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/ pooley-2006-7 CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 confocal microscopy confocal microscopy immunostaining immunostaining Pooley RD., Reddy S., Soukoulis V., Roland JT., Goldenring JR., Bader DM. Synapse - Interactions of proteins with an established role in the presynapse. Tagged proteins were visualised by tag fluorescence. david.bader@vanderbilt.edu mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 Only protein-protein interactions imex curation pooley-2006-1 CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. 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 Pooley RD., Reddy S., Soukoulis V., Roland JT., Goldenring JR., Bader DM. Synapse - Interactions of proteins with an established role in the presynapse. david.bader@vanderbilt.edu mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 Only protein-protein interactions imex curation pooley-2006-2 CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Pooley RD., Reddy S., Soukoulis V., Roland JT., Goldenring JR., Bader DM. Synapse - Interactions of proteins with an established role in the presynapse. david.bader@vanderbilt.edu mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 Only protein-protein interactions imex curation pooley-2006-3 CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. 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 Pooley RD., Reddy S., Soukoulis V., Roland JT., Goldenring JR., Bader DM. Synapse - Interactions of proteins with an established role in the presynapse. david.bader@vanderbilt.edu mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 Only protein-protein interactions imex curation pooley-2006-4 CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. mouse-nih_3t3 Mus musculus Swiss NIH 3t3 embryo fibroblast cells nih_3t3 Mouse Swiss NIH embryo contact inhibited fibroblast cells confocal microscopy confocal microscopy immunostaining immunostaining Pooley RD., Reddy S., Soukoulis V., Roland JT., Goldenring JR., Bader DM. Synapse - Interactions of proteins with an established role in the presynapse. david.bader@vanderbilt.edu mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 Only protein-protein interactions imex curation pooley-2006-5 CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. mouse-c2c12 Mouse C3H muscle myoblast c2c12 Mouse C3H muscle myoblast confocal microscopy confocal microscopy immunostaining immunostaining Pooley RD., Reddy S., Soukoulis V., Roland JT., Goldenring JR., Bader DM. Synapse - Interactions of proteins with an established role in the presynapse. david.bader@vanderbilt.edu mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 Only protein-protein interactions imex curation pooley-2006-6 CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. mouse-nih_3t3 Mus musculus Swiss NIH 3t3 embryo fibroblast cells nih_3t3 Mouse Swiss NIH embryo contact inhibited fibroblast cells anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Pooley RD., Reddy S., Soukoulis V., Roland JT., Goldenring JR., Bader DM. Synapse - Interactions of proteins with an established role in the presynapse. david.bader@vanderbilt.edu mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 Only protein-protein interactions imex curation pooley-2006-8 CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Pooley RD., Reddy S., Soukoulis V., Roland JT., Goldenring JR., Bader DM. Synapse - Interactions of proteins with an established role in the presynapse. david.bader@vanderbilt.edu mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 Only protein-protein interactions imex curation q155p7_mouse Cenpf Lek1 protein protein mouse Mus musculus Mouse MSWALEEWKEGLPSRALQKIQELEGQLEKLKKEKQQRQFQLDSLEAALQKQKQKVEDGKTEGADLKRENQRLMEICEHLEKSRQKLSHELQVKESQVNLQESQLSSCKKQIEKLEQELKRCKSEFERSQQVAQSADVSLNPCSTPQKLFATPLTPSSTYEDLKEKYNKEVEERKRLEEEVKALHAKKVSLPVSQATMNHRDIARHQASSSVFPWQQENTPSRLSSDALKTPLRRDGSAAHFLGEEVSPNKSSMKTGRGDCSSLPGEPHSAQLLHQAKAQNQDLKSKMTELELRLQGQEKEMRSQVNKCQDLQLQLEKTKVELIEKERILNKTRDEVVRSTAQYDQAAAKCTTLEQKLKTLTEELSCHRQNAESAKRSLEQRIKEKEKELQEELSRQHQSFQALDSEYTQMKTRLTQELQQVKHLHSTLQLELEKVTSVKQQLERNLEEIRLKLSRAEQALQASQVAENELRRSSEEMKKENSLIRSQSEQRTREVCHLEEELGKVKVSLSKSQNFAEEMKAKNTSQEIMLRDLQEKLNQQENSLTLEKLKLALADLERQRNCSQDLLKKREHHIDQLNNKLNKIEKEFETLLSALELKKKECEELKEEKNQISFWKIDSEKLINQIESEKEILLGKINHLETSLKTQQVSPDSNERIRTLEMERENFTVEIKNLQSMLDSKMVEIKTQKQAYLELQQKSESSDQKHQKEIENMCLKANKLTGQVESLECKLQLLSSEVVTKDQQYQDLRMEYETLRDLLKSRGSSLVTNEDNQRSSEDNQRSSEDNQRGSLAFEQQPAVSDSFANVMGRKGSINSERSDCSVDGGRSPEHIAILQNRVTSLESSLESQNQMNSDLQMRCEELLQIKGEVEENLSKAEQIHQNFVAETNQCISKLQEDAAVHQNIVAETLATLESKEKELQLLKEKLEAQQTEVQKLNKNNCLLEGTLKELQLLSDTLSSEKKEMNSIISLSKKNIEELTQANEALKEVNEALEQEKMNLLQKHEKITSCIAEQERSIAELSDQYKQERLQLLQRCEETEAVLEDLRGNYKTAQENNAKLECMLSECTALCENRKNELEQLKETFAKEQQEFLTKLAFAEEQNRKLMLELEIEQQTVRSEITNTNKHSMSATDGLRQECLTLNEEQNEQQNEVSNLTHENEQLMELTQTKHDSYLAVEPVENSVKATEDEIGKSSSQYQMDIDTKDISLDSYKAQLVHLEALVRILEVQLDQSEEENKKLHLELQTIREELETKSSQDPQSQARTGLKDCDTAEEKYVSMLQELSASQNENAHLQCSLQTAVNKLNELGKMCDVLRVEKLQLESELNDSRTECITATSQMTAEVEKLVSEMKMLNHESALSQNELMKDTSGGEFHDKANHSSVFLTPLDSSNFCEQMTLSSKEVRVHFAELQEKFSCLQSEHKILHDQHCEVSSKMSALRSYVDTLKAENSALSMSLRTLQGDLVKEGEPAAEGGHGLPLSFCGADSPSLTNFGETSFYKDVLEQTGDTCHLSLEGNASANSCDLDEEFSSSLEEETLTEKESPPAPGRTVEGLEVLCQVYLQSLKNLEEKTESQRIMKNKEIEELEQLLSSERKELSCLRKQYLSEKEQWQQKLTSVTLEMESKLAEEKQQTKTLSLELEVARLQLQELDLSSRSLLGTDLESVVRCQNDNYDIKESEVYISETTEKTPKQDTDQTCDKDIQQDLGLETSVTESETTRLTGEGCEEQPPKTNCEAPAEDKTQDCSECISELCSSSNVLVPMDVLEDQGSIQNLQLQKDTLNENLRLLPEVEDWDKKVESLLNEIMEADSKLSLQEVQLKMKIATCIQLEKIVKDLRKEKADLSEKLESLPCNQEVCLRVERSEEDLGFNLDMGANELLSKSTKDNATNTEDNYKEKFLDMERELTRIKSEKANIEHHILSVETNLEVVQAEKLCLERDTESKQKVIIDLKEELFTVISERNRLREELDNVSKESKALDQMSKKMKEKIEELESHQRESLRHIGAVESEVKDKADLIQTLSFNVGELTKDKAHLQEQLQNLQNDSQELSLAIGELEIQIGQLNKEKESLVKESQNFQIKLTESECEKQTISKALEVALKEKGEFAVQLSSAQEEVHQLRRGIEKLSVRIEADEKKHLSAVAKLKESQRESDSLKDTVETLERELERSEENQELAILDSENLKAEVETLKAQKDEMTKSLRIFELDLVTVRTERENLAKQLQEKQSRVSELDERCSSLRRLLEEKEQARVQMEEDSKSAMLMLQMQLKELREEVAALCNDQETLKAQEQSLDQPGEEVHHLKSSIRKLKVHIDADEKKHQNILEQLKESKHHADLLKDRVENLEQELILSEKNMIFQAEKSKAEIQTLKSEIQRMAQNLQDLQLELISTRSENENLIKELKKEQERVSDLETINPSIENLLKDKEQEKVQMKEEAKITVEMLQTQLKELNETVVSLCNDQEVSKTKEQNLGSQVQTLELEKAQLLQDLGEAKNKYIIFQSSVNALTQEVEAGKQKLEKGEKEIRTLKEQLKSQEQLVCKLAQVEGEQELWQKQKLELRNVTMALEQKVQVLQSENNTLQSTYEALQNSHKSLESELGLIKLEKVALVERVSTISGKEAELQRELRDMLQKTTQLSEDYNKEKNRLTEEVEVLREELQNTKAAHLKSVNQLEKELQRAQGKIKLMLKSCRQLEGEKEMLQKELSQLEAAQQQRAGSLVDSNVDEVMTENKALKETLEEKVKEADKYLDKYCSLLISHEELEKAKEILEIEVARLKSRQSRQDLQSSPLLNSSIPGPSPNTSVSEMKSASGQNKASGKRQRSSGVWEHGKRAAPSTAETFSKKSRKSDSKSTRPAEHEQETEFEPEGLPEVVKKGFADIPTGKTSPYILRRTTMATRTSPRFATQKLVGSSPSLGKENVVESSKPTAGGSRSQKVKVVQESSADSHTAFQELPAKSLTASNIPGRNSTESPREGLRAKRAYPASSPAAGPDPTNNENCRVQ snp25_mouse Synaptosomal-associated protein 25 Synaptosomal-associated 25 kDa protein Super protein Snap25 Snap protein protein mouse Mus musculus Mouse MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG q9xsv7_chlae rab11 protein protein chlae Chlorocebus aethiops Cercopithecus aethiops Grivet Green monkey AGSNIVIMLVGNKSDLRHLRAVPTDEARAFAEKNGLSFIETSALDSTNVEAAFQTILTEIYRIVSQKQMSDRRENDMSPSNNVVPIHVPPTTENKPKVQCCQNI stx4_cerae_protein Syntaxin-4 protein protein chlae Chlorocebus aethiops Cercopithecus aethiops Grivet Green monkey An amino acid sequence which is not represented in UniProt Similar to STX4_HUMAN (Q12846). No sequence avaiilable yet. vamp2_cerae_protein Vesicle-associated membrane protein 2 protein protein chlae Chlorocebus aethiops Cercopithecus aethiops Grivet Green monkey An amino acid sequence which is not represented in UniProt Similar to VAMP2_HUMAN (P63027). No sequence available myo5b_cerae_protein Myosin-Vb protein protein chlae Chlorocebus aethiops Cercopithecus aethiops Grivet Green monkey An amino acid sequence which is not represented in UniProt Similar to MYO5B_HUMAN (Q9ULV0). No sequence available. cenpf-snap25-7 Colocalization of of 5'LSD of cenpf and snap25 in the cytoplasm and perinuclear region of cos-7 cells 793167 n/a 793175 unspecified role unspecified role neutral component neutral component n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false n/a 793176 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 colocalization colocalization false false false Fig 3, Fig 4a-i, Fig 6. Coexpression of the cytLEK SNAP25 binding domain (5' LSD) and GFP-SNAP-25 demonstrated an intense overlap in a perinuclear locus. These findings demonstrate a specific interaction between 5'LSD and GFP-SNAP-25, which is not a consequence of simple protein overexpression. cenpf-snap25rab11a Colocalization of of 5'LSD of cenpf, snap25 and rab11a in the cytoplasm and perinuclear region of cos-7 cells 793167 n/a 793176 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 n/a 793175 unspecified role unspecified role neutral component neutral component n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false n/a 793177 unspecified role unspecified role neutral component neutral component colocalization colocalization false false false Fig 4a. The endonenous SNARE protein RAB11a colocalized in the perinuclear region in cotransfected cells coexpressed with the cytLEK SNAP-25 binding domain (5'LSD) and GFP-SNAP-25. cenpf-snap25stx4 Colocalization of of 5'LSD of cenpf, snap25 and syntaxin 4 in the cytoplasm and perinuclear region of cos-7 cells 793167 n/a 793178 unspecified role unspecified role neutral component neutral component n/a 793176 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 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 n/a 793175 unspecified role unspecified role neutral component neutral component n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 colocalization colocalization false false false Fig 4c. The endogenous SNARE protein syntaxin 4 colocalized in the perinuclear region in cotransfected cells coexpressed with cytLEK SNAP25 binding domain (5'LSD) and GFP-SNAP-25. Surprisingly, syntaxin 13 did not colocalize at the perinuclear region. cenpf-snap25vmp2 Colocalization of of 5'LSD of cenpf, snap25 and vamp2 in the cytoplasm and perinuclear region of cos-7 cells. 793167 n/a 793179 unspecified role unspecified role neutral component neutral component n/a 793176 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 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 n/a 793175 unspecified role unspecified role neutral component neutral component n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 colocalization colocalization false false false Fig 4d. The endogenous SNARE protein VAMP2 colocalized in the perinuclear region in cotransfected cells coexpressed with cytLEK SNAP25 binding domain (5'LSD) and GFP-SNAP-25. Surprisingly Vamp8 and Vamp3 did not colocalize at the perinuclear region. cenpf-snap25 Y2H interaction between cenpf and snap25 793168 n/a 793175 unspecified role unspecified role bait bait n-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false n/a 793176 unspecified role unspecified role prey prey physical association physical association false false false Fig 1a cenpf-snap25-3 CoIP showing interaction between cenpf and snap25 793169 n/a 793175 unspecified role unspecified role prey prey n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 n/a 793176 unspecified role unspecified role bait bait 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 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 physical association physical association false false false Fig 1c and 1d The n-terminus (aa 1-474) SNAP-25 binding domain (5'LSD) of cytLEK1 is required for SNAP-25 interaction. cenpf-snap25-8 Y2H interaction between cenpf and snap25 793170 n/a 793176 unspecified role unspecified role bait bait n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false n/a 793175 unspecified role unspecified role prey prey n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false 793218 793216 physical association physical association false false false Fig 1a and 1b cenpf-snap25-4 Colocalization of cenpf and snap25 in the cytoplasm and perinuclear region of nih-3t3 cells 793171 n/a 793176 unspecified role unspecified role neutral component neutral component n/a 793175 unspecified role unspecified role neutral component neutral component colocalization colocalization false false false Fig 2a cenpf-snap25-5 Colocalization of cenpf and snap25 in the cytoplasm and perinuclear region of c2c12 cells 793172 n/a 793175 unspecified role unspecified role neutral component neutral component n/a 793176 unspecified role unspecified role neutral component neutral component colocalization colocalization false false false Fig 2b and 2c cenpf-snap25-6 CoIP showing interaction between cenpf and snap25 793173 n/a 793175 unspecified role unspecified role bait bait n/a 793176 unspecified role unspecified role prey prey physical association physical association false false false Fig 2d SNAP-25 forms an endogenous complex containing cytLEK1/CENPF. cenpf-snap25rab11a-1 CoIP showing existence of a complex between 5'LSD of cenpf, snap25 and endogenous rab11a/myovb/vamp2 in cos-7 cells 793174 n/a 793177 unspecified role unspecified role prey prey n/a 793175 unspecified role unspecified role bait bait n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a 793176 unspecified role unspecified role prey prey 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 n/a 793180 unspecified role unspecified role prey prey n/a 793179 unspecified role unspecified role prey prey association association false false false Fig 5. This demonstrates the critical proteins in the complex that links the cytLEK SNAP-25 binding domain (5'LSD) with recycling endosomes. Although the authors have not directly proven that SNAP-25 is in the Cenf immunoprecipitate, the endogenous proteins (rab11a, myoVb and vamp2) did not coimmunoprecipitate in the absence of this protein.