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/ Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev. (0890-9369) 2009 imex curation Qiao H., Chang KN., Yazaki J., Ecker JR. ecker@salk.edu 23-Dec-2009: Contacted by JYOTI. orchard Only protein-protein interactions 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 Qiao H., Chang KN., Yazaki J., Ecker JR. ecker@salk.edu Accepted 2009-NOV-29 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Genes Dev. (0890-9369) 2009 orchard Only protein-protein interactions imex curation Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev. (0890-9369) 2009 imex curation Qiao H., Chang KN., Yazaki J., Ecker JR. ecker@salk.edu 23-Dec-2009: Contacted by JYOTI. orchard Only protein-protein interactions 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 Qiao H., Chang KN., Yazaki J., Ecker JR. ecker@salk.edu Accepted 2009-NOV-29 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Genes Dev. (0890-9369) 2009 orchard Only protein-protein interactions imex curation Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev. (0890-9369) 2009 imex curation Qiao H., Chang KN., Yazaki J., Ecker JR. ecker@salk.edu 23-Dec-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Qiao H., Chang KN., Yazaki J., Ecker JR. ecker@salk.edu Accepted 2009-NOV-29 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Genes Dev. (0890-9369) 2009 orchard Only protein-protein interactions imex curation Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev. (0890-9369) 2009 imex curation Qiao H., Chang KN., Yazaki J., Ecker JR. ecker@salk.edu 23-Dec-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Qiao H., Chang KN., Yazaki J., Ecker JR. ecker@salk.edu Accepted 2009-NOV-29 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Genes Dev. (0890-9369) 2009 orchard Only protein-protein interactions imex curation fb161_arath F-box protein ETP2 ETP2 EIN2 targeting protein 2 K13E13.1 At3g18910 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MKTIQEQLPNDLVEEILCRVPATSLRRLRSTCKAWNRLFKGDRILASKHFEKSAKQFRSLSLRNDYRIFPISFNLHGNSPSLELKSELIDPHSKNSAAPFEISRVIHCEGLLLCSSQLDESRVVVWNPLTGETRWIRTGDFRQKGRSFDVGYYYQKDKRSWIKSYKLLCYYRGTKYFEIYDFDSDSWRILDDIIAPRGSIGYSELSVSLKGNTYWFAKGVTEERPRTISLLKFDFYTEKSVPVLLPYQSRRLFQASSLSVVREDKLSVLLQLDQSSKTEIWVTNVIDETTKGAVSWTKVLALDLSPHLQIGNDGSFFLGEDKKVVMFCEKLIDENKVKDMVYIVGEDNVVTEVGFGVDEMDGCRAVILNYVPSLVQIERAGGNRKRGH 059A4B219DE2AF0C ein2_arath Ethylene-insensitive protein 2 EIN2 CKR1 Cytokinin-resistant protein AtCKR1 ORE3 Protein ORESARA 3 F12E4.10 MOK16.19 At5g03280 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEAEIVNVRPQLGFIQRMVPALLPVLLVSVGYIDPGKWVANIEGGARFGYDLVAITLLFNFAAILCQYVAARISVVTGKHLAQICNEEYDKWTCMFLGIQAEFSAILLDLTMVVGVAHALNLLFGVELSTGVFLAAMDAFLFPVFASFLENGMANTVSIYSAGLVLLLYVSGVLLSQSEIPLSMNGVLTRLNGESAFALMGLLGASIVPHNFYIHSYFAGESTSSSDVDKSSLCQDHLFAIFGVFSGLSLVNYVLMNAAANVFHSTGLVVLTFHDALSLMEQVFMSPLIPVVFLMLLFFSSQITALAWAFGGEVVLHDFLKIEIPAWLHRATIRILAVAPALYCVWTSGADGIYQLLIFTQVLVAMMLPCSVIPLFRIASSRQIMGVHKIPQVGEFLALTTFLGFLGLNVVFVVEMVFGSSDWAGGLRWNTVMGTSIQYTTLLVSSCASLCLILWLAATPLKSASNRAEAQIWNMDAQNALSYPSVQEEEIERTETRRNEDESIVRLESRVKDQLDTTSVTSSVYDLPENILMTDQEIRSSPPEERELDVKYSTSQVSSLKEDSDVKEQSVLQSTVVNEVSDKDLIVETKMAKIEPMSPVEKIVSMENNSKFIEKDVEGVSWETEEATKAAPTSNFTVGSDGPPSFRSLSGEGGSGTGSLSRLQGLGRAARRHLSAILDEFWGHLYDFHGQLVAEARAKKLDQLFGTDQKSASSMKADSFGKDISSGYCMSPTAKGMDSQMTSSLYDSLKQQRTPGSIDSLYGLQRGSSPSPLVNRMQMLGAYGNTTNNNNAYELSERRYSSLRAPSSSEGWEHQQPATVHGYQMKSYVDNLAKERLEALQSRGEIPTSRSMALGTLSYTQQLALALKQKSQNGLTPGPAPGFENFAGSRSISRQSERSYYGVPSSGNTDTVGAAVANEKKYSSMPDISGLSMSARNMHLPNNKSGYWDPSSGGGGYGASYGRLSNESSLYSNLGSRVGVPSTYDDISQSRGGYRDAYSLPQSATTGTGSLWSRQPFEQFGVAERNGAVGEELRNRSNPINIDNNASSNVDAEAKLLQSFRHCILKLIKLEGSEWLFGQSDGVDEELIDRVAAREKFIYEAEAREINQVGHMGEPLISSVPNCGDGCVWRADLIVSFGVWCIHRVLDLSLMESRPELWGKYTYVLNRLQGVIDPAFSKLRTPMTPCFCLQIPASHQRASPTSANGMLPPAAKPAKGKCTTAVTLLDLIKDVEMAISCRKGRTGTAAGDVAFPKGKENLASVLKRYKRRLSNKPVGMNQDGPGSRKNVTAYGSLG 1A806303B8B8E71B fb162_arath F-box protein ETP1 ETP1 EIN2 targeting protein 1 K13E13.9 At3g18980 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MTIPDLCNDLVDEILCRVPARNLKRLRSTSKRWNRLFKDDRRFAREHMHKAPKEYLPLMLTSEYRICPVSINLQGDVPSVVLKRELSLPDPDYSHQFDIGRVFHCDGLLVCNHVGKNPRYGSKIVVWNPLTGQTRWIEAGYRWKEYEVRFVLGYCYQQDENNSCSKKIYKILCFYPNGQDTEIYELNYSDRWTRTIPDGDLTPGWTLIYSEQTVSMNGNLYLFASEKSKPHLGVSLLRFDFSTEKSSLCVTLPYQRPRYEILSISAVRGGENLSLLLQLDFESKTEIWVTNKIDDTTTKGAAVSWTKVLAFDLSPDLQLFSEEVNFLLDEDKKVAVCCERWLEPQEHHRYQCRREYKITDKIYILGEDNKVDEVGSGEGEATDSLEGISQVILNYAPSLVQIEQAGGGKTKRGDD F5792A61198CE408 ein2-etp1 1 6 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 7 unspecified role unspecified role prey prey physical association physical association To identify EIN2-interacting proteins and potentially fill in gaps within the ethylene signaling pathway, we used EIN2-CEND to perform a yeast two-hybrid screen. Among the potential interactors, an F-box protein, ETP1, was identified. ein2-etp2 1 5 unspecified role unspecified role prey prey 6 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 physical association physical association Figure 2B Figure 2D The authors concluded that he " results demonstrated that the most highly conserved region of EIN2 (EIN2-C5), the last ∼250 amino acids (Supplemental Fig. S3), is both necessary and sufficient for the interaction of EIN2 with ETP1 and ETP2. Additionally, these results suggested that other portions of EIN2-CEND are not essential for these interactions but may enhance them". ein2-etp1-1 3 6 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 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) 7 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 in vitro In vitro physical association physical association Figure 2C EIN2 interacts with ETP1 and ETP2 in vitro ein2-etp2-1 3 5 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 in vitro In vitro 6 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 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) physical association physical association Figure 2C ein2-etp1-2 4 6 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 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 myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress physical association physical association Total protein extracts from 35S::MYC-ETP1 transgenic plants with or without ethylene treatment were incubated with equal excessive amounts of 35S-methionine-labeled EIN2-C2 protein from in vitro transcriptional/translational system, and subsequently immunoprecipitated with an anti-MYC antibody. The level of ETP1 protein was decreased by added ethylene, resulting in less EIN2 coimmunoprecipitating with ETP1. Fig. 6B ein2-etp1-3 2 6 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 7 unspecified role unspecified role prey prey physical association physical association To further understand the post-transcriptional regulation of EIN2 levels, we performed a yeast two-hybrid screen to identify proteins that potentially interact with the EIN2-CEND. As a result, a novel F-box protein ETP1 (At3g18980, GB:Q9LJ68; GB:Q8LB99) was identified.