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/ A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1. Cell (0092-8674) 2009 imex curation Ren Y., Yip CK., Tripathi A., Huie D., Jeffrey PD., Walz T., Hughson FM. hughson@princeton.edu 29-Mar-2010: Contacted by JYOTI. jyoti Only protein-protein interactions 8 in vitro In vitro electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy predetermined predetermined participant predetermined Ren Y., Yip CK., Tripathi A., Huie D., Jeffrey PD., Walz T., Hughson FM. hughson@princeton.edu Accepted 2010-MAR-9 by JYOTI. 29-Mar-2010: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 8 imex curation A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1. Cell (0092-8674) 2009 imex curation Ren Y., Yip CK., Tripathi A., Huie D., Jeffrey PD., Walz T., Hughson FM. hughson@princeton.edu 29-Mar-2010: Contacted by JYOTI. jyoti Only protein-protein interactions 8 in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Ren Y., Yip CK., Tripathi A., Huie D., Jeffrey PD., Walz T., Hughson FM. hughson@princeton.edu Accepted 2010-MAR-9 by JYOTI. 29-Mar-2010: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 8 imex curation A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1. Cell (0092-8674) 2009 imex curation Ren Y., Yip CK., Tripathi A., Huie D., Jeffrey PD., Walz T., Hughson FM. hughson@princeton.edu 29-Mar-2010: Contacted by JYOTI. jyoti Only protein-protein interactions 8 in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Ren Y., Yip CK., Tripathi A., Huie D., Jeffrey PD., Walz T., Hughson FM. hughson@princeton.edu Accepted 2010-MAR-9 by JYOTI. 29-Mar-2010: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 8 imex curation sec20_yeast Protein transport protein SEC20 SEC20 D9719.4 YDR498C protein protein yeast Saccharomyces cerevisiae Baker's yeast MVVTFLQDLEVLQDALLNNLQKLSAISRRKESGESKHDNKDSFAAIANEHNDEEEEIEFEDLVNIIESKVSDFESVLKCSIVEMTYKYPELKLQWEKSPRYDQCDKLHIVKLDKQMNEDIYAQLVEELDFVLQFVDWFYCYRLKVKEILRQHHKRDLAWNDEKRDRAIKFHAVDYDKLHQGTSSSSSLTSTSMEKASTREKLLSKTKQLTNNLVRGNQILQSGILQSDLNLDELRAQTNSLTQIDDKYTQFETVFKKTADLVKVLENASHQEKRDVYLSLGFLLCCVSWVLWRRIFKLPVKLGLWLLFKFFKGILVTLGLVKSYAGSSSSLQAPSLVLNAPILATTTTSSATSVEPFASVSAVSSIQRAVDEAVDRIVSHDEL 9BF8C748B8A8F589 9BF8C748B8A8F589 sec39_yeast Protein transport protein SEC39 Dependent on SLY1-20 protein 3 SEC39 DSL3 YLR440C protein protein yeast Saccharomyces cerevisiae Baker's yeast MLEEQLYLLACIFASRADTRNIKKLSTRLGSQSKYLEILCVLWPELDDPKNLLFLRELEEEVQSPEGEETTDEDVIVELLESDSSLIPLIESDTTTRSNRYHELQEFISKKLNNKTLENFEEWLRERILICNEMIPETPLLYSVLWETAKSKVLSTKFIGWVEGVLKPLDHLNKRLHLIFKINEWEKMPDSELFKIIFDGVEDMQGYIGIADVIEDELAPTLSYGKKWETFITEFFNKQQFSLKSDTNYQLFIKLYYSLEKGVKDNSEASRKLQSNVVDILFHNSENLFNLSSLTHKLDELWSILSGFPDEITIEEQKTITALEMKQFMEFFIKCSTKFSFKEIFAITQEEESAQLAHFSSLCHEEFNKANEISSFLQAMYETVLDISKDDKIFTRISMDEKLYSILEILLQMNEFAYIEAIIERFDYSNNTQIYELLVKFFWHFFNNASNGLRKEPEMKKASQTLQIIQKHMSQRAGTNLTKLEVLLEISDKLSHYSINLNKSHNGARDTAFKPSNILEYRDCPLDIISNLLELNPRLYKDLPTTKSLLFGIYDSLSINREGQTGKVEVDLMVLHIDYALVNLDFGTAYELGKQVFEICQEAGQHMMKALGDEHWLTFYQMGKFVDPNWVDNEIPTEIIVLQMSILGRLLEVCPLEEVEIVTSQWSTLELELSARDLVKDKYALDGQNDNKSKVGGIAREIFHNVTNF 8ABD59328A006863 8ABD59328A006863 tip20_yeast Protein transport protein TIP20 YGL145W TIP20 TIP1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MNGIDDLLNINDRIKQVQNERNELASKLQNLKQSLASNDTEVALSEVIAQDIIEVGASVEGLEQLRAKYGDLQILNKLEKVAVQQTQMQAGVDKLDSFERQLDELAEQPPDQFTLDDVKALHSKLTSVFATVPQINNIDSQYAAYNKLKSKVTGKYNDVIIQRLATNWSNTFDQKLLEAQWDTQKFASTSVGLVKCLRENSTKLYQLSLLYLPLEEETQNGDSERPLSRSNNNQEPVLWNFKSLANNFNVRFTYHFHATSSSSKIETYFQFLNDYLAENLYKCINIFHDDCNGLTKPVIHEQFINYVLQPIRDKVRSTLFQNDLKTLIVLISQILATDKNLLNSFHYHGLGLVSLISDEVWEKWINYEVEMANRQFINITKNPEDFPKSSQNFVKLINKIYDYLEPFYDLDFDLLVRYKLMTCSLIFMNLTSSYLDYILTVDSLNETRTKEQELYQTMAKLQHVNFVYRKIKSLSSNFIFIQLTDIVNSTESKKYNSLFQNVENDYEKAMSTDMQNSIVHRIQKLLKETLRNYFKISTWSTLEMSVDENIGPSSVPSAELVNSINVLRRLINKLDSMDIPLAISLKVKNELLNVIVNYFTESILKLNKFNQNGLNQFLHDFKSLSSILSLPSHATNYKCMSLHELVKILKLKYDPNNQQFLNPEYIKTGNFTSLKEAYSIKYLKDTKIQDALYRIIYGNIL 3E561DD4C0B9C176 3E561DD4C0B9C176 sec22_yeast Protein transport protein SEC22 SEC22 SLY2 YLR268W L8479.3 TSL26 Suppressor of loss of YPT1 protein 2 protein protein yeast Saccharomyces cerevisiae Baker's yeast MIKSTLIYREDGLPLCTSVDNENDPSLFEQKQKVKIVVSRLTPQSATEATLESGSFEIHYLKKSMVYYFVICESGYPRNLAFSYLNDIAQEFEHSFANEYPKPTVRPYQFVNFDNFLQMTKKSYSDKKVQDNLDQLNQELVGVKQIMSKNIEDLLYRGDSLDKMSDMSSSLKETSKRYRKSAQKINFDLLISQYAPIVIVAFFFVFLFWWIFLK 9FA3E34EAAFB9536 9FA3E34EAAFB9536 use1_yeast Protein transport protein USE1 USE1 SLT1 Unconventional SNARE in the endoplasmic reticulum protein 1 SNARE-like tail-anchored protein 1 YGL098W protein protein yeast Saccharomyces cerevisiae Baker's yeast MAETSNDPFLSYVLSSKQLTNLNRLRRKAVTKQLGSSDDNKVSEEFLRYQHTYQREAFEYLQTKHDAHKIMESQYEQYQSSSKTRRYSIDLDSVDAVDTESQTEYPNEEFIDRNEDSEAVMELRKRLLGKGQNKGLGYETTKSVDRQIEDQDTLQQDLIQDMSKLVGSLKQGAVAFQSALDEDKQVLGAAEIGIQVASQGLMDVSGKLRKYDKSKLSYLFYITVFIFMILGLVFTFIIIQLFPAL 213ABBF0492E80EF 213ABBF0492E80EF ufe1_yeast Syntaxin UFE1 UFE1 YOR29-26 YOR075W protein protein yeast Saccharomyces cerevisiae Baker's yeast MMSDLTPIFRKYVAVIDDARNEQNGIDDHVERKQEDFGNSNETCEMFRDSFIKECARLLKFLVELNKVIKQIEKNYLDDFNMSDAEKDEFDMECRLQIQQYFKKFEFLENYEMERHNLSLKRFQSKSHRWSKILSNKNDNTKHVIHPQDIENGVYEFRLGVLRCLNLWIKYVSSKFTTIQQERLILENKMNFNSTPMPTLSNNADDFSADAIDISVSQSAPVETVQDEVKHYEETISKLTQEQLQVLETEHSELLNQKNEQLKKVETINKTILDIVNIQNELSNHLTVQSQNINLMLNNQDDIELNIKKGNKELRKAKRAAGRTAKMTTYGAIIMGVFILFLDYVG DDC164793510A897 dsl1_yeast Protein transport protein DSL1 Dependent on SLY1-20 protein 1 YNL258C N0842 DSL1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MESLFPNKGEIIRELLKDPLILKNDSKRSNGSELELDSSDLLQREAILANELNILDNLKTFLNLIKEVKTNLNILELENCYYSLQSLRKKMRNNAAYLKQSFNFQQSISTYVDTLHLELVSTLYKILTNGFWKITENSIQFTPTVEWGKDKVHIEYDTFMDFVAQQYFPKGSLDNQAWFILDMTSADSQEQVRAKLNTIMKEYMNLSRIVSMIKNSIFISGKEISYENEKNILVFSKSSSHGQHCVSTVLTSFEAVCDFMLDGLAFRDRKTLSYELGPLFNTEFTKFVKNNASIILESLDSPLKNLVSVINNKLTRLVAKSEVTNWTHSGKEIQDLLMNKQLYYNLLLDKVLESHISEIRSIFEDPKKSWQNLEVVELTTSNTNTMSEKIGKNDSDVQNEKELHNAVSKDDDWNWEVEDDDADAWGDEIDVNIDDEEEKTNQEKEKEPEEEENAWDEAWAIDENIDDASLENGKEHLKAHDVGSLDKDHIEVTQLPKLFLAISQNFKSSFADSHVDEQYFAYKYNLLQTSYMAMCTANFSHNWCQLYVDMRYLIERDEKLYRIKELTRNLLETKLNMKYRIVCQLIRHQLTEFRENERNPSWDATIEKLLPYILKEIVRPLQKIRGEEGSRYLLSFLNFLYNDCVTKEILKWQIISEVNSENLGELVSLLVNNTDIQLLAKEPSYKKMREKFATMGKFLPLHLKEIMEMFYNGDFYLFATDELIQWIELLFADTPLRRNAIDDIYEIRGTALDD 51EDB17F2C55A917 51EDB17F2C55A917 q6cus2_klula KLLA0_C02695g protein protein klula Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) Yeast Candida sphaerica MATLEKLLNDLLSDTSALSQAIEADPQFKSSDSVHPKPNSKLLEEVLREDSDLTKDLAKYNQYKILSDLLKEFNTNYELNELENCYYSLQNIHKKFKDVNISGESFRFQQSVATYIDCLHLNVLERFKEVSNMFYTISGNEMMFKKTIELDSVEFNFDEMVELFENNFFDHNMIDQTHWFIASTNLETYKDKEIVLLQEILDDTVKLNPLIKFFKEFVFDEKCIMVRAEPGKLTLATSEYNSLDQIKSYQTLTTFIENDLSKHAKESVMIHFGNVLLTELLKIIRANPELLSNDKEQTTEIISSIHDSLTKISEETEWAYDGFELNRILKDENIYTTLKFESMMQQRVIQIRSIPEEEYHELVSVQFKTDGGKYEKGEKQDLELSEKKTENGKDTESWGWNENQDSDEHDGWDEELDIDVDNVPIQVSVFVQSAAKVFTEFEQGCDTIGRSKVESIYLYKFNLLQTAFFAMVSEKVNDWTQLYKDVRYLYTENPKLLQLMELNSRRLDLNLNLIKKTIYKLVNDQLQELKDNERTPDWDITISSLLPYLKKTALPTLYKLEDNTILVALIRYIVHDLVIDNILHWRVISEKSSENLSEFIMLLLSGLEIPRLNLIETYRHSREKLGILSKILTAHLKDILEMFYEGEFFLFETDEIVQWIILLFADTPTRRDCIDEIRRVREEATD 0D094B3F20EB033B dsl1-sec39-1 3 11 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction X-ray Crystal Structure of a Dsl1-Sec39 Complex 2.6A R working 20.5%, R free 27.1 Supp. Table 1 dsl1-sec39-2 1 5 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) direct interaction direct interaction Negative stain EM of the Dsl1�Sec39 complex. Fig. 2B dsl1-sec39-3 1 10 unspecified role unspecified role neutral component neutral component dsl1_yeast binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain dsl1_yeast binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component tip20_yeast binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role neutral component neutral component sec39_yeast binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 26 21 24 22 direct interaction direct interaction Next, we examined the Tip20�Dsl1�Sec39 sample by negative stain EM Supp. Fig. 2C The Tip20-Dsl1 interaction was stabilized by an engineered disulphide bond between Tip20 L28C and Dsl1 L48C. sec39-use1 2 5 unspecified role unspecified role neutral component neutral component 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) 8 unspecified role unspecified role neutral component neutral component n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region trigger factor fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 29 31 direct interaction direct interaction The N-terminal region of Sec39 (residues 1-392) binds TF-Use1 (residues 1-212 fused to trigger factor). Fig. 3A sec39-use1-1 2 8 unspecified role unspecified role neutral component neutral component n-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region trigger factor fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction Binding of Sec39 to Use1 residues 1�35, but not to Use1 residues 1�212 (data not shown), appears to induce oligomerization as judged by performance on gel filtration. Fig. 3B, 3C tip20-sec20-2 2 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role neutral component neutral component region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Tip20 binds the N-terminal regulatory region of Sec20 (residues 1�174) but not the SNARE motif (residues 198�275 Fig. 3D, 3E use1-sec20-1 2 4 unspecified role unspecified role neutral component neutral component cytoplasmic region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component cytoplasmic region 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 unspecified role unspecified role neutral component neutral component snare region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role neutral component neutral component snare region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association To reconstitute a minimal complex containing all four SNAREs, we combined the SNARE motifs of Ufe1 and Sec22 with the full-length cytoplasmic domains of Use1 and Sec20; Fig. 4A Sec39 was added to the pre-mix to solubulise Use1 but was not shown to be present in the assembled complex. use1-sec20-2 2 8 unspecified role unspecified role neutral component neutral component cytoplasmic region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role neutral component neutral component snare region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role neutral component neutral component cytoplasmic region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component snare region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Indeed, SNARE complexes assembled in the presence of the Dsl1 complex, and together they formed a stable heptameric complex, Tip20�Dsl1�Sec39�Use1�Sec20�Ufe1�Sec22 (Figure 4C). Fig. 4C