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/ HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2001 imex curation Wu J., Carmen AA., Kobayashi R., Suka N., Grunstein M. mg@mbi.ucla.edu 21-Dec-2010: Contacted by JYOTI. mgt Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Wu J., Carmen AA., Kobayashi R., Suka N., Grunstein M. mg@mbi.ucla.edu Accepted 2010-OCT-22 by MGT. 21-Dec-2010: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2001 mgt Only protein-protein interactions imex curation HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2001 imex curation Wu J., Carmen AA., Kobayashi R., Suka N., Grunstein M. mg@mbi.ucla.edu 21-Dec-2010: Contacted by JYOTI. mgt Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Wu J., Carmen AA., Kobayashi R., Suka N., Grunstein M. mg@mbi.ucla.edu Accepted 2010-OCT-22 by MGT. 21-Dec-2010: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2001 mgt Only protein-protein interactions imex curation HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2001 imex curation Wu J., Carmen AA., Kobayashi R., Suka N., Grunstein M. mg@mbi.ucla.edu 21-Dec-2010: Contacted by JYOTI. mgt Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Wu J., Carmen AA., Kobayashi R., Suka N., Grunstein M. mg@mbi.ucla.edu Accepted 2010-OCT-22 by MGT. 21-Dec-2010: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2001 mgt Only protein-protein interactions imex curation HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2001 imex curation Wu J., Carmen AA., Kobayashi R., Suka N., Grunstein M. mg@mbi.ucla.edu 21-Dec-2010: Contacted by JYOTI. mgt 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 Wu J., Carmen AA., Kobayashi R., Suka N., Grunstein M. mg@mbi.ucla.edu Accepted 2010-OCT-22 by MGT. 21-Dec-2010: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2001 mgt Only protein-protein interactions imex curation hda3_yeast HDA1 complex subunit 3 Histone deacetylase complex 1 subunit 3 HDA3 PLO1 YPR179C protein protein yeast Saccharomyces cerevisiae Baker's yeast MDLLRILDTKPIPTIVDATTLGISGNTSGDYWLPTTMSLYQKELTDQIVSLHYSDILRYFETSHYKEDVILESMKTMCLNGSLVATHPYLLIDHYMPKSLITRDVPAHLAENSGKFSVLRDLINLVQEYETETAIVCRPGRTMDLLEALLLGNKVHIKRYDGHSIKSKQKANDFSCTVHLFSSEGINFTKYPIKSKARFDMLICLDTTVDTSQKDIQYLLQYKRERKGLERYAPIVRLVAINSIDHCRLFFGKKFDKNSREYLENVTAAMVILRDRLGTLPPDLRPIYSQKLHYLVEWLENPTVPWPLPDIYPLKQYTSMDVERSLLTEVHFKKSDDQLEDAFSNCSKKRGRHGANKAASSTVAGIEDNITPSFYSTKRLKNDYYTNPLKQDMTQLTGITTADNSSNVNYHLSSGIITHKLIQSMGEVYMDICVQKQELDDYSCLDDLQNDHLKFFSNEDEKIIKEYETVLRTNNENLNRSHELEVENNLKFSQIETLEKDIETLKGSLMAQGETLSKLKDAFVKTDNVQDEIEKEERVSVSRDTEKKYMEQEIKRAVDAIRENEEETHKLNEKQNGLESELKLKFEKSEISTKELNEKIGFLKKELKLENDLNEELVGQLSKTMDNLENLTIPRVRTQNGNTKKKSRAKKPGNV CC8F8F73D6B78E45 hda2_yeast HDA1 complex subunit 2 HDA2 Histone deacetylase complex 1 subunit 2 YDR295C PLO2 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSRKNSKKLKVYYLPVTLTQFQKDLSEILISLHAKSFKASIIGEPQADAVNKPSGLPAGPETHPYPTLSQRQLTYIFDSNIRAIANHPSLLVDHYMPRQLLRMEPTESSIAGSHKFQVLNQLINSICFRDREGSPNEVIKCAIIAHSIKELDLLEGLILGKKFRTKRLSGTSLYNEKHKFPNLPTVDSTINKDGTPNSVSSTSSNSNSTSYTGYSKDDYDYSVKRNLKKRKINTDDWLFLATTKHLKHDQYLLANYDIDMIISFDPMLEVELPALQVLRNNANKDIPIIKLLVQNSPDHYLLDSEIKNSSVKSSHLSNNGHVDDSQEYEEIKSSLLYFLQARNAPVNNCEIDYIKLVKCCLEGKDCNNILPVLDLITLDEASKDSSDSGFWQPQLTKLQYSSTELPLWDGPLDIKTYQTELMHRAVIRLRDIQDEYAKGTVPLYEKRLNETQRQNQLDEIKNSVGLTFKKKQEVEKSINDSEKRLKHAMTESTKLQNKINHLLKNRQELENFNKLPSNTISSENHLEEGSALADKLKEYIDKNATLFNKLKELQQANAEKSKLNDELRSKYQIESSKAAESAQTLKILQESMKSLENEVNGPLTKFSTESLKKELERLQNDFQSLKARNKFLKNYITLMNRQYDLKNKNNVQVEKAAANGTRFRSTRSNTPNYT CE768CBCC84CCD97 hda1_yeast Histone deacetylase HDA1 HDA1 N2819 YNL021W protein protein yeast Saccharomyces cerevisiae Baker's yeast MDSVMVKKEVLENPDHDLKRKLEENKEEENSLSTTSKSKRQVIVPVCMPKIHYSPLKTGLCYDVRMRYHAKIFTSYFEYIDPHPEDPRRIYRIYKILAENGLINDPTLSGVDDLGDLMLKIPVRAATSEEILEVHTKEHLEFIESTEKMSREELLKETEKGDSVYFNNDSYASARLPCGGAIEACKAVVEGRVKNSLAVVRPPGHHAEPQAAGGFCLFSNVAVAAKNILKNYPESVRRIMILDWDIHHGNGTQKSFYQDDQVLYVSLHRFEMGKYYPGTIQGQYDQTGEGKGEGFNCNITWPVGGVGDAEYMWAFEQVVMPMGREFKPDLVIISSGFDAADGDTIGQCHVTPSCYGHMTHMLKSLARGNLCVVLEGGYNLDAIARSALSVAKVLIGEPPDELPDPLSDPKPEVIEMIDKVIRLQSKYWNCFRRRHANSGCNFNEPINDSIISKNFPLQKAIRQQQQHYLSDEFNFVTLPLVSMDLPDNTVLCTPNISESNTIIIVVHDTSDIWAKRNVISGTIDLSSSVIIDNSLDFIKWGLDRKYGIIDVNIPLTLFEPDNYSGMITSQEVLIYLWDNYIKYFPSVAKIAFIGIGDSYSGIVHLLGHRDTRAVTKTVINFLGDKQLKPLVPLVDETLSEWYFKNSLIFSNNSHQCWKENESRKPRKKFGRVLRCDTDGLNNIIEERFEEATDFILDSFEEWSDEE 4E7069E66D03264D 4E7069E66D03264D hda1-hda2-hda3 1 5 unspecified role unspecified role prey prey 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait association association We found that in strain YDS2 a-HDA1 antibody immunoprecipitates HDA1 (Fig. 2A, lane 1), HDA2 (Fig. 2A, lane 4), and HDA3 (Fig. 2A, lane 7). Fig. 2A hda2-hda3-hda1 4 5 unspecified role unspecified role prey prey 6 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 association association Using WJY201, the HDA2-myc containing strain, we found that a-myc antibody immunoprecipitates HDA2-myc (Fig. 2A, lane 5), HDA1 (Fig. 2A, lane 2), and HDA3 (Fig. 2A, lane 8). Fig. 2A hda3-hda2-hda1 4 7 unspecified role unspecified role prey prey 5 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 6 unspecified role unspecified role prey prey association association Finally, in WJY202 the HDA3-myc containing strain, we found that a-myc antibody immunoprecipitates HDA3-myc (Fig. 2A, lane 9), HDA1 (Fig. 2A, lane 3), and HDA2 (Fig. 2A, lane 6) Fig. 2A hda2-hda3-1 4 6 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 5 unspecified role unspecified role prey prey physical association physical association Fig. 2D Interaction observed in Hda1 -/- cells. hda1-hda1-1 2 7 unspecified role unspecified role prey prey region GAD tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 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 Fig. 2E Interaction observed in Hda2 -/- and Hda3 -/- cells. hda1-hda1-2 3 7 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 7 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 3 hda1-hda3-1 3 5 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 7 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 3 hda3-hda2-1 3 5 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction Fig. 3 It was found that GST-HDA3 precipitates '60% of 35S-labeled HDA2, whereas GST alone cannot retain HDA2 (Fig. 3, lanes 10 and 12). hda1-hda2-4 3 7 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction Fig. 3