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/ http://www.ebi.ac.uk/ ballon-2006-3 DEP-domain-mediated regulation of GPCR signaling responses. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast confocal microscopy confocal microscopy predetermined predetermined participant predetermined Ballon DR., Flanary PL., Gladue DP., Konopka JB., Dohlman HG., Thorner J. Sst2-GFP was introduced into strains in which chromosomally expressed Ste2 was fused to mCherry, a variant of mRFP. jthorner@berkeley.edu Cell (0092-8674) 2006 Only protein-protein interactions 6 imex curation ballon-2006-1 DEP-domain-mediated regulation of GPCR signaling responses. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Ballon DR., Flanary PL., Gladue DP., Konopka JB., Dohlman HG., Thorner J. jthorner@berkeley.edu Cell (0092-8674) 2006 Only protein-protein interactions 6 imex curation ballon-2006-2 DEP-domain-mediated regulation of GPCR signaling responses. 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 Ballon DR., Flanary PL., Gladue DP., Konopka JB., Dohlman HG., Thorner J. One protein was fused to an N-terminal (N-UbG) fragment defective for association with a C-terminal (C-Ub) fragment that is fused to the other protein. Interaction between the proteins forces ubiquitin reassembly. If reassembled, ubiquitin C-terminal hydrolases clip off a transcription factor (LexA-VP16) fused to the C terminus of C-Ub. Once freed, LexA-VP16 enters the nucleus and induces expression of a lacZ reporter gene with LexA binding sites. jthorner@berkeley.edu Cell (0092-8674) 2006 Only protein-protein interactions 6 imex curation ste2_yeasx Pheromone alpha factor receptor STE2 protein protein yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast MSDAAPSLSNLFYDPTYNPGQSTINYTSIYGNGSTITFDELQGLVNSTVTQAIMFGVRCGAAALTLIVMWMTSRSRKTPIFIINQVSLFLIILHSALYFKYLLSNYSSVTYALTGFPQFISRGDVHVYGATNIIQVLLVASIETSLVFQIKVIFTGDNFKRIGLMLTSISFTLGIATVTMYFVSAVKGMIVTYNDVSATQDKYFNASTILLASSINFMSFVLVVKLILAIRSRRFLGLKQFDSFHILLIMSCQSLLVPSIIFILAYSLKPNQGTDVLTTVATLLAVLSLPLSSMWATAANNASKTNTITSDFTTSTDRFYPGTLSSFQTDSINNDAKSSLRSRLYDLYPRRKETTSDKHSERTFVSETADDIEKNQFYQLPTPTSSKNTRIGPFADASYKEGEVEPVDMYTPDTAADEEARKFWTEDNNNL sst2_yeast Protein SST2 SST2 L9324.9 YLR452C protein protein yeast Saccharomyces cerevisiae Baker's yeast MVDKNRTLHELSSKNFSRTPNGLIFTNDLKTVYSIFLICLDLKEKKHSSDTKSFLLTAFTKHFHFTFTYQEAIKAMGQLELKVDMNTTCINVSYNIKPSLARHLLTLFMSSKLLHTPQDRTRGEPKEKVLFQPTPKGVAVLQKYVRDIGLKTMPDILLSSFNSMKLFTFERSSVTDSIIHSDYLIHILFIKMMGAKPNVWSPTNADDPLPCLSSLLEYTNNDDTFTFEKSKPEQGWQAQIGNIDINDLERVSPLAHRFFTNPDSESHTQYYVSNAGIRLFENKTFGTSKKIVIKYTFTTKAIWQWIMDCTDIMHVKEAVSLAALFLKTGLIVPVLLQPSRTDKKKFQISRSSFFTLSKRGWDLVSWTGCKSNNIRAPNGSTIDLDFTLRGHMTVRDEKKTLDDSEGFSQDMLISSSNLNKLDYVLTDPGMRYLFRRHLEKELCVENLDVFIEIKRFLKKMTILKKLIDSKHCDKKSNTSTSKNNIVKTIDSALMKQANECLEMAYHIYSSYIMIGSPYQLNIHHNLRQNISDIMLHPHSPLSEHFPTNLYDPSPASAESAASSISSTEADTLGEPPEVSLKPSKNLSNENCSFKKQGFKHQLKEYKPAPLTLAETHSPNASVENSHTIVRYGMDNTQNDTKSVESFPATLKVLRKLYPLFEIVSNEMYRLMNNDSFQKFTQSDVYKDASALIEIQEKC gpa1-ste2_protein Ste2(297-431) fused to Gpa1(1-10) protein protein yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast GCTVSTQTIGTAANNASKTNTITSDFTTSTDRFYPGTLSSFQTDSINNDAKSSLRSRLYDLYPRRKETTSDKHSERTFVSETADDIEKNQFYQLPTPTSSKNTRIGPFADASYKEGEVEPVDMYTPDTAADEEARKFWTEDNNNL An amino acid sequence which is not represented in UniProt ste3_yeast Pheromone a factor receptor STE3 YKL178C protein protein yeast Saccharomyces cerevisiae Baker's yeast MSYKSAIIGLCLLAVILLAPPLAWHSHTKNIPAIILITWLLTMNLTCIVDAAIWSDDDFLTRWDGKGWCDIVIKLQVGANIGISCAVTNIIYNLHTILKADSVLPDLSSWTKIVKDLVISLFTPVMVMGFSYLLQVFRYGIARYNGCQNLLSPTWITTVLYTMWMLIWSFVGAVYATLVLFVFYKKRKDVRDILHCTNSGLNLTRFARLLIFCFIIILVMFPFSVYTFVQDLQQVEGHYTFKNTHSSTIWNTIIKFDPGRPIYNIWLYVLMSYLVFLIFGLGSDALHMYSKFLRSIKLGFVLDMWKRFIDKNKEKRVGILLNKLSSRKESRNPFSTDSENYISTCTENYSPCVGTPISQAHFYVDYRIPDDPRKSQNKSKKYLFADKETDDILDEIDLKESRHIPYVTQGQSFDDEISLGGFSKVTLDYSEKLHNSASSNFEGESLCYSPASKEENSSSNEHSSENTAGP 5DBF611076B8CA91 sst2-ste2-2 541594 N/A 541597 unspecified role unspecified role neutral component neutral component n-terminal c-terminal cytoplasmic tail truncation binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false c-terminal Ste2 fused to mCherry, a variant of mRFP fluorescent prot tag fluorescent protein tag fluorescent prot tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false P0CI39:p.[Lys337Arg;Lys352Arg;Lys358Arg;Lys374Arg;Lys387Arg;Lys400Arg;Lys422Arg] 7K-to-R mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P0CI39:p.[Ser331Ala;Lys337Arg;Ser338Ala;Ser339Ala;Lys352Arg;Thr354Ala;Thr355Ala;Ser356Ala;Lys358Arg;Thr363Ala;Ser366Ala;Thr368Ala;Lys374Arg;Thr382Ala;Thr384Ala;Ser385Ala;Ser386Ala;Lys387Arg;Thr389Ala;Ser398Ala;Lys400Arg;Thr411Ala;Lys422Arg] Ste2(7K-to-R, 19S/T-to-A) mutation increasing mutation increasing interaction mutation increasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false N/A 541598 unspecified role unspecified role neutral component neutral component P11972:p.Gln304Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false 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 false colocalization colocalization false false false 5 B, C, 6 C, S3 The colocalisation of SSt2 with Ste2 requires the function of the Sst2 DEP domain. The authors do not give the position of the 7 lysines in the cytoplasmic tail of Sste2 that were mutated to prevent endocytosis. The positions given refer to the 7 most C-terminal lysines found in the UniprotKb sequence. This interaction took place in an exponentially growing culture of a Gpa1(gly302ser) mutant. In naive cells the proteins colocalise all around the perimeter of the cell. However, by the time cells form prominent shmoos after exposure to pheromone, Sst2-GFP concentrates at the tip of the shmoo and does not associate with Ste2(7K-to-R)-mCherry elsewhere in the plasma membrane. In cells exposed to pheromone and expressing Ste2(7K-to-R, 19S&T-to-A)-mCherry there was colocalisation in every cells; whereas in cells expressing Ste2(7K-to-R)-mCherry colocalisation only occurs at the shmoo tip. In cells exposed to pheromone, where Yck1- and Yck2-mediated phosphorylation was ablated by mutations, Sst2-GFP associated with Ste2(7K-toR)around the whole perimeter of the cell. The Ste2(7K-to-R, 19S/R-to-A) construct carried all 7 lys-to-arg mutations listed above and all 19 ser&thr to ala listed above. sst2-ste2-3 541594 N/A 541599 unspecified role unspecified role neutral component neutral component c-terminal mCherry ( a variant of mRFP) tagged protein fluorescent prot tag fluorescent protein tag fluorescent prot tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 541598 unspecified role unspecified role neutral component neutral component P11972:p.Gln304Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false 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 false colocalization colocalization false false false 6 B The Ste2(297-431) fusion protein recruited Sst2 to the cell membrane, especially in the bud. sst2-ste2-4 541594 N/A 541600 unspecified role unspecified role neutral component neutral component c-terminal mCherry ( a variant of mRFP) tagged protein fluorescent prot tag fluorescent protein tag fluorescent prot tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false P06783:p.[Lys424Arg;Lys432Arg;Lys453Arg] 3K-to-R mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false N/A 541598 unspecified role unspecified role neutral component neutral component 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 false P11972:p.Gln304Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false colocalization colocalization false false false S4 The authors do not give the residue position of the 3 mutated lysines but the positions given are the 3 most c-terminal lysines. sst2-ste2 541595 N/A 541598 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 false P11972:p.Gln304Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast N/A 541597 unspecified role unspecified role prey prey yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association false false false 4 A No detectable Gpa1 was present in the pull-down, as judged by immunoblotting with an anti-Gpa1 antibody. ste2-sst2 541595 N/A 541598 unspecified role unspecified role prey prey 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 false N/A 541597 unspecified role unspecified role bait bait 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 false yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association false false false 4 B The Sst2 protein was expressed in insect cells. sst2-ste2-1 541596 N/A 541598 unspecified role unspecified role unspecified role unspecified role sst2-n-ubg Sst2 fused to an N-terminal (N-UbG) fragment fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P11972:p.Gln304Glu mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P11972:p.Gln304Leu mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P11972:p.Gln304Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false N/A 541597 unspecified role unspecified role unspecified role unspecified role ste2-c-ub-lv Ste2 fused to C-terminal ubiqutin fragment and transcription factor LexA-VP16 fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false 4 C, D