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/ An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell (1040-4651) 2008 imex curation Hala M., Cole R., Synek L., Drdova E., Pecenkova T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler JE., Zarsky V. zarsky@ueb.cas.cz, fowler@science.oregonstate.edu 09-April-2009: Contacted by JYOTI. orchard Only protein-protein interactions arath-pollen_tube Arabidopsis thaliana pollen_tube Pollen tube confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Hala M., Cole R., Synek L., Drdova E., Pecenkova T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler JE., Zarsky V. zarsky@ueb.cas.cz, fowler@science.oregonstate.edu Accepted 2009-FEB-26 by ORCHARD. 09-April-2009: Contacted by JYOTI. Plant Cell (1040-4651) 2008 orchard Only protein-protein interactions imex curation An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell (1040-4651) 2008 imex curation Hala M., Cole R., Synek L., Drdova E., Pecenkova T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler JE., Zarsky V. zarsky@ueb.cas.cz, fowler@science.oregonstate.edu 09-April-2009: Contacted by JYOTI. orchard Only protein-protein interactions 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 Hala M., Cole R., Synek L., Drdova E., Pecenkova T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler JE., Zarsky V. zarsky@ueb.cas.cz, fowler@science.oregonstate.edu Accepted 2009-FEB-26 by ORCHARD. 09-April-2009: Contacted by JYOTI. Plant Cell (1040-4651) 2008 orchard Only protein-protein interactions imex curation An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell (1040-4651) 2008 imex curation Hala M., Cole R., Synek L., Drdova E., Pecenkova T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler JE., Zarsky V. zarsky@ueb.cas.cz, fowler@science.oregonstate.edu 09-April-2009: Contacted by JYOTI. orchard Only protein-protein interactions arath-cell-culture Arabidopsis thaliana cell culture plant_cell_culture plant cellular culture The culture conditions should always be cited in the experimental modifications annotation Either whole plant or undifferentiated plant cells maintained in culture. bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Hala M., Cole R., Synek L., Drdova E., Pecenkova T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler JE., Zarsky V. The antibodies used to detect Sec3 and Sec5 are thought likely to cross react with both isoforms of the proteins. Therefore detection of these proteins is ambiguous and they were not curated. Different ratios of total protein to detergent(dodecyl-beta-d-maltoside, DDM) during solubilization were used in this assay, as indicated in the annotations of each interaction entry. zarsky@ueb.cas.cz, fowler@science.oregonstate.edu Accepted 2009-FEB-26 by ORCHARD. 09-April-2009: Contacted by JYOTI. Plant Cell (1040-4651) 2008 orchard Only protein-protein interactions imex curation An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell (1040-4651) 2008 imex curation Hala M., Cole R., Synek L., Drdova E., Pecenkova T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler JE., Zarsky V. zarsky@ueb.cas.cz, fowler@science.oregonstate.edu 09-April-2009: Contacted by JYOTI. orchard Only protein-protein interactions arath-cell-culture Arabidopsis thaliana cell culture plant_cell_culture plant cellular culture The culture conditions should always be cited in the experimental modifications annotation Either whole plant or undifferentiated plant cells maintained in culture. molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Hala M., Cole R., Synek L., Drdova E., Pecenkova T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler JE., Zarsky V. The antibodies used to detect Sec3 and Sec5 are thought likely to cross react with both isoforms of the proteins. Therefore detection of these proteins is ambiguous and they were not curated. A three-step purification protocol was used. High-capacity affinity chromatography on polyphosphate hydroxyapatite matrix (hydroxyapatite Bio- Gel) with discontinuous phosphate elution. All fractions were subjected to SDS-PAGE, protein gel blotting, and analyzed by anti-SEC6 and anti-SEC8 antibodies. Corresponding fractions containing both SEC6 and SEC8 were collected and prepared for the second purification step by dialysis. In the second purification step, the dialyzed fractions from the affinity chromatography were loaded onto an ion exchange chromatography column (Fractogel EMD-TMAE). Anex resin was then used to isolate the exocyst complex in the buffer with almost neutral pH. Size exclusion gel chromatography was then carried out and western blotting used to identify protein components of the isolated complex. zarsky@ueb.cas.cz, fowler@science.oregonstate.edu Accepted 2009-FEB-26 by ORCHARD. 09-April-2009: Contacted by JYOTI. Plant Cell (1040-4651) 2008 orchard Only protein-protein interactions imex curation e70a1_arath Exocyst complex component EXO70A1 EXO70A1 Exocyst subunit Exo70 family protein A1 F12E4.320 At5g03540 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAVDSRMDLLSERAVLMRASLQKSQTITDNVVSILGSFDSRLSALETAMRPTQIRTHAIRKAHENIDRTLKAAEVILSQFDLLRQAETKVLKGPHEDLESYLDAIAQLRKIIRYFMSNKSFKSSDGVLNHANSLLAKAQSKLEEEFKQLLASYSKAVEPDRLFDGLPNSLRPSSDGDGGGKPHGGHHNDDAETAAYTLPILIPSRVLPLLHDLAQQMVQAGHQQQLLQIYRDTRSFVLEESLKKLGVEKLSKEDVQRMQWEVLEAKIGNWIHFMRIAVKLLFAGERQVCDQIFRGFDSLSDQCFAEVTVSSVSMLLSFGDAIARSKRSPEKLFVLLDMYEIMRELHTEIETIFKGKACLEIRDSATGLTKRLAQTAQETFGDFEEAVEKDATKTAVLDGTVHPLTSYVINYVKFLFDYQTTLKQLFLEFGNGDDSNSQLASVTMRIMQALQNNLDGKSKQYKDPALTHLFLMNNIHYMVRSVRRSEAKDLLGDDWVQRHRRIVQQHANQYKRVAWTKILQSSSAQGLTSSGGGSLEGGNSSGVSRGLLKERFKMFNMQFDELHQRQSQWTVPDTELRESLRLAVAEVLLPAYRSFLKRFGPLVESGKNPQKYIKYTAEDLERLLGELFEGKSMNEPRR 4660A819EA0C8BB2 sec10_arath Exocyst complex component SEC10 Exocyst complex component 5 T2L20.2 SEC10 At5g12370 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MTEGIRARGPRSSSVNSVPLILDIEDFKGDFSFDALFGNLVNDLLPSFLDEEADSGDGHGNIAGVDGLTNGHLRGQSAPLSSAPFFPEVDGLLSLFKDACKELVDLRKQVDGRLNTLKKEVSTQDSKHRKTLTEIEKGVDGLFESFARLDGRISSVGQTAAKIGDHLQSADAQRETASQTIDLIKYLMEFNGSPGDLMELSALFSDDSRVAEAASIAQKLRSFAEEDIGRQGASAAAGNATPGRGLEVAVANLQDYCNELENRLLSRFDAASQRRDLSTMSECAKILSQFNRGTSAMQHYVATRPMFIDVEVMNSDIRLVLGDHGSQPSPSNVARGLSALFKEITDTVRKEAATITAVFPTPNEVMAILVQRVLEQRVTGILDKILAKPSLMSPPPVQEGGLLLYLRMLAVAYERTQELAKDLRAVGCGDLDVEDLTESLFSSHKDEYPEHERASLKQLYQAKMEELRAESQQVSESSGTIGRSKGASISSSLQQISVTVVTDFVRWNEEAITRCTLFSSQPATLAANVKAIFTCLLDQVSVYITEGLERARDSLSEAAALRERFVLGRRVAAAAASAAEAAAAAGESSFKSFMVAVQRCGSSVAIVQQYFANSISRLLLPVDGAHAASCEEMSTALSKAEAAAYKGLQQCIETVMAEVDRLLSSEQKSTDYRSTDDGIASDHRPTNACIRVVAYLSRVLESAFTALEGLNKQAFLTELGNRLEKLLLTHWQKFTFNPSGGLRLKRDLNEYVGFVKSFGAPSVDEKFELLGIIANVFIVAPDSLPTLFEGSPSIRKDAQRFIQLREDYKSAKLATKLSSLWPSLS 0D02882FC81501EB sc15b_arath Exocyst complex component SEC15B SEC15B T14P8.16 At4g02350 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MQSSKGRRKVGSTTAGAGIDSAEKLDELLISSAICNGEDLGPFVRKTFGTGKPETLLHHLKFFARSKESEIEEVCKAHYQDFIHAVDDLKSLLSDVESLKSALSDSNSKLQSVAAPLLSSLDSLVEAQTVSKNVDLAIGAVTHCVRVMELVSRANQHLQSGNFYMALKCVDSIESDFMEKTPSSTLKRMLENRIPAIRSYVERKVNKEFGDWLVEIRVVSRNLGQLAIGEASAARQREEELRIKQRQAEEQSRLSLRDCVYALNEEEDDEFGSGHEGSDGGSSGGGLLGFDLTPLYRAYHIHQTLSLGDTFKQYYYNNRDLQLTSDFQIAGFFIVEDRVLRTGGGLISKLEVETLWDTAVTKMCAVLEDQFSRMQTANHLLLIKDYVSLLGVSLRRYGYAVDSLLEVLSKHRDKYHELLLSDCRKQITEALSADKFEQMLMKKEYEYSMNVLSFQLQTSEIVPAFPFIAPFSTTVPDCCRIVRSFIEDSVSFMSHGGQLDFYDVVKKYLDRLLGEVLDEALLKLISTSVHGVSQAMQVAANMAVFERACDFFFRHAAHLSGVPLRMAERGRRHFPLTKSQNTAEDTLSGMLKKKIDGFMTLLENVNWTSDDIPQGGNEYMNEVLIYLETLVSTAQQILPAKVLKRVLRDVLAHISEKIVGTLCGDLVKRLSMAAIKGLDVDIQLLDSFTENLTPLLTDKEAREMKKAFVEIRQMINLLLSSHPENFVNPVIRERSYNALDYRKVATVSEKFRDPSDSIFGTFGTRGSRQNPKNKSLDALIKRLKDVS DC91B59EA3203D49 sec6_arath Exocyst complex component SEC6 SEC6 F14O23.20 At1g71820 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MMVEDLGVEAKEAAVREVAKLLPLPELLQSISSIKADYIARQQANDAQLSTMVAEQVEQAQAGLESLSSSEKTIYELRDNFISIDKLCQECQTLIDNHDQIKLLSNARNNLNKTLKDVEGMMSISVEAAAARDSLSDDKEIVNTYERLTALDGKRRFALAAAGEEVGRLREYFEDVDRTWETFEKTLWGHVSNYYKLSKESPQTLVRALRVVEMQEILDQQLAEEAAEAEGEGAMASVANPRRPGKKSTTTSASSKGLAQQKLKVQGKGYKDKCYEQIRKAVEDRFNRLLTLVFEDLKAALEEARMIGEELGDIYDYVAPCFPPRYEIFQLMVNLYTERFIQMLRLLSDRANDLTNIEILKVTGWVVEYQENLIALGVDDSLAQVCSESGSMDPLMNAYVERMQATTKKWYMNILEADKVQPPKKTEEGKLYTPAAVDLFRILGEQVQIVRDNSTDVMLYRIALAIIQVMIDFQAAEKKRVDEPASDIGLEPLCAMINNNLRCYDLAMELSNSTLEALPQNYAEQVNFEDTCKGFLEVAKEAVHQTVRVIFEDPGVQELLVKLYQKEWCEGQVTEYLVATFGDYFTDVKMYVEERSFRRFVEACLEETVVVYVDHLLTQKNYIKEETIERMRLDEEVLMDFFREYISASKVESRIRIMSDLRELASAESLDAFTLVYSNILEHQPDCPAEVVEKLVSLREGIPRKDTKEVVQECKEIYENTLVDGNPPKTGFVFPRVKCLTASKGSMWRKLT 4A775EB27FC24CFA sec3a_arath Exocyst complex component SEC3A F16N3.18 At1g47550 SEC3A protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAKSSADDEELRRACEAAIEGTKQSIVMSIRVAKSRGVWGKSGKLGRQMAKPRVLALSVKSKGPRKKAFLRVMKYSSGGVLEPAKMYKLKHLSKVEVITNDPSGCTFTLGFDNLRSQSVAPPQWTMRNTDDRNRLLVCILNICKDVLGRLPKVVGIDIVEMALWAKDNTPVVTTQRSTEDGEPVAESVTESDLKVTVEKELVSQAEEEDMEALLGTYVMGIGEAEAFSERLKRELQALEAANVHAILESEPLVDEVLNGLEAATNIVDDMDEWLGIFNIKLRHMREDIESIETRNNKLEMQSVNNKALIEELDKVIERLRVPSEYAASLTGGSFDEADMLQNIEACEWLAKALRGLEVPNLDPIYANMRAVKEKRAELEKLKATFVRRASEFLRNYFASLVDFMVSDKSYFSQRGQLKRPDHADLRYKCRTYARLLQHLKGLDKNCLGPLRKAYCSSLNLLLRREAREFANELRASTKVSRNPTVWLEGSTGSSQNANTDTSAVSDAYAKMLTIFIPLLVDESSFFAHFMCFEVPALAPPGGAGNDKKSQSNNDDGNDDDDLGIMDIDETDKKPGKNSPDLTALNESLQDLLDGIQEDFYAVVDWAYKIDPLRCISMHGITERYLSGQKADAAGFVRLLLGDLESRVSMQFSRFVDEACHQIERNERNVRQMGVLPYIPRFAALATRMEQYIQGQSRDLVDQAYTKFVSIMFVTLEKIAQQDPKYADILLLENYAAFQNSLYDLANVVPTLAKFYHQASEAYEQACTRHISMIIYYQFERLFQFAKKIEDFMYTITPEEIPFQLGLSKVELRKMLKSSLSGVDKSIAAMYKKLQKNLASEELLPSLWDKCKKEFLDKYESFVQLVAKVYPSENVPGVTEMRGLLASM 533510830A4A5099 sec8_arath Exocyst complex component SEC8 SEC8 Exocyst complex component 4 F14P13.2 At3g10380 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MGIFNGLPVPSDKTYLREELARIDESWAAARFDSLPHVVHILTSKDREADIHILKEQSDVVEEVVDEVVHAYHGGFNKAIQNYSQILRLFSESTEKIGDLKHDLAEAKQSLGARNKQLHQLWYRSVTLRHIISLLDQIEGIAKVPSRIEKLIADKQFYAAIQVYLQSSLMLEREGLQTVGALQDVRSELTKLRGALFFKILDDLHAHLYNRGEYSSVASSIYERDDEVPTTTAVAASRMSSQPLSRRTRTLKGDSQFGVRGLTNGSYRTASNDESSSFDGHDEEDSVEHDESTADTARNGTDSKLLSHQLPPWLSDSTPDEFIEAVRKSDDPLHVKYLQTLVQCLCMLGKVAAAGAIICQKLRPTIHEIIISKIKAHLETTNLSKSACSQGDRSVAAGLHLIKGQSEAYRLSKEKPQNGISNSGTHLAVSPVSPLMAPGGKAQAAAKELLDSILDTIVKIFENHVVIGELLEMKASQHDINTPRSLPTDVNWNTESEASQATGGYTISFPLTVLQSECQQLICEILRATPEAASADAAAQTAKLAKKAPKKDKRDAPEDGLTFTFRFTDATVSISNQGADLIRQGWGKRAPNASLEGYGSAAVLPEQGIYLAASIYRPVLQFTDKITSMLPKKHSQLVNDGLLTFTENFVKDHLLPTMFVDYRKGVQQAISSAAAFRPRAHTTTYTATVEKGRPILQGLLAIDLLAKEVLGWAQAMPKFATDLVKYVQTFLERTFERCRTSYMEAVLEKLSYMLIGRHDIEKLMRLDAASACLPSTLGHAVSHSEAVGTEVELSDLFLSLRPIKQDNLIRDDNKLILLASLSDSLEYVADSIERLGQAVPRVASQAEGNSRNQAASPRNLASFADEYRKLATDCLKVLRVEMQLETVFHLQEMTNREYLEDEDAEEPDDFVISLTSQITRREEGMAPFISGEKRNYVFGGISGIAANASIKALADMRSINLFGVQQICRNTIAVEQAMAAIPYIDGETVQQNLDRVRTYFELLNMPFEALLAFIAEHDQMFTPTEYSNLLKVNVPGRDTPSDAQSRLLEILSH A8D6559FFDA0E039 at1g71820-at3g10380 2 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association Figure 6 The interaction was weaker than the murine p53 (bait) SV40 large (prey) positive control interaction. we observed three pairs of strongly interacting subunits: EXO70A1/ SEC3a, SEC15b/SEC10, and SEC6/SEC8 at3g10380-at1g71820 2 10 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association Figure 6 The interaction was weaker than the murine p53 (bait) SV40 large (prey) positive control interaction. we observed three pairs of strongly interacting subunits: EXO70A1/ SEC3a, SEC15b/SEC10, and SEC6/SEC8 at4g02350-at5g12370 2 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association Figure 6 The interaction was slightly weaker than the positive control interaction between murine p53 (bait) and SV40 (prey). we observed three pairs of strongly interacting subunits: EXO70A1/ SEC3a, SEC15b/SEC10, and SEC6/SEC8 at5g03540-att5g03540 2 9 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait physical association physical association Figure 6 The interaction was weaker than the murine p53 (bait) SV40 (prey) positive control interaction. we observed three pairs of strongly interacting subunits: EXO70A1/ SEC3a, SEC15b/SEC10, and SEC6/SEC8 at1g71820-at3g10380- 3 5 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component physical association physical association Supplementary Figure 7 After the solubilization with the mild detergent dodecyl-b-D-maltoside (DDM) in concentrations of total protein to DDM ratio of 1.33 and low NaCl concentrations (10 and 30 mM), a range of high molecular mass complexes was detected for four of the five exocyst complex components tested: SEC3, SEC6, SEC8, and EXO70A1 at1g71820-at5g03540 3 5 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component physical association physical association Supplementary Figure 7 Under the conditions of higher detergent concentrations (total protein:DDM ratio of 0.67) and no additional salt, SEC6 and EXO70A1 were predominantly present in small, rather than in high, molecular complexes This interaction was observed when total protein to detergent (dodecyl-beta-d-maltoside, DDM) ratio was 0.67. at1g71820 3 8 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component physical association physical association Supplementary Figure 7 Notably, we observed a well defined complex detected only by anti-SEC6 that could represent SEC6 dimers. This interaction was observed when total protein to detergent (dodecyl-beta-d-maltoside, DDM) ratio was 0.67. The authors demonstrated the presence of a well defined complex detected by only Sec6 antibodies, that they proposed as Sec6 homodimers. at1g71820-at3g1038-1 4 10 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component 5 unspecified role unspecified role neutral component neutral component physical association physical association Figure 5 Importantly, cofractionation of five exocyst subunits (SEC3, SEC5, SEC6, SEC8, and EXO70A1) was maintained through all three steps of purification. at1g71820-at3g1038-2 1 10 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component colocalization colocalization Figure 3 We took advantage of the distinct sources for anti-SEC8 (rabbit) versus anti-EXO70A1 and anti-SEC6 (mouse) and demonstrated colocalization of SEC8 and EXO70 proteins and SEC8 and SEC6 proteins, respectively, by double labeling at5g03540-at3g1038-1 1 10 unspecified role unspecified role neutral component neutral component 5 unspecified role unspecified role neutral component neutral component colocalization colocalization Figure 3 We took advantage of the distinct sources for anti-SEC8 (rabbit) versus anti-EXO70A1 and anti-SEC6 (mouse) and demonstrated colocalization of SEC8 and EXO70 proteins and SEC8 and SEC6 proteins, respectively, by double labeling