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/ Identification of PamA as a PII-binding membrane protein important in nitrogen-related and sugar-catabolic gene expression in Synechocystis sp. PCC 6803. Identification of PamA as a PII-binding membrane protein important in nitrogen-related and sugar-catabolic gene expression in Synechocystis sp. PCC 6803. J. Biol. Chem. (0021-9258) 2005 imex curation Osanai T., Sato S., Tabata S., Tanaka K. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species kntanaka@iam.u-tokyo.ac.jp Yeast two-hybrid screening of 3.8x10e6 independent clones of a Synechocystis genomic library tested versus a single bait glnB Only protein-protein interactions in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting antibody detection identification by antibody antibody detection Osanai T., Sato S., Tabata S., Tanaka K. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species Fig. 3 kntanaka@iam.u-tokyo.ac.jp Accepted 2006-JUN-9 by ORCHARD. Yeast two-hybrid screening of 3.8x10e6 independent clones of a Synechocystis genomic library tested versus a single bait glnB In these experiments, rabbit antiserum against the preys GlnB and sll0985 is used. J. Biol. Chem. (0021-9258) 2005 Only protein-protein interactions imex curation Identification of PamA as a PII-binding membrane protein important in nitrogen-related and sugar-catabolic gene expression in Synechocystis sp. PCC 6803. Identification of PamA as a PII-binding membrane protein important in nitrogen-related and sugar-catabolic gene expression in Synechocystis sp. PCC 6803. J. Biol. Chem. (0021-9258) 2005 imex curation Osanai T., Sato S., Tabata S., Tanaka K. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species kntanaka@iam.u-tokyo.ac.jp Yeast two-hybrid screening of 3.8x10e6 independent clones of a Synechocystis genomic library tested versus a single bait glnB 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 Osanai T., Sato S., Tabata S., Tanaka K. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species kntanaka@iam.u-tokyo.ac.jp Accepted 2006-JUN-9 by ORCHARD. Yeast two-hybrid screening of 3.8x10e6 independent clones of a Synechocystis genomic library tested versus a single bait glnB J. Biol. Chem. (0021-9258) 2005 Only protein-protein interactions imex curation Identification of PamA as a PII-binding membrane protein important in nitrogen-related and sugar-catabolic gene expression in Synechocystis sp. PCC 6803. Identification of PamA as a PII-binding membrane protein important in nitrogen-related and sugar-catabolic gene expression in Synechocystis sp. PCC 6803. J. Biol. Chem. (0021-9258) 2005 imex curation Osanai T., Sato S., Tabata S., Tanaka K. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species kntanaka@iam.u-tokyo.ac.jp Yeast two-hybrid screening of 3.8x10e6 independent clones of a Synechocystis genomic library tested versus a single bait glnB Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Osanai T., Sato S., Tabata S., Tanaka K. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species Fig. 2 kntanaka@iam.u-tokyo.ac.jp Accepted 2006-JUN-9 by ORCHARD. Yeast two-hybrid screening of 3.8x10e6 independent clones of a Synechocystis genomic library tested versus a single bait glnB J. Biol. Chem. (0021-9258) 2005 Only protein-protein interactions imex curation glnb_syny3 Nitrogen regulatory protein P-II glnB ssl0707 PII signal transducing protein protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MKKVEAIIRPFKLDEVKIALVNAGIVGMTVSEVRGFGRQKGQTERYRGSEYTVEFLQKLKIEIVVDEGQVDMVVDKLVSAARTGEIGDGKIFISPVDSVVRIRTGEKDTEAI F9ABD0F5C173B799 p73148_syny3 sll0985 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MIIFMKFYSLPIVITSLWNLFVVRVLPTNRKNYIFRHQRLNLSQLIALTLLTLLLSLGFAQAQSAQTLLSQLPPQIDLVDPVKSDFPLQNKAFNLGFESDLSGRIGNLDYTTVRVDGEPIFLIAVPTGNASKEGDSSLSPIVYRQQRIESKFKEIVQRGFDPETLNVASSILNGAIIIQISDDKNLRPQILRTITKADADLYALTPEDLASETVDQVRNALMRARRERQPEALRRQIKVALVIVTIVVTVSLVVLFIYRWLGHRIHLLKMAINCLVKAEEEDPNFDYNYIDASIFKNIAAIPRYSFAFQILDRIAIFLYYLSAYSKTILFGNVGWGDSKKNPPLTFSLDNQKIFENILELFQGKDFYDFLKNRLKQVVFTRRILIFFLFIIWIRGSAFFLQVFPNTRRLGRQLSGTPISLTLIWLSAIILIKISDFLIESFFHTIENDYAILQTAKYTRRKIRINTVSSAVRGINLVVCIFTAFVFSLSLFDVPVATVLAGAGIIGFAVSFGSQNLIKDLIAGISNLMSDAFAINDFVLIGEFEGVVEDTNLFVTRIRCPNGDLVTIPNGAIGTVCNQTKDWSRVDYSVMVAADADPKKAIAVLQQVALGLYHDPWWHPKMLQAPELKGIEEVSHQGILIRIWLKTLPGEQWDVARELRLRVKVAFESQEIAIGVPQQQLLMTGTGTSPLYGDEKERASKDGEK 63C45B016F0AE3F3 glnb-glnb-11 2 4 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait physical association physical association GlnB is known to form a trimeric structure Yeast two-hybrid screening of 3.8x106 independent clones of a Synechocystis genomic library with Synechocystis PII as bait yielded four positive clones. One of these four clones contained the PII gene (glnB) itself, consistent with the trimeric nature of PII, and the other three clones were found to harbor the 3' terminal region of a predicted gene sll0985. glnb-sll0985-1 2 4 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey c-terminus binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain physical association physical association Yeast two-hybrid screening of 3.8x106 independent clones of a Synechocystis genomic library with Synechocystis PII as bait yielded four positive clones. One of these four clones contained the PII gene (glnB) itself, consistent with the trimeric nature of PII, and the other three clones were found to harbor the 3' terminal region of a predicted gene sll0985. glnb-sll0985-2 3 5 unspecified role unspecified role bait bait c-terminus binding region binding-associated region binding region 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 ecobb Escherichia coli (strain B / BL21) 4 unspecified role unspecified role prey prey cellulose-bp-tag fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Interaction of PII with PamA in Vitro� P73148 purified Q55247 purified The binding is disrupted by simultanous treatment with both 1 mM ATP and 1 mM 2-oxoglutarate, the treatment with only one of these two chemicals does not affect the binding. sll0985-glnb-1 1 4 unspecified role unspecified role prey prey syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) 5 unspecified role unspecified role bait bait 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 c-terminus binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) physical association physical association Both nonphosphorylated (ammonia-grown) and fully phosphorylated (nitrogen-starved) PII could interact with GST-PamA-(475�680) but not with GST (Fig. 3A). P73148 purified Q55247 cell lysate The interaction occurs independently of the phosphorylation state of PII (GlnB) glnb-sll0985-4 1 4 unspecified role unspecified role bait bait cellulose-bp-tag fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) 5 unspecified role unspecified role prey prey syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) physical association physical association Cell lysates prepared from GT cells were incubated with this filter, and endogenous PamA, interacting with the filter bound PII, was detected with the PamA antiserum. Q55247 purified P73148 cell lysate Endogenous sll0985 also binds PII (GlnB) denying an artificial interaction by the sll0985 truncation.