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/ Characterization of the Arabidopsis heterotrimeric G protein. Characterization of the Arabidopsis heterotrimeric G protein. J. Biol. Chem. (0021-9258) 2008 imex curation Wang S., Assmann SM., Fedoroff NV. The authors confirmed the identities of the proteins used in this study. nvf1@psu.edu 14-May-2008: Contacted by JYOTI. Only protein-protein interactions arath-leaf Arabidopsis thaliana leaf leaf leaf confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Wang S., Assmann SM., Fedoroff NV. The authors confirmed the identities of the proteins used in this study. nvf1@psu.edu Accepted 2008-MAY-25 by JYOTI. 14-May-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation Characterization of the Arabidopsis heterotrimeric G protein. Characterization of the Arabidopsis heterotrimeric G protein. J. Biol. Chem. (0021-9258) 2008 imex curation Wang S., Assmann SM., Fedoroff NV. The authors confirmed the identities of the proteins used in this study. nvf1@psu.edu 14-May-2008: Contacted by JYOTI. Only protein-protein interactions arath-leaf Arabidopsis thaliana leaf leaf leaf anti bait coip anti bait coimmunoprecipitation anti bait coip weight identificat confirmation by molecular weight weight identificat Wang S., Assmann SM., Fedoroff NV. As the anti-body used in western blotting was not specific , mouse monoclonal anti-G/C/Y antibodies (anti-G/C/Y antibody detects GFP, CFP and YFP proteins)), protein indentification was made on the basis of protein size using electrophoresis through a discontinuous polyacrylamide gel. The authors confirmed the identities of the proteins used in this study. Transformed Arabidopsis mesophyll protoplasts used. nvf1@psu.edu Accepted 2008-MAY-25 by JYOTI. 14-May-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation Characterization of the Arabidopsis heterotrimeric G protein. Characterization of the Arabidopsis heterotrimeric G protein. J. Biol. Chem. (0021-9258) 2008 imex curation Wang S., Assmann SM., Fedoroff NV. The authors confirmed the identities of the proteins used in this study. nvf1@psu.edu 14-May-2008: Contacted by JYOTI. Only protein-protein interactions arath-leaf Arabidopsis thaliana leaf leaf leaf fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret tag fluorescence tag visualisation by fluorescence tag fluorescence Wang S., Assmann SM., Fedoroff NV. The authors confirmed the identities of the proteins used in this study. Transformed Arabidopsis mesophyll protoplasts used for in vivo FRET. nvf1@psu.edu Accepted 2008-MAY-25 by JYOTI. 14-May-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation 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 DIP protein P18064 original sequence version: 80 46BF8650E067F968 gbb_arath Guanine nucleotide-binding protein subunit beta GB1 T4L20.40 At4g34460 transducin AGB1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSVSELKERHAVATETVNNLRDQLRQRRLQLLDTDVARYSAAQGRTRVSFGATDLVCCRTLQGHTGKVYSLDWTPERNRIVSASQDGRLIVWNALTSQKTHAIKLPCAWVMTCAFSPNGQSVACGGLDSVCSIFSLSSTADKDGTVPVSRMLTGHRGYVSCCQYVPNEDAHLITSSGDQTCILWDVTTGLKTSVFGGEFQSGHTADVLSVSISGSNPNWFISGSCDSTARLWDTRAASRAVRTFHGHEGDVNTVKFFPDGYRFGTGSDDGTCRLYDIRTGHQLQVYQPHGDGENGPVTSIAFSVSGRLLFAGYASNNTCYVWDTLLGEVVLDLGLQQDSHRNRISCLGLSADGSALCTGSWDSNLKIWAFGGHRRVI DIP protein P49177 original sequence version: 63 06673A2F707A2F44 gg1_arath Guanine nucleotide-binding protein subunit gamma 1 GG1 AGG1 Ggamma-subunit 1 Heterotrimeric G protein gamma-subunit 1 MAA21.50 At3g63420 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MREETVVYEQEESVSHGGGKHRILAELARVEQEVAFLEKELKEVENTDIVSTVCEELLSVIEKGPDPLLPLTNGPLNLGWDRWFEGPNGGEGCRCLIL F032D1883E59538B gb1-agg1 1 5 unspecified role unspecified role neutral component neutral component n-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 6 unspecified role unspecified role neutral component neutral component colocalization colocalization Figure 2 We infer that Gγ1 is required for plasma membrane localization of Gβ, Localisation of GB1 to the plasma membrane was dependent on the presence of AGG1. Q9FDX9 over-expressed Since endogenous Q9FDX9 was insufficient to localise GB1 to the plasma membrane , only 35S promoter driven Q9FDX9 expression was sufcient , the expression-level annotation was considered appropriate. gb1-agg1-1 3 5 unspecified role unspecified role neutral component neutral component n-terminal cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 6 unspecified role unspecified role neutral component neutral component n-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range direct interaction direct interaction Figure 3A This result clearly shows that FRET occurs in the plasma membrane between CFP-Gβ and YFP-Gγ1. We conclude that Gβ and Gγ1 form a complex, which is required for membrane localization of the Gβ protein. Colocalisation occurs in the plasma membrane. gb1-agg1-gpa1 3 6 unspecified role unspecified role neutral component neutral component n-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 5 unspecified role unspecified role neutral component neutral component n-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 4 unspecified role unspecified role neutral component neutral component region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag certain certain sequence position certain certain certain sequence position certain proximity proximity close-contact colocalization neighbourhood interaction Figure 3D Figure 3I The presence of FRET between L-CFP-Gα, YFP-Gγ1 and YFP-Gβ (Fig. 3D and 3I) supports the inference that the subunits of Arabidopsis G protein form a heterotrimer in vivo. The cfp tag was positioned between amino acid residues 97 and 98 in the GPA1 protein The authors state that "the G alpha subunit does not interact with either G beta or G gamma1 alone in protoplasts, but interacts with the G beta /gamma complex". P18064 over-expressed P49177 over-expressed Q9FDX9 over-expressed The authors state "the FRET construct pairs were transfected into wildtype protoplasts containing all three subunits, the endogenous levels of the third subunit were insufficient to allow FRET detection when only two of the three subunits were overexpressed", there fore I annotated all proteins as overexpressed. gpa1-agg1 2 5 unspecified role unspecified role ancillary ancillary 6 unspecified role unspecified role prey prey n-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 4 unspecified role unspecified role bait bait region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag certain certain sequence position certain certain certain sequence position certain association association Figure 4 Thus, our biochemical results support the conclusions from FRET analysis that the subunits of the Arabidopsis G protein form a heterotrimeric complex in vivo and that the interaction of Gα and Gγ1 requires Gβ. The cfp tag was positioned between amino acid residues 97 and 98 in the GPA1 protein The authors state that " the subunits of the Arabidopsis G protein form a heterotrimeric complex in vivo and that the interaction of G alpha and G gamma 1 requires G beta". Q9FDX9 over-expressed P49177 over-expressed P18064 over-expressed Protein indentification was made on the basis of protein size using electrophoresis through a 12 percent discontinuous polyacrylamide gel. gb1-agg1-gpa1-1 3 5 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component n-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 4 unspecified role unspecified role neutral component neutral component region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag certain certain sequence position certain certain certain sequence position certain proximity proximity close-contact colocalization neighbourhood interaction Figure 3F Figure 3I We detected FRET signal and a spectral shift in protoplasts transfected with L-CFP-Gα-(35S)-Gβ-(35S)-YFP-Gγ1 (Fig. 3F and 3I), but not in protoplasts transfected with the L-CFP-Gα-(35S)-YFP-Gβ-(35S)-Gγ1 construct (Fig. 3E and 3I). The cfp tag was positioned between amino acid residues 97 and 98 in the GPA1 protein The authors concluded that "the G alpha subunit does not interact with either Gbeta or G gamma1 alone in protoplasts, but interacts with the G beta/gamma complex.". P18064 over-expressed P49177 over-expressed Q9FDX9 over-expressed The authors state "the FRET construct pairs were transfected into wildtype protoplasts containing all three subunits, the endogenous levels of the third subunit were insufficient to allow FRET detection when only two of the three subunits were overexpressed", there fore I annotated all proteins as overexpressed.