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 architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. Cell (0092-8674) 2006 imex curation Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. bhoh@postech.ac.kr,msacher@alcor.concordia.ca 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions 17 in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. bhoh@postech.ac.kr,msacher@alcor.concordia.ca Accepted 2006-DEC-8 by DAVET. 21-Dec-2006: Contacted by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 17 imex curation The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. Cell (0092-8674) 2006 imex curation Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. bhoh@postech.ac.kr,msacher@alcor.concordia.ca 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions 17 ecoli Escherichia coli (strain K12) electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy predetermined predetermined participant predetermined Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. The compelxes were negatively stained with uranyl formate. bhoh@postech.ac.kr,msacher@alcor.concordia.ca Accepted 2006-DEC-8 by DAVET. 21-Dec-2006: Contacted by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 17 imex curation The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. Cell (0092-8674) 2006 imex curation Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. bhoh@postech.ac.kr,msacher@alcor.concordia.ca 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions 17 in vitro In vitro pull down pull down weight identificat confirmation by molecular weight weight identificat Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. Molecular weight of the purified proteins participating in interaction determined by SDS PAGE. bhoh@postech.ac.kr,msacher@alcor.concordia.ca Accepted 2006-DEC-8 by DAVET. 21-Dec-2006: Contacted by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 17 imex curation The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. Cell (0092-8674) 2006 imex curation Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. bhoh@postech.ac.kr,msacher@alcor.concordia.ca 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions 17 in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight identificat confirmation by molecular weight weight identificat Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. Subcomplexes were formed by expressing individual proteins or heterodimers, mixing them in vitro, and purifying the subcomplex by size-exclusion chromatography. Molecular weight of the proteins participating in interaction determined by SDS PAGE. bhoh@postech.ac.kr,msacher@alcor.concordia.ca Accepted 2006-DEC-8 by DAVET. 21-Dec-2006: Contacted by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 17 imex curation The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. Cell (0092-8674) 2006 imex curation Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. bhoh@postech.ac.kr,msacher@alcor.concordia.ca 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions 17 ecoli Escherichia coli (strain K12) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight identificat confirmation by molecular weight weight identificat Kim YG., Raunser S., Munger C., Wagner J., Song YL., Cygler M., Walz T., Oh BH., Sacher M. Molecular weight of the proteins participating in interaction determined by SDS PAGE. bhoh@postech.ac.kr,msacher@alcor.concordia.ca Accepted 2006-DEC-8 by DAVET. 21-Dec-2006: Contacted by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 17 imex curation tppc2_mouse Trafficking protein particle complex subunit 2 Trappc2 Sedl Sedlin protein protein mouse Mus musculus Mouse MSGSFYFVIVGHHDNPVFEMEFLPPGKAESKDDHRHLNQFIAHAALDLVDENMWLSNNMYLKTVDKFNEWFVSAFVTAGHMRFIMLHDVRQEDGIKNFFTDVYDLYIKFAMNPFYEPNSPIRSSAFDRKVQFLGKKHLLS 6EE89427D223FCB0 tppc5_mouse Trafficking protein particle complex subunit 5 Trappc5 protein protein mouse Mus musculus Mouse MEARFTRGKSALLERALVRPRTEVSLSAFALLFSELVQHCQSRVFSVAELQARLAALGRQVGARVLDALVAREKGARRETKVLGALLFVKGAVWKALFGKEADKLEQANDDARTFYIIEREPLINTYISVPKENSTLNCASFTAGIVEAVLTHSGFPAKVTAHWHKGTTLMIKFEEAVIARDRALEGR AA5E3CA45DDD02C9 trs31_yeast Trafficking protein particle complex subunit 31 TRS31 YDR472W Transport protein particle 31 kDa subunit protein protein yeast Saccharomyces cerevisiae Baker's yeast MSQRIIQPSASDQQFPGKSDGYEYTVGPKQAITSEASTTYIPSRIYSESLLFKRQEASLSAMAFLFQEMISQLHRTCKTAGDFETKLSDYGHNIGIRLLELLNFRASVSPSSLPRASAFLSQNESSSKLSNASNSPGMLANSSTATSASANERLQEKQTESLSNYITKMRRRDLKILDILQFIHGTLWSYLFNHVSDDLVKSSERDNEYMIVDNFPTLTQFIPGENVSCEYFVCGIIKGFLFNAGFPCGVTAHRMPQGGHSQRTVYLIQFDRQVLDREGLRFG 857BBA3E03423D42 857BBA3E03423D42 tppc3_mouse Trafficking protein particle complex subunit 3 BET3 homolog Trappc3 Bet3 protein protein mouse Mus musculus Mouse MSRQANRGTESKKMSSELFTLTYGALVTQLCKDYENDEDVNKQLDRMGYNIGVRLIEDFLARSNVGRCHDFRETADVIAKVAFKMYLGITPSITNWSPAGDEFSLILENNPLVDFVELPDNHSALIYSNLLCGVLRGALEMVQMAVEAKFVQDTLKGDGVTEIRMRFIRRIEDNLPAGEE B8D57A33D68C0D94 trs31_brare_protein Trs31 homologue protein protein danre Danio rerio (Zebrafish) Brachydanio rerio Zebrafish An amino acid sequence which is not represented in UniProt Similar to Q9P7N9 Similar to Q03337 trs20_yeast Trafficking protein particle complex subunit 20 TRS20 YBR254C YBR1722 Transport protein particle 20 kDa subunit protein protein yeast Saccharomyces cerevisiae Baker's yeast MPQYFAIIGKKDNPVYEIEFTNAENPQGFPQDLKELNPFILHASLDIVEDLQWQINPTSQLNGNGGNGSNGGGGFLRSRAVNNTDNCYLGKVDHFYGLAITAYISYSGMKFVMIHGNSANSSVVIDDNNMRSFYQEVHELYVKTLMNPFYKITDPIRSPAFDSRVRTLARKHLSK 82285CAEAC2735D9 82285CAEAC2735D9 trs33_yeast Trafficking protein particle complex subunit 33 TRS33 YOR115C O3251 YOR3251C Transport protein particle 33 kDa subunit protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSTHSNNVGHPQSSPQGPLTEQQRAQQQYQIFENSLPKVSQSVYQMLLNEMVPLAMGIERQISGDVISSDSNVTSENGNINNMIKRLKIEEHHTVDIIRSHNLIHELYKADEEEKEKVLARLRNIGFQIGLKLSELLIFSNNPNLKFKEMDLLLIMKFICRDVWKQIFGKQIDNLKTNHRGTFYLLDYDYRPIQSFSLEEDAKNEELKMIEPFLEIPVGIIRGVLSSLGYSSEEVICLASFIDRPTDRPKTAFPKGVSFHVQVTMPQ 2480F69D7A48D363 2480F69D7A48D363 bet3_yeast Trafficking protein particle complex subunit BET3 YKR068C BET3 Transport protein particle 22 kDa subunit protein protein yeast Saccharomyces cerevisiae Baker's yeast MVSTTQSRSLKAMGEEIWKNKTEKINTELFTLTYGSIVAQLCQDYERDFNKVNDHLYSMGYNIGCRLIEDFLARTALPRCENLVKTSEVLSKCAFKIFLNITPNITNWSHNKDTFSLILDENPLADFVELPMDAMKSLWYSNILCGVLKGSLEMVQLDCDVWFVSDILRGDSQTEIKVKLNRILKDEIPIGED C802D8A3563DE82B C802D8A3563DE82B tppc4_human Trafficking protein particle complex subunit 4 Synbindin TRS23 homolog Hematopoietic stem/progenitor cell protein 172 TRAPPC4 SBDN CGI-104 HSPC172 PTD009 protein protein human Homo sapiens Human MAIFSVYVVNKAGGLIYQLDSYAPRAEAEKTFSYPLDLLLKLHDERVLVAFGQRDGIRVGHAVLAINGMDVNGRYTADGKEVLEYLGNPANYPVSIRFGRPRLTSNEKLMLASMFHSLFAIGSQLSPEQGSSGIEMLETDTFKLHCYQTLTGIKFVVLADPRQAGIDSLLRKIYEIYSDFALKNPFYSLEMPIRCELFDQNLKLALEVAEKAGTFGPGS C6ADE525B7CFB731 bet5_yeast Trafficking protein particle complex subunit BET5 YML077W BET5 Transport protein particle 18 kDa subunit protein protein yeast Saccharomyces cerevisiae Baker's yeast MGIYSFWIFDRHCNCIFDREWTLASNSASGTINSKQNEEDAKLLYGMIFSLRSITQKLSKGSVKNDIRSISTGKYRVHTYCTASGLWFVLLSDFKQQSYTQVLQYIYSHIYVKYVSNNLLSPYDFAENENEMRGQGTRKITNRNFISVLESFLAPMVNQ 4821217270F516E9 4821217270F516E9 trs23_yeast Trafficking protein particle complex subunit 23 TRS23 YDR246W YD8419.13 Transport protein particle 23 kDa subunit protein protein yeast Saccharomyces cerevisiae Baker's yeast MAIETILVINKSGGLIYQRNFTNDEQKLNSNEYLILASTLHGVFAIASQLTPKALQLTQQTNIENTIPYIPYVGMSSNRSDTRNGGGNNNKHTNNEKLGSFKGDDFFKEPFTNWNKSGLRQLCTDQFTMFIYQTLTGLKFVAISSSVMPQRQPTIATTDKPDRPKSTSNLAIQIADNFLRKVYCLYSDYVMKDPSYSMEMPIRSNLFDEKVKKMVENLQ 8B32B1D5FE3846ED 8B32B1D5FE3846ED tpc6a_human Trafficking protein particle complex subunit 6A TRAPPC6A HSPC289 protein protein human Homo sapiens Human MADTVLFEFLHTEMVAELWAHDPDPGPGGQKMSLSVLEGMGFRVGQALGERLPRETLAFREELDVLKFLCKDLWVAVFQKQMDSLRTNHQGTYVLQDNSFPLLLPMASGLQYLEEAPKFLAFTCGLLRGALYTLGIESVVTASVAALPVCKFQVVIPKS E7AF768013E7E031 bet3-bet5-2 5 12 unspecified role unspecified role neutral component neutral component 15 unspecified role unspecified role neutral component neutral component 13 unspecified role unspecified role neutral component neutral component 16 unspecified role unspecified role neutral component neutral component physical association physical association When each trimeric complex was coexpressed with Trs23p, stable tetrameric complexes of Bet3p-Trs33p-Bet5p-Trs23p and Bet3p-Trs31p-Trs20p-Trs23p were formed (Figure 1D), and both exhibited a 1:1:1:1 stoichiometry. 1D bet3-trs31-1 5 16 unspecified role unspecified role neutral component neutral component 11 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component 13 unspecified role unspecified role neutral component neutral component physical association physical association 1D When each trimeric complex was coexpressed with Trs23p, stable tetrameric complexes of Bet3p-Trs33p-Bet5p-Trs23p and Bet3p-Trs31p-Trs20p-Trs23p were formed (Figure 1D), and both exhibited a 1:1:1:1 stoichiometry. bet3-trs31-2 5 13 unspecified role unspecified role neutral component neutral component 11 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component physical association physical association 1C upon coexpression of TRAPP I subunits, stable Bet3p-Trs33p-Bet5p and Bet3p-Trs31p-Trs20p complexes were produced (Figure 1C). Quantitation of the bands on the SDS gels indicated a 1:1:1 stoichiometry for both subcomplexes. trappc5-trappc6a 3 9 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 17 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 7 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 ecoli Escherichia coli (strain K12) association association 1A GST-bet5 fusion protein interacted with the bet3-trs33 heterodimer trappc5-trappc4 3 14 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 7 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction 1A GST-bet5 fusion protein interacted with the bet3-trs33 heterodimer and trs23 trappc2-trappc3 3 10 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 6 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 ecoli Escherichia coli (strain K12) association association 1A Quantitation of the bands on the gel indicates a 1:1:1 stoichiometry for the bet3-trs31-sedlin complex trappc5-trappc4-1 3 14 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 ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction 1A trappc2-trappc3-1 4 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association 1B Quantitation of the bands on the gel indicates a 1:1:1 stoichiometry for the bet3-trs31-sedlin complex and a 1:1:2 stoichiometry for the bet3-trs33-bet5 complex, consistent with their molecular sizes on a size-exclusion column (51 kDa and 71 kDa, respectively; (1B) Complex was 51kDa in size. trappc5-trappc6a-1 4 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 17 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association 1B Incubation of trs23 with the bet3-trs33-bet5 complex resulted in the production of a four-subunit complex. Gel filtration followed by electrophoretic analysis of the mixture indicated that one copy of bet5 in the three-subunit complex was slowly replaced by trs23 (Figure 1B). A time dependent electrophoretic analysis of the mixture indicated that one copy of bet5 in the three-subunit complex was slowly replaced by trs23. trappc5-trappc6a-2 4 17 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association 1B Molecular size of the complex was 71kDa. Quantitation of the bands on the gel indicates a 1:1:1 stoichiometry for the bet3-trs31-sedlin complex and a 1:1:2 stoichiometry for the bet3-trs33-bet5 complex, consistent with their molecular sizes on a size-exclusion column (51 kDa and 71 kDa, respectively; (1B) bet3-bet5-3 5 13 unspecified role unspecified role neutral component neutral component 12 unspecified role unspecified role neutral component neutral component 15 unspecified role unspecified role neutral component neutral component physical association physical association upon coexpression of TRAPP I subunits, stable Bet3p-Trs33p-Bet5p and Bet3p-Trs31p-Trs20p complexes were produced (Figure 1C). Quantitation of the bands on the SDS gels indicated a 1:1:1 stoichiometry for both subcomplexes. 1C bet3-trs31-3 5 12 unspecified role unspecified role neutral component neutral component 16 unspecified role unspecified role neutral component neutral component 13 unspecified role unspecified role neutral component neutral component 11 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component 15 unspecified role unspecified role neutral component neutral component physical association physical association 2A all six yeast proteins were coexpressed, a stable complex was formed. Quantitation of the bands on the SDS gel indicated a stoichiometry of 2:1:1:1:1:1 with respect to Bet3p:Trs33p:Trs31p:Trs23p:Trs20p:Bet5p Complex of about 170kDa formed. bet3-trs31-4 5 16 unspecified role unspecified role neutral component neutral component 15 unspecified role unspecified role neutral component neutral component 13 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component physical association physical association 2D However, we could coexpress and purify the heterotetramer Bet5p-Trs23p-Bet3p-Trs31p trappc5-trappc6a-3 1 7 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 17 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction 3B table S1 The structure of the bet3-trs33-bet5-trs23 subcomplex was determined at 2.4 � resolution. 2.4A 20.7 work, 24.9 free. trappc2-trappc3-2 1 10 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction 3A, 4A table S1 The structure of the bet3-trs31-sedlin subcomplex was determined at 2.1 � resolution. 2.1A 23.1 work, 27.7 free. trappc2-trappc3-3 1 10 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction table S1 2.6A 21.9 work, 25.6 free. bet3-trs31-5 2 11 unspecified role unspecified role neutral component neutral component 15 unspecified role unspecified role neutral component neutral component 12 unspecified role unspecified role neutral component neutral component 16 unspecified role unspecified role neutral component neutral component 13 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component physical association physical association 6 As the next step in the structural characterization of TRAPP I, we examined the morphology of rTRAPP I by single-particle electron microscopy (EM).