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/ LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. Nature (0028-0836) 2003 imex curation Seo HS., Yang JY., Ishikawa M., Bolle C., Ballesteros ML., Chua NH. Note: It was not possible to identify the source of ubiquitin used in this papre and the ubiquitin assays were not curated. All fusion genes or genes for YFP and CFP were expressed from a CamV 35S promoter. Onion epidermal cells were bombarded with different combinations of plasmids with a helium biolistic gun and analysed by confocal microscopy after incubation in the dark for 12 h. chua@mail.rockefeller.edu 14-Nov-2008: Contacted by JYOTI. orchard Only protein-protein interactions allce Allium cepa (Onion) Onion confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Seo HS., Yang JY., Ishikawa M., Bolle C., Ballesteros ML., Chua NH. Note: It was not possible to identify the source of ubiquitin used in this papre and the ubiquitin assays were not curated. All fusion genes or genes for YFP and CFP were expressed from a CamV 35S promoter. Onion epidermal cells were bombarded with different combinations of plasmids with a helium biolistic gun and analysed by confocal microscopy after incubation in the dark for 12 h. chua@mail.rockefeller.edu Accepted 2008-NOV-7 by ORCHARD. Nature (0028-0836) 2003 orchard Only protein-protein interactions imex curation LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. Nature (0028-0836) 2003 imex curation Seo HS., Yang JY., Ishikawa M., Bolle C., Ballesteros ML., Chua NH. Note: It was not possible to identify the source of ubiquitin used in this papre and the ubiquitin assays were not curated. All fusion genes or genes for YFP and CFP were expressed from a CamV 35S promoter. Onion epidermal cells were bombarded with different combinations of plasmids with a helium biolistic gun and analysed by confocal microscopy after incubation in the dark for 12 h. chua@mail.rockefeller.edu 14-Nov-2008: Contacted by JYOTI. orchard Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress pull down pull down anti tag western anti tag western blot anti tag western Seo HS., Yang JY., Ishikawa M., Bolle C., Ballesteros ML., Chua NH. Note: It was not possible to identify the source of ubiquitin used in this papre and the ubiquitin assays were not curated. All fusion genes or genes for YFP and CFP were expressed from a CamV 35S promoter. Onion epidermal cells were bombarded with different combinations of plasmids with a helium biolistic gun and analysed by confocal microscopy after incubation in the dark for 12 h. chua@mail.rockefeller.edu Accepted 2008-NOV-7 by ORCHARD. 14-Nov-2008: Contacted by JYOTI. Nature (0028-0836) 2003 orchard Only protein-protein interactions imex curation LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. Nature (0028-0836) 2003 imex curation Seo HS., Yang JY., Ishikawa M., Bolle C., Ballesteros ML., Chua NH. Note: It was not possible to identify the source of ubiquitin used in this papre and the ubiquitin assays were not curated. All fusion genes or genes for YFP and CFP were expressed from a CamV 35S promoter. Onion epidermal cells were bombarded with different combinations of plasmids with a helium biolistic gun and analysed by confocal microscopy after incubation in the dark for 12 h. chua@mail.rockefeller.edu 14-Nov-2008: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Seo HS., Yang JY., Ishikawa M., Bolle C., Ballesteros ML., Chua NH. Note: It was not possible to identify the source of ubiquitin used in this papre and the ubiquitin assays were not curated. All fusion genes or genes for YFP and CFP were expressed from a CamV 35S promoter. Onion epidermal cells were bombarded with different combinations of plasmids with a helium biolistic gun and analysed by confocal microscopy after incubation in the dark for 12 h. chua@mail.rockefeller.edu Accepted 2008-NOV-7 by ORCHARD. 14-Nov-2008: Contacted by JYOTI. Nature (0028-0836) 2003 orchard Only protein-protein interactions imex curation spa1_arath Protein SUPPRESSOR OF PHYA-105 1 SPA1 F11C10.3/F11C10.4 At2g46340/At2g46350 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MPVMERVAEETVATNNIQLKARVDDVPCNKLDARHNDMVIQSETANSDCPGSSAHRNVDLTKPPPPEEAAGAKLSVEELTLGNYRIVQGSNNTNVDSPRAGKFEHLYRLARGSAFRAGDGDLDSQPRDMDQMLSRIRQQLAGAPSERQNLKPFMSRRSDQNLEAFSERLRAAGENSIMNAPALISEGVQMKTPVSSSNFSQLLLKRAMKGKGVVGKNQETPPEFVSDQDLGSKEKKLDISKSPTPHDVLPLKSSPKGNGMVSHGDGNHSKSSIGISLREFLRSSYAKREKRHGLCLFRQLVELVDSAHSKRLFLLDLRPSLFTLVPSKKLRYIGNFGKNDLESDVDEDLNRRRPVVEESSSGGRDSKKRKMDLHLNSPGNQLQATSTGRPFKRKSPVIDLNMVDARNPDSCELQQQDYIKNLSVSSVSRKQSMSTWLEEQWYTCPEEINGEDIGEKSNIYALGVLLFELLCHCESGEMHAAMMADLRHRILPPTFLSKYPKEAGFCLWLLHPEPSSRPSARDILKSELICEDDSVKSTAAAEEISELLLHFLSSLEVQKKKKASKLLQDIQTLEDDIKEAERRYSSNVSLVRSHGAIEKRVQSSPLDEHCTTSSALFVPTANTDRLMSNIRQLEDAYFFMRSQINLSSSAATARSDKTLKDRDRCSENQNENQDMSTKGKSSDQLEVFFEGLCKFARYSKFETCGTIRSGDLLNSASVVCSLSFDPDEEHIAAAGISKKIKIFDFNAFMNESVGVHYPLVEMVNKSKLSCVCWNSYIKNYLASTDYDGVVQIWDAGTGQGFSQYTEHQKRAWSVDFSPSDPTKFVSGSDDCSVKLWSINEKRSLGTIWSPANVCCVQFSSYSNHLLAFGSADYKVYCYDLRYVKTPWCTLAGHEKAVSYVKFMDSETIVSASTDNSLKLWNLNKTNSSGLSPGACSLTYKGHTNQKNFVGLSVLDGYIACGSETNEVYSYYKSLPMPMTSYKFGSVDPISGNEYFDDNGQFVSSVCWRKKSNMLVAANSTGNMKLLKLV DIP protein Q9SYX2 original sequence version: 102 8DEDF32BB06B4A18 cip8_arath E3 ubiquitin-protein ligase CIP8 CIP8 COP1-interacting protein 8 RING-type E3 ubiquitin transferase CIP8 MXK3.15 At5g64920 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSDAPSSSPDATASHWCYHCNKRVVVETLDDFVVCCECNKGFVESIQPTPAAYSSPAPPQPLSPDLNVEDSSIGSHFLQMLRLLAHAPSQRSPPRHLDVLSYEDDFFRLELNSRNEIDDDEDEDEDDGDEEEEDEEENLTVNDEEDEEDDLRRRNRFPLTTTQSRTGRNRILDWAEILMGIEDNSIEFRMESDRYAGNPADYIDDAAGYEALLQNLAEGDGGGGGGRRGAPPAAKSAIEALETFEVSSSEGEMVMVCAVCKDGMVMGETGKKLPCGHCYHGDCIVPWLGTRNSCPVCRFQLETDDAEYEEERKKRTSTVSDSAAASSSSSTSRY 59EADBA18782E0B8 cop1_arath E3 ubiquitin-protein ligase COP1 COP1 Constitutive photomorphogenesis protein 1 RING-type E3 ubiquitin transferase COP1 T21L14.11 At2g32950 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEEISTDPVVPAVKPDPRTSSVGEGANRHENDDGGSGGSEIGAPDLDKDLLCPICMQIIKDAFLTACGHSFCYMCIITHLRNKSDCPCCSQHLTNNQLYPNFLLDKLLKKTSARHVSKTASPLDQFREALQRGCDVSIKEVDNLLTLLAERKRKMEQEEAERNMQILLDFLHCLRKQKVDELNEVQTDLQYIKEDINAVERHRIDLYRARDRYSVKLRMLGDDPSTRNAWPHEKNQIGFNSNSLSIRGGNFVGNYQNKKVEGKAQGSSHGLPKKDALSGSDSQSLNQSTVSMARKKRIHAQFNDLQECYLQKRRQLADQPNSKQENDKSVVRREGYSNGLADFQSVLTTFTRYSRLRVIAEIRHGDIFHSANIVSSIEFDRDDELFATAGVSRCIKVFDFSSVVNEPADMQCPIVEMSTRSKLSCLSWNKHEKNHIASSDYEGIVTVWDVTTRQSLMEYEEHEKRAWSVDFSRTEPSMLVSGSDDCKVKVWCTRQEASVINIDMKANICCVKYNPGSSNYIAVGSADHHIHYYDLRNISQPLHVFSGHKKAVSYVKFLSNNELASASTDSTLRLWDVKDNLPVRTFRGHTNEKNFVGLTVNSEYLACGSETNEVYVYHKEITRPVTSHRFGSPDMDDAEEEAGSYFISAVCWKSDSPTMLTANSQGTIKVLVLAA DIP protein P43254 original sequence version: 138 DIP protein P43254 original sequence version: 113 DIP protein P43254 original sequence version: 114 E6D9A1B702EAE8F0 laf1_arath Transcription factor LAF1 LAF1 MYB18 M7J2.70 At4g25560 Protein LONG AFTER FAR-RED LIGHT 1 Myb-related protein 18 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAKTKYGERHRKGLWSPEEDEKLRSFILSYGHSCWTTVPIKAGLQRNGKSCRLRWINYLRPGLKRDMISAEEEETILTFHSSLGNKWSQIAKFLPGRTDNEIKNYWHSHLKKKWLKSQSLQDAKSISPPSSSSSSLVACGKRNPETLISNHVFSFQRLLENKSSSPSQESNGNNSHQCSSAPEIPRLFFSEWLSSSYPHTDYSSEFTDSKHSQAPNVEETLSAYEEMGDVDQFHYNEMMINNSNWTLNDIVFGSKCKKQEHHIYREASDCNSSAEFFSPSTTT A04054C846CF2411 laf1-cop1 1 7 unspecified role unspecified role neutral component neutral component c-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 6 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 colocalization colocalization Figure 1a To this end, we transiently expressed LAF1 tagged with yellow fluorescent protein (YFP) and COP1 tagged with cyan fluorescent protein (CFP) in onion epidermal cells. Figure 1a shows that the two proteins were indeed localized in the same nuclear bodies, although COP1 was also found in cytoplasmic regions not containing LAF1, which is strictly localized in the nucleus. laf1-cop1-1 3 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 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) physical association physical association Figure 1b As expected, COP1 interacted with CIP8 (ref. 15) but not luciferase (Fig. 1b) and, under the same conditions, LAF1 translated in vitro purified together with COP1 (Fig. 1b). cop1-cip8 3 5 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 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) physical association physical association Figure 1b As expected, COP1 interacted with CIP8 (ref. 15) but not luciferase (Fig. 1b) and, under the same conditions, LAF1 translated in vitro purified together with COP1 (Fig. 1b). cop1-spa1-3 1 6 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 4 unspecified role unspecified role prey prey c-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range colocalization colocalization Figure 1a Similar experiments showed that SPA1 was distributed in the nucleus and the cytosol but it could be relocalized by COP1 to the same LAF1/COP1 nuclear bodies, which is consistent with an interaction between SPA1 and COP1 in vitro cop1-laf1 2 6 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 ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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 in vitro In vitro physical association physical association Figure 1c The interaction between LAF1 and COP1 also occurred in vivo, as confirmed by co-purification experiments