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/ A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. Plant Mol. Biol. (0167-4412) 2004 Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu orchard in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu Accepted 2007-DEC-23 by ORCHARD. Plant Mol. Biol. (0167-4412) 2004 orchard A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. Plant Mol. Biol. (0167-4412) 2004 Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu orchard yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu Accepted 2007-DEC-23 by ORCHARD. Arabidopsis cDNA library Plant Mol. Biol. (0167-4412) 2004 orchard A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. Plant Mol. Biol. (0167-4412) 2004 Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu orchard in vitro In vitro affinity techniques affinity technology affinity techniques weight by comassie molecular weight estimation by coomasie staining weight by comassie Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu Accepted 2007-DEC-23 by ORCHARD. Plant Mol. Biol. (0167-4412) 2004 orchard A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. Plant Mol. Biol. (0167-4412) 2004 Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu orchard in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu Accepted 2007-DEC-23 by ORCHARD. Plant Mol. Biol. (0167-4412) 2004 orchard A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. Plant Mol. Biol. (0167-4412) 2004 Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu orchard yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Du L., Poovaiah BW. pET-3b carrying potato CaM6 (PCaM6) was expressed in E. coli strain BL21/pLysS, and the 35Slabeled CaM was prepared. The proteins were mixed in calcium containing and calcium free binding buffer. The proteins separated by gel electrophoresis and visulaised by autoradiography. poovaiah@wsu.edu Accepted 2007-DEC-23 by ORCHARD. Plant Mol. Biol. (0167-4412) 2004 orchard calm1_soltu Calmodulin-1 PCM1 protein protein soltu Solanum tuberosum (Potato) Potato MAEQLTEEQIAEFKEAFSLFDKDGDGCITTKELGTVMRSLGQNPTEAELQDMISEADADQNGTIDFPEFLNLMARKMKDTDSEEELKEAFKVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVRMMLAK 218809B99164671E gte9_arath Transcription factor GTE9 At5g14270 GTE9 BET9 BROMODOMAIN AND EXTRATERMINAL DOMAIN PROTEIN 9 Bromodomain-containing protein GTE9 Protein GLOBAL TRANSCRIPTION FACTOR GROUP E9 F18O22.60 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MTERNGGFPGDYCFEAPGGDYDEGSDSPRVSEGSNCSKRKVGETFGVSKMVLPLSGLSSSDRKELIRRLRQELEQIRVFQKNFELSRTVALTSSSASGLTRVKSFGMSRCSTGPGKTVNPISAASKPTPVTTAVMLLMKQCEALLKRLMSHQYGWVFNTPVDVVKLNILDYFNVIEHPMDLGTVKNKLTSGTYSCPSEFAADVRLTFSNAMTYNPPGNDVYVMADTLRKFFEVRWKTLEKKLSGTKVHTEPSNLDAHKEKHIVIPVPMAKKRKTTAVDCENVVDPAKRVMTDEDRLKLGKDLESLTEFPAQLINFLRDHNSNEGGIGDDEIEIDINDLSDHALFQLRDLLDEHLREIQNKKSSVEPCEIELLHGSVPGNSSMQHCDGSELDDEVVDIGENEHPTSSISPVTIEKDLVLGNSNGNSLGSVSGDPKMSSLPRASKGLGTIDLEPMLDGATSASPTRGSSVGGLDQLESASPEKISSVEADCQQDGNSAQNEKQLPPEKSYRAAILKNRFADIILKAREKPLNQNDTRDPEKLQREREELELQKKKEKARLQAEAKAAEEARRKAEAQAAAEAAAEAKRKLELEREAARQALMEMEQSVELNENAKFLEDLELLKTVDTDHLTNTIEEEDGPDVGLRSFSFGGSNPLEQLGLFMKQDEDEEEADPLTSPAPEIDIEEGEID B6D08D263A243033 bt5_arath BTB/POZ and TAZ domain-containing protein 5 BT5 BTB and TAZ domain protein 5 F19F18.100 At4g37610 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MENMDDFSPENVLAPPPPPPPMKKSTDLFMQRSNSFVSKATRDSWDRMFDEAHGADVLIHTDDNGLIYAHSNVIGMASDVIRGMMKQHKRKSHRKSISILGVPHHALRVFIRFLYSSCYEKQDMEDFAIHLLVLSHVYVVPHLKRVCESEFESSLLNKENVIDVFQLALLCDAPRLGLLCHRMILNNFEEVSTSEGWQAMKESHPRLQKELLRSVAYELNSLKQRNRKQKEIQTYTQLYEAMEAFVHICRDGCREIGPTKTETPHMSCGFQACNGLEQLLKHLAGCKLRSIPGGCSRCKRMWQLLELHSRICVDSEQCKVPLCSSLKERMKTQSRKDEKRWKLLVRNVLSTKRIGGSPFFLQAIDVTL CB531228DAB7212B bt4_arath BTB/POZ and TAZ domain-containing protein 4 BT4 At5g67480 BTB and TAZ domain protein 4 K9I9.4 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MVTGCVDLHQSFKSADSSSVPIPPPLPSKSDGLKKKLGHSSVSTATRDMWDRLFNDGYKADVVIYTDNGSIIYAHANILGTASTVIKGMLKQAKRHGKWHTISIRGVPHDAVRVFIRFLYSSCYEKEEMNEFIMHLLLLSHAYVVPQLKRVCEWHLEHGLLTTENVVDVFQLALLCDFPRLSLISHRMIMKHFNELSATEAWTAMKKSHPFLEKEVRDSVIIEANTRKERMRKRNDQRIYSQLYEAMEALVHICRDGCKTIGPHDKDFKPNHATCNYEACKGLESLIRHFAGCKLRVPGGCVHCKRMWQLLELHSRVCAGSDQCRVPLCRNLKEKMEKQSKKDESRWKLLVKNVLGSKKIGGSPFFLPVTNC 99A73DB48E66652F bt1_arath BTB/POZ and TAZ domain-containing protein 1 At5g63160 BT1 BTB and TAZ domain protein 1 MDC12.13 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAITATQNDGVSLNANKISYDLVETDVEIITSGRRSIPAHSGILASVSPVLTNIIEKPRKIHGGSSKKVIKILGVPCDAVSVFVRFLYSPSVTENEMEKYGIHLLALSHVYMVTQLKQRCTKGVGERVTAENVVDILQLARLCDAPDLCLKCMRFIHYKFKTVEQTEGWKFLQEHDPFLELDILQFIDDAESRKKRRRRHRREQNLYLQLSEAMECIEHICTEGCTLVGPSSNLDNKSTCQAKPGPCSAFSTCYGLQLLIRHFAVCKKRVDGKGCVRCKRMIQLLRLHSSICDQSESCRVPLCRQYKNRGEKDKKMVEDTKWKVLVRRVASAKAMSSLSQSKKKKSEVLFKEEAEDLIRIRNKLM 53142FB681946BE5 bt3_arath BTB/POZ and TAZ domain-containing protein 3 BT3 F3F20.14 At1g05690 BTB and TAZ domain protein 3 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSSSTKNIPKPPPLPCITYQRFQSSTRKPSSLMRLVPKEALETWDKLFKEGSGADTYVETDNKSHFPAHSSVLAAASPVIATLLNQSRDKNGNTYLKIHGVPCEAVYMFIRFLYSSCYEEEEMKKFVLHLLVLSHCYSVPSLKRLCVEILDQGWINKENVIDVLQLARNCDVTRICFVCLSMVIKDFKSVSSTEGWKVMKRSNPLLEQELIEAVIESDSRKQERRRKLEEREVYLQLYEAMEALVHICREGCGTIGPRDKALKGSHTVCKFPACKGLEGALRHFLGCKSRASCSHCKRMWQLLQLHSCICDDSNSCKVSLCWNFKEKMKKLSKKEQSKWRLLVENIIRARNSLGPFSSRSSGLI 7011FA737154DF34 gte11_arath Transcription factor GTE11 At3g01770 GTE11 BET10 BROMODOMAIN AND EXTRATERMINAL DOMAIN PROTEIN 10 Bromodomain-containing protein GTE11 Protein GLOBAL TRANSCRIPTION FACTOR GROUP E11 F4P13.32 F28J7.10 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MTVRNGGFPGDYNRNSFDSPGGCDDSPNASKDDETFGVPRIVLPLSDLSSSERRKWIHTLRQELEQLRSFQKSVGDLLPISKIVTSTPASNVSRPKSFGMSRCSTGPGKRVLPFTATKPEPVTTSTMLRMKQCESLLKRLMSQQHCWLFNTPVDVVKLNIPDYFTIIKHPMDLGTVKSKLTSGTYSSPSEFSADVRLTFRNAMTYNPSDNNVYRFADTLSKFFEVRWKTIEKKSSGTKSEPSNLATLAHKDIAIPEPVAKKRKMNAVKRNSLLEPAKRVMTDEDRVKLGRDLGSLTEFPVQIINFLRDHSSKEERSGDDEIEIDINDLSHDALFQLRDLFDEFLRENQKKDSNGEPCVLELLHGSGPGNSLTQHCDGSELEDEDVDIGNYEHPISHISTVRTEKDSVGGLNQMEDASRGKLSLIEGADGHQDGNSAPKEKELPPEKRYRAALLKNRFADIILKAQEITLNQNEKRDPETLQREKEELELQKKKEKARLQAEAKEAEEARRKAEAQEAKRKLELEREAARQALLEMEKSVEINENTRFLKDLELLKTVNTDQLRNLRDVGSESDGLAVFGFGGSNPLEQLGLFMKHEEDEDESDMLAFPDPGNEVEEGEID CCB5504E708845A3 bt2_arath BTB/POZ and TAZ domain-containing protein 2 BT2 At3g48360 BTB and TAZ domain protein 2 T29H11.120 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEAVLVAMSVPATTEDDGFSLITDKLSYNLTPTSDVEIVTSDNRRIPAHSGVLASASPVLMNIMKKPMRRYRGCGSKRVIKILGVPCDAVSVFIKFLYSSSLTEDEMERYGIHLLALSHVYMVTQLKQRCSKGVVQRLTTENVVDVLQLARLCDAPDVCLRSMRLIHSQFKTVEQTEGWKFIQEHDPFLELDILQFIDDAESRKKRRRRHRKEQDLYMQLSEAMECIEHICTQGCTLVGPSNVVDNNKKSMTAEKSEPCKAFSTCYGLQLLIRHFAVCKRRNNDKGCLRCKRMLQLFRLHSLICDQPDSCRVPLCRQFRKRGEQDKKMGEDTKWKLLVTRVVSAKAMTSLCQSKKNKCEQAQGV 59235A953EC36CD6 atb1-calm 1 10 unspecified role unspecified role bait bait c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 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 direct interaction direct interaction 3 AtBT1 is a Ca2+-dependent CaM-binding protein. AtBT1 binding to Calmodulin was Ca2+-dependent. atb1-calm-1 3 6 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) 10 unspecified role unspecified role neutral component neutral component c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain in vitro In vitro direct interaction direct interaction 3 These binding tests demonstrated that the CaMBD of the AtBT1 protein binds to three conserved CaM isoforms AtBT1 binding to Calmodulin was Ca2+-dependent. atb2-calm 1 13 unspecified role unspecified role bait bait 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 direct interaction direct interaction 5 To verify this prediction, the full length cds of all five AtBT genes were cloned into expression vector pET32b (Figure 5A) for a 35S CaM-binding assay. The CaM-binding assay was performed in the presence of Ca2+ and demonstrated that all five AtBTs are Ca2+/CaMbinding proteins. atb3-calm 1 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 11 unspecified role unspecified role bait bait direct interaction direct interaction 5 To verify this prediction, the full length cds of all five AtBT genes were cloned into expression vector pET32b (Figure 5A) for a 35S CaM-binding assay. The CaM-binding assay was performed in the presence of Ca2+ and demonstrated that all five AtBTs are Ca2+/CaMbinding proteins. atb4-calm 1 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 9 unspecified role unspecified role bait bait direct interaction direct interaction 5 To verify this prediction, the full length cds of all five AtBT genes were cloned into expression vector pET32b (Figure 5A) for a 35S CaM-binding assay. The CaM-binding assay was performed in the presence of Ca2+ and demonstrated that all five AtBTs are Ca2+/CaMbinding proteins. atb5-calm 1 8 unspecified role unspecified role bait bait 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 direct interaction direct interaction 5 To verify this prediction, the full length cds of all five AtBT genes were cloned into expression vector pET32b (Figure 5A) for a 35S CaM-binding assay. The CaM-binding assay was performed in the presence of Ca2+ and demonstrated that all five AtBTs are Ca2+/CaMbinding proteins. atb1-atbet10 2 10 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 12 unspecified role unspecified role prey prey physical association physical association AtBT1 protein interacts with two Arabidopsis BET proteins which are members of fsh/Ring3 class transcriptional regulator family atb1-atbet9 2 7 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association AtBT1 protein interacts with two Arabidopsis BET proteins which are members of fsh/Ring3 class transcriptional regulator family atb1-atbet10-1 5 12 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role bait bait n-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain physical association physical association The protein� protein interaction confirmation results between AtBT1 and AtBET10/GTE11 in yeast cell are shown in Figure 8A. 8 atb1-atbet10-2 4 12 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 10 unspecified role unspecified role bait bait n-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 54 57 direct interaction direct interaction The results show that GST-AtBET10 recombinant protein pulled down a GUS(1�360)-AtBT1(1�192) recombinant protein band. 8 atb4-atbet10 5 9 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey physical association physical association and a very weak interaction between AtBT4 and AtBET10/GTE11 was also observed (Figure 8D). 8 atb2-atbet10-3 5 13 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 8 Growth of transformants on the synthetic dropout medium without His Leu and Trp (SD/-HLW) indicated that AtBT2 and AtBET10/GTE11 exhibit an interaction comparable