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/ Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. EMBO J. (0261-4189) 2001 imex curation Fraschini R., Beretta A., Sironi L., Musacchio A., Lucchini G., Piatti S. simonetta.piatti@unimib.it Only protein-protein interactions 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 Accepted 2007-AUG-13 by JYOTI. EMBO J. (0261-4189) 2001 Only protein-protein interactions imex curation Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. EMBO J. (0261-4189) 2001 imex curation Fraschini R., Beretta A., Sironi L., Musacchio A., Lucchini G., Piatti S. simonetta.piatti@unimib.it Only protein-protein interactions 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 Accepted 2007-AUG-13 by JYOTI. EMBO J. (0261-4189) 2001 Only protein-protein interactions imex curation cdc20_yeast APC/C activator protein CDC20 YGL116W CDC20 Cell division control protein 20 protein protein yeast Saccharomyces cerevisiae Baker's yeast MPESSRDKGNAAISGNRSVLSIASPTKLNILSSDWSRNQGKVSKNSLKRSSSLNIRNSKRPSLQASANSIYSRPKITIGAPPLIRRDSSFFKDEFDAKKDKATFSAYSSRSYPTIGSESVVSQTSLSQPTTSREVDEQFTVAADRYIPILQGASQNKVDPETLHEALPPPNASPISHLRAQTKIVFKQNVAEACGLDMNKRILQYMPEPPKCSSLRQKSYIMKKRTHYSYQQEQKIPDLIKLRKINTNPERILDAPGFQDDFYLNLLSWSKKNVLAIALDTALYLWNATTGDVSLLTDFENTTICSVTWSDDDCHISIGKEDGNTEIWDVETMSLIRTMRSGLGVRIGSLSWLDTLIATGSRSGEIQINDVRIKQHIVSTWAEHTGEVCGLSYKSDGLQLASGGNDNTVMIWDTRTSLPQFSKKTHTAAVKALSWCPYSPNILASGGGQTDKHIHFWNSITGARVGSINTGSQVSSLHWGQSHTSTNGGMMNKEIVATGGNPENAISVYNYETKFKVAEVVHAHEARICCSQLSPDGTTLATVGGDENLKFYKIFDPRCTGRSREDGLMDGMLGLIGKEGCRTNDKENRSKNSSEIHTRRPSSTSQYLIR CCE7CD149C1F5ACF CCE7CD149C1F5ACF bub3_yeast Cell cycle arrest protein BUB3 OR26.16 YOR026W BUB3 protein protein yeast Saccharomyces cerevisiae Baker's yeast MQIVQIEQAPKDYISDIKIIPSKSLLLITSWDGSLTVYKFDIQAKNVDLLQSLRYKHPLLCCNFIDNTDLQIYVGTVQGEILKVDLIGSPSFQALTNNEANLGICRICKYGDDKLIAASWDGLIEVIDPRNYGDGVIAVKNLNSNNTKVKNKIFTMDTNSSRLIVGMNNSQVQWFRLPLCEDDNGTIEESGLKYQIRDVALLPKEQEGYACSSIDGRVAVEFFDDQGDDYNSSKRFAFRCHRLNLKDTNLAYPVNSIEFSPRHKFLYTAGSDGIISCWNLQTRKKIKNFAKFNEDSVVKIACSDNILCLATSDDTFKTNAAIDQTIELNASSIYIIFDYEN 0BDFB8697935BCEB 0BDFB8697935BCEB mad1_yeast Spindle assembly checkpoint component MAD1 MAD1 Mitotic arrest deficient protein 1 YGL086W protein protein yeast Saccharomyces cerevisiae Baker's yeast MDVRAALQCFFSALSGRFTGKKLGLEIYSIQYKMSNSGGSSPFLESPGGSPDVGSTNGQSNRQIQALQFKLNTLQNEYEIEKLQLQKQTNILEKKYKATIDELEKALNDTKYLYESNDKLEQELKSLKERSANSMNDKDKCIEELRTTLQNKDLEMETLRQQYDSKLSKVTNQCDHFKLEAESSHSLLMKYEKEIKRQSVDIKDLQHQVMEKDDELSSVKASKMINSHPNYSTEEFNELTEMNKMIQDQVQYTKELELANMQQANELKKLKQSQDTSTFWKLENEKLQNKLSQLHVLESQYENLQLENIDLKSKLTKWEIYNDSDDDDDNNVNNNDNNNNNKNDNNNDNNNDTSNNNNINNNNRTKNNIRNNPEEIIRDWKLTKKECLILTDMNDKLRLDNNNLKLLNDEMALERNQILDLNKNYENNIVNLKRLNHELEQQKSLSFEECRLLREQLDGLYSAQNNALLEVENSETHASNKNVNEDMNNLIDTYKNKTEDLTNELKKLNDQLLSNSNDVETQRKKRKLTSDQIGLNYSQRLNELQLENVSVSRELSKAQTTIQLLQEKLEKLTKLKEKKIRILQLRDGPFIKDQFIKKNKLLLLEKENADLLNELKKNNPAVETVPISVYDSLNFELKQFEQEVFKSNKRFSRLKQVFNNKSLEFIDVVNSLLGFKLEFQQDSRVKIFSCFKPEKYLIADLNENTLKSNLDADIEGWDDLMNLWVEDRGQLPCFLATITLRLWEQRQAK 312DDE1BE241610D bub1_yeast Checkpoint serine/threonine-protein kinase BUB1 YGR188C G7542 BUB1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MNLDLGSTVRGYESDKDTFPQSKGVSSSQKEQHSQLNQTKIAYEQRLLNDLEDMDDPLDLFLDYMIWISTSYIEVDSESGQEVLRSTMERCLIYIQDMETYRNDPRFLKIWIWYINLFLSNNFHESENTFKYMFNKGIGTKLSLFYEEFSKLLENAQFFLEAKVLLELGAENNCRPYNRLLRSLSNYEDRLREMNIVENQNSVPDSRERLKGRLIYRTAPFFIRKFLTSSLMTDDKENRANLNSNVGVGKSAPNVYQDSIVVADFKSETERLNLNSSKQPSNQRLKNGNKKTSIYADQKQSNNPVYKLINTPGRKPERIVFNFNLIYPENDEEFNTEEILAMIKGLYKVQRRGKKHTEDYTSDKNRKKRKLDVLVERRQDLPSSQPPVVPKSTRIEVFKDDDNPSQSTHHKNTQVQVQTTTSILPLKPVVDGNLAHETPVKPSLTSNASRSPTVTAFSKDAINEVFSMFNQHYSTPGALLDGDDTTTSKFNVFENFTQEFTAKNIEDLTEVKDPKQETVSQQTTSTNETNDRYERLSNSSTRPEKADYMTPIKETTETDVVPIIQTPKEQIRTEDKKSGDNTETQTQLTSTTIQSSPFLTQPEPQAEKLLQTAEHSEKSKEHYPTIIPPFTKIKNQPPVIIENPLSNNLRAKFLSEISPPLFQYNTFYNYNQELKMSSLLKKIHRVSRNENKNPIVDFKKTGDLYCIRGELGEGGYATVYLAESSQGHLRALKVEKPASVWEYYIMSQVEFRLRKSTILKSIINASALHLFLDESYLVLNYASQGTVLDLINLQREKAIDGNGIMDEYLCMFITVELMKVLEKIHEVGIIHGDLKPDNCMIRLEKPGEPLGAHYMRNGEDGWENKGIYLIDFGRSFDMTLLPPGTKFKSNWKADQQDCWEMRAGKPWSYEADYYGLAGVIHSMLFGKFIETIQLQNGRCKLKNPFKRYWKKEIWGVIFDLLLNSGQASNQALPMTEKIVEIRNLIESHLEQHAENHLRNVILSIEEELSHFQYKGKPSRRF 6D76FC980775D3F9 6D76FC980775D3F9 mad3_yeast Spindle assembly checkpoint component MAD3 MAD3 YJL013C J1341 Mitotic MAD3 protein protein protein yeast Saccharomyces cerevisiae Baker's yeast MKAYAKKRISYMPSSPSQNVINFEEIETQKENILPLKEGRSAAALSKAIHQPLVEINQVKSSFEQRLIDELPALSDPITLYLEYIKWLNNAYPQGGNSKQSGMLTLLERCLSHLKDLERYRNDVRFLKIWFWYIELFTRNSFMESRDIFMYMLRNGIGSELASFYEEFTNLLIQKEKFQYAVKILQLGIKNKARPNKVLEDRLNHLLRELGENNIQLGNEISMDSLESTVLGKTRSEFVNRLELANQNGTSSDVNLTKNNVFVDGEESDVELFETPNRGVYRDGWENFDLKAERNKENNLRISLLEANTNLGELKQHEMLSQKKRPYDEKLPIFRDSIGRSDPVYQMINTKDQKPEKIDCNFKLIYCEDEESKGGRLEFSLEEVLAISRNVYKRVRTNRKHPREANLGQEESANQKEAEAQSKRPKISRKALVSKSLTPSNQGRMFSGEEYINCPMTPKGRSTETSDIISAVKPRQLTPILEMRESNSFSQSKNSEIISDDDKSSSSFISYPPQR 36550249D81BB6D1 36550249D81BB6D1 mad2_yeast Mitotic spindle checkpoint component MAD2 MAD2 J1256 YJL030W protein protein yeast Saccharomyces cerevisiae Baker's yeast MSQSISLKGSTRTVTEFFEYSINSILYQRGVYPAEDFVTVKKYDLTLLKTHDDELKDYIRKILLQVHRWLLGGKCNQLVLCIVDKDEGEVVERWSFNVQHISGNSNGQDDVVDLNTTQSQIRALIRQITSSVTFLPELTKEGGYTFTVLAYTDADAKVPLEWADSNSKEIPDGEVVQFKTFSTNDHKVGAQVSYKY EFE59916C5720644 bub3-cdc20-1 1 4 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 3 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 physical association physical association As shown in Figure 1A, Bub3HA3 immunoprecipitates from extracts of the latter strain contained Cdc20myc18 and vice versa, suggesting that the two proteins physically interact in vivo. 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 1 4 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 3 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 physical association physical association As shown in Figure 1A, Bub3HA3 immunoprecipitates from extracts of the latter strain contained Cdc20myc18 and vice versa, suggesting that the two proteins physically interact in vivo. Figure 1A. Interaction observed in cycling cells and when cells are arrested in G2 but not when arrested in G1 bub3-cdc20-3 1 3 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 8 unspecified role unspecified role prey prey 4 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 5 unspecified role unspecified role prey prey association association We also found that, in addition to Cdc20myc18, both Mad1 and Mad2 associated with Bub3HA3 and inter action increased upon nocodazole treatment (Figure 1B). 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 1 8 unspecified role unspecified role prey prey 3 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 4 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 association association Conversely, Cdc20myc18 immunoprecipitates contained Bub3HA3 and Mad2, but not Mad1, suggesting that either Mad1 is not part of the Cdc20�Bub3 complex, or the levels of Mad1 bound to Cdc20 are below the detection limit. Figure 1B. Interaction increase when cells are in G2 arrest. bub3-cdc20-5 1 3 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 7 unspecified role unspecified role prey prey 8 unspecified role unspecified role prey prey 4 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 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 W G certain certain sequence position certain certain certain sequence position certain W G Wed May 02 16:51:29 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'trp31gly,trp120gly' Tue Jun 30 18:03:50 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Trp31Gly;Trp120Gly]' association association Cdc20myc18, Mad2 and Mad3 co-immunoprecipitated with Bub3HA3 in all kinetochore mutants, whereas, as expected, Bub3HA3�Cdc20myc18 interaction was abolished by deletion of MAD2, which did not affect association of Bub3HA3 to Mad3, as previously shown Figure 3B, 5C bub3-cdc20-6 2 8 unspecified role unspecified role neutral component neutral component 6 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 7 unspecified role unspecified role neutral component neutral component 4 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 3 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 5 unspecified role unspecified role neutral component neutral component physical association physical association As shown in Figure 4A, we found that Mad2, Mad3, Bub1myc18, Bub3HA3 and Cdc20myc18 co-fractionated in two peaks (corresponding to fractions 2�6 and 14�19, respectively), which might identify two distinct complexes, both eluting with apparent molecular weights >670 kDa. Figure 4A Authors believe this may comprise two separate complexes, both with MW = 670kDa. bub3-cdc20-7 1 7 unspecified role unspecified role prey prey 4 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 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 W G certain certain sequence position certain certain certain sequence position certain W G Wed May 02 16:03:21 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'trp31gly,trp120gly' Tue Jun 30 18:28:09 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Trp31Gly;Trp120Gly]' 8 unspecified role unspecified role prey prey 3 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 association association As shown in Figure 5B, HA-tagged Bub3-WDd mutant proteins were no longer able to interact with Mad2, Mad3 and Cdc20 in co-immunoprecipitation experiments, although the mutant protein levels were comparable to those of wild-type Bub3 (Figure 5B). Figure 5B