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/ CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. Mol. Biol. Cell (1059-1524) 2006 imex curation 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 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA orchard Only protein-protein interactions 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 Accepted 2009-MAR-17 by ORCHARD. 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 orchard Only protein-protein interactions imex curation CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. Mol. Biol. Cell (1059-1524) 2006 imex curation 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 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA orchard 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 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 Accepted 2009-MAR-17 by ORCHARD. 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 orchard Only protein-protein interactions imex curation CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. Mol. Biol. Cell (1059-1524) 2006 imex curation 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 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA orchard Only protein-protein interactions 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 Accepted 2009-MAR-17 by ORCHARD. 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 orchard Only protein-protein interactions imex curation CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. Mol. Biol. Cell (1059-1524) 2006 imex curation 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 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA orchard Only protein-protein interactions 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 Accepted 2009-MAR-17 by ORCHARD. 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 orchard Only protein-protein interactions imex curation CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. Mol. Biol. Cell (1059-1524) 2006 imex curation 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 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA orchard Only protein-protein interactions 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 Accepted 2009-MAR-17 by ORCHARD. 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 orchard Only protein-protein interactions imex curation CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. Mol. Biol. Cell (1059-1524) 2006 imex curation 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 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA orchard Only protein-protein interactions 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 Accepted 2009-MAR-17 by ORCHARD. 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 orchard Only protein-protein interactions imex curation CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. Mol. Biol. Cell (1059-1524) 2006 imex curation 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 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA orchard Only protein-protein interactions 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 Accepted 2009-MAR-17 by ORCHARD. 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 orchard Only protein-protein interactions imex curation CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. Mol. Biol. Cell (1059-1524) 2006 imex curation 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 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA orchard 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 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 Accepted 2009-MAR-17 by ORCHARD. 09-April-2009: Contacted by JYOTI. mouse embryonic day 17.5 cDNA Mol. Biol. Cell (1059-1524) 2006 orchard Only protein-protein interactions imex curation snp25_mouse Synaptosomal-associated protein 25 Synaptosomal-associated 25 kDa protein Super protein Snap25 Snap protein protein mouse Mus musculus Mouse MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG DIP protein P60879 original sequence version: 38 DIP protein P60879-1 original sequence version: 41 DIP protein P60879-1 original sequence version: 109 DIP protein P60879-1 original sequence version: 81 DIP protein P60879 original sequence version: 111 FBED2B082A4CB6A6 q9xsv7_chlae rab11 protein protein chlae Chlorocebus aethiops Cercopithecus aethiops Grivet Green monkey AGSNIVIMLVGNKSDLRHLRAVPTDEARAFAEKNGLSFIETSALDSTNVEAAFQTILTEIYRIVSQKQMSDRRENDMSPSNNVVPIHVPPTTENKPKVQCCQNI 90A119B8F5EA30F4 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. q155p7_mouse Cenpf Lek1 protein protein mouse Mus musculus Mouse MSWALEEWKEGLPSRALQKIQELEGQLEKLKKEKQQRQFQLDSLEAALQKQKQKVEDGKTEGADLKRENQRLMEICEHLEKSRQKLSHELQVKESQVNLQESQLSSCKKQIEKLEQELKRCKSEFERSQQVAQSADVSLNPCSTPQKLFATPLTPSSTYEDLKEKYNKEVEERKRLEEEVKALHAKKVSLPVSQATMNHRDIARHQASSSVFPWQQENTPSRLSSDALKTPLRRDGSAAHFLGEEVSPNKSSMKTGRGDCSSLPGEPHSAQLLHQAKAQNQDLKSKMTELELRLQGQEKEMRSQVNKCQDLQLQLEKTKVELIEKERILNKTRDEVVRSTAQYDQAAAKCTTLEQKLKTLTEELSCHRQNAESAKRSLEQRIKEKEKELQEELSRQHQSFQALDSEYTQMKTRLTQELQQVKHLHSTLQLELEKVTSVKQQLERNLEEIRLKLSRAEQALQASQVAENELRRSSEEMKKENSLIRSQSEQRTREVCHLEEELGKVKVSLSKSQNFAEEMKAKNTSQEIMLRDLQEKLNQQENSLTLEKLKLALADLERQRNCSQDLLKKREHHIDQLNNKLNKIEKEFETLLSALELKKKECEELKEEKNQISFWKIDSEKLINQIESEKEILLGKINHLETSLKTQQVSPDSNERIRTLEMERENFTVEIKNLQSMLDSKMVEIKTQKQAYLELQQKSESSDQKHQKEIENMCLKANKLTGQVESLECKLQLLSSEVVTKDQQYQDLRMEYETLRDLLKSRGSSLVTNEDNQRSSEDNQRSSEDNQRGSLAFEQQPAVSDSFANVMGRKGSINSERSDCSVDGGRSPEHIAILQNRVTSLESSLESQNQMNSDLQMRCEELLQIKGEVEENLSKAEQIHQNFVAETNQCISKLQEDAAVHQNIVAETLATLESKEKELQLLKEKLEAQQTEVQKLNKNNCLLEGTLKELQLLSDTLSSEKKEMNSIISLSKKNIEELTQANEALKEVNEALEQEKMNLLQKHEKITSCIAEQERSIAELSDQYKQERLQLLQRCEETEAVLEDLRGNYKTAQENNAKLECMLSECTALCENRKNELEQLKETFAKEQQEFLTKLAFAEEQNRKLMLELEIEQQTVRSEITNTNKHSMSATDGLRQECLTLNEEQNEQQNEVSNLTHENEQLMELTQTKHDSYLAVEPVENSVKATEDEIGKSSSQYQMDIDTKDISLDSYKAQLVHLEALVRILEVQLDQSEEENKKLHLELQTIREELETKSSQDPQSQARTGLKDCDTAEEKYVSMLQELSASQNENAHLQCSLQTAVNKLNELGKMCDVLRVEKLQLESELNDSRTECITATSQMTAEVEKLVSEMKMLNHESALSQNELMKDTSGGEFHDKANHSSVFLTPLDSSNFCEQMTLSSKEVRVHFAELQEKFSCLQSEHKILHDQHCEVSSKMSALRSYVDTLKAENSALSMSLRTLQGDLVKEGEPAAEGGHGLPLSFCGADSPSLTNFGETSFYKDVLEQTGDTCHLSLEGNASANSCDLDEEFSSSLEEETLTEKESPPAPGRTVEGLEVLCQVYLQSLKNLEEKTESQRIMKNKEIEELEQLLSSERKELSCLRKQYLSEKEQWQQKLTSVTLEMESKLAEEKQQTKTLSLELEVARLQLQELDLSSRSLLGTDLESVVRCQNDNYDIKESEVYISETTEKTPKQDTDQTCDKDIQQDLGLETSVTESETTRLTGEGCEEQPPKTNCEAPAEDKTQDCSECISELCSSSNVLVPMDVLEDQGSIQNLQLQKDTLNENLRLLPEVEDWDKKVESLLNEIMEADSKLSLQEVQLKMKIATCIQLEKIVKDLRKEKADLSEKLESLPCNQEVCLRVERSEEDLGFNLDMGANELLSKSTKDNATNTEDNYKEKFLDMERELTRIKSEKANIEHHILSVETNLEVVQAEKLCLERDTESKQKVIIDLKEELFTVISERNRLREELDNVSKESKALDQMSKKMKEKIEELESHQRESLRHIGAVESEVKDKADLIQTLSFNVGELTKDKAHLQEQLQNLQNDSQELSLAIGELEIQIGQLNKEKESLVKESQNFQIKLTESECEKQTISKALEVALKEKGEFAVQLSSAQEEVHQLRRGIEKLSVRIEADEKKHLSAVAKLKESQRESDSLKDTVETLERELERSEENQELAILDSENLKAEVETLKAQKDEMTKSLRIFELDLVTVRTERENLAKQLQEKQSRVSELDERCSSLRRLLEEKEQARVQMEEDSKSAMLMLQMQLKELREEVAALCNDQETLKAQEQSLDQPGEEVHHLKSSIRKLKVHIDADEKKHQNILEQLKESKHHADLLKDRVENLEQELILSEKNMIFQAEKSKAEIQTLKSEIQRMAQNLQDLQLELISTRSENENLIKELKKEQERVSDLETINPSIENLLKDKEQEKVQMKEEAKITVEMLQTQLKELNETVVSLCNDQEVSKTKEQNLGSQVQTLELEKAQLLQDLGEAKNKYIIFQSSVNALTQEVEAGKQKLEKGEKEIRTLKEQLKSQEQLVCKLAQVEGEQELWQKQKLELRNVTMALEQKVQVLQSENNTLQSTYEALQNSHKSLESELGLIKLEKVALVERVSTISGKEAELQRELRDMLQKTTQLSEDYNKEKNRLTEEVEVLREELQNTKAAHLKSVNQLEKELQRAQGKIKLMLKSCRQLEGEKEMLQKELSQLEAAQQQRAGSLVDSNVDEVMTENKALKETLEEKVKEADKYLDKYCSLLISHEELEKAKEILEIEVARLKSRQSRQDLQSSPLLNSSIPGPSPNTSVSEMKSASGQNKASGKRQRSSGVWEHGKRAAPSTAETFSKKSRKSDSKSTRPAEHEQETEFEPEGLPEVVKKGFADIPTGKTSPYILRRTTMATRTSPRFATQKLVGSSPSLGKENVVESSKPTAGGSRSQKVKVVQESSADSHTAFQELPAKSLTASNIPGRNSTESPREGLRAKRAYPASSPAAGPDPTNNENCRVQ 3CB74C4F24537155 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 8 12 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 9 unspecified role unspecified role prey prey physical association physical association Fig 1a A Y2H screen was used to identify novel cytLEK1 binding partners and further characterize cytLEK1 function. The region chosen as bait to screen an embryonic whole mouse cDNA library consisted of the N-terminal most 689 aa of cytLEK1 beginning at the translation start site (base pairs 1-2067; termed 5' LCR for cytLEK Coil Region; Figure 1). cenpf-snap25-8 2 9 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 12 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 21 23 physical association physical association Fig 1a and 1b Next, we analyzed the region within SNAP-25 that was responsible for cytLEK1 interaction. SNAP-25 deletion studies revealed that the N-terminal 75 aa of the protein, termed SNAP-25 Lek1 binding Domain (SNLD), were sufficient and required for the interaction of the 5'LSD domain of cytLEK1 (Figure 1, A and B). cenpf-snap25-3 6 12 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 region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 9 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 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 physical association physical association Fig 1c and 1d As seen in Figure 1C, coimmunoprecipitations of GFP-SNAP-25 revealed interaction with myc-tagged 5'LCR. The n-terminus (aa 1-474) SNAP-25 binding domain (5'LSD) of cytLEK1 is required for SNAP-25 interaction. cenpf-snap25-4 7 9 unspecified role unspecified role neutral component neutral component 12 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig 2a As seen in confocal and deconvolution images in Figure 2, there was significant colocalization of cytLEK1 with SNAP-25 in NIH 3T3 fibroblast and C2C12 myoblast cells. cenpf-snap25-5 1 12 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig 2b and 2c As seen in confocal and deconvolution images in Figure 2, there was significant colocalization of cytLEK1 with SNAP-25 in NIH 3T3 fibroblast and C2C12 myoblast cells. cenpf-snap25-6 3 12 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association Fig 2d We next tested whether endogenous cytLEK1/SNAP-25 complexes could be isolated from cells. A series of coimmunoprecipitation studies, with the same NIH 3T3 cell line that demonstrated immunofluorescent colocalization, was conducted with an antibody previously used to recover SNAP-25 and its interacting partners (Kolk et al., 2000). As seen in Figure 2D (lane 2), SNAP-25 forms an endogenous complex containing cytLEK1. SNAP-25 forms an endogenous complex containing cytLEK1/CENPF. cenpf-snap25-7 5 12 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 9 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 colocalization colocalization Fig 3, Fig 4a-i, Fig 6. Interestingly, when COS-7 cells were cotransfected with 5'LSD and GFP-SNAP-25, a dramatic redistribution of transiently expressed protein localization was observed. 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-1 4 10 unspecified role unspecified role prey prey 12 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 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 9 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 14 unspecified role unspecified role prey prey 13 unspecified role unspecified role prey prey association association Fig 5. To test whether Rab11a is contained within the same 5'LSD/GFP-SNAP-25 complex, whole protein lysates from cotransfected COS-7 cells were collected and analyzed. Lysates containing transfected 5'LSD and GFP-SNAP-25 proteins were probed for Rab11a and were subsequently found to contain endogenous Rab11a in the 5'LSD/GFP-SNAP-25 complex (Figure 5). Myosin Vb, a key regulator of Rab11a-containing recycling vesicles (Lapierre et al., 2001), was also found in the complex (Figure 5). We coimmunoprecipitated the same lysates and identified VAMP2 to be associated in the complex (Figure 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. cenpf-snap25rab11a 5 9 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 12 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 10 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig 4a. To determine whether components of the endosomal recycling pathway were also present in the 5'LSD/GFP-SNAP-25 complex, we assesed the distribution of Rab11a in cotransfected cells (Ullrich et al., 1996; Green et al., 1997). 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 5 11 unspecified role unspecified role neutral component neutral component 9 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 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 12 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 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 colocalization colocalization Fig 4c. We next examined whether these proteins were also in the perinuclear focus in cotransfected cells. We examined and detected both VAMP2 and syntaxin 4 at the perinuclear focus in cells coexpressing 5' LSD and GFP-SNAP-25 (Figure 4, C and D). 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 5 13 unspecified role unspecified role neutral component neutral component 9 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 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 12 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 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 colocalization colocalization Fig 4d. We next examined whether these proteins were also in the perinuclear focus in cotransfected cells. We examined and detected both VAMP2 and syntaxin 4 at the perinuclear focus in cells coexpressing 5'LSD and GFP-SNAP-25 (Figure 4, C and D). 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.