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/ Two F-box/WD-repeat proteins Pop1 and Pop2 form hetero- and homo-complexes together with cullin-1 in the fission yeast SCF (Skp1-Cullin-1-F-box) ubiquitin ligase. Two F-box/WD-repeat proteins Pop1 and Pop2 form hetero- and homo-complexes together with cullin-1 in the fission yeast SCF (Skp1-Cullin-1-F-box) ubiquitin ligase. Genes Cells (1356-9597) 1998 imex curation Kominami K., Ochotorena I., Toda T. toda@cancer.org.uk 12-MAR-2007: Contacted by JYOTI. Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast pull down pull down predetermined predetermined participant predetermined Kominami K., Ochotorena I., Toda T. toda@cancer.org.uk Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Genes Cells (1356-9597) 1998 Only protein-protein interactions imex curation Two F-box/WD-repeat proteins Pop1 and Pop2 form hetero- and homo-complexes together with cullin-1 in the fission yeast SCF (Skp1-Cullin-1-F-box) ubiquitin ligase. Two F-box/WD-repeat proteins Pop1 and Pop2 form hetero- and homo-complexes together with cullin-1 in the fission yeast SCF (Skp1-Cullin-1-F-box) ubiquitin ligase. Genes Cells (1356-9597) 1998 imex curation Kominami K., Ochotorena I., Toda T. toda@cancer.org.uk 12-MAR-2007: Contacted by JYOTI. Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast anti tag coip anti tag coimmunoprecipitation anti tag coip predetermined predetermined participant predetermined Kominami K., Ochotorena I., Toda T. toda@cancer.org.uk Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Genes Cells (1356-9597) 1998 Only protein-protein interactions imex curation cul1_schpo Cullin-1 cul1 pcu1 SPAC17G6.12 Cell division control 53 homolog protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MTTLNTNDKDLPIVKKYDSLNGTWDFLKTGVSQIFERLDEGMTITKYMELYTAIHNYCADASKTITVDNFNDQTANVLGEALYNNLVLYLEEYLARLRKECISQTNHEEQLAAYAKYWTRFTTSARFINHLFGYLNRYWVKLKNRFTETLVYDIYTLCLVSWHHHVFSHIRDSLLQNLLYMFTKKRLYEPTDMKYVEVCVDSITSLSFDKTDMTKPNLSSYKTFFETNFIENTKNFYAKESSEYLASHSITDYLKKAEIRLAEEEELVRLYLHESTLKPLLEATEDVLIAQHEEVLHNDFARMLDQNCSEDIIRMYRLMSRTPNGLQPLRQTFEEFVKRSGFAAVAKIVPQVGGEADVDPKEYMEMLLSTYKASKELVNTAFHGDTDFTKSLDTAFRELVNRNVVCQRSSSRSPELLAKYADSILRKSNKNVDIDDVEDCLSSIIIIFRYVEDKDVFQNFYTKLLAKRLVNGTSNSQDAESSMLSKLKEVCGFEYTSKLQRMFQDISLSQEITEAFWQLPQSRAGNIDFSALVLGTSFWPLSPNNVNFHLPEELVPLYEGFQNYYYSCHNGRKLSWLFHLSKGEIKARINPQTNVTYVFQVSTYQMGVLLLYNHRDSYTYEELAKITGLSTDFLTGILNIFLKAKVLLLGDNDKLGDPNSTYKINENFRMKKIRVQLNLPIRSEQKQESLETHKTIEEDRKLLLQSAIVRIMKARRTLKHVVLVKETIDQIKSRFTPKVSDIKQCIDMLIEKEYLERQGRDEYIYLA 458CA31B5D97FAA9 pop1_schpo WD repeat-containing protein pop1 WD repeat-containing protein ste16 pop1 SPBC1718.01 SPBC2G2.18 ste16 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MNEKKKDSLSVLDSNEFFGETTMVSPSIDVSSSPRPNVERFSPCSTKKDLLEGNNIMTRIPEELSRVSLQFDSKGSQQSMIFTNNRCLSDKENLENLQNLLYLHCDLNRPHLSCELPSEHREKCLKRRNSSLSSNLHANKRFLFNSQSDGNKKNETFPSTNYSNVFYPNNCDSKEVASETTFSLDAPNNSVNYSYFSPNLLGNDSKTRQSFPPHSSSSSHNSLHEPVIYDFSSENPSIHPSNHLSSQKNAVLKLAQLISSFEKLPESVRQYLLFHLLSRCGKHAVQNIHKILLPIFQKNFLTGFPAEITNLVLTHLDAPSLCAVSQVSHHWYKLVSSNEELWKSLFLKDGFFWDSIDSKIRTMCLEQSLSACAIMKRVYFRHFNLRERWLHAPEKIKRCSFPIHGVRLITKLQFDDDKIIVSTCSPRINIYDTKTGVLIRSLEEHEGDVWTFEYVGDTLVTGSTDRTVRVWDLRTGECKQVFYGHTSTIRCIKIVQGNQSTTDTDDVEKENRPASNDANSMPPYIISSSRDCTIRLWSLPCLDDPPFVNVNENPDQNNDFTSATTNPFYIRTLRGHTDSVREVACLGDLIVSASYDGTLRVWKASTGVCLHVLRGHVGRVYSVTINPSRQQCISAGTDAKIRIWNLESGELLQTLHGHSNLVSQVTFNQNILVSASAPPDTSLRVWDLNTGSCRDILKCPLGHIFFQHDESKVVSGSHSTLQLWDIRSGKLVRDLLTDLDIIWQVAYNENVCVAAVLRNNRFWIEVLEFGSTKSS B06EDBA46553EEC1 pop2_schpo WD repeat-containing protein pop2 Proteolysis factor sud1 pop2 sud1 SPAC4D7.03 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MSLSRCPTDNSSSRINSSVPLINSSSPATPPESFDPQVFPSSLIHGDNLLPQDDQIASDPRSESNSCNGNTSSSLPCTDSYQYPLKHSCTPSFLRKFNESIENVSYKCLDHSPPDSVPGDFSISLVPQRNFLYSHSSLPPKIISIDRNNRIKLDNSISSNSDNFPPSPKVDTSNTVSPGSKPISEDLEDLNLQSIVQTFEDLPEGIQSYAFFQLLRSCNRQSMRLLLNECEPLLKKDILSNLPFSIVQSILLNLDIHSFLSCRLVSPTWNRILDVHTSYWKHMFSLFGFQINENDWKYANPNLNRPPFLHNDQISDDYFPEIFKRHFLNRKRWLFPSIPPSHLSFPIHVPNFMITSLLLHKDRIITTSGSGTIQIHNAITGVLEARLEGHKEGVWAVKIHENTLVSGSIDKTVRVWNIEKAKCTHIFRGHISIIRCLEILVPSRLIRHGVEIVEPDQPYIVSGSRDHTLRVWKLPKNTDPPYLPDNTNSIDRWEKNPYFVHTLIGHTDSVRTISGYGDILVSGSYDSSIRIWRVSTGECLYHLRGHSLRIYSVLYEPERNICISGSMDKSIRVWDLSTGTCKYVLEGHDAFVTLLNVFQNRLISGSADSTIRIWDLNTGKPLMVLPSNSGYISSFVSDEHKIISGNDGSVKLWDVRTGKLLRFLLTDLTKIWHVDFDAMRCVAAVQRDDQAYLEVINFSGSRP 0336A0568C152396 pop1-pop1 2 4 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 4 unspecified role unspecified role prey prey 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association To address the sugeestion as to how Pop1 and Pop2 interact in the cell, a series of combinations of tagged pop1� and pop2� genes were constructed (they were all functional, see Experimental procedures), expressed in the fission yeast, and a physical interaction between Pop1 and Pop2 was examined. As shown in Fig. 4, the myc-Pop1 protein was specifically co-immunoprecipitated with anti-HA (haemagglutinin) antibody (lanes 3) but not upon mock treatment (lane 2). Likewise Pop1- HA and GST (glutathione S-transferase)-Pop2 were co-immunoprecipitated with anti-HA antibody (lane 5). Conversely pull-down experiments using GSH (glutathione)-coupled beads also precipitated both myc-Pop1 and GST-Pop2 (lane 6). These results strongly suggest that Pop1 and Pop2 act as heterodimers or hetero-multimers in the cell. Figure 4 pop1 is able to interact with itself forming a homodimer in the cell. pop1-pop2-1 2 5 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 4 unspecified role unspecified role prey prey 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association To address the sugeestion as to how Pop1 and Pop2 interact in the cell, a series of combinations of tagged pop1� and pop2� genes were constructed (they were all functional, see Experimental procedures), expressed in the fission yeast, and a physical interaction between Pop1 and Pop2 was examined. As shown in Fig. 4, the myc-Pop1 protein was specifically co-immunoprecipitated with anti-HA (haemagglutinin) antibody (lanes 3) but not upon mock treatment (lane 2). Likewise Pop1- HA and GST (glutathione S-transferase)-Pop2 were co-immunoprecipitated with anti-HA antibody (lane 5). Conversely pull-down experiments using GSH (glutathione)-coupled beads also precipitated both myc-Pop1 and GST-Pop2 (lane 6). These results strongly suggest that Pop1 and Pop2 act as heterodimers or hetero-multimers in the cell. Figure 4 pop1 is able to interact with pop2 forming a heterodimer in the cell. pop2-pop2-2 2 5 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 5 unspecified role unspecified role prey prey region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association To address the sugeestion as to how Pop1 and Pop2 interact in the cell, a series of combinations of tagged pop1� and pop2� genes were constructed (they were all functional, see Experimental procedures), expressed in the fission yeast, and a physical interaction between Pop1 and Pop2 was examined. As shown in Fig. 4, the myc-Pop1 protein was specifically co-immunoprecipitated with anti-HA (haemagglutinin) antibody (lanes 3) but not upon mock treatment (lane 2). Likewise Pop1- HA and GST (glutathione S-transferase)-Pop2 were co-immunoprecipitated with anti-HA antibody (lane 5). Conversely pull-down experiments using GSH (glutathione)-coupled beads also precipitated both myc-Pop1 and GST-Pop2 (lane 6). These results strongly suggest that Pop1 and Pop2 act as heterodimers or hetero-multimers in the cell. Figure 4 pop2 is able to interact with itself forming a homodimer within the cell. pop1-pop2-2 2 4 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 5 unspecified role unspecified role prey prey region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association To address the sugeestion as to how Pop1 and Pop2 interact in the cell, a series of combinations of tagged pop1� and pop2� genes were constructed (they were all functional, see Experimental procedures), expressed in the fission yeast, and a physical interaction between Pop1 and Pop2 was examined. As shown in Fig. 4, the myc-Pop1 protein was specifically co-immunoprecipitated with anti-HA (haemagglutinin) antibody (lanes 3) but not upon mock treatment (lane 2). Likewise Pop1- HA and GST (glutathione S-transferase)-Pop2 were co-immunoprecipitated with anti-HA antibody (lane 5). Conversely pull-down experiments using GSH (glutathione)-coupled beads also precipitated both myc-Pop1 and GST-Pop2 (lane 6). These results strongly suggest that Pop1 and Pop2 act as heterodimers or hetero-multimers in the cell. Figure 4 pop2 is able to interact with pop1 forming a heterodimer within the cell. pop1-pop2-3 1 4 unspecified role unspecified role prey prey 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 5 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association To address the sugeestion as to how Pop1 and Pop2 interact in the cell, a series of combinations of tagged pop1� and pop2� genes were constructed (they were all functional, see Experimental procedures), expressed in the fission yeast, and a physical interaction between Pop1 and Pop2 was examined. As shown in Fig. 4, the myc-Pop1 protein was specifically co-immunoprecipitated with anti-HA (haemagglutinin) antibody (lanes 3) but not upon mock treatment (lane 2). Likewise Pop1- HA and GST (glutathione S-transferase)-Pop2 were co-immunoprecipitated with anti-HA antibody (lane 5). Conversely pull-down experiments using GSH (glutathione)-coupled beads also precipitated both myc-Pop1 and GST-Pop2 (lane 6). These results strongly suggest that Pop1 and Pop2 act as heterodimers or hetero-multimers in the cell. Figure 4 pop2 is able to interact with pop1 forming a heterodimer in the cell. cul1-pop2 2 3 unspecified role unspecified role prey prey 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 5 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association To analyse a functional relationship between cullin and the SCF complex in the fission yeast, a physical interaction between Pcu1, Pop1 and Pop2 was examined. For this purpose, a tagged version of pcu1� (myc-pcu1�) was constructed (Experimental procedures). As a control for the specificity of an interaction, the tagged pcu3� gene (GST-pcu3�) was also constructed. pop1�-HA or pop2�-HA was co-expressed with either myc-pcu1� or GST-pcu3� and immunoprecipitation was performed using anti-HA antibody. As shown in Fig. 6, myc-Pcu1 was co-immunoprecipitated with Pop2-HA (lane 2) also with Pop1-HA (data not shown). This binding was specific to anti-HA antibody, as neither of the two proteins was precipitated upon mock treatment (lane 1). Remarkably myc-Pcu1 was still capable of forming a complex with Pop2-HA in Dpop1 cells (lanes 3 and 4). These results indicate that Pcu1 is a component of SCFPop2, and that complex formation is not abrogated in the absence of Pop1. In sharp contrast to the result for Pcu1, GST-Pcu3 was not co-precipitated with Pop1-HA (lanes 6 and 7) although the protein was expressed (lane 5). Figure 6 pop2 is able to interact with cul1/pcu1 forming a SCF-POP2 complex within the cell, even in a pop1 depleted strain. However, pop1 was unable to coprecipitate with pcu3.