IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ fraschini-2001-1 Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Fraschini R., Beretta A., Sironi L., Musacchio A., Lucchini G., Piatti S. simonetta.piatti@unimib.it EMBO J. (0261-4189) 2001 Only protein-protein interactions imex curation fraschini-2001-2 Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Fraschini R., Beretta A., Sironi L., Musacchio A., Lucchini G., Piatti S. Superose 6 column simonetta.piatti@unimib.it EMBO J. (0261-4189) 2001 Only protein-protein interactions imex curation bub3_yeast Cell cycle arrest protein BUB3 OR26.16 YOR026W BUB3 protein protein yeast Saccharomyces cerevisiae Baker's yeast MQIVQIEQAPKDYISDIKIIPSKSLLLITSWDGSLTVYKFDIQAKNVDLLQSLRYKHPLLCCNFIDNTDLQIYVGTVQGEILKVDLIGSPSFQALTNNEANLGICRICKYGDDKLIAASWDGLIEVIDPRNYGDGVIAVKNLNSNNTKVKNKIFTMDTNSSRLIVGMNNSQVQWFRLPLCEDDNGTIEESGLKYQIRDVALLPKEQEGYACSSIDGRVAVEFFDDQGDDYNSSKRFAFRCHRLNLKDTNLAYPVNSIEFSPRHKFLYTAGSDGIISCWNLQTRKKIKNFAKFNEDSVVKIACSDNILCLATSDDTFKTNAAIDQTIELNASSIYIIFDYEN 0BDFB8697935BCEB cdc20_yeast APC/C activator protein CDC20 YGL116W CDC20 Cell division control protein 20 protein protein yeast Saccharomyces cerevisiae Baker's yeast MPESSRDKGNAAISGNRSVLSIASPTKLNILSSDWSRNQGKVSKNSLKRSSSLNIRNSKRPSLQASANSIYSRPKITIGAPPLIRRDSSFFKDEFDAKKDKATFSAYSSRSYPTIGSESVVSQTSLSQPTTSREVDEQFTVAADRYIPILQGASQNKVDPETLHEALPPPNASPISHLRAQTKIVFKQNVAEACGLDMNKRILQYMPEPPKCSSLRQKSYIMKKRTHYSYQQEQKIPDLIKLRKINTNPERILDAPGFQDDFYLNLLSWSKKNVLAIALDTALYLWNATTGDVSLLTDFENTTICSVTWSDDDCHISIGKEDGNTEIWDVETMSLIRTMRSGLGVRIGSLSWLDTLIATGSRSGEIQINDVRIKQHIVSTWAEHTGEVCGLSYKSDGLQLASGGNDNTVMIWDTRTSLPQFSKKTHTAAVKALSWCPYSPNILASGGGQTDKHIHFWNSITGARVGSINTGSQVSSLHWGQSHTSTNGGMMNKEIVATGGNPENAISVYNYETKFKVAEVVHAHEARICCSQLSPDGTTLATVGGDENLKFYKIFDPRCTGRSREDGLMDGMLGLIGKEGCRTNDKENRSKNSSEIHTRRPSSTSQYLIR CCE7CD149C1F5ACF mad2_yeast Mitotic spindle checkpoint component MAD2 MAD2 J1256 YJL030W protein protein yeast Saccharomyces cerevisiae Baker's yeast MSQSISLKGSTRTVTEFFEYSINSILYQRGVYPAEDFVTVKKYDLTLLKTHDDELKDYIRKILLQVHRWLLGGKCNQLVLCIVDKDEGEVVERWSFNVQHISGNSNGQDDVVDLNTTQSQIRALIRQITSSVTFLPELTKEGGYTFTVLAYTDADAKVPLEWADSNSKEIPDGEVVQFKTFSTNDHKVGAQVSYKY mad1_yeast Spindle assembly checkpoint component MAD1 MAD1 Mitotic arrest deficient protein 1 YGL086W protein protein yeast Saccharomyces cerevisiae Baker's yeast MDVRAALQCFFSALSGRFTGKKLGLEIYSIQYKMSNSGGSSPFLESPGGSPDVGSTNGQSNRQIQALQFKLNTLQNEYEIEKLQLQKQTNILEKKYKATIDELEKALNDTKYLYESNDKLEQELKSLKERSANSMNDKDKCIEELRTTLQNKDLEMETLRQQYDSKLSKVTNQCDHFKLEAESSHSLLMKYEKEIKRQSVDIKDLQHQVMEKDDELSSVKASKMINSHPNYSTEEFNELTEMNKMIQDQVQYTKELELANMQQANELKKLKQSQDTSTFWKLENEKLQNKLSQLHVLESQYENLQLENIDLKSKLTKWEIYNDSDDDDDNNVNNNDNNNNNKNDNNNDNNNDTSNNNNINNNNRTKNNIRNNPEEIIRDWKLTKKECLILTDMNDKLRLDNNNLKLLNDEMALERNQILDLNKNYENNIVNLKRLNHELEQQKSLSFEECRLLREQLDGLYSAQNNALLEVENSETHASNKNVNEDMNNLIDTYKNKTEDLTNELKKLNDQLLSNSNDVETQRKKRKLTSDQIGLNYSQRLNELQLENVSVSRELSKAQTTIQLLQEKLEKLTKLKEKKIRILQLRDGPFIKDQFIKKNKLLLLEKENADLLNELKKNNPAVETVPISVYDSLNFELKQFEQEVFKSNKRFSRLKQVFNNKSLEFIDVVNSLLGFKLEFQQDSRVKIFSCFKPEKYLIADLNENTLKSNLDADIEGWDDLMNLWVEDRGQLPCFLATITLRLWEQRQAK mad3_yeast Spindle assembly checkpoint component MAD3 MAD3 YJL013C J1341 Mitotic MAD3 protein protein protein yeast Saccharomyces cerevisiae Baker's yeast MKAYAKKRISYMPSSPSQNVINFEEIETQKENILPLKEGRSAAALSKAIHQPLVEINQVKSSFEQRLIDELPALSDPITLYLEYIKWLNNAYPQGGNSKQSGMLTLLERCLSHLKDLERYRNDVRFLKIWFWYIELFTRNSFMESRDIFMYMLRNGIGSELASFYEEFTNLLIQKEKFQYAVKILQLGIKNKARPNKVLEDRLNHLLRELGENNIQLGNEISMDSLESTVLGKTRSEFVNRLELANQNGTSSDVNLTKNNVFVDGEESDVELFETPNRGVYRDGWENFDLKAERNKENNLRISLLEANTNLGELKQHEMLSQKKRPYDEKLPIFRDSIGRSDPVYQMINTKDQKPEKIDCNFKLIYCEDEESKGGRLEFSLEEVLAISRNVYKRVRTNRKHPREANLGQEESANQKEAEAQSKRPKISRKALVSKSLTPSNQGRMFSGEEYINCPMTPKGRSTETSDIISAVKPRQLTPILEMRESNSFSQSKNSEIISDDDKSSSSFISYPPQR 36550249D81BB6D1 bub1_yeast Checkpoint serine/threonine-protein kinase BUB1 YGR188C G7542 BUB1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MNLDLGSTVRGYESDKDTFPQSKGVSSSQKEQHSQLNQTKIAYEQRLLNDLEDMDDPLDLFLDYMIWISTSYIEVDSESGQEVLRSTMERCLIYIQDMETYRNDPRFLKIWIWYINLFLSNNFHESENTFKYMFNKGIGTKLSLFYEEFSKLLENAQFFLEAKVLLELGAENNCRPYNRLLRSLSNYEDRLREMNIVENQNSVPDSRERLKGRLIYRTAPFFIRKFLTSSLMTDDKENRANLNSNVGVGKSAPNVYQDSIVVADFKSETERLNLNSSKQPSNQRLKNGNKKTSIYADQKQSNNPVYKLINTPGRKPERIVFNFNLIYPENDEEFNTEEILAMIKGLYKVQRRGKKHTEDYTSDKNRKKRKLDVLVERRQDLPSSQPPVVPKSTRIEVFKDDDNPSQSTHHKNTQVQVQTTTSILPLKPVVDGNLAHETPVKPSLTSNASRSPTVTAFSKDAINEVFSMFNQHYSTPGALLDGDDTTTSKFNVFENFTQEFTAKNIEDLTEVKDPKQETVSQQTTSTNETNDRYERLSNSSTRPEKADYMTPIKETTETDVVPIIQTPKEQIRTEDKKSGDNTETQTQLTSTTIQSSPFLTQPEPQAEKLLQTAEHSEKSKEHYPTIIPPFTKIKNQPPVIIENPLSNNLRAKFLSEISPPLFQYNTFYNYNQELKMSSLLKKIHRVSRNENKNPIVDFKKTGDLYCIRGELGEGGYATVYLAESSQGHLRALKVEKPASVWEYYIMSQVEFRLRKSTILKSIINASALHLFLDESYLVLNYASQGTVLDLINLQREKAIDGNGIMDEYLCMFITVELMKVLEKIHEVGIIHGDLKPDNCMIRLEKPGEPLGAHYMRNGEDGWENKGIYLIDFGRSFDMTLLPPGTKFKSNWKADQQDCWEMRAGKPWSYEADYYGLAGVIHSMLFGKFIETIQLQNGRCKLKNPFKRYWKKEIWGVIFDLLLNSGQASNQALPMTEKIVEIRNLIESHLEQHAENHLRNVILSIEEELSHFQYKGKPSRRF 6D76FC980775D3F9 bub3-cdc20-1 Interaction between Bub3 and Cdc20 identified by coimmunoprecipitation 653761 N/A 653763 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653764 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false Figure 1A, 2A, 3A The amount of the Bub3-Cdc20 complex varied during the cell cycle, peaking during G1-S transition, and did not exactly reflect the periodicity of Cdc20 protein levels. bub3-cdc20-2 Interaction between Bub3 and Cdc20 identified by coimmunoprecipitation 653761 N/A 653763 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653764 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false Figure 1A. Interaction observed in cycling cells and when cells are arrested in G2 but not when arrested in G1 bub3-cdc20-3 Interaction between Mad1, Mad2, Bub3 and Cdc20 identified by coimmunoprecipitation 653761 N/A 653764 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653765 unspecified role unspecified role prey prey N/A 653763 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653766 unspecified role unspecified role prey prey association association false false false Figure 1B, 2B. Interaction increases when cells are in G2 arrest or by Mps1 which activates the kinetochore checkpoint in the absence of spindle damage bub3-cdc20-4 Interaction between Mad2, Bub3 and Cdc20 identified by coimmunoprecipitation 653761 N/A 653765 unspecified role unspecified role prey prey N/A 653764 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653763 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false association association false false false Figure 1B. Interaction increase when cells are in G2 arrest. bub3-cdc20-5 Interaction between Mad2, Mad3, Bub3 and Cdc20 identified by coimmunoprecipitation 653761 N/A 653764 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653767 unspecified role unspecified role prey prey N/A 653765 unspecified role unspecified role prey prey N/A 653763 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P26449:p.[Trp31Gly;Trp120Gly] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false association association false false false Figure 3B, 5C bub3-cdc20-7 Interaction between Mad2, Mad3, Bub3 and Cdc20 identified by coimmunoprecipitation 653761 N/A 653767 unspecified role unspecified role prey prey N/A 653763 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P26449:p.[Trp31Gly;Trp120Gly] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false N/A 653765 unspecified role unspecified role prey prey N/A 653764 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false association association false false false Figure 5B bub3-cdc20-6 Coelution of Mad1, Mad2, Mad3, Bub3 Bub3 and Cdc20 653762 N/A 653765 unspecified role unspecified role neutral component neutral component N/A 653768 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653767 unspecified role unspecified role neutral component neutral component N/A 653763 unspecified role unspecified role neutral component neutral component region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653764 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 653766 unspecified role unspecified role neutral component neutral component physical association physical association false false false Figure 4A Authors believe this may comprise two separate complexes, both with MW = 670kDa.