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/ DEP-domain-mediated regulation of GPCR signaling responses. DEP-domain-mediated regulation of GPCR signaling responses. Cell (0092-8674) 2006 imex curation Ballon DR., Flanary PL., Gladue DP., Konopka JB., Dohlman HG., Thorner J. jthorner@berkeley.edu Only protein-protein interactions 6 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 Accepted 2006-OCT-9 by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 6 imex curation DEP-domain-mediated regulation of GPCR signaling responses. DEP-domain-mediated regulation of GPCR signaling responses. Cell (0092-8674) 2006 imex curation Ballon DR., Flanary PL., Gladue DP., Konopka JB., Dohlman HG., Thorner J. jthorner@berkeley.edu Only protein-protein interactions 6 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 Accepted 2006-OCT-9 by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 6 imex curation DEP-domain-mediated regulation of GPCR signaling responses. DEP-domain-mediated regulation of GPCR signaling responses. Cell (0092-8674) 2006 imex curation Ballon DR., Flanary PL., Gladue DP., Konopka JB., Dohlman HG., Thorner J. jthorner@berkeley.edu Only protein-protein interactions 6 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 Accepted 2006-OCT-9 by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 6 imex curation sst2_yeast Protein SST2 SST2 L9324.9 YLR452C protein protein yeast Saccharomyces cerevisiae Baker's yeast MVDKNRTLHELSSKNFSRTPNGLIFTNDLKTVYSIFLICLDLKEKKHSSDTKSFLLTAFTKHFHFTFTYQEAIKAMGQLELKVDMNTTCINVSYNIKPSLARHLLTLFMSSKLLHTPQDRTRGEPKEKVLFQPTPKGVAVLQKYVRDIGLKTMPDILLSSFNSMKLFTFERSSVTDSIIHSDYLIHILFIKMMGAKPNVWSPTNADDPLPCLSSLLEYTNNDDTFTFEKSKPEQGWQAQIGNIDINDLERVSPLAHRFFTNPDSESHTQYYVSNAGIRLFENKTFGTSKKIVIKYTFTTKAIWQWIMDCTDIMHVKEAVSLAALFLKTGLIVPVLLQPSRTDKKKFQISRSSFFTLSKRGWDLVSWTGCKSNNIRAPNGSTIDLDFTLRGHMTVRDEKKTLDDSEGFSQDMLISSSNLNKLDYVLTDPGMRYLFRRHLEKELCVENLDVFIEIKRFLKKMTILKKLIDSKHCDKKSNTSTSKNNIVKTIDSALMKQANECLEMAYHIYSSYIMIGSPYQLNIHHNLRQNISDIMLHPHSPLSEHFPTNLYDPSPASAESAASSISSTEADTLGEPPEVSLKPSKNLSNENCSFKKQGFKHQLKEYKPAPLTLAETHSPNASVENSHTIVRYGMDNTQNDTKSVESFPATLKVLRKLYPLFEIVSNEMYRLMNNDSFQKFTQSDVYKDASALIEIQEKC 48CF5BB6E456C11C ste2_yeasx Pheromone alpha factor receptor STE2 protein protein yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast MSDAAPSLSNLFYDPTYNPGQSTINYTSIYGNGSTITFDELQGLVNSTVTQAIMFGVRCGAAALTLIVMWMTSRSRKTPIFIINQVSLFLIILHSALYFKYLLSNYSSVTYALTGFPQFISRGDVHVYGATNIIQVLLVASIETSLVFQIKVIFTGDNFKRIGLMLTSISFTLGIATVTMYFVSAVKGMIVTYNDVSATQDKYFNASTILLASSINFMSFVLVVKLILAIRSRRFLGLKQFDSFHILLIMSCQSLLVPSIIFILAYSLKPNQGTDVLTTVATLLAVLSLPLSSMWATAANNASKTNTITSDFTTSTDRFYPGTLSSFQTDSINNDAKSSLRSRLYDLYPRRKETTSDKHSERTFVSETADDIEKNQFYQLPTPTSSKNTRIGPFADASYKEGEVEPVDMYTPDTAADEEARKFWTEDNNNL 011F604DC2A6907E ste3_yeast Pheromone a factor receptor STE3 YKL178C protein protein yeast Saccharomyces cerevisiae Baker's yeast MSYKSAIIGLCLLAVILLAPPLAWHSHTKNIPAIILITWLLTMNLTCIVDAAIWSDDDFLTRWDGKGWCDIVIKLQVGANIGISCAVTNIIYNLHTILKADSVLPDLSSWTKIVKDLVISLFTPVMVMGFSYLLQVFRYGIARYNGCQNLLSPTWITTVLYTMWMLIWSFVGAVYATLVLFVFYKKRKDVRDILHCTNSGLNLTRFARLLIFCFIIILVMFPFSVYTFVQDLQQVEGHYTFKNTHSSTIWNTIIKFDPGRPIYNIWLYVLMSYLVFLIFGLGSDALHMYSKFLRSIKLGFVLDMWKRFIDKNKEKRVGILLNKLSSRKESRNPFSTDSENYISTCTENYSPCVGTPISQAHFYVDYRIPDDPRKSQNKSKKYLFADKETDDILDEIDLKESRHIPYVTQGQSFDDEISLGGFSKVTLDYSEKLHNSASSNFEGESLCYSPASKEENSSSNEHSSENTAGP 5DBF611076B8CA91 5DBF611076B8CA91 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 8E57A9C4D5801640 sst2-ste2 1 4 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 P11972:p.Gln304Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q N Wed May 02 14:41:51 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln304asn' Tue Jun 30 17:04:46 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln304Asn' yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 5 unspecified role unspecified role prey prey yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association endogenous Ste2 detergent solubilized from purified plasma membranes, regardless of its state of N-glycosylation, binds to c-Myc epitope-tagged Sst2 immobilized on beads using anti-myc mAb 9E10 (Figure 4A). Interaction was specific because no bound species were observed if beads lacked Sst2 or if membrane extracts were prepared from ste2Dcells. Most importantly, no Ste2 was retained when beads were coated with an equivalent amount of Sst2(Q304N) 4 A No detectable Gpa1 was present in the pull-down, as judged by immunoblotting with an anti-Gpa1 antibody. ste2-sst2 1 4 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 5 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 yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association 4 B We found, second, that His6-Sst2 (produced by baculovirus expression and purified by FPLC) bound to FLAG-tagged Ste2 (detergent solubilized from plasma membranes of sst2D ste2D gpa1D ste4D cells) immobilized on beads with anti-FLAG epitope mAb The Sst2 protein was expressed in insect cells. sst2-ste2-1 3 4 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 P11972:p.Gln304Glu mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q E Wed May 02 20:08:15 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln304glu' Tue Jun 30 16:07:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln304Glu' P11972:p.Gln304Leu mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q L Wed May 02 16:44:00 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln304leu' Tue Jun 30 17:55:46 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln304Leu' P11972:p.Gln304Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q N Wed May 02 18:49:25 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln304asn' Tue Jun 30 18:57:46 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln304Asn' 5 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 physical association physical association 4 C, D Every transformant expressing Sst2-N-UbG and Ste2-C-Ub-LV showed robust reporter-gene expression at a level equivalent to that of the positive control (Figure 4D). For this control, cells were cotransformed with an Ste2-N-UbI chimera that binds Ste2-C-Ub-LV for two reasons: N-UbI interacts constitutively with C-Ub, and Ste2 self-associates to form dimers (Overton et al., 2003). Most importantly, like the negative control expressing N-UbG alone, no reporter expression was seen when Sst2-N-UbG carried any Q304 mutation sst2-ste2-2 2 5 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 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 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 K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R Wed May 02 17:02:22 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys337arg,lys352arg,lys358arg,lys374arg,lys387arg,lys400arg,lys422arg' Tue Jun 30 19:16:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Lys337Arg;Lys352Arg;Lys358Arg;Lys374Arg;Lys387Arg;Lys400Arg;Lys422Arg]' 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 S A certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain T A Fri Jul 22 11:12:45 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'ser331ala,ser338ala,ser339ala,ser356ala,ser366ala,ser385ala,ser386ala,ser398ala,thr354ala,thr355ala,thr356ala,thr363ala,thr368ala,thr382ala,thr384ala,thr389ala,thr411ala,lys337arg,lys352arg,lys358arg,lys374arg,lys387arg,lys400arg,lys422arg' Wed May 02 20:27:00 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser331ala,lys337arg,ser338ala,ser339ala,lys352arg,thr354ala,thr355ala,ser356ala,lys358arg,thr363ala,ser366ala,thr368ala,lys374arg,thr382ala,thr384ala,ser385ala,ser386ala,lys387arg,thr389ala,ser398ala,lys400arg,thr411ala,lys422arg' Tue Jun 30 18:33:37 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser331Ala;Lys337Arg;Ser338Ala;Ser339Ala;Lys352Arg;Thr354Ala;Thr355Ala;Ser356Ala;Lys358Arg;Thr363Ala;Ser366Ala;Thr368Ala;Lys374Arg;Thr382Ala;Thr384Ala;Ser385Ala;Ser386Ala;Lys387Arg;Thr389Ala;Ser398Ala;Lys400Arg;Thr411Ala;Lys422Arg]' 4 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 Q N Wed May 02 16:54:52 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln304asn' Tue Jun 30 19:53:53 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln304Asn' 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 colocalization colocalization 5 B, C, 6 C, S3 Ste2(7K-to-R)-mCherry displayed prominent decoration of the plasma membrane (Figure 5B, lower panels). Most strikingly, the Sst2-GFP signal now was also most pronounced at the plasma membrane (Figure 5B, lower panels). Thus, localization of Sst2- GFP tracked with localization of Ste2-mCherry (Figure S3), as expected for two proteins that associate rather stably and with high affinity. When identical experiments were conducted using Sst2(Q304N)-GFP (Figure 5C), green fluorescence remained exclusively in the cytosol, even in the cells expressing Ste2(7K-to-R)-mCherry 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 2 7 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 4 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 Q N Wed May 02 17:12:27 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln304asn' Tue Jun 30 18:14:44 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln304Asn' 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 colocalization colocalization 6 B In cells lacking native Ste2, GAL1-promoter-expressed Ste2(297�431)-mCherry, tethered to the plasma membrane via its fusion to Gpa1(1�10), recruited endogenously expressed Sst2-GFP to the cell membrane, especially in the bud The Ste2(297-431) fusion protein recruited Sst2 to the cell membrane, especially in the bud. sst2-ste2-4 2 6 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 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 K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R Wed May 02 19:38:10 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys424arg,lys432arg,lys453arg' Tue Jun 30 15:47:43 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Lys424Arg;Lys432Arg;Lys453Arg]' 4 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 P11972:p.Gln304Asn mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q N Wed May 02 20:10:14 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln304asn' Tue Jun 30 18:09:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln304Asn' colocalization colocalization S4 As observed for Ste2 (Figure 5B), preventing ubiquitylation and endocytosis of Ste3 with 3K-to-R mutations greatly increased the amount of Sst2 recruited to the plasma membrane The authors do not give the residue position of the 3 mutated lysines but the positions given are the 3 most c-terminal lysines.