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/ The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. Mol. Genet. Genomics (1617-4615) 2001 imex curation Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de 23-Dec-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de Accepted 2009-OCT-14 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Mol. Genet. Genomics (1617-4615) 2001 orchard Only protein-protein interactions imex curation The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. Mol. Genet. Genomics (1617-4615) 2001 imex curation Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de 23-Dec-2009: Contacted by JYOTI. orchard Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 1 hybrid one hybrid yeast one-hybrid yeast one hybrid one-hybrid 1 hybrid yeast one-hybrid one-hybrid yeast one hybrid predetermined predetermined participant predetermined Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de Accepted 2009-OCT-14 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Mol. Genet. Genomics (1617-4615) 2001 orchard Only protein-protein interactions imex curation The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. Mol. Genet. Genomics (1617-4615) 2001 imex curation Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de 23-Dec-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de Accepted 2009-OCT-14 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Mol. Genet. Genomics (1617-4615) 2001 orchard Only protein-protein interactions imex curation The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. Mol. Genet. Genomics (1617-4615) 2001 imex curation Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de 23-Dec-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro dnase 1 footprinting DNase I footprinting dnase 1 footprinting predetermined predetermined participant predetermined Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de Accepted 2009-OCT-14 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Mol. Genet. Genomics (1617-4615) 2001 orchard Only protein-protein interactions imex curation The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. Mol. Genet. Genomics (1617-4615) 2001 imex curation Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de 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 Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Brennicke A., Schafer E., Kudla J., Harter K. harterkl@ruf.uni-freiburg.de Accepted 2009-OCT-14 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Mol. Genet. Genomics (1617-4615) 2001 orchard Only protein-protein interactions imex curation tyky_arath_gene NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial gene gene arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress arr2_arath Two-component response regulator ARR2 ARR2 ARP5 At4g16110 dl4095w FCAALL.297 Receiver-like protein 5 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MVNPGHGRGPDSGTAAGGSNSDPFPANLRVLVVDDDPTCLMILERMLMTCLYRVTKCNRAESALSLLRKNKNGFDIVISDVHMPDMDGFKLLEHVGLEMDLPVIMMSADDSKSVVLKGVTHGAVDYLIKPVRIEALKNIWQHVVRKKRNEWNVSEHSGGSIEDTGGDRDRQQQHREDADNNSSSVNEGNGRSSRKRKEEEVDDQGDDKEDSSSLKKPRVVWSVELHQQFVAAVNQLGVDKAVPKKILEMMNVPGLTRENVASHLQKYRIYLRRLGGVSQHQGNMNHSFMTGQDQSFGPLSSLNGFDLQSLAVTGQLPPQSLAQLQAAGLGRPTLAKPGMSVSPLVDQRSIFNFENPKIRFGDGHGQTMNNGNLLHGVPTGSHMRLRPGQNVQSSGMMLPVADQLPRGGPSMLPSLGQQPILSSSVSRRSDLTGALAVRNSIPETNSRVLPTTHSVFNNFPADLPRSSFPLASAPGISVPVSVSYQEEVNSSDAKGGSSAATAGFGNPSYDIFNDFPQHQQHNKNISNKLNDWDLRNMGLVFSSNQDAATATATAAFSTSEAYSSSSTQRKRRETDATVVGEHGQNLQSPSRNLYHLNHVFMDGGSVRVKSERVAETVTCPPANTLFHEQYNQEDLMSAFLKQEGIPSVDNEFEFDGYSIDNIQV F930558EB55549F4 psst_arath_gene PSST gene gene gene arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress nadh_arath_gene NADH dehydrogenase subunit gene gene arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress ahp1_arath Histidine-containing phosphotransfer protein 1 AHP1 ATHP3 At3g21510 MIL23.8 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDLVQKQKSLQDYTKSLFLEGILDSQFLQLQQLQDESNPDFVSQVVTLFFQDSDRILNDLSLSLDQQVVDFKKVDPHVHQLKGSSSSIGAQRVKNACVVFRSFCEQQNVEACHRCLQQVKQEYYLVKNRLETLFKLEQQIVASGGMIPAVELGF BA0C026EA19AF83D ahp2_arath Histidine-containing phosphotransfer protein 2 AHP2 ATHP1 At3g29350 MUO10.6 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDALIAQLQRQFRDYTISLYQQGFLDDQFTELKKLQDDGSPDFVSEVLSLFFEDCVKLISNMARALDTTGTVDFSQVGASVHQLKGSSSSVGAKRVKTLCVSFKECCEAKNYEGCVRCLQQVDIEYKALKTKLQDMFNLEKQIIQAGGIVPQVDIN 9A8FA1184F0E4462 arr2-dna 2 7 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait direct interaction direct interaction Figure 3 ARR2 binds to a PSST promoter fragment in vivo in a yeast one-hybrid system The PSST promoter construct consisted of a tandem repeat of -235 to -126 base pairs of the promoter region. arr2-psst-1 4 7 unspecified role unspecified role prey prey region Strep tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role bait bait direct interaction direct interaction Figure 5A Recombinant Strep-tagged ARR2 was purified from E. coli (Fig. 4, lane 2) and incubated with the monomeric PSST promoter fragment extending from nucleotide -268 to -129. As shown in Fig. 5A, the DNase I protection analysis defines two regions within the PSST promoter fragment which are protected from digestion by DNase I. The PSST promoter region used was -268 base pairs to -129 base pairs. The authors demonstrated that ARR2 protein interacted with regions -224 to -164 base pairs -259 to -240 base pairs of the PSST promoter region. arr2-dna-2 1 8 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey region Strep tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 5B In addition, we performed EMSA with purified Strep-tagged ARR2 and Strep-tagged ARR4 (Fig. 4, lane 1 for recombinant proteins), using as a probe a PSST promoter fragment that contains the entire pollen box and extends from nucleotide -215 to -171 (see Fig. 5A, probe I). ARR2 clearly bound to this promoter fragment The PSST promoter region used was -215 base pairs to -171 base pairs. arr2-dna-3 1 7 unspecified role unspecified role prey prey c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 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 ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role bait bait direct interaction direct interaction Figure 5D C-terminal peptide bound to the PSST promoter fragment even more strongly than full-length ARR2 The PSST promoter region used was -215 base pairs to -171 base pairs. arr2-dna-4 1 6 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 5C To test whether ARR2 binds to promoter fragments of other Arabidopsis nCI genes, we included DNA probes derived from the promoters of the genes TYKY and 55 kDa protein (Zabaleta et al. 1998), which show homology to the PSST probe, in our EMSA analysis. As shown in Fig. 5C, lanes 5-7, ARR2 efficiently associated with the pollen box-containing promoter fragments of all tested nCI genes. The TYKY promoter region used was -144 base pairs to -100 base pairs. arr2-dna-5 1 9 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 5C To test whether ARR2 binds to promoter fragments of other Arabidopsis nCI genes, we included DNA probes derived from the promoters of the genes TYKY and 55 kDa protein (Zabaleta et al. 1998), which show homology to the PSST probe, in our EMSA analysis. As shown in Fig. 5C, lanes 5-7, ARR2 efficiently associated with the pollen box-containing promoter fragments of all tested nCI genes. The NADH dehydrogenase promoter region used was -176 base pairs to -132 base pairs. ahp1-arr2 5 7 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association Figure 8A ARR2 interacts with the HPt proteins AHP1 and AHP2. A Interaction of ARR2 with AHP1 and AHP2 in yeast cells. ahp1-arr2-1 3 10 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 ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait region Strep tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 8C The interactions were also seen in vitro with Strep-tagged ARR2 and (His)6-tagged AHP1 or (His)6-tagged AHP2, respectively ahp2-arr2-1 3 7 unspecified role unspecified role bait bait region Strep tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 11 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 8C The interactions were also seen in vitro with Strep-tagged ARR2 and (His)6-tagged AHP1 or (His)6-tagged AHP2, respectively ahp2-arr2-3 5 11 unspecified role unspecified role bait bait ecobb Escherichia coli (strain B / BL21) 7 unspecified role unspecified role prey prey n-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 8A, 8D To determine the sequence requirements for the ability of ARR2 to interact with HPt proteins, the receiver module and the output domain fused to BD were co-expressed with AHP2. Whereas the controls, as well as the output domain of ARR2, failed to activate the lacZ reporter gene, the receiver module clearly interacted with AHP2 (Fig. 8D). These results suggest that ARR2 could be an element of a plant two-component signalling system that includes at least one HPt protein.