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/ HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling Genes Dev. (0890-9369) 2005 Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu 22-Jan-2008: Contacted by JYOTI. arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress enzymatic study enzymatic study predetermined predetermined participant predetermined Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu Accepted 2007-DEC-26 by JYOTI. 22-Jan-2008: Contacted by JYOTI. Genes Dev. (0890-9369) 2005 HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling Genes Dev. (0890-9369) 2005 Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu 22-Jan-2008: Contacted by JYOTI. arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu Accepted 2007-DEC-26 by JYOTI. 22-Jan-2008: Contacted by JYOTI. Genes Dev. (0890-9369) 2005 HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling Genes Dev. (0890-9369) 2005 Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu 22-Jan-2008: Contacted by JYOTI. 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 Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu Accepted 2007-DEC-26 by JYOTI. 22-Jan-2008: Contacted by JYOTI. Genes Dev. (0890-9369) 2005 HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling Genes Dev. (0890-9369) 2005 Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu 22-Jan-2008: Contacted by JYOTI. allce Allium cepa (Onion) Onion confocal microscopy confocal microscopy tag visualisation tag visualisation Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu Accepted 2007-DEC-26 by JYOTI. Genes Dev. (0890-9369) 2005 HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling Genes Dev. (0890-9369) 2005 Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu 22-Jan-2008: Contacted by JYOTI. in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Jang IC., Yang JY., Seo HS., Chua NH. For in vitro ubiquitination assays each reaction mixture contained protein substrate [MBPHFR1-3HA or MBP-HFR1(deltaN)-3HA, 20 ng rabbit E1 protein 20 ng human E2 (UbcH5b) protein 10 micro gram His6-ubiquitin and 200 ng E3 (COP1 or SINAT5). Reactions were carried out at 30 degree celsius for 2 h. chua@mail.rockefeller.edu Accepted 2007-DEC-26 by JYOTI. 22-Jan-2008: Contacted by JYOTI. Genes Dev. (0890-9369) 2005 ub2d2_human Ubiquitin-conjugating enzyme E2 D2 UBC5B UBCH4 UBE2D2 UBC4 UBCH5B Ubiquitin-protein ligase D2 Ubiquitin carrier protein D2 Ubiquitin-conjugating enzyme E2-17 kDa 2 Ubiquitin-conjugating enzyme E2(17)KB 2 PUBC1 p53-regulated ubiquitin-conjugating enzyme 1 (E3-independent) E2 ubiquitin-conjugating enzyme D2 E2 ubiquitin-conjugating enzyme D2 protein protein human Homo sapiens Human MALKRIHKELNDLARDPPAQCSAGPVGDDMFHWQATIMGPNDSPYQGGVFFLTIHFPTDYPFKPPKVAFTTRIYHPNINSNGSICLDILRSQWSPALTISKVLLSICSLLCDPNPDDPLVPEIARIYKTDREKYNRIAREWTQKYAM DIP protein P62837 original sequence version: 81 DIP protein P62837-1 original sequence version: 134 DIP protein P62837 original sequence version: 95 DIP protein P62837 original sequence version: 139 DIP protein P62837 original sequence version: 37 DIP protein P62837 original sequence version: 115 DIP protein P62837 original sequence version: 58 DIP protein P62837 original sequence version: 74 DIP protein P62837 original sequence version: 103 DIP protein P62837 original sequence version: 101 C942BE7853CBC355 hfr1_arath Transcription factor HFR1 Transcription factor EN 68 bHLH transcription factor bHLH026 HFR1 BHLH26 EN68 Basic helix-loop-helix protein 26 Reduced sensitivity to far-red light Protein LONG HYPOCOTYL IN FAR-RED 1 FBI1 Protein REDUCED PHYTOCHROME SIGNALING REP1 RSF1 At1g02340 T6A9.4 T6A9_13 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSNNQAFMELGWRNDVGSLAVKDQGMMSERARSDEDRLINGLKWGYGYFDHDQTDNYLQIVPEIHKEVENAKEDLLVVVPDEHSETDDHHHIKDFSERSDHRFYLRNKHENPKKRRIQVLSSDDESEEFTREVPSVTRKGSKRRRRDEKMSNKMRKLQQLVPNCHKTDKVSVLDKTIEYMKNLQLQLQMMSTVGVNPYFLPATLGFGMHNHMLTAMASAHGLNPANHMMPSPLIPALNWPLPPFTNISFPHSSSQSLFLTTSSPASSPQSLHGLVPYFPSFLDFSSHAMRRL BC75C5EB26E10A20 uba1_rabit Ubiquitin-like modifier-activating enzyme 1 Ubiquitin-activating enzyme E1 UBA1 UBE1 protein protein rabit Oryctolagus cuniculus (Rabbit) Rabbit MSSSPLSKKRRVSGPDPKPGSNCSPAQSVLPQVPSAPTNGMAKNGSEADIDEGLYSRQLYVLGHEAMKRLQTSSVLVSGLRGLGVEIAKNIILGGVKAVTLHDQGTAQWADLSSQFYLREEDIGKNRAEVSQPRLAELNSYVPVTAYTGPLVEDFLSGFQVVVLTNSPLEDQLRVGEFCHSRGIKLVVADTRGLFGQLFCDFGEEMILTDSNGEQPLSTMVSMVTKDNPGVVTCLDEARHGFESGDFVSFSEVQGMTELNGNQPIEIKVLGPYTFSICDTSNFSDYIRGGIVSQVKVPKKISFKSLSASLAEPDFVMTDFAKFSRPAQLHIGFQALHKFCAQHSRPPRPRNEEDAAELVTLARAVNSKASSAVQQDSLDEDLIRNLAFVAAGDLAPINAFIGGLAAQEVMKACSGKFMPIMQWLYFDALECLPEDKESLTEDKCLPRQNRYDGQVAVFGSDLQEKLGRQKYFLVGAGAIGCELLKNFAMIGLGCGENGEIIVTDMDTIEKSNLNRQFLFRPWDVTKLKSDTAAAAVHQMNPHIRVTSHQNRVGPDTERIYDDDFFQTLDGVANALDNVDARMYMDRRCVYYRKPLLESGTLGTKGNVQVVIPFLTESYSSSQDPPEKSIPICTLKNFPNAIEHTLQWARDEFEGLFKQPAENVNQYLTDPKFVERTLRLAGTQPLEVLEAVQRSLVLQLPQSWADCVTWACHHWHTQYSNNIRQLLHNFPPDQLTSSGAPFWSGPKRCPHPLTFDVSNPLHLDYVMAAANLFAQTYGLAGSQDRAAVATLLQSVQVPEFTPKSGVKIHVSDQELQSANASVDDSRLEELKATLPSPDKLPGFKMYPIDFEKDDDSNFHMDFIVAASNLRAENYDIPPADRHKSKLIAGKIIPAIATTTAAVVGLVCLELYKVVQGHRHLDSYKNGFLNLALPFFGFSEPLAAPRHQYYNQEWTLWDRFEVQGLQPNGEEMTLKQFLDYFKTEHKLEITMLSQGVSMLYSFFMPAAKLKERLDQPMTEIVSRVSKRKLGRHVRALVLELCCNDESGEDVEVPYVRYTIR 1196EAC5EEEC06A5 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 hfr1-cop1-4 4 9 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 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role neutral component neutral component region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1543479]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 675-675, sequence length 292 [EBI-1543479]102-675 colocalization colocalization 1 Colocalization of HFR1 or HFR1( N) with COP1 in nuclear bodies hfr1-cop1-1 3 7 unspecified role unspecified role bait bait n-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) 9 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 arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress physical association physical association 2A HFR1 and COP1 interact in vitro and in vivo. hfr1-cop1-5 5 9 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag 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 arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress 7 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 n-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) physical association physical association 2B To further investigate HFR1�COP1 interaction, we carried out in vitro pull-down assays. Figure 2B shows that HFR1 interacted with the COP1 WD40 domain, but not the COP1 deletion mutant containing only the RING and coiled-coil domains, confirming results obtained with yeast two-hybrid assays. cop1-hfr1 2 9 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2C Coimmunoprecipitation of HFR1 with COP1 protein. Degradation of HFR1 was achieved using treatment of transgenic plants with the proteasomal inhibitor MG132. The coimmunoprecipitation of HFR1 was low the authors conclude that this could be explained by "(1) Only a minor proportion of the total cellular HFR1-3HA was associated with COP1-6Myc, and (2) the interaction between COP1-6Myc and HFR13HA may be weak, and did not survive the stringent wash conditions used in our experiments". cop1-hfr1-1 1 6 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) 7 enzyme target enzyme target substrate substrate neutral component neutral component 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 9 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination 3 Figure 3 shows that HFR1 was polyubiquitinated by COP1 E3 ligase in a reaction dependent on E1 and E2 activities. ubiquitination of HFR1 The source organism of the unbiquitin used in this study was not given. The protein was purchased from Sigma which list a number ubiquitin products from different source organisms. Therefore ubiquiyin does not appear in the interaction , although implied by the interaction type annotation.