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/ Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. J. Biol. Chem. (0021-9258) 2004 imex curation Zhao J., Wang X. wangs@ksu.edu. 25-Jun-2008: Contacted by JYOTI. orchard Only protein-protein interactions ecobb Escherichia coli (strain B / BL21) pull down pull down western blot western blot Immuno blot Immuno blot Zhao J., Wang X. wangs@ksu.edu. Accepted 2008-JUN-12 by ORCHARD. J. Biol. Chem. (0021-9258) 2004 orchard Only protein-protein interactions imex curation Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. J. Biol. Chem. (0021-9258) 2004 imex curation Zhao J., Wang X. wangs@ksu.edu. 25-Jun-2008: Contacted by JYOTI. orchard Only protein-protein interactions ecobb Escherichia coli (strain B / BL21) pull down pull down predetermined predetermined participant predetermined Zhao J., Wang X. In this case PLD alpha was shown to be present with GST-GPA1 by assaying for phospholipase activity. wangs@ksu.edu. Accepted 2008-JUN-12 by ORCHARD. 25-Jun-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2004 orchard Only protein-protein interactions imex curation Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. J. Biol. Chem. (0021-9258) 2004 imex curation Zhao J., Wang X. wangs@ksu.edu. 25-Jun-2008: Contacted by JYOTI. orchard Only protein-protein interactions arath-leaf Arabidopsis thaliana leaf leaf leaf pull down pull down western blot western blot Immuno blot Immuno blot Zhao J., Wang X. wangs@ksu.edu. Accepted 2008-JUN-12 by ORCHARD. 25-Jun-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2004 orchard Only protein-protein interactions imex curation plda1_arath Phospholipase D alpha 1 PLDALPHA1 PLD1 At3g15730 MSJ11.13 Choline phosphatase 1 Phosphatidylcholine-hydrolyzing phospholipase D 1 PLDalpha protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAQHLLHGTLHATIYEVDALHGGGVRQGFLGKILANVEETIGVGKGETQLYATIDLQKARVGRTRKIKNEPKNPKWYESFHIYCAHLASDIIFTVKDDNPIGATLIGRAYIPVDQVINGEEVDQWVEILDNDRNPIQGGSKIHVKLQYFHVEEDRNWNMGIKSAKFPGVPYTFFSQRQGCKVSLYQDAHIPDNFVPRIPLAGGKNYEPQRCWEDIFDAISNAKHLIYITGWSVYAEIALVRDSRRPKPGGDVTIGELLKKKASEGVRVLLLVWDDRTSVDVLKKDGLMATHDEETENFFRGSDVHCILCPRNPDDGGSIVQSLQISTMFTHHQKIVVVDSEMPSRGGSEMRRIVSFVGGIDLCDGRYDTPFHSLFRTLDTVHHDDFHQPNFTGAAITKGGPREPWHDIHSRLEGPIAWDVMYNFEQRWSKQGGKDILVKLRDLSDIIITPSPVMFQEDHDVWNVQLFRSIDGGAAAGFPESPEAAAEAGLVSGKDNIIDRSIQDAYIHAIRRAKDFIYVENQYFLGSSFAWAADGITPEDINALHLIPKELSLKIVSKIEKGEKFRVYVVVPMWPEGLPESGSVQAILDWQRRTMEMMYKDVIQALRAQGLEEDPRNYLTFFCLGNREVKKDGEYEPAEKPDPDTDYMRAQEARRFMIYVHTKMMIVDDEYIIIGSANINQRSMDGARDSEIAMGGYQPHHLSHRQPARGQIHGFRMSLWYEHLGMLDETFLDPSSLECIEKVNRISDKYWDFYSSESLEHDLPGHLLRYPIGVASEGDITELPGFEFFPDTKARILGTKSDYLPPILTT 87A8692E43BD73CE 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 gpa1-pld_alpha1 1 5 unspecified role unspecified role bait bait n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 4 unspecified role unspecified role prey prey n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range Q38882:p.Glu563Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain E A Wed May 02 20:26:07 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu563ala' Tue Jun 30 18:22:41 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu563Ala' Q38882:p.Lys564Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain K A Wed May 02 14:55:34 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys564ala' Tue Jun 30 16:18:16 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys564Ala' Q38882:p.Phe565Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain F A Wed May 02 18:28:01 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe565ala' Tue Jun 30 16:56:58 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe565Ala' physical association physical association Figure 2 Figure 4 Binding of G{alpha} with PLD{alpha}1 expressed in E. coli The authors state that "To estimate the stoichiometry of the interaction, G{alpha} and PLD{alpha}1 were purified and co-incubated at different molar ratios (Fig. 2B). Increasing the ratio of PLD{alpha}1 to G{alpha} from 0.5 to 1 led to a doubling of the PLD co-precipitated with G{alpha}, whereas an increase in the molar ratio to 2:1 did not lead to a further increase in PLD{alpha} complexed with G{alpha}. These results indicate that PLD{alpha}1 binds to G{alpha} in a 1:1 ratio". From the data presented in Figure 4 the authors conclude that "the residue Lys564 is essential for PLD{alpha}1 to interact with G{alpha}, whereas the flanking Glu563 and Phe565 are important for enhancing this interaction". Note the authors state that "expression and purification of the mutated PLD{alpha}1 proteins in E. coli were performed with the same procedure as that for the original PLD{alpha}1" indicating the constructs used had an N-terminal 6XHis tag. gpa1-pld_alpha1-1 2 5 unspecified role unspecified role bait bait n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 4 unspecified role unspecified role prey prey n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range Q38882:p.Glu563Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain E A Wed May 02 17:44:26 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu563ala' Tue Jun 30 18:54:27 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu563Ala' Q38882:p.Lys564Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain K A Wed May 02 16:32:13 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys564ala' Tue Jun 30 18:54:26 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys564Ala' Q38882:p.Phe565Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain F A Wed May 02 18:49:49 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe565ala' Tue Jun 30 19:16:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe565Ala' physical association physical association Figure 2 Figure 4 The presence of PLD{alpha}1 in the precipitates was measured by immunoblotting with PLD{alpha}1-specific antibodies and assaying PLD{alpha}1 activity The authors state that "To estimate the stoichiometry of the interaction, G{alpha} and PLD{alpha}1 were purified and co-incubated at different molar ratios (Fig. 2B). Increasing the ratio of PLD{alpha}1 to G{alpha} from 0.5 to 1 led to a doubling of the PLD co-precipitated with G{alpha}, whereas an increase in the molar ratio to 2:1 did not lead to a further increase in PLD{alpha} complexed with G{alpha}. These results indicate that PLD{alpha}1 binds to G{alpha} in a 1:1 ratio". From the data presented in Figure 4 the authors conclude that "the residue Lys564 is essential for PLD{alpha}1 to interact with G{alpha}, whereas the flanking Glu563 and Phe565 are important for enhancing this interaction". Note: The authors state that "expression and purification of the mutated PLD{alpha}1 proteins in E. coli were performed with the same procedure as that for the original PLD{alpha}1" ; indicating that the constructs used to express the mutated forms of PLD alpha contaned an N-terminal 6XHis tag. gpa1-pld_alpha1-2 3 5 unspecified role unspecified role bait bait n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 4 unspecified role unspecified role prey prey physical association physical association Figure 3 Binding of G{alpha} with PLD{alpha}1 from Arabidopsis leaf extracts The authors state "the amount of PLD{alpha}1 co-precipitated with G{alpha} increased as the amount of G{alpha} was increased (Fig. 3), indicating that the G{alpha} and PLD{alpha}1 binding is dose-dependent". The identification was confirmed by enzymic assay as described in the text.