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/ Rapid E2-E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Rapid E2-E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Cell (0092-8674) 2009 imex curation Kleiger G., Saha A., Lewis S., Kuhlman B., Deshaies RJ. I have not tried to capture the ubiquitination reactions in Figs. 2 + 5 as sequence/source of beta-catenin unknown. CUL1 construct is cleaved into two parts to ensure solubility. Peptides then reform by binding to Rbx1. Additional details of FRET system given in PMID: 18851830 Additional details of constructs taken from PMID: 16275325. deshaies@caltech.edu 23-Feb-2010: Contacted by JYOTI. jyoti Only protein-protein interactions 3 in vitro In vitro crosslink cross-linking study crosslink predetermined predetermined participant predetermined Kleiger G., Saha A., Lewis S., Kuhlman B., Deshaies RJ. I have not tried to capture the ubiquitination reactions in Figs. 2 + 5 as sequence/source of beta-catenin unknown. CUL1 construct is cleaved into two parts to ensure solubility. Peptides then reform by binding to Rbx1. Additional details of FRET system given in PMID: 18851830 Additional details of constructs taken from PMID: 16275325. deshaies@caltech.edu Accepted 2010-FEB-12 by JYOTI. 23-Feb-2010: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 3 imex curation Rapid E2-E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Rapid E2-E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Cell (0092-8674) 2009 imex curation Kleiger G., Saha A., Lewis S., Kuhlman B., Deshaies RJ. I have not tried to capture the ubiquitination reactions in Figs. 2 + 5 as sequence/source of beta-catenin unknown. CUL1 construct is cleaved into two parts to ensure solubility. Peptides then reform by binding to Rbx1. Additional details of FRET system given in PMID: 18851830 Additional details of constructs taken from PMID: 16275325. deshaies@caltech.edu 23-Feb-2010: Contacted by JYOTI. jyoti Only protein-protein interactions 3 in vitro In vitro fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret tag fluorescence tag visualisation by fluorescence tag fluorescence Kleiger G., Saha A., Lewis S., Kuhlman B., Deshaies RJ. I have not tried to capture the ubiquitination reactions in Figs. 2 + 5 as sequence/source of beta-catenin unknown. CUL1 construct is cleaved into two parts to ensure solubility. Peptides then reform by binding to Rbx1. Additional details of FRET system given in PMID: 18851830 Additional details of constructs taken from PMID: 16275325. deshaies@caltech.edu Accepted 2010-FEB-12 by JYOTI. 23-Feb-2010: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 3 imex curation fbw1a_human F-box/WD repeat-containing protein 1A BTRC BTRCP FBW1A FBXW1A F-box and WD repeats protein beta-TrCP E3RSIkappaB pIkappaBalpha-E3 receptor subunit Epididymis tissue protein Li 2a protein protein human Homo sapiens Human MDPAEAVLQEKALKFMCSMPRSLWLGCSSLADSMPSLRCLYNPGTGALTAFQNSSEREDCNNGEPPRKIIPEKNSLRQTYNSCARLCLNQETVCLASTAMKTENCVAKTKLANGTSSMIVPKQRKLSASYEKEKELCVKYFEQWSESDQVEFVEHLISQMCHYQHGHINSYLKPMLQRDFITALPARGLDHIAENILSYLDAKSLCAAELVCKEWYRVTSDGMLWKKLIERMVRTDSLWRGLAERRGWGQYLFKNKPPDGNAPPNSFYRALYPKIIQDIETIESNWRCGRHSLQRIHCRSETSKGVYCLQYDDQKIVSGLRDNTIKIWDKNTLECKRILTGHTGSVLCLQYDERVIITGSSDSTVRVWDVNTGEMLNTLIHHCEAVLHLRFNNGMMVTCSKDRSIAVWDMASPTDITLRRVLVGHRAAVNVVDFDDKYIVSASGDRTIKVWNTSTCEFVRTLNGHKRGIACLQYRDRLVVSGSSDNTIRLWDIECGACLRVLEGHEELVRCIRFDNKRIVSGAYDGKIKVWDLVAALDPRAPAGTLCLRTLVEHSGRVFRLQFDEFQIVSSSHDDTILIWDFLNDPAAQAEPPRSPSRTYTYISR DIP protein Q9Y297 original sequence version: 142 DIP protein Q9Y297 original sequence version: 153 DIP protein Q9Y297 original sequence version: 144 DIP protein Q9Y297 original sequence version: 145 DIP protein Q9Y297 original sequence version: 106 DIP protein Q9Y297 original sequence version: 82 DIP protein Q9Y297 original sequence version: 111 DIP protein Q9Y297 original sequence version: 125 4C67F3B7E400FD37 cul1_human Cullin-1 CUL1 protein protein human Homo sapiens Human MSSTRSQNPHGLKQIGLDQIWDDLRAGIQQVYTRQSMAKSRYMELYTHVYNYCTSVHQSNQARGAGVPPSKSKKGQTPGGAQFVGLELYKRLKEFLKNYLTNLLKDGEDLMDESVLKFYTQQWEDYRFSSKVLNGICAYLNRHWVRRECDEGRKGIYEIYSLALVTWRDCLFRPLNKQVTNAVLKLIEKERNGETINTRLISGVVQSYVELGLNEDDAFAKGPTLTVYKESFESQFLADTERFYTRESTEFLQQNPVTEYMKKAEARLLEEQRRVQVYLHESTQDELARKCEQVLIEKHLEIFHTEFQNLLDADKNEDLGRMYNLVSRIQDGLGELKKLLETHIHNQGLAAIEKCGEAALNDPKMYVQTVLDVHKKYNALVMSAFNNDAGFVAALDKACGRFINNNAVTKMAQSSSKSPELLARYCDSLLKKSSKNPEEAELEDTLNQVMVVFKYIEDKDVFQKFYAKMLAKRLVHQNSASDDAEASMISKLKQACGFEYTSKLQRMFQDIGVSKDLNEQFKKHLTNSEPLDLDFSIQVLSSGSWPFQQSCTFALPSELERSYQRFTAFYASRHSGRKLTWLYQLSKGELVTNCFKNRYTLQASTFQMAILLQYNTEDAYTVQQLTDSTQIKMDILAQVLQILLKSKLLVLEDENANVDEVELKPDTLIKLYLGYKNKKLRVNINVPMKTEQKQEQETTHKNIEEDRKLLIQAAIVRIMKMRKVLKHQQLLGEVLTQLSSRFKPRVPVIKKCIDILIEKEYLERVDGEKDTYSYLA DIP protein Q13616 original sequence version: 144 DIP protein Q13616 original sequence version: 133 DIP protein Q13616 original sequence version: null DIP protein Q13616 original sequence version: 135 DIP protein Q13616 original sequence version: 98 DIP protein Q13616 original sequence version: 110 DIP protein Q13616 original sequence version: 146 DIP protein Q13616 original sequence version: 148 DIP protein Q13616 original sequence version: 100 DIP protein Q13616 original sequence version: 85 DIP protein Q13616 original sequence version: 114 DIP protein Q13616 original sequence version: 119 DIP protein Q13616 original sequence version: 136 DIP protein Q13616 original sequence version: 117 DIP protein Q13616 original sequence version: 99 DIP protein Q13616 original sequence version: 108 6625A1FFA7799BBA rbx1_mouse E3 ubiquitin-protein ligase RBX1 RING finger protein 75 RING-box protein 1 Rbx1 E3 ubiquitin-protein transferase RBX1 protein protein mouse Mus musculus Mouse MAAAMDVDTPSGTNSGAGKKRFEVKKWNAVALWAWDIVVDNCAICRNHIMDLCIECQANQASATSEECTVAWGVCNHAFHFHCISRWLKTRQVCPLDNREWEFQKYGH 30FC5ADF66096C0E ub2r1_human Ubiquitin-conjugating enzyme E2 R1 Ubiquitin-protein ligase R1 Ubiquitin-conjugating enzyme E2-32 kDa complementing Ubiquitin-conjugating enzyme E2-CDC34 CDC34 UBE2R1 UBCH3 (E3-independent) E2 ubiquitin-conjugating enzyme R1 E2 ubiquitin-conjugating enzyme R1 protein protein human Homo sapiens Human MARPLVPSSQKALLLELKGLQEEPVEGFRVTLVDEGDLYNWEVAIFGPPNTYYEGGYFKARLKFPIDYPYSPPAFRFLTKMWHPNIYETGDVCISILHPPVDDPQSGELPSERWNPTQNVRTILLSVISLLNEPNTFSPANVDASVMYRKWKESKGKDREYTDIIRKQVLGTKVDAERDGVKVPTTLAEYCVKTKAPAPDEGSDLFYDDYYEDGEVEEEADSCFGDDEDDSGTEES DIP protein P49427 original sequence version: 101 DIP protein P49427 original sequence version: 134 258960666B589DB3 skp1_human S-phase kinase-associated protein 1 SKP1 EMC19 OCP2 SKP1A TCEB1L Cyclin-A/CDK2-associated protein p19 p19skp1 RNA polymerase II elongation factor-like protein Organ of Corti protein 2 Organ of Corti protein II SIII Transcription elongation factor B polypeptide 1-like protein protein human Homo sapiens Human MPSIKLQSSDGEIFEVDVEIAKQSVTIKTMLEDLGMDDEGDDDPVPLPNVNAAILKKVIQWCTHHKDDPPPPEDDENKEKRTDDIPVWDQEFLKVDQGTLFELILAANYLDIKGLLDVTCKTVANMIKGKTPEEIRKTFNIKNDFTEEEEAQVRKENQWCEEK C794D62AFB75528A cdc34-cul1-1 2 5 unspecified role unspecified role ancillary ancillary n-terminal 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 ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept 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 ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role fluorescence donor fluorescence donor region Lumio Green fluorescent dye fluorescent dye label fluorescent dye undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association pH 7.5, temp=20-22C Using a FRET-based read-out (Saha and Deshaies, 2008), we measured a dissociation rate constant (koff) of 35 +/- 1 s-1 and a Kd of 7.4 +/- 0.7x10−8 M for the Cdc34-RCCFP (Rbx1 plus Cul1-CFP) complex at 50 mM NaCl Fig. 1, Supp. Fig. 2, Supp. Fig. 3 Neddylation of the Rbx1-CUL1 complex had minimal effect on kinetic parameters. Pre-loading of the CDC34 E2 protein with ubiquitin also had minimal kinetic effect. Increasing the ionic strength from 10 mM to 100 mM resulted in a 470-fold change in KD which was unaffected by the N- or C-terminal positioning of the Lumino Green tag on CDC34. cdc34-skp1-1 2 7 unspecified role unspecified role ancillary ancillary trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. 3 unspecified role unspecified role ancillary ancillary n-terminal 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 trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. 6 unspecified role unspecified role fluorescence donor fluorescence donor region Lumio Green fluorescent dye fluorescent dye label fluorescent dye undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept 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 trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. physical association physical association pH 7.5, temp=20-22C Supp. Fig. 2 Koff=27sec-1 cdc34-cul1-2 1 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role neutral component neutral component c-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) direct interaction direct interaction The arrow indicates the crosslinked product between Cul1 CTD and WT Cdc34. Fig. 4D