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/ Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Dev. Cell (1534-5807) 2005 imex curation Martin SG., McDonald WH., Yates JR., Chang F. fc99@columbia.edu 01-DEC-2006: Contacted by JYOTI. 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 Martin SG., McDonald WH., Yates JR., Chang F. fc99@columbia.edu Accepted 2006-NOV-17 by ORCHARD. 01-DEC-2006: Contacted by JYOTI. Dev. Cell (1534-5807) 2005 orchard Only protein-protein interactions imex curation Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Dev. Cell (1534-5807) 2005 imex curation Martin SG., McDonald WH., Yates JR., Chang F. fc99@columbia.edu 01-DEC-2006: Contacted by JYOTI. orchard Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 2h fragment pooling two hybrid fragment pooling approach 2h fragment pooling predetermined predetermined participant predetermined Martin SG., McDonald WH., Yates JR., Chang F. fc99@columbia.edu Accepted 2006-NOV-17 by ORCHARD. 01-DEC-2006: Contacted by JYOTI. Dev. Cell (1534-5807) 2005 orchard Only protein-protein interactions imex curation Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Dev. Cell (1534-5807) 2005 imex curation Martin SG., McDonald WH., Yates JR., Chang F. fc99@columbia.edu 01-DEC-2006: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Martin SG., McDonald WH., Yates JR., Chang F. fc99@columbia.edu Accepted 2006-NOV-17 by ORCHARD. 01-DEC-2006: Contacted by JYOTI. Dev. Cell (1534-5807) 2005 orchard Only protein-protein interactions imex curation Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Dev. Cell (1534-5807) 2005 imex curation Martin SG., McDonald WH., Yates JR., Chang F. fc99@columbia.edu 01-DEC-2006: Contacted by JYOTI. orchard Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Martin SG., McDonald WH., Yates JR., Chang F. fc99@columbia.edu Accepted 2006-NOV-17 by ORCHARD. 01-DEC-2006: Contacted by JYOTI. Dev. Cell (1534-5807) 2005 orchard Only protein-protein interactions imex curation tip1_schpo Tip elongation protein 1 tip1 noc1 SPAC3C7.12 Protein noc1 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MFPLGSVVEVITGERGFVRYAGEVENRKGVYVGLELLPEFAEFGKNRGVVDGREYFKTKNNEKTGIFVPFDKCKLASSISSSPSPKIDGTAASIGMGFPPMSPNLQSSIPRLTNVSSSSNLSMNTISSTALTPTEKILQKRIEDLLYERQNHQQQLEEVLATVDQLQSLVTNFNDQQDEVDELRERITLKEERIQQMRNEASQRRFEFKTTIECLEESSNRAIETYENRIAELEAQLEMYMSGKSEDDLLFSLQQERDYALNQVEILQERVDTLMKQKANSSTANEKLSHMESSSPTLTNASFESPKRGKGSNDLPENHPQRRQTLEFYEIEIEVLREKVEKLQALSDEKDFYISKLEKSLDRNDTTPVPSDEKLSNYAAEKENLVSRISELEHTIEQLTINNERDNERMSPAEFELETTQEVEENDSDSHDDEETWCEVCETNNHSLQECPTVFGSTDEA 66CCDB96146F86D6 66CCDB96146F86D6 for3_schpo Formin-3 for3 SPCC895.05 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MASKMPEGSPPTSRSIQSRNSSYSTSSNERIGTPSTISLSENSDLSKLQSTNDFESREDLSLTSDDNNDPEYVMCYNTVYHQKTKINDKLLSETEQLRKIYPLESRVFPKPTIVKEITNERTKRYTFYDDDAPLTNQHTVLDEATYNRILKRIDFFIEKVVEVPPTYHFLSLLNVQLRIQPIWWMDEFAERNGIESLLSALRNLGHYPERASKTPLESQIIQSLFHCLNSENCRRRYQSSAKCSVPGFNALGTIAETVLSKSLNSARMATFLLKFLCNKKGLSYFKAVIRAFEWLVEQKLSKTRFSAWMHSFNDVITGVRVCADSSPQAIVHMDEFEDTDCLIDYLVATLALIRDLCAAPPDLQLRCALRHELLSAGLQKAIDSLLKWRNRHVRDALQLLIKEHNADARRFRSGSDVNNVDRKCVKKQMNYREESHTPHGNTTRKTSTPVSNNRPTTPEQQAVWDVFQRIYTRFTGSEGSKESFIKLLEYFVTEPDNGKIQKSMQLLTHTLEALEGFKTAKADTNVGLTILSQRLLDKLGTAEEIAEYKTKYNGAMLENKHLKEQVESMLSQLNVGPRDPMQFLKKQLDELKAELNLRDNLLASMQREFETRYRAQIQAYNKLQSQMEHVQNSNEQHLQPGLLNKVSKSFDSVHRRNLSQDSLDAMTEQFSYHVEPNILSGSGIPVRVHTPSKTEDLDESFSGSEISSSPSPLLPDVSDTVEEQQKLLLKSPPPPPPAVIVPTPAPAPIPVPPPAPIMGGPPPPPPPPGVAGAGPPPPPPPPPAVSAGGSRYYAPAPQAEPEPKIDETSLTEEQKIQLEEARKQRKAADDAARAAIEKYTSIPSLRDLHKPTRPLKRVHWQRVDPLPGPNVFTKFCLNFDITAKVFIDNGLLDFLDEKFDNTPREDFVAVEISDQRSSLLPDTVEQNMAIILRSVSNMPVEDLVQKFLVEPDFLPASILYFDRASLASTNAYTDPFIPYSTDYTKKNPKEPTADVNSLSYFEKFFVLFVVNLRHYFQERMKALKFRSTLFGDLEILEVRMKEVIDTSDSIMEDKNFAEFFQVLLIIGNYFNEPYDRASAFSLYMIYRLETLRDSSSALTLMHYFDEIIRTRFPELLQAESTFKKIQSVSGYNIDAMVAGVDGAYDEFCDFQTSLKDGALSKCDQHHPDDKAYDILSEWLPEAKERIRNIKKLKTDMLTKLENTVKYLCEYDSIDKVRNSFFKNLNSFYEMYSIAKAENEERFEKEKRRIMSEDRDKLIRGRQKTSIVAKYRNKRELPEDSDDKQDTASKDKNSLETIDEKMEDASKIEGDAKTGDDNEMEDLDKMEDLEKPDYAEEKDPYITVMSELRSRIQNVPKRTVTVYSDEGVATLEPGAQGDDVVDKAKMILEKMEGHSQLLTSSANPDEEVLRAKLKAAERLQKPAIPRTRRKGHTEPKSAKSLLAELTNGSNASNLVENDRQKQ C34E2C9672DA01CE tea4_schpo Tip elongation aberrant protein Tea4 SPBC1706.01 tea4 wsh3 Cell polarity protein tea4 Win1-interacting SH3 domain protein protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MLHMNSASSADSMEIMESHFDPTQQNDSTIIESRYSPEEYLEQSFEIQRIISGENSEPQTVASQEISDSQEEDTTLTSSQFEDCGTEYNEVVEDDEFRSEDEDDFMDEEEEYALYEAELSSSPSIHEEVIDCNFVHAIRGFEATVEGQVDATKGDMMILLDDSNSYWWLVKMCKNLAIGYLPAEYIETPSERLARLNKYKNSETSNSQQSVTLPPLDIVEKTLEAPSPNFRIKRVTFTCSSNSSDDEMDSENDYEAMVNRTVAENGLEIEFSDSSDSSLSAEYRSESEDHVTDSPAYVDLTELEGGFNQFNSTSFQSTSPLGLEIVETEINGSSTTADSKNSHSPYSKFSSAYPDAENSNISKINISIAGNKELYGNATQSDPSLYSTWIANKHKTASSATVDSPLRRSLSVDAMQSNASFSSYSSTSNTDKSLRPSSYSAVSESSNFTHDVSRDNKEISLNAPKSIIVSQSDSFDTSNVTQDAPNDVEKEPISGQMPNNLSVQSLKQLEVYPIRHSVSIEMPSEKLLSPRLYSSSTPSSPTKGFQKDDEEDSENRKQADKVELSPSSLLRQMSLPVDSSSQSDAQCTTSSVYITAERKAFSQSSIDLSTLSNHHVNNEINRRSFAGGFTSLADELSEMRELLHESPAPLECNEEMVIPTPELDASSAIPSSSISHDEDLLPRKNTEESTSSSSFSSLITSPASLQYDENPFKQSVVAELNNNSSSVPFVDSAHASDIHAYDNDHVSTKNKEFNRRLREFILDPDSLSGLYWSVKSAGVRASRRVSRNIEGESVSSDLDDIFANVLKGLSDEMASLLNTNR B1DC53794E56D73E tea1_schpo Tip elongation aberrant protein 1 Cell polarity protein tea1 Altered polarity protein 8 tea1 SPCC1223.06 alp8 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MSFLFKRNKGSAHKPTKPNFSKTSTTPSTSQLKHSHESNVKMSTSTVTEHRKKPTGSGSHITASPWSKLTVRGSSNVLPRYSHASHLYAEGGQEIYIFGGVASDSQPKNDLWVLNLATSQFTSLRSLGETPSPRLGHASILIGNAFIVFGGLTNHDVADRQDNSLYLLNTSSLVWQKANASGARPSGRYGHTISCLGSKICLFGGRLLDYYFNDLVCFDLNNLNTSDSRWELASVVNDPPPARAGHVAFTFSDKLYIFGGTDGANFFNDLWCYHPKQSAWSKVETFGVAPNPRAGHAASVVEGILYVFGGRASDGTFLNDLYAFRLSSKHWYKLSDLPFTPSPRSSHTLSCSGLTLVLIGGKQGKGASDSNVYMLDTSRFRLGSVPTTSGRQRNTSFFSNSTGNTNPSAFNGLLTSSRIPSYNGSKVRSTSHPSRQQYIGSSNSRFNTRHQTISTPVSGRASNDLPSPVVPTRSNSSSILQPSYNLNSHSSDRRNTNDDDQSSLNSQQLSNQAKAQGEVSPTLSFVPSSHSMEQGNGSVASANNAQSEAATRSINSISEVSEVRFPEQSSVKTVDERKSLDGRITSVTLETLVEKYSELSKQQIVEWFKSKLYEILRDSASKIDSLTEKLKVANAEKNAALCEAALEKVPLAKHNKLSDGTFSTPDKENVQSTNDAHIMQENFSLHKALEVMRETSSDLDKQLKDATASQKELIVQTSSFQKELVEERERHNAISKRLQEIESLYRDRELLVTNLEDQLVDQTVTINKFAFERDQFRERSMGFENTIKDLTRKMEATDMLNVSLHESLRSVQTENSELVTEMALLKAELVKKQAIIDANANIYDKLTADHTNYETVSADINQNLKETLDKLLNGSSDFKNNEIELLHDQIRITNAKLEKREKLINASKYIEDTLRSEIQEAAEKVSNLEFSNFNLKEENSNMQLQLMKALEQRNTGAKQLVNLRMQLSTATSELDMLKLKLRTTALALEESPDDYSDILSILRADMSPFHDLHKQCGVLIDTLNGVKRGFGIFEKKFTDYHKFLENISDKLKSEEDTSLETPIHENQSIQSDQIKEVGEVLSAIKSLSDSVMLLKNQIDDLAKEKLPLSSSDDEKVNIKEKTDFMKLLVKSGLSNPPAKEPVHDNEN 7BE65F6C666EF4F8 tea4-tea1-2 1 8 unspecified role unspecified role bait bait c-terminus region required to bind necessary binding region required to bind deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1100304]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 1148-1148, sequence length 1147 [EBI-1100304]538-1148 7 unspecified role unspecified role prey prey physical association physical association Shows that the C-terminal half of tea1 was necessary and sufficient to bind to tea4. Tea1p and tea4p also showed a strong interaction in the two-hybrid system (Figures 1C and 1D), which allowed us to map the regions of the proteins important for these interactions. Figure 1C tea4-tea1-3 1 7 unspecified role unspecified role bait bait c-terminus region required to bind necessary binding region required to bind deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1100376]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 810-810, sequence length 809 [EBI-1100376]515-810 8 unspecified role unspecified role prey prey physical association physical association The C-terminal third of tea4 mediated the interaction with tea1. Tea1p and tea4p also showed a strong interaction in the two-hybrid system (Figures 1C and 1D), which allowed us to map the regions of the proteins important for these interactions. Figure 1D tea4-tea1-4 4 7 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 8 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association The tea1-HA coimmunoprecipitation with Tea4-GFP in yeast extracts was determined with an an anti-HA antibody. We confirmed the interaction between tea1p and tea4p by using coimmunoprecipitation and two-hybrid approaches. Tea1p-HA and tea4p-GFP coimmunoprecipitated in yeast extracts with either an anti-HA or an anti-GFP antibody (Figure 1E). Figure 1E tea4-tea1-5 4 7 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 8 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association The tea1-HA coimmunoprecipitation with Tea4-GFP in yeast extracts was determined with an an anti-GFP antibody. We confirmed the interaction between tea1p and tea4p by using coimmunoprecipitation and two-hybrid approaches. Tea1p-HA and tea4p-GFP coimmunoprecipitated in yeast extracts with either an anti-HA or an anti-GFP antibody (Figure 1E). Figure 1E tea4-tea1-6 3 7 unspecified role unspecified role prey prey c-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1102430]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 810-810, sequence length 809 [EBI-1102430]229-810 ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role bait bait c-terminus mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1102428]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 1148-1148, sequence length 1147 [EBI-1102428]538-1148 ecoli Escherichia coli (strain K12) physical association physical association Bacterially expressed tea1-MBP bound to tea4-His in in-vitro pull-down experiment. We then showed that tea1p and tea4p bind directly by using bacterially expressed 6His-tagged fragments in in vitro assays. The C-terminal half of tea4p specifically bound to MBP-tea1C, but not to MBP alone (Figure 1F). Figure 1F tea1-tea1-1 3 8 unspecified role unspecified role prey prey c-terminus binding region binding-associated region binding region deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1102442]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 1148-1148, sequence length 1147 [EBI-1102442]538-1148 region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role bait bait c-terminus binding region binding-associated region binding region deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1102440]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 1148-1148, sequence length 1147 [EBI-1102440]538-1148 region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association Bacterially expressed tea1-MBP bound to tea1-His in in-vitro pull-down experiment as shown in Figures 1F and 4B. We also noted that tea1C can dimerize through its coiled-coil region (Figure 1F). Figure 1F tea1-tip1-1 1 5 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait n-terminus binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association The N-terminus of tea1 containing kelch repeats, is necessary for the interaction with tip1. An interaction between tea1p and the CLIP170 tip1p has been shown to concentrate tea1p at the MT plus end (Feierbach et al., 2004). The kelch repeats at the N terminus of tea1p were necessary and sufficient for this interaction (Figure 2E). Figure 2E tea4-for3-1 1 6 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 7 unspecified role unspecified role prey prey physical association physical association We mapped the interaction domains between for3p and tea4p. Deletion analysis defined amino acids 137�515 in for3p as the minimal sufficient region for interaction with tea4p (Figure 4D). This region overlaps with the predicted FH3 domain of for3p (aa 306�507, as defined by SMART) and the rho binding region (Nakano et al., 2002). This N-terminal region is necessary and sufficient for localization of for3p at cell tips (Nakano et al., 2002), suggesting that tea4p binding to this for3p region may regulate for3p localization. Figure 4D tea4-for3-2 1 7 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey 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 physical association physical association No specific domain of tea4p could be defined in for3p binding. Neither the tea4p C-terminal region, which mediates the interaction with tea1p, nor the N-terminal half interacted with for3p reproducibly (Figure 4E), suggesting that multiple parts of the protein may be required for strong interaction. What is clear though is that the SH3 domain of tea4p was dispensable for binding, as shown by deletion or specific point mutation in the SH3 domain. No specific domain of tea4p could be defined in for3p binding. Neither the tea4p C-terminal region, which mediates the interaction with tea1p, nor the N-terminal half interacted with for3p reproducibly (Figure 4E), suggesting that multiple parts of the protein may be required for strong interaction. What is clear though is that the SH3 domain of tea4p was dispensable for binding, as shown by deletion or specific point mutation in the SH3 domain. Figure 4E tea4-tip1-1 4 5 unspecified role unspecified role prey prey schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 7 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Tip1 coimmunoprecipitates with tea4 in a tea1-dependent manner. This experiment was performed using cell extracts from tea1 containing and tea4 depleted cells. tip1p coimmunoprecipitated with tea4p from tea1+ yeast extracts, but not from tea1 yeast extracts (Figure 2F) Figure 2F tea1-tip1-2 4 8 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 5 unspecified role unspecified role prey prey schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Tip1 coimmunoprecipitates with tea1 in a tea4-indedependent manner. This experiment was performed using cell extracts from tea4 containing and tea4 depleted cells. In contrast, the association of tea1p with tip1p was tea4 independent (Figure 2G). Therefore, tea1p functions to link tip1p and tea4p in a common complex. Figure 2G tea4-for3-3 4 6 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 7 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Endogenous tagged for3-HA coimmunoprecipitated with Tea4-GFP in yeast extracts; determined with an an anti-HA and anti-myc antibody. Indicating that tea4p and for3p are present in the same complex in vivo. We tested whether tea4p interacts with for3p. In soluble yeast extracts, for3p-myc coimmunoprecipitated with tea4p-GFP (Figure 4A), Figure 4A tea1-for3-1 4 7 unspecified role unspecified role ancillary ancillary schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 8 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 6 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast association association Endogenous tagged for3-HA interacted with tea1-HA in yeast extracts from tea4-containing cells and not in tea4-depleted cells; determined with an an anti-HA and anti-myc antibody. Indicating that tea4 is required for tea1 and for3 interaction. Furthermore, the interaction between tea1p and for3p in yeast extracts was not present in extracts from tea4 cells (Figure 4C), showing that tea4p is required for for3p-tea1p interaction. Together, these results show that tea4p directly binds to for3p and tea1p and links these proteins together. Figure 4C tea4-tea1-8 2 7 unspecified role unspecified role neutral component neutral component region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 8 unspecified role unspecified role neutral component neutral component region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Tea4-YFP colocalizes precisely with tea1-CFP in vivo. Tea4-YFP colocalizes precisely with tea1-CFP both at cell tips and at MT plus ends (Figure 2B). Figure 2B tea4-tea1-9 3 7 unspecified role unspecified role bait bait region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role prey prey c-terminus binding region binding-associated region binding region deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1104255]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 1148-1148, sequence length 1147 [EBI-1104255]538-1148 region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association Bacterially expressed tea1-His bound to tea4-MBP in in-vitro pull-down experiment. Figure 4B tea4-for3-4 3 6 unspecified role unspecified role bait bait n-terminus binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association Bacterially expressed N-terminus for3-His bound to tea4-MBP in an in-vitro pull-down experiment. Importantly, a bacterially expressed N-terminal fragment of for3p (6His-for3N) was able to bind tea4p in vitro (Figure 4B), showing that this interaction between for3p and tea4p is likely to be direct. In contrast, for3p did not bind to the C-terminal region of tea1p in this assay (Figure 4B), suggesting that the interaction between for3p and tea1p is probably indirect. Figure 4B