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/ STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell (0092-8674) 2009 imex curation Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu 23-Dec-2009: 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 weight by comassie molecular weight estimation by coomasie staining weight by comassie Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Purified proteins were fractionated on a Superose 6 gel filtration column. Molecular weight determination of the STIM1 tetramer was confrimed by multiangle light scattering (MALS). Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu Accepted 2009-DEC-20 by JYOTI. 23-Dec-2009: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 8 imex curation STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell (0092-8674) 2009 imex curation Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions 8 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu Accepted 2009-DEC-20 by JYOTI. 23-Dec-2009: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 8 imex curation STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell (0092-8674) 2009 imex curation Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions 8 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu Accepted 2009-DEC-20 by JYOTI. 23-Dec-2009: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 8 imex curation STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell (0092-8674) 2009 imex curation Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions 8 yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast ub reconstruction ubiquitin reconstruction ub reconstruction predetermined predetermined participant predetermined Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu Accepted 2009-DEC-20 by JYOTI. 23-Dec-2009: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 8 imex curation STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell (0092-8674) 2009 imex curation Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions 8 in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Park CY., Hoover PJ., Mullins FM., Bachhawat P., Covington ED., Raunser S., Walz T., Garcia KC., Dolmetsch RE., Lewis RS. Source of STIM1 taken from PMID: 16966423 and of ORAI1 from the work of the contributing author described in PMID:17293345 ricardo.dolmetsch@stanford.edu, rslewis@stanford.edu Accepted 2009-DEC-20 by JYOTI. 23-Dec-2009: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 8 imex curation stim1_human Stromal interaction molecule 1 STIM1 GOK protein protein human Homo sapiens Human MDVCVRLALWLLWGLLLHQGQSLSHSHSEKATGTSSGANSEESTAAEFCRIDKPLCHSEDEKLSFEAVRNIHKLMDDDANGDVDVEESDEFLREDLNYHDPTVKHSTFHGEDKLISVEDLWKAWKSSEVYNWTVDEVVQWLITYVELPQYEETFRKLQLSGHAMPRLAVTNTTMTGTVLKMTDRSHRQKLQLKALDTVLFGPPLLTRHNHLKDFMLVVSIVIGVGGCWFAYIQNRYSKEHMKKMMKDLEGLHRAEQSLHDLQERLHKAQEEHRTVEVEKVHLEKKLRDEINLAKQEAQRLKELREGTENERSRQKYAEEELEQVREALRKAEKELESHSSWYAPEALQKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIHSLVAALNIDPSWMGSTRPNPAHFIMTDDVDDMDEEIVSPLSMQSPSLQSSVRQRLTEPQHGLGSQRDLTHSDSESSLHMSDRQRVAPKPPQMSRAADEALNAMTSNGSHRLIEGVHPGSLVEKLPDSPALAKKALLALNHGLDKAHSLMELSPSAPPGGSPHLDSSRSHSPSSPDPDTPSPVGDSRALQASRNTRIPHLAGKKAVAEEDNGSIGEETDSSPGRKKFPLKIFKKPLKK DIP protein Q13586 original sequence version: 141 DIP protein Q13586 original sequence version: 99 DIP protein Q13586 original sequence version: 108 DIP protein Q13586 original sequence version: 121 DIP protein Q13586 original sequence version: 125 DIP protein Q13586 original sequence version: 100 FFB5E7CB3D68A7A6 crcm1_human Calcium release-activated calcium channel protein 1 ORAI1 CRACM1 TMEM142A Protein orai-1 Transmembrane protein 142A protein protein human Homo sapiens Human MHPEPAPPPSRSSPELPPSGGSTTSGSRRSRRRSGDGEPPGAPPPPPSAVTYPDWIGQSYSEVMSLNEHSMQALSWRKLYLSRAKLKASSRTSALLSGFAMVAMVEVQLDADHDYPPGLLIAFSACTTVLVAVHLFALMISTCILPNIEAVSNVHNLNSVKESPHERMHRHIELAWAFSTVIGTLLFLAEVVLLCWVKFLPLKKQPGQPRPTSKPPASGAAANVSTSGITPGQAAAIASTTIMVPFGLIFIVFAVHFYRSLVSHKTDRQFQELNELAEFARLQDQLDHRGDHPLTPGSHYA DIP protein Q96D31 original sequence version: 95 DIP protein Q96D31 original sequence version: 38 DIP protein Q96D31 original sequence version: 58 DIP protein Q96D31 original sequence version: 55 DIP protein Q96D31 original sequence version: 57 DIP protein Q96D31 original sequence version: 53 DIP protein Q96D31 original sequence version: 63 DIP protein Q96D31 original sequence version: 76 DIP protein Q96D31 original sequence version: 79 DIP protein Q96D31 original sequence version: 56 F85F7C06F36628D4 orai1-stim1-1 3 6 unspecified role unspecified role prey prey cad region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 7 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 region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Anti-Flag antibodies coimmunoprecipitate GFP-myc-Orai1 with Flag-myc-CAD (B) and coimmunoprecipitate YFP-CAD with Flag-myc-Orai1 (C). Fig. 3B, Supp. Fig. 8 orai1-stim1-2 3 6 unspecified role unspecified role bait bait cad region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 7 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Anti-Flag antibodies coimmunoprecipitate GFP-myc-Orai1 with Flag-myc-CAD (B) and coimmunoprecipitate YFP-CAD with Flag-myc-Orai1 (C). Fig. 3C, 5B, 5D orai1-stim1-3 4 6 unspecified role unspecified role bait bait cad region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 7 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association CAD associates with Orai1 in the split ubiquitin assay. Fig. 3F, 5A, 5C orai1-stim1-4 5 6 unspecified role unspecified role bait bait cad region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal EEYMPME tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 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 We incubated purified GST-CAD or GST alone with purified EE-Orai1-His8 and used glutathione beads to precipitate the complexes. GST-CAD coprecipitated Orai1, while GST alone did not, indicating that CAD binds directly to Orai1 in vitro (Figure 4A). Fig. 4A orai1-5 1 7 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal EEYMPME tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 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. direct interaction direct interaction On SDS-PAGE (right), the band corresponding to the dimer of EE-Orai1-His8 (arrow) was identified by western blotting. Fig. 4C stim1-5 1 6 unspecified role unspecified role neutral component neutral component cad region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction Multiangle light scattering (MALS) indicates a molecular weight of not, vert, similar58 kDa for purified CAD-His6, approximately four times the predicted mass of the monomer Fig. 4D orai1-stim1-5 1 7 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal EEYMPME tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 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 neutral component neutral component cad region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-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 In contrast to Orai1 alone, EE-Orai1-His8 purified from cells coexpressing CAD-His6 eluted largely in the void volume, consistent with the formation of a large protein complex of molecular weight >10 MDa (Figure 4C). Fig. 4C The complex was stable in 0.5 M NaCl, suggesting a high-affinity hydrophobic interaction of multiple CAD and Orai1 proteins. orai1-stim1-6 2 7 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role neutral component neutral component region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization When expressed together, STIM1 and Orai1 form colocalized puncta after store depletion Fig. 1C