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/ EIN2, the central regulator of ethylene signalling, is localised at the ER membrane where it interacts with the ethylene receptor ETR1. EIN2, the central regulator of ethylene signalling, is localised at the ER membrane where it interacts with the ethylene receptor ETR1. Biochem. J. (0264-6021) 2009 imex curation Bisson MM., Bleckmann A., Allekotte S., Groth G. georg.groth@uni-duesseldorf.de 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions nicbe Nicotiana benthamiana fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret tag fluorescence tag visualisation by fluorescence tag fluorescence Bisson MM., Bleckmann A., Allekotte S., Groth G. georg.groth@uni-duesseldorf.de Accepted 2009-NOV-2 by JYOTI. Biochem. J. (0264-6021) 2009 jyoti Only protein-protein interactions imex curation EIN2, the central regulator of ethylene signalling, is localised at the ER membrane where it interacts with the ethylene receptor ETR1. EIN2, the central regulator of ethylene signalling, is localised at the ER membrane where it interacts with the ethylene receptor ETR1. Biochem. J. (0264-6021) 2009 imex curation Bisson MM., Bleckmann A., Allekotte S., Groth G. georg.groth@uni-duesseldorf.de 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr predetermined predetermined participant predetermined Bisson MM., Bleckmann A., Allekotte S., Groth G. georg.groth@uni-duesseldorf.de Accepted 2009-NOV-2 by JYOTI. 27-Oct-2007: Contacted by JYOTI. Biochem. J. (0264-6021) 2009 jyoti Only protein-protein interactions imex curation 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 etr1_arath Ethylene receptor 1 ETR1 T27F4.9 At1g66340 Protein ETHYLENE RESPONSE 1 Protein ETR1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEVCNCIEPQWPADELLMKYQYISDFFIAIAYFSIPLELIYFVKKSAVFPYRWVLVQFGAFIVLCGATHLINLWTFTTHSRTVALVMTTAKVLTAVVSCATALMLVHIIPDLLSVKTRELFLKNKAAELDREMGLIRTQEETGRHVRMLTHEIRSTLDRHTILKTTLVELGRTLALEECALWMPTRTGLELQLSYTLRHQHPVEYTVPIQLPVINQVFGTSRAVKISPNSPVARLRPVSGKYMLGEVVAVRVPLLHLSNFQINDWPELSTKRYALMVLMLPSDSARQWHVHELELVEVVADQVAVALSHAAILEESMRARDLLMEQNVALDLARREAETAIRARNDFLAVMNHEMRTPMHAIIALSSLLQETELTPEQRLMVETILKSSNLLATLMNDVLDLSRLEDGSLQLELGTFNLHTLFREVLNLIKPIAVVKKLPITLNLAPDLPEFVVGDEKRLMQIILNIVGNAVKFSKQGSISVTALVTKSDTRAADFFVVPTGSHFYLRVKVKDSGAGINPQDIPKIFTKFAQTQSLATRSSGGSGLGLAISKRFVNLMEGNIWIESDGLGKGCTAIFDVKLGISERSNESKQSGIPKVPAIPRHSNFTGLKVLVMDENGVSRMVTKGLLVHLGCEVTTVSSNEECLRVVSHEHKVVFMDVCMPGVENYQIALRIHEKFTKQRHQRPLLVALSGNTDKSTKEKCMSFGLDGVLLKPVSLDNIRDVLSDLLEPRVLYEGM D6ED3C4BBA87E96E ein2-etr1 1 3 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept c-terminal rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 4 unspecified role unspecified role fluorescence donor fluorescence donor c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range direct interaction direct interaction Figure 2 Detection of EIN2-ETR1 interactions via FRET microscopy ein2-etr1-1 2 3 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 4 unspecified role unspecified role fluorescence donor fluorescence donor direct interaction direct interaction Figure 4 When wild type EIN2 was titrated with the tryptophan-less ETR1 mutant a Kd of 360 nM was obtained (see Figure 4A), while titration of ETR1 by a tryptophan-less EIN2 mutant resulted in a slightly higher Kd of 440 nM as shown in Figure 4B. Taken together both studies underline that ETR1 and EIN2 form a highly specific interaction. The authors do not provide details of the substitution of endogenous tryptophan residues in both proteins. For information the authors state "endogenous tryptophan residues at positions 11, 53, 74, 182, 265, 288 and 563 in ETR1 and at positions 622, 682, 812, 948, 1012, 1075, 1129, 1140 and 1158 in EIN2 were substituted either by phenylalanine or leucine by site-directed mutagenesis using sequential PCR steps".