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/ http://www.ebi.ac.uk/ huang-2006-1 The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis. 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 Huang J., Taylor JP., Chen JG., Uhrig JF., Schnell DJ., Nakagawa T., Korth KL., Jones AM. alan_jones@unc.edu Arabidopsis cDNA libraries Plant Cell (1040-4651) 2006 Only protein-protein interactions imex curation huang-2006-2 The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Huang J., Taylor JP., Chen JG., Uhrig JF., Schnell DJ., Nakagawa T., Korth KL., Jones AM. Poly-His-GPA1 was loaded with GDP or GTPgamma S before addiyion to GST-THF1. alan_jones@unc.edu Arabidopsis cDNA libraries Plant Cell (1040-4651) 2006 Only protein-protein interactions imex curation huang-2006-3 The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis. arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Huang J., Taylor JP., Chen JG., Uhrig JF., Schnell DJ., Nakagawa T., Korth KL., Jones AM. alan_jones@unc.edu Arabidopsis cDNA libraries Plant Cell (1040-4651) 2006 Only protein-protein interactions imex curation huang-2006-4 The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis. arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret predetermined predetermined participant predetermined Huang J., Taylor JP., Chen JG., Uhrig JF., Schnell DJ., Nakagawa T., Korth KL., Jones AM. Epitope tagged proteins were expressed in transgenic arabidopsis which was deficient in GPA1 and THF1 through mutation. alan_jones@unc.edu Arabidopsis cDNA libraries Plant Cell (1040-4651) 2006 Only protein-protein interactions imex curation thf1_arath Protein THYLAKOID FORMATION 1, chloroplastic THF1 At2g20890 F5H14.14 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAATAISSLSFPALGQSDKISNFASSRPLASAIRICTKFSRLSLNSRSTSKSLIHCMSNVTADVPPVSETKSKFLKAYKRPIPSIYNTVLQELIVQQHLMRYKKTYRYDPVFALGFVTVYDQLMEGYPSDQDRDAIFKAYIEALNEDPKQYRIDAQKMEEWARSQTSASLVDFSSKEGDIEAVLKDIAGRAGSKEGFSYSRFFAVGLFRLLELASATDPTVLDKLCASLNINKKSVDRDLDVYRNLLSKLVQAKELLKEYVEREKKKQGERAQSQKANETISKCLGDTLYNPSFLVERKS gpa1_arath Guanine nucleotide-binding protein alpha-1 subunit GPA1 At2g26300 T1D16.6 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MGLLCSRSRHHTEDTDENTQAAEIERRIEQEAKAEKHIRKLLLLGAGESGKSTIFKQIKLLFQTGFDEGELKSYVPVIHANVYQTIKLLHDGTKEFAQNETDSAKYMLSSESIAIGEKLSEIGGRLDYPRLTKDIAEGIETLWKDPAIQETCARGNELQVPDCTKYLMENLKRLSDINYIPTKEDVLYARVRTTGVVEIQFSPVGENKKSGEVYRLFDVGGQRNERRKWIHLFEGVTAVIFCAAISEYDQTLFEDEQKNRMMETKELFDWVLKQPCFEKTSFMLFLNKFDIFEKKVLDVPLNVCEWFRDYQPVSSGKQEIEHAYEFVKKKFEELYYQNTAPDRVDRVFKIYRTTALDQKLVKKTFKLVDETLRRRNLLEAGLL gpa1-thf1 Yeast 2 hybrid analysis reveals interaction between GPA1 protein and THF1 protein 496722 N/A 496726 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 496727 unspecified role unspecified role bait bait physical association physical association false false false Figure 2 A The GPA1 protein rendered constituatively active through the Gln222Leu muation and was used in library screenings. gpa1-thf1-1 GST pulldown analysis reveals interaction between GPA1 protein and THF1 protein 496723 N/A 496726 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 false N/A 496727 unspecified role unspecified role prey prey 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 false ecobb Escherichia coli (strain B / BL21) physical association physical association false false false Both the GDP- and GTPgammaS-bound forms of GPA1 interacted with THF1 in vitro. Figure 2 B gpa1-thf1-2 Coimmunoprecipitation analysis reveals interaction between GPA1 protein and THF1 protein 496724 N/A 496726 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 496727 unspecified role unspecified role bait bait physical association physical association false false false Both GDP- and GTPgammaS-bound forms of GPA1 interact with THF1 in vivo. Figure 2 C gpa1-thf1-3 FRET analysis reveals interaction between GPA1 protein and THF1 protein 496725 N/A 496726 unspecified role unspecified role fluorescence donor fluorescence donor region yellow fluorescent protein yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 496727 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region cyan fluorescent protein yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecobb Escherichia coli (strain B / BL21) proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 5