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/ Interaction between Arabidopsis Ca2+/H+ exchangers CAX1 and CAX3. Interaction between Arabidopsis Ca2+/H+ exchangers CAX1 and CAX3. J. Biol. Chem. (0021-9258) 2009 imex curation Zhao J., Shigaki T., Mei H., Guo YQ., Cheng NH., Hirschi KD. Transgenic Arabidopsis (ecotype Columbia) expressing epitope tagged proteins were used in these studies. Microsomal preparations were obtained from transgenic plant leaves. kendalh@bcm.edu jyoti Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Zhao J., Shigaki T., Mei H., Guo YQ., Cheng NH., Hirschi KD. Transgenic Arabidopsis (ecotype Columbia) expressing epitope tagged proteins were used in these studies. Microsomal preparations were obtained from transgenic plant leaves. kendalh@bcm.edu Accepted 2009-MAY-20 by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation Interaction between Arabidopsis Ca2+/H+ exchangers CAX1 and CAX3. Interaction between Arabidopsis Ca2+/H+ exchangers CAX1 and CAX3. J. Biol. Chem. (0021-9258) 2009 imex curation Zhao J., Shigaki T., Mei H., Guo YQ., Cheng NH., Hirschi KD. Transgenic Arabidopsis (ecotype Columbia) expressing epitope tagged proteins were used in these studies. Microsomal preparations were obtained from transgenic plant leaves. kendalh@bcm.edu jyoti Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Zhao J., Shigaki T., Mei H., Guo YQ., Cheng NH., Hirschi KD. Transgenic Arabidopsis (ecotype Columbia) expressing epitope tagged proteins were used in these studies. Microsomal preparations were obtained from transgenic plant leaves. kendalh@bcm.edu Accepted 2009-MAY-20 by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation Interaction between Arabidopsis Ca2+/H+ exchangers CAX1 and CAX3. Interaction between Arabidopsis Ca2+/H+ exchangers CAX1 and CAX3. J. Biol. Chem. (0021-9258) 2009 imex curation Zhao J., Shigaki T., Mei H., Guo YQ., Cheng NH., Hirschi KD. Transgenic Arabidopsis (ecotype Columbia) expressing epitope tagged proteins were used in these studies. Microsomal preparations were obtained from transgenic plant leaves. kendalh@bcm.edu jyoti Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast ub reconstruction ubiquitin reconstruction ub reconstruction predetermined predetermined participant predetermined Zhao J., Shigaki T., Mei H., Guo YQ., Cheng NH., Hirschi KD. Transgenic Arabidopsis (ecotype Columbia) expressing epitope tagged proteins were used in these studies. Microsomal preparations were obtained from transgenic plant leaves. kendalh@bcm.edu Accepted 2009-MAY-20 by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation cax3_arath Vacuolar cation/proton exchanger 3 CAX3 Ca(2+)/H(+) exchanger 3 Ca(2+)/H(+) antiporter CAX3 Protein CATION EXCHANGER 3 ATEM1.11 At3g51860 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MGSIVEPWAAIAENGNANVTAKGSSRELRHGRTAHNMSSSSLRKKSDLRLVQKVPCKTLKNILSNLQEVILGTKLTLLFLAIPLAILANSYNYGRPLIFGLSLIGLTPLAERVSFLTEQLAFYTGPTVGGLLNATCGNATELIIAILALANNKVAVVKYSLLGSILSNLLLVLGTSLFFGGIANIRREQRFDRKQADVNFFLLLMGLLCHLLPLLLKYAATGEVSTSMINKMSLTLSRTSSIVMLIAYIAYLIFQLWTHRQLFEAQQDDDDAYDDEVSVEETPVIGFWSGFAWLVGMTIVIALLSEYVVDTIEDASDSWGLSVSFISIILLPIVGNAAEHAGAIIFAFKNKLDISLGVALGSATQISLFVVPLSVIVAWILGIKMDLNFNILETSSLALAIIITAFTLQDGTSHYMKGLVLLLCYVIIAACFFVDQIPQPNDLDVGLQPMNNLGEVFSA 33B8AB06C3101FE1 cax1_arath Vacuolar cation/proton exchanger 1 CAX1 RCI4 Ca(2+)/H(+) exchanger 1 Ca(2+)/H(+) antiporter CAX1 Protein RARE COLD INDUCIBLE 4 F16M14.10 At2g38170 Protein CATION EXCHANGER 1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAGIVTEPWSVAENGNPSITAKGSSRELRLGRTAHNMSSSSLRKKSDLRVIQKVPYKGLKDFLSNLQEVILGTKLAILFPAIPAAIICTYCGVSQPWIFGLSLLGLTPLAERVSFLTEQLAFYTGPTLGGLLNATCGNATELIIAILALTNNKVAVVKYSLLGSILSNLLLVLGTSLFCGGIANIRREQRFDRKQADVNFFLLLLGFLCHLLPLLVGYLKNGEASAAVLSDMQLSISRGFSIVMLISYIAYLVFQLWTHRQLFDAQEQEDEYDDDVEQETAVISFWSGFAWLVGMTLVIALLSEYVVATIEEASDKWNLSVSFISIILLPIVGNAAEHAGAVIFAFKNKLDISLGVALGSATQIGLFVVPLTIIVAWILGINMDLNFGPLETGCLAVSIIITAFTLQDGSSHYMKGLVLLLCYFIIAICFFVDKLPQKQNAIHLGHQAMNNVVTATGGGVFSS C7502A62AA51DF0A cax1-cax3 2 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 5 unspecified role unspecified role bait bait physical association physical association Figure 5A Figure 5D In planta coimmunoprecipitation of CAX1 and CAX3 Accession numbers for CAX1 and CAX3 were obtained from references in the text. Lithium Chloride (15mM) treatment for 8 hours. cax1-cax3-1 1 5 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 physical association physical association Figure 5B In planta coimmunoprecipitation of CAX1 and CAX3 Accession numbers for CAX1 and CAX3 were obtained from references in the text. cax1-cax3-2 3 4 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait physical association physical association Figure 6C Figure 6D Yeast Split Ubiquitin Assay of CAX1 and CAX3 Interaction�To test whether CAX1 and CAX3 can physically interact in yeast, an optimized split ubiquitin system was used (13). This system allows detection of interactions between membrane proteins in vivo. Protein fusions of ubiquitin were constructed (Fig. 6, A and B), and the interaction of CAX1 and CAX3 was monitored by the release of the artificial transcription factor PLV, activating lexA-driven reporter genes in the nucleus. As a positive control, we demonstrated the interaction between AtKAT1 subunits (13, 23) (Fig. 6). As negative controls, CAX1 and CAX3 did not interact with the membrane protein AtKAT1. Using this system, interactions were observed between CAX1 and CAX3 (Fig. 6). The interaction of CAX1 (Cub or Nub) and CAX3 (Cub or Nub) was also detected by measuring β-galactosidase activity of LacZ (Fig. 6D). These results provided independent evidence that CAXs were capable of assembling in a heterodimeric protein complex. Accession numbers for CAX1 and CAX3 were obtained from references in the text. cax1-cax3-3 3 4 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association Figure 6C Figure 6D Yeast Split Ubiquitin Assay of CAX1 and CAX3 Interaction�To test whether CAX1 and CAX3 can physically interact in yeast, an optimized split ubiquitin system was used (13). This system allows detection of interactions between membrane proteins in vivo. Protein fusions of ubiquitin were constructed (Fig. 6, A and B), and the interaction of CAX1 and CAX3 was monitored by the release of the artificial transcription factor PLV, activating lexA-driven reporter genes in the nucleus. As a positive control, we demonstrated the interaction between AtKAT1 subunits (13, 23) (Fig. 6). As negative controls, CAX1 and CAX3 did not interact with the membrane protein AtKAT1. Using this system, interactions were observed between CAX1 and CAX3 (Fig. 6). The interaction of CAX1 (Cub or Nub) and CAX3 (Cub or Nub) was also detected by measuring β-galactosidase activity of LacZ (Fig. 6D). These results provided independent evidence that CAXs were capable of assembling in a heterodimeric protein complex. Accession numbers for CAX1 and CAX3 were obtained from references in the text.