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 C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. J. Biol. Chem. (0021-9258) 2006 imex curation Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. orchard Only protein-protein interactions tobac-by_2 Tobacco cell line by_2 Tobacco cell line An undifferentiated cell line derived from tobacco callus tissue. confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. The authors incorrectly state that " as shown in Arabidopsis and BY2 cells", when the BY2 cell line is derived from Tobacco. faure@versailles.inra.fr Accepted 2009-JUN-8 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. J. Biol. Chem. (0021-9258) 2006 orchard Only protein-protein interactions imex curation The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. J. Biol. Chem. (0021-9258) 2006 imex curation Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. orchard Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr Accepted 2009-JUN-8 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. J. Biol. Chem. (0021-9258) 2006 orchard Only protein-protein interactions imex curation The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. J. Biol. Chem. (0021-9258) 2006 imex curation Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr Accepted 2009-JUN-8 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. J. Biol. Chem. (0021-9258) 2006 orchard Only protein-protein interactions imex curation The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. J. Biol. Chem. (0021-9258) 2006 imex curation Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. orchard 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 Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr Accepted 2009-JUN-8 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. J. Biol. Chem. (0021-9258) 2006 orchard Only protein-protein interactions imex curation The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. The C terminus of the immunophilin PASTICCINO1 is required for plant development and for interaction with a NAC-like transcription factor. J. Biol. Chem. (0021-9258) 2006 imex curation Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. orchard Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Smyczynski C., Roudier F., Gissot L., Vaillant E., Grandjean O., Morin H., Masson T., Bellec Y., Geelen D., Faure JD. faure@versailles.inra.fr Accepted 2009-JUN-8 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Arabidopsis cDNA library. J. Biol. Chem. (0021-9258) 2006 orchard Only protein-protein interactions imex curation nac89_arath NAC domain-containing protein 89 NAC089 FAN FSQ6 Protein FRUCTOSE-SENSING QUANTITATIVE TRAIT LOCUS 6 MWD9.7 T6G21.9 At5g22290 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDTKAVGVSKDTAASMEASTVFPGFKFSPTDVELISYYLKRKMDGLERSVEVIPDLEIYNFEPWDLPDKSIVKSDSEWFFFCARGKKYPHGSQNRRATKMGYWKATGKERDVKSGSEVIGTKRTLVFHIGRAPKGERTDWIMHEYCVKGVSLDDAMVVCRVRRNKEYNSGTSQKAPKPNSSAEKHAKVQNGATSSGSPSDWDNLVDFYLAGESGEKLLAEMAESSENLQVDNDEDFFADILRDEIINLDEAVMTGNTPNEVPTLESASMEIRVLPLPNMIDKQMSSLLEERPSQKKKGKDATESLSSCFVGLYSIKSVNKARWDVIIGVVALIAMLFYLE 14DB0290E32E15C1 pas1_arath Peptidyl-prolyl cis-trans isomerase PASTICCINO1 PAS1 F5K20.310 At3g54010 Rotamase 70 kDa peptidyl-prolyl isomerase DEI1 FK506-binding protein 72 FKBP70 FKBP72 Immunophilin FKBP72 Peptidyl-prolyl cis-trans isomerase FKBP72 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAVGDQTEQNYLPKKKKSETEDDKRRKKIVPGSLLKAVVRPGGGDSSPVDGDQVIYHCTVRTLDGVVVESTRSESGGRGVPIRDVLGNSKMILGLLEGIPTMHKGEIAMFKMKPEMHYAEIDCPVSAPENFPKDDELHFEIELLDFSKAKIASDDLGVIKKILNEGEGWESPREPYEVKARISAKSGDGHVIFSHTEEPYFFTFGKSEVPKGLEIGIGTMARKEKAVIYVRKQYLTESPLLHIDQDLEEVHFEVELVHFIQVRDMLGDGRLIKRRIRDGRGEFPMDCPLQDSRLSVHYKGMLLNEEKTVFYDSKIDNNDQPLEFSSGEGLVPEGFEMCTRLMLPGEIALVTCPPDYAYDKFPRPPGVSEGAHVQWEIELLGFETPRDWTGLNFQSIMDEADKIRSTGNRLFKEGKFELAKAKYEKVLREFNHVNPQDEDEGKIFGDTRNMLHLNVAACLLKMGEWRKSIETCNKVLEAKPGHVKGLYRRGMAYIAGGEYDDARNDFNMMIKVDKSSEADATAALLKLKQKEQEAESKARKQFKGLFDKRPGEITEVGSEIREESKTIEEVDETKDNDDDETLEEEGATTVSTERKRKWSEKAWPFLKNVMLQIGIQLGVVLIGILIFQFVSAKFT 561EEBAC77BFE241 pas1-fan 3 6 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 7 unspecified role unspecified role bait bait physical association physical association We used a yeast two-hybrid approach to identify proteins interacting with PAS1. We used the full-length PAS1 protein as bait and screened 16 million clones from two A. thaliana cDNA libraries. We identified seven independent clones, one of which had a sequence similar to those of the NAC family of transcription factors and was subsequently named FAN (FKBP-associated NAC). pas1-fan-1 2 7 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region Strep tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 6 unspecified role unspecified role bait bait region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction PAS1 interacts with FAN in vitro. Figure 3A pas1-fan-2 5 7 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress 6 unspecified role unspecified role bait bait region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association This interaction was further confirmed in a pulldown assay with an MBP:FAN column and crude protein extract from BY2 cells expressing the 35S-PAS1: GFP construct. PAS1:GFP bound to the MBP:FAN column but not to the maltose matrix alone Figure 3A pas1-fan-3 5 6 unspecified role unspecified role prey prey region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp 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 region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction in vitro interaction between FAN and PAS1 requires the C-terminal domain of PAS1. Figure 3B pas1-fan-4 1 6 unspecified role unspecified role neutral component neutral component region DsRed tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role neutral component neutral component region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain colocalization colocalization DsRED:FAN was also localized in the cytoplasm and colocalized with PAS1:GFP Figure 3E pas1-fan-5 4 7 unspecified role unspecified role fluorescence donor fluorescence donor region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region DsRed tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined proximity proximity close-contact colocalization neighbourhood interaction Colocalization and FRET analysis demonstrated that PAS1 interacted in vivo with FAN and that its C terminus was required for this interaction. Figure 3E