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 yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. J. Biol. Chem. (0021-9258) 2006 imex curation Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library Only protein-protein interactions in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de Accepted 2006-SEP-13 by JYOTI. 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. J. Biol. Chem. (0021-9258) 2006 imex curation Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library 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 Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology In some studies BCS (bathophenantroline) at 3mM was included in the yeast two hybrid selection media. b.schwappach@zmbh.uni-heidelberg.de Accepted 2006-SEP-13 by JYOTI. 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. J. Biol. Chem. (0021-9258) 2006 imex curation Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology Proteins were identified by Coomassie blue staining after SDS PAGE . revisit when electrophoresis in place. Detection of protiens by SDS PAGE b.schwappach@zmbh.uni-heidelberg.de Accepted 2006-SEP-13 by JYOTI. 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. J. Biol. Chem. (0021-9258) 2006 imex curation Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library Only protein-protein interactions in vitro In vitro bn-page blue native page bn-page predetermined predetermined participant predetermined Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de Accepted 2006-SEP-13 by JYOTI. 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. J. Biol. Chem. (0021-9258) 2006 imex curation Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de Accepted 2006-SEP-13 by JYOTI. Yeast two hybrid genomic library J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. J. Biol. Chem. (0021-9258) 2006 imex curation Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library Only protein-protein interactions in vitro In vitro anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Metz J., Wachter A., Schmidt B., Bujnicki JM., Schwappach B. BioCreative - Critical Assessment of Information Extraction systems in Biology b.schwappach@zmbh.uni-heidelberg.de Accepted 2006-SEP-13 by JYOTI. 24-JAN-2007: Contacted by JYOTI. Yeast two hybrid genomic library J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation aakg_yeast 5'-AMP-activated protein kinase subunit gamma SNF4 CAT3 Regulatory protein CAT3 SCI1 Sucrose non-fermenting protein 4 YGL115W protein protein yeast Saccharomyces cerevisiae Baker's yeast MKPTQDSQEKVSIEQQLAVESIRKFLNSKTSYDVLPVSYRLIVLDTSLLVKKSLNVLLQNSIVSAPLWDSKTSRFAGLLTTTDFINVIQYYFSNPDKFELVDKLQLDGLKDIERALGVDQLDTASIHPSRPLFEACLKMLESRSGRIPLIDQDEETHREIVVSVLTQYRILKFVALNCRETHFLKIPIGDLNIITQDNMKSCQMTTPVIDVIQMLTQGRVSSVPIIDENGYLINVYEAYDVLGLIKGGIYNDLSLSVGEALMRRSDDFEGVYTCTKNDKLSTIMDNIRKARVHRFFVVDDVGRLVGVLTLSDILKYILLGSN 51B387E346EE9561 51B387E346EE9561 gef1_yeast Anion/proton exchange transporter GEF1 CLCY1 GEF1 YJR040W J1616 Voltage-gated chloride channel ClC-Y1 ClC-A CLC protein GEF1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MPTTYVPINQPIGDGEDVIDTNRFTNIPETQNFDQFVTIDKIAEENRPLSVDSDREFLNSKYRHYREVIWDRAKTFITLSSTAIVIGCIAGFLQVFTETLVNWKTGHCQRNWLLNKSFCCNGVVNEVTSTSNLLLKRQEFECEAQGLWIAWKGHVSPFIIFMLLSVLFALISTLLVKYVAPMATGSGISEIKVWVSGFEYNKEFLGFLTLVIKSVALPLAISSGLSVGKEGPSVHYATCCGYLLTKWLLRDTLTYSSQYEYITAASGAGVAVAFGAPIGGVLFGLEEIASANRFNSSTLWKSYYVALVAITTLKYIDPFRNGRVILFNVTYDRDWKVQEIPIFIALGIFGGLYGKYISKWNINFIHFRKMYLSSWPVQEVLFLATLTALISYFNEFLKLDMTESMGILFHECVKNDNTSTFSHRLCQLDENTHAFEFLKIFTSLCFATVIRALLVVVSYGARVPAGIFVPSMAVGATFGRAVSLLVERFISGPSVITPGAYAFLGAAATLSGITNLTLTVVVIMFELTGAFMYIIPLMIVVAITRIILSTSGISGGIADQMIMVNGFPYLEDEQDEEEEETLEKYTAEQLMSSKLITINETIYLSELESLLYDSASEYSVHGFPITKDEDKFEKEKRCIGYVLKRHLASKIMMQSVNSTKAQTTLVYFNKSNEELGHRENCIGFKDIMNESPISVKKAVPVTLLFRMFKELGCKTIIVEESGILKGLVTAKDILRFKRIKYREVHGAKFTYNEALDRRCWSVIHFIIKRFTTNRNGNVI 544C751402D32E19 544C751402D32E19 snf1_yeast Carbon catabolite-derepressing protein kinase SNF1 CAT1 CCR1 GLC2 PAS14 Sucrose nonfermentating protein 1 D8035.20 YDR477W protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSNNNTNTAPANANSSHHHHHHHHHHHHHGHGGSNSTLNNPKSSLADGAHIGNYQIVKTLGEGSFGKVKLAYHTTTGQKVALKIINKKVLAKSDMQGRIEREISYLRLLRHPHIIKLYDVIKSKDEIIMVIEYAGNELFDYIVQRDKMSEQEARRFFQQIISAVEYCHRHKIVHRDLKPENLLLDEHLNVKIADFGLSNIMTDGNFLKTSCGSPNYAAPEVISGKLYAGPEVDVWSCGVILYVMLCRRLPFDDESIPVLFKNISNGVYTLPKFLSPGAAGLIKRMLIVNPLNRISIHEIMQDDWFKVDLPEYLLPPDLKPHPEEENENNDSKKDGSSPDNDEIDDNLVNILSSTMGYEKDEIYESLESSEDTPAFNEIRDAYMLIKENKSLIKDMKANKSVSDELDTFLSQSPPTFQQQSKSHQKSQVDHETAKQHARRMASAITQQRTYHQSPFMDQYKEEDSTVSILPTSLPQIHRANMLAQGSPAASKISPLVTKKSKTRWHFGIRSRSYPLDVMGEIYIALKNLGAEWAKPSEEDLWTIKLRWKYDIGNKTNTNEKIPDLMKMVIQLFQIETNNYLVDFKFDGWESSYGDDTTVSNISEDEMSTFSAYPFLHLTTKLIMELAVNSQSN F5C63565C986C4E3 F5C63565C986C4E3 get3_yeast ATPase GET3 YDL100C GET3 ARR4 D2371 Arsenical pump-driving ATPase Arsenite-stimulated ATPase Guided entry of tail-anchored proteins 3 Golgi to ER traffic protein 3 protein protein yeast Saccharomyces cerevisiae Baker's yeast MDLTVEPNLHSLITSTTHKWIFVGGKGGVGKTTSSCSIAIQMALSQPNKQFLLISTDPAHNLSDAFGEKFGKDARKVTGMNNLSCMEIDPSAALKDMNDMAVSRANNNGSDGQGDDLGSLLQGGALADLTGSIPGIDEALSFMEVMKHIKRQEQGEGETFDTVIFDTAPTGHTLRFLQLPNTLSKLLEKFGEITNKLGPMLNSFMGAGNVDISGKLNELKANVETIRQQFTDPDLTTFVCVCISEFLSLYETERLIQELISYDMDVNSIIVNQLLFAENDQEHNCKRCQARWKMQKKYLDQIDELYEDFHVVKMPLCAGEIRGLNNLTKFSQFLNKEYNPITDGKVIYELEDKE F795C359B5A4A461 F795C359B5A4A461 gef1-get3 5 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association In search for proteins that would bind to the C terminus (CT) of Gef1p, we screened 6 million co-transformants of the bait plasmid and a yeast genomic two-hybrid library constructed in the prey plasmid. gef1-get3-1 2 8 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role prey prey physical association physical association Figure 1A Figure 4A FIGURE 1. Arr4p interacts with the CT of Gef1p. Interaction was not seen when 3mM BCS (bathophenantroline sulphate) (a copper chelating agent) was present in the yeast culture media. snf1-snf4 2 7 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association BCS was not used in the selection media in this study. Figure 1 A Positive control used in directed yeast two hybrid assay. although Snf4p did interact with its partner subunit Snf1p (Fig. 1A). gef1-get3-2 6 10 unspecified role unspecified role bait bait yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 8 unspecified role unspecified role prey prey n terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association Figure 1B Detergent- solubilized extracts from yeast cells expressing the N-terminally HA-tagged Gef1p channel were immunoprecipitated using an antiserum that we raised against Arr4p. gef1-get3-3 1 10 unspecified role unspecified role prey prey region Protein C tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 8 unspecified role unspecified role bait bait n terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association Figure 1C In the inverse experiment we were able to specifically coimmunoprecipitate a tagged form of Arr4p (Arr4p-4PC) with Gef1p (Fig. 1C). We conclude that Arr4p interacts with Gef1p. Gef1 protein was overexpressed in the yeast strain analysed in this study. gef1-get3-4 3 8 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region IGg A protein tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association Figure 2 B and 3A This interaction was only observed when yeast lysate, or trace mineral solution or CuSo4 (160nm) and ascorbate (10 micro M) was present in the binding assay reaction buffer. Next, we tried to reconstitute the interaction in vitro employing recombinant proteins. Arr4p N-terminally fused to a decahistidine tag was purified from E. coli by nickel chromatography. get3-get3 2 10 unspecified role unspecified role bait bait Q12154:p.[Cys285Thr;Cys288Thr] mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain C T certain certain sequence position certain certain certain sequence position certain C T Wed May 02 20:27:49 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys285thr,cys288thr' Tue Jun 30 18:32:49 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Cys285Thr;Cys288Thr]' 10 unspecified role unspecified role prey prey physical association physical association BCS was not used in the selcetion media in this study. Figure 6B homotypic Arr4p interaction assayed by the two-hybrid system. Bait and prey fusions contained the indicated double mutation. get3-get3-2 4 10 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component Q12154:p.[Cys285Thr;Cys288Thr] mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain C T certain certain sequence position certain certain certain sequence position certain C T Wed May 02 15:45:31 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys285thr,cys288thr' Tue Jun 30 20:20:04 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Cys285Thr;Cys288Thr]' physical association physical association Figure 6C We further tested this result by blue native gel electrophoresis (Fig. 6C), where recombinant Arr4p purified from E. coli migrated in two species, presumably representing the monomer (lower band) and the homodimer (upper band), which was absent for the C285T/C288T and not the C240T/C242T double mutant. gef1-get3-5 6 10 unspecified role unspecified role bait bait yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 8 unspecified role unspecified role prey prey n terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association Figure 1B Detergent- solubilized extracts from yeast cells expressing the N-terminally HA-tagged Gef1p channel were immunoprecipitated using an antiserum that we raised against Arr4p. Over-expression of Arr4p Q12154