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/ A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. J. Bacteriol. (0021-9193) 2008 imex curation Cot SS., So AK., Espie GS. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species george.espie@utoronto.ca davet Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Cot SS., So AK., Espie GS. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species george.espie@utoronto.ca Accepted 2009-SEP-5 by DAVET. J. Bacteriol. (0021-9193) 2008 davet Only protein-protein interactions imex curation A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. J. Bacteriol. (0021-9193) 2008 imex curation Cot SS., So AK., Espie GS. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species george.espie@utoronto.ca davet Only protein-protein interactions syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) density sedimentation cosedimentation through density gradient density sedimentation western blot western blot Immuno blot Immuno blot Cot SS., So AK., Espie GS. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species Cell extracts separated on a Percol (silica particle) density gradient. george.espie@utoronto.ca Accepted 2009-SEP-5 by DAVET. J. Bacteriol. (0021-9193) 2008 davet Only protein-protein interactions imex curation A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. J. Bacteriol. (0021-9193) 2008 imex curation Cot SS., So AK., Espie GS. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species george.espie@utoronto.ca davet Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Cot SS., So AK., Espie GS. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species george.espie@utoronto.ca Accepted 2009-SEP-5 by DAVET. J. Bacteriol. (0021-9193) 2008 davet Only protein-protein interactions imex curation A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria. J. Bacteriol. (0021-9193) 2008 imex curation Cot SS., So AK., Espie GS. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species george.espie@utoronto.ca davet 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 Cot SS., So AK., Espie GS. Cyanobacteria - Interaction dataset based on Cyanobacteria proteins and related species george.espie@utoronto.ca Accepted 2009-SEP-5 by DAVET. J. Bacteriol. (0021-9193) 2008 davet Only protein-protein interactions imex curation ccmk2_syny3 Carboxysome shell protein CcmK2 ccmK2 Carbon dioxide-concentrating mechanism protein CcmK2 sll1028 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MSIAVGMIETRGFPAVVEAADSMVKAARVTLVGYEKIGSGRVTVIVRGDVSEVQASVSAGIEAANRVNGGEVLSTHIIARPHENLEYVLPIRYTEEVEQFRTY 6A59E01CD4CE54CE rbl_syny3 Ribulose bisphosphate carboxylase large chain cbbL rbcL slr0009 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MVQAKAGFKAGVQDYRLTYYTPDYTPKDTDLLACFRMTPQPGVPAEEAAAAVAAESSTGTWTTVWTDNLTDLDRYKGRCYDLEAVPNEDNQYFAFIAYPLDLFEEGSVTNVLTSLVGNVFGFKALRALRLEDIRFPVALIKTFQGPPHGITVERDKLNKYGRPLLGCTIKPKLGLSAKNYGRAVYECLRGGLDFTKDDENINSQPFMRWRDRFLFVQEAIEKAQAETNEMKGHYLNVTAGTCEEMMKRAEFAKEIGTPIIMHDFFTGGFTANTTLARWCRDNGILLHIHRAMHAVVDRQKNHGIHFRVLAKCLRLSGGDHLHSGTVVGKLEGERGITMGFVDLMREDYVEEDRSRGIFFTQDYASMPGTMPVASGGIHVWHMPALVEIFGDDSCLQFGGGTLGHPWGNAPGATANRVALEACVQARNEGRNLAREGNDVIREACRWSPELAAACELWKEIKFEFEAMDTL 45B9322482745B2B ccmm_syny3 Carboxysome assembly protein CcmM ccmM Carbon dioxide concentrating mechanism protein CcmM sll1031 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MLAKSLGWLLAVSRRNYCMGSRTALASRPWSKHLADPQIDPTAYVHSFANVVGDVRIQPGVSVAPGSSIRADEGTPFWIGGNVLIQHGVVIHGLETGRVLGDDDQEYSVWIGPGTCVAHLALVHGPVYLGANCFIGFRSTVLNARVGDGAVVMMHSLVQDVEIPPNKLVPSGAMITQQHQADSLPDVQAGDRHFVQQIAAMHGQSASPTQGTDPTVCVLPESLPAVTPVTETPYINSIDNMSINSDITNQIRSLLAQGYGIGAEHANERRFKTKSWQSCGTADGFRPDQVIATVEGWLQEFAGEYVRLIGIDQGAKRRVVEVIIQRPGDVPGSPSRGTTTTKALSSGGSGRSAVAHQTGNLAGDSANQLRALLHQGYKIGLEYASARRFKTGSWLTGGTIGSHREGEALQELNRFLADHTNEYVRIIGIDPAGKRRVAEIVVHRPNGNGNGKPSSSSSSVGYKSAPVSSAGGSSAGGLTPEVIATVRGLLANGHSIGTEHTDKRRFKAKSWDTCPTIDGGREAEVLAKLEACLADHAGEYVRIIGIDRVGKRRVLEQIIQRPGDNVVAGRSPSSSSASTSSSASSNGFGSGNGGGYSNSAVRLDNSVVTQVRSLLAQGYKIGTEHTDKRRFKAKSWQSCAPITSTHESEVLRALEGCLADHNGEYVRLLGIDPTAKRRVLETIIQRP 2CD79B6058DCCECF rbs_syny3 Ribulose bisphosphate carboxylase small subunit cbbS rbcS slr0012 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MKTLPKERRYETLSYLPPLTDQQIAKQVEFLLDQGFIPGVEFEEDPQPETHFWTMWKLPFFGGATANEVLAEVRECRSENPNCYIRVIGFDNIKQCQTVSFIVHKPNQNQGRY 20C762A777F4E623 ccml_syny3 Carboxysome shell vertex protein CcmL ccmL Carbon dioxide concentrating mechanism protein CcmL sll1030 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MQLAKVLGTVVSTSKTPNLTGVKLLLVQFLDTKGQPLERYEVAGDVVGAGLNEWVLVARGSAARKERGNGDRPLDAMVVGIIDTVNVASGSLYNKRDDGR 745B80ED084E7680 cynt_syny3 Carbonic anhydrase ccaA Carbonate dehydratase icfA slr1347 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MQRLIEGLQKFREGYFSSHRDLFEQLSHGQHPRILFICCSDSRVDPNLITQSEVGDLFVIRNAGNIIPPYGAANGGEGAAMEYALVALEINQIIVCGHSHCGAMKGLLKLNSLQEKLPLVYDWLKHTEATRRLVLDNYSHLEGEDLIEVAVAENILTQLKNLQTYPAIHSRLHRGDLSLHGWIYRIEEGEVLAYDGVLHDFVAPQSRINALEPEDEYAPHPNSPLISYDAFKVPGKERPGREKATESPAPQLSPLPGFGHLPREQAERIYRGSR 2F4F634246C0D5A9 2F4F634246C0D5A9 ccmk1_syny3 Carboxysome shell protein CcmK1 ccmK1 Carbon dioxide-concentrating mechanism protein CcmK1 sll1029 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MSIAVGMIETLGFPAVVEAADSMVKAARVTLVGYEKIGSGRVTVIVRGDVSEVQASVTAGIENIRRVNGGEVLSNHIIARPHENLEYVLPIRYTEAVEQFREIVNPSIIRR 4C3B3E207A600609 ccmk4_syny3 Carboxysome shell protein CcmK4 ccmK4 Carbon dioxide-concentrating mechanism protein CcmK4 slr1839 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MSAQSAVGSIETIGFPGILAAADAMVKAGRITIVGYIRAGSARFTLNIRGDVQEVKTAMAAGIDAINRTEGADVKTWVIIPRPHENVVAVLPIDFSPEVEPFREAAEGLNRR F483C04B1FEBC150 ccmn_syny3 Carboxysome assembly protein CcmN ccmN Carbon dioxide concentrating mechanism protein CcmN sll1032 protein protein syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) MQLPPVHSVSLSEYFVSGNVIIHETAVIAPGVILEAAPDCQITIEAGVCIGLGSVISAHAGDVKIQEQTAIAPGCLVIGPVTIGATACLGSRSTVFQQDIDAQVLIPPGSLLMNRVADVQTVGASSPTTDSVTEKKSPSTANPIAPIPSPWDNEPPAKGTDSPSDQAKESIARQSRPSTAEAAEQISSNRSPGESTPTAPTVVTTAPLVSEEVQEKPPVVGQVYINQLLLTLFPERRYFSS 2555A88B4EB8B9D4 ifca-ccmm-1 4 7 unspecified role unspecified role prey prey n-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role bait bait physical association physical association In yeast transformants expressing recombinant CcaA as bait, significant β-galactosidase reporter activity was detected in cells coexpressing, as prey, the full-length 687-amino-acid (73.1 kDa) CcmM polypeptide (CcmM73), indicating a positive protein-protein interaction. Fig. 1B ifca-ccmm-2 4 10 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association A similar positive reaction was obtained when CcaA and CcmM were alternatively coexpressed as prey and bait. Figs. 1B and 2 ccmm-ccmm-1 4 7 unspecified role unspecified role bait bait n-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 7 unspecified role unspecified role prey prey n-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 24 26 physical association physical association Fig. 1B CcmM73 also interacts with itself when the protein was coexpressed as both bait and prey, suggesting that, like CcaA, CcmM73 exists in an oligomeric state in vivo. ifca-ifca-1 4 10 unspecified role unspecified role prey prey region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role bait bait physical association physical association Figs. 1B and 2 We have previously shown that the truncation of 63 or more C-terminal amino acids led to the loss of CcaA-CcaA self-interaction (dimerization) ccmm-ccmk1-1 4 7 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey physical association physical association When CcmM73 was expressed as bait and the other carboxysome components were individually expressed as prey, significant levels of β-galactosidase activity were observed in cells expressing CcmL, CcmN, and the K1, K2, and K4 variants of CcmK (Fig. 1B). Fig. 1B ccmm-ccmk2-1 4 7 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association When CcmM73 was expressed as bait and the other carboxysome components were individually expressed as prey, significant levels of β-galactosidase activity were observed in cells expressing CcmL, CcmN, and the K1, K2, and K4 variants of CcmK (Fig. 1B). Fig. 1B ccmm-ccmk4-1 4 7 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey physical association physical association When CcmM73 was expressed as bait and the other carboxysome components were individually expressed as prey, significant levels of β-galactosidase activity were observed in cells expressing CcmL, CcmN, and the K1, K2, and K4 variants of CcmK (Fig. 1B). Fig. 1B ccmm-ccml-1 4 9 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association When CcmM73 was expressed as bait and the other carboxysome components were individually expressed as prey, significant levels of β-galactosidase activity were observed in cells expressing CcmL, CcmN, and the K1, K2, and K4 variants of CcmK (Fig. 1B). Fig. 1B ccmm-ccmn-1 4 7 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 13 unspecified role unspecified role prey prey physical association physical association When CcmM73 was expressed as bait and the other carboxysome components were individually expressed as prey, significant levels of β-galactosidase activity were observed in cells expressing CcmL, CcmN, and the K1, K2, and K4 variants of CcmK (Fig. 1B). Fig. 1B ccmm-ccmn-2 3 7 unspecified role unspecified role bait bait region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 13 unspecified role unspecified role prey prey 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 ecoli Escherichia coli (strain K12) physical association physical association Similarly, immunoblot analysis revealed that CcmM73 was also able to interact with and precipitate CcmN from E. coli protein extracts (Fig. 3, lane 4). Fig. 3 ifca-ccmm-4 1 10 unspecified role unspecified role prey prey 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) 10 unspecified role unspecified role bait bait region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association Tagged and non-tagged icfA differentiated by molecular weight. Fig. 4 ccmm-icfa-1 1 10 unspecified role unspecified role prey prey syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) 7 unspecified role unspecified role bait bait region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association When resin-bound T7 tag-CcmM73 or T7 tag-CcmM NT was used to probe the Synechocystis lysate, CcaA-specific antibodies detected the presence of the 30.7-kDa native form of CcaA associated with both tagged polypeptides (Fig. 4). Fig. 3, Fig. 4 ccmm-cbbl-1 1 7 unspecified role unspecified role prey prey syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) 8 unspecified role unspecified role prey prey syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) 6 unspecified role unspecified role prey prey syny3 Synechocystis sp. (strain PCC 6803 / Kazusa) 7 unspecified role unspecified role bait bait region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) association association In contrast, the T7 tag-CcmM CT probe did not form a complex with CcaA, but Rubisco antibodies identified a complex of RbcL and RbcS associated with the T7 tag-CcmM CT. Fig. 4 Tagged and untagged ccmM differentiated by molecular weight. ccmm-cbbl-2 2 10 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component physical association physical association In contrast, the T7 tag-CcmM CT probe did not form a complex with CcaA, but Rubisco antibodies identified a complex of RbcL and RbcS associated with the T7 tag-CcmM CT. Fig. 5A, 5B Tagged and untagged ccmM differentiated by molecular weight.