 1,4-piperazinediethanesulfonic acid hemagglutinin 4′,6-diamidino-2-phenylindole. spindle assembly checkpoint anaphase-promoting complex/cyclosome mitotic checkpoint complex mitotic arrest-defective tandem affinity purification green fluorescent protein His6-TEV-biotin tobacco etch virus 1,4-piperazinediethanesulfonic acid hemagglutinin 4′,6-diamidino-2-phenylindole. Cdc20 is the key target of the SAC (9Kim S.H. Lin D.P. Matsumoto S. Kitazono A. Matsumoto T. Science. 1998; 279: 1045-1047Crossref PubMed Scopus (318) Google Scholar, 10Hwang L.H. Lau L.F. Smith D.L. Mistrot C.A. Hardwick K.G. Hwang E.S. Amon A. Murray A.W. Science. 1998; 279: 1041-1044Crossref PubMed Scopus (461) Google Scholar). The Mad2 and Mad3/BubR1 checkpoint proteins bind to and inhibit Cdc20, but their molecular mechanism(s) of inhibition remain incompletely understood. They could act by simply sequestering the APC/C activator or by otherwise preventing processive polyubiquitination of mitotic APC/C substrates. The APC/C substrates that must be polyubiquitinated and destroyed for mitosis to be completed are securin (Cut2/Pds1) and cyclin (Cdc13/Clb2) (11Cohen-Fix O. Genes Dev. 1996; 10: 3081-3093Crossref PubMed Scopus (677) Google Scholar, 12Shirayama M. Toth A. Galova M. Nasmyth K. Nature. 1999; 402: 203-207Crossref PubMed Scopus (293) Google Scholar, 13Thornton B.R. Toczyski D.P. Nat. Cell Biol. 2003; 5: 1090-1094Crossref PubMed Scopus (149) Google Scholar). APC/C substrates contain destruction signals, usually referred to as destruction (D) boxes, and KEN boxes (14Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1890) Google Scholar, 15Pfleger C.M. Kirschner M.W. Genes Dev. 2000; 14: 655-665Crossref PubMed Google Scholar). These signals are recognized by APC/C activators (16Burton J.L. Solomon M.J. Genes Dev. 2001; 15: 2381-2395Crossref PubMed Scopus (148) Google Scholar, 17Hilioti Z. Chung Y.S. Mochizuki Y. Hardy C.F. Cohen-Fix O. Curr. Biol. 2001; 11: 1347-1352Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Burton J.L. Tsakraklides V. Solomon M.J. Mol. Cell. 2005; 18: 533-542Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 19Kraft C. Vodermaier H.C. Maurer-Stroh S. Eisenhaber F. Peters J.M. Mol. Cell. 2005; 18: 543-553Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), but it has also been shown that the vertebrate APC/C is capable of recognizing destruction motifs directly, in a Cdc20/Cdh1-independent manner (20Yamano H. Gannon J. Mahbubani H. Hunt T. Mol. Cell. 2004; 13: 137-147Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Current models of SAC mechanism highlight the role of Mad2 in Cdc20 inhibition. A stable kinetochore-bound Mad1-Mad2 complex recruits additional Mad2 molecules, which then become primed for Cdc20 binding and inhibition (21De Antoni A. Pearson C.G. Cimini D. Canman J.C. Sala V. Nezi L. Mapelli M. Sironi L. Faretta M. Salmon E.D. Musacchio A. Curr. Biol. 2005; 15: 214-225Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 22Nasmyth K. Cell. 2005; 120: 739-746Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 23Yu H. J. Cell Biol. 2006; 173: 153-157Crossref PubMed Scopus (89) Google Scholar, 24Mapelli M. Massimiliano L. Santaguida S. Musacchio A. Cell. 2007; 131: 730-743Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). However, it is clear that the Mad3/BubR1 spindle checkpoint component also has a central role to play in Cdc20 inhibition (25Hardwick K.G. Johnston R.C. Smith D.L. Murray A.W. J. Cell Biol. 2000; 148: 871-882Crossref PubMed Scopus (211) Google Scholar, 26Sudakin V. Chan G.K. Yen T.J. J. Cell Biol. 2001; 154: 925-936Crossref PubMed Scopus (659) Google Scholar, 27Tang Z. Bharadwaj R. Li B. Yu H. Dev. Cell. 2001; 1: 227-237Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 28Millband D.N. Hardwick K.G. Mol. Cell. Biol. 2002; 22: 2728-2742Crossref PubMed Scopus (114) Google Scholar, 29Mao Y. Abrieu A. Cleveland D.W. Cell. 2003; 114: 87-98Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In fission yeast, Mad3 is the only checkpoint protein required for the overexpression of Mad2 to induce a metaphase arrest (28Millband D.N. Hardwick K.G. Mol. Cell. Biol. 2002; 22: 2728-2742Crossref PubMed Scopus (114) Google Scholar), indicating that a Mad2-Cdc20 complex is insufficient for checkpoint arrest in vivo. In addition, it has been shown that the mitotic checkpoint complex (MCC, typically Mad3/BubR1-Bub3-Mad2-Cdc20) is a far more potent inhibitor than Mad2 in vitro (26Sudakin V. Chan G.K. Yen T.J. J. Cell Biol. 2001; 154: 925-936Crossref PubMed Scopus (659) Google Scholar, 30Fang G. Mol. Biol. Cell. 2002; 13: 755-766Crossref PubMed Scopus (255) Google Scholar). Here we show that both Mad3 and Mad2 can stably interact with the APC/C during fission yeast mitosis. The Mad3-APC/C interaction is dependent on Cdc20 and Mad2. Mutation of an N-terminal Mad3 KEN box prevents stable Mad3-Cdc20 binding, as well as Mad3 and Mad2 binding to APC/C, and abrogates the spindle checkpoint. We demonstrate that the N-terminal Mad3 KEN box, which is conserved from yeast Mad3 to human BubR1, is a critical link required for the concerted actions of Mad3 and Mad2 in checkpoint inhibition of Cdc20-APC/C. Mitotic Arrests—Yeast strains are listed in Table 1. nda3-KM311(cs) cells in to a of and then to for the cells on and cells in and with and overexpression in Schizosaccharomyces with for then with the lacking and and for Mad2 and or in a The used has all of the with as to and cells in metaphase it is H. C. Gannon J. Hunt T. EMBO J. 1998; 17: PubMed Scopus (94) Google Scholar). is the box of the cyclin and as a substrates as are in an of to their of for used for these of of cells in of and of and and with of for and to a of A with or and tandem affinity purification to for and and proteins from the by for with by on on Mad2 its with His6-TEV-biotin C. H. B. L. P. 2006; PubMed Scopus Google The of and in and by by and on an of by and as A. L. C. Y. Z. T. Mol. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). tandem the S. protein the and then by and the of The for for for and and for of all by cells in with a and of and by for of then on by for and by of an with and in and cell with then with in for and with for by used for Mitotic an a and cells for the of protein during mitosis, of S. the H. C. Gannon J. Hunt T. EMBO J. 1998; 17: PubMed Scopus (94) Google Scholar). A and and the on to the to a of to protein or and and for by cells with in S. APC/C with Mad2 and of the APC/C from S. A. J.L. A.J. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). the we APC/C from cells in the The has and with chromosomes to spindle checkpoint activation Y. Toda T. M. Cell. Full Text PDF PubMed Scopus Google Scholar). APC/C A. J.L. A.J. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google with with the Cdc20 activator Strikingly, this mitotic APC/C also the Mad2 and Mad3 spindle checkpoint In a Cdc20, and all APC/C in a from or by proteins in these These which by and that both Mad2 and Mad3 can stably interact with the APC/C in S. The stable we by and to the mechanism by which Mad2 and Mad3 interact with the APC/C in S. Mad3 and Mad2 with the APC/C also that Mad3 and Mad2 are stably with the APC/C in an arrest A. L. C. Y. Z. T. Mol. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). in a to and the of securin and cyclin C. G. M. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). These cells are to and of the SAC on kinetochores in cells that Mad3 and Mad2 associate with the APC/C in a mitotic arrest that is of spindle checkpoint by cells in mitosis overexpression of the N-terminal of S. cyclin that with APC/C substrates H. C. Gannon J. Hunt T. EMBO J. 1998; 17: PubMed Scopus (94) Google Scholar), and cells in metaphase of spindle checkpoint We could both Mad2 and Mad3 to APC/C in mitotic delays and mutation abolishes checkpoint are strains metaphase overexpression of the of cyclin and by mitotic spindle from mitotic by and then for and Mad2. the strains and and then for and Mad2. that the mutation Mad2-Cdc20 strains and metaphase overexpression of the of cyclin The mitotic by mitotic spindle with from mitotic by and then for and Mad2. The are where and of the for from checkpoint and for and Mad2. The mitotic by mitotic spindle To the complex and during every cell we used the to in then from the and Mad2 and we for Cdc20 and APC/C proteins by Mad2 to associate with the APC/C during mitosis during an in a Mad2 until the of of from of and to with To of the APC/C with checkpoint we from and Mad2 its with and the of with the and in the We that to of the APC/C with Mad2 the of checkpoint proteins with APC/C in mitosis and are with of the APC/C by Mad2. Cdc20, and Mad2 the Mad3-APC/C is the Mad3-APC/C interaction Mad3 interaction with the APC/C to be dependent on The in a the protein is to bind the APC/C T. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar). In a from this and Mad2 all by A. L. C. Y. Z. T. Mol. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). these from cells both Mad3 and but the from mitotic cells We that stable checkpoint protein with the mitotic APC/C are Cdc20 destruction (D) boxes and KEN boxes of APC/C substrates. of Mad3 from two conserved KEN boxes To their role in an interaction with we in which the KEN boxes by a and The proteins are and can be to kinetochores and mutation of these does to in of the Mad3 protein or its The in mitosis with and the proteins by The and also association with the APC/C, the and the KEN box APC/C and association is dependent on the conserved N-terminal KEN box that is in Mad3 and Mad2 interaction with the APC/C is by interaction with Cdc20 (9Kim S.H. Lin D.P. Matsumoto S. Kitazono A. Matsumoto T. Science. 1998; 279: 1045-1047Crossref PubMed Scopus (318) Google Scholar, 10Hwang L.H. Lau L.F. Smith D.L. Mistrot C.A. Hardwick K.G. Hwang E.S. Amon A. Murray A.W. Science. 1998; 279: 1041-1044Crossref PubMed Scopus (461) Google Scholar). To Mad3 requires Mad2 to stably bind the APC/C, we in cells that in mitosis, and for Mad3-APC/C binding Mad3 is dependent on its N-terminal KEN box, and Cdc20 for APC/C Mad2 is also dependent on the N-terminal Mad3 KEN box for stable APC/C association Mad2 and Mad3 are for stable APC/C Cdc20 and is of the which in fission yeast is of and In is also a component of the However, S. Mad3 the that is and for Mad3 to bind J. S. A. 2007; PubMed Scopus Google Scholar). is for the of Mad3 (28Millband D.N. Hardwick K.G. Mol. Cell. Biol. 2002; 22: 2728-2742Crossref PubMed Scopus (114) Google Scholar, V. R. Hardwick K.G. Mol. Cell. 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