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/ F-box/WD-repeat proteins pop1p and Sud1p/Pop2p form complexes that bind and direct the proteolysis of cdc18p. F-box/WD-repeat proteins pop1p and Sud1p/Pop2p form complexes that bind and direct the proteolysis of cdc18p. Curr. Biol. (0960-9822) 1999 imex curation Wolf DA., McKeon F., Jackson PK. pjackson@cmgm.stanford.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast anti bait coip anti bait coimmunoprecipitation anti bait coip predetermined predetermined participant predetermined Wolf DA., McKeon F., Jackson PK. pjackson@cmgm.stanford.edu Accepted 2007-MAR-28 by DAVET. 09-MAR-2007: Contacted by JYOTI. Curr. Biol. (0960-9822) 1999 Only protein-protein interactions imex curation 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 cdc18_schpo Cell division control protein 18 cdc18 SPBC14C8.07c protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MCETPIGCHTPRRCNRFIDSAALIDCTNKTNQREHSPSFSIEIPTTPSRKRTLASSHFQTPTKRIKYELGELQEEKTDLYPNFPAQLKENKKPKLPTTPQTPKTPKRTIQIVTPKSLNRTCNPVPFATRLLQSTPHRQLFPPTPSTPSTPSYNSTAKLSLRKSYRSAGVVGRENEKSIVESFFRQHLDANAGGALYVSGAPGTGKTVLLHNVLDHVVSDYPKVNVCYINCMTINEPKAIFEKIHSKIVKEEILENEDHHINFQCELESHFTQSANELYNPVIIVLDEMDHLIAREQQVLYTLFEWPSRPTSRLILVGIANALDMTDRFLPRLRTKHITPKLLSFTPYTAQEISTIIKARLKTAATTSEKNNPFTPIKSISEVSDDSINVVSQHADETPFIHPAAIELCARKVAASSGDLRKALDICRHAIELAEREWKAQHDNTLSSVDIPRASIAHVVRATSAMSQSASARLKNLGLQQKAILCTLVVCEKTSLSVADVFEKYSSLCLRDRLIYPLTSSEFCDVANSLETLAIIRLRTKQRNGKPQDRIISLLVPEMDVITAVGDIGTLKRFFDRR 74DBE9D5CF23504D pop1-pop2-4 1 2 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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 3 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 Figure 2a pop2 is able to interact with pop1 forming a heterodimer within the cell. A specific interaction was readily detectable in lysates from cells co-overexpressing Pop1p and Pop2p, however. Histidine (His)-tagged Pop1p co-purified with hemagglutinin (HA)-epitope-tagged Pop2p (HA-Pop2p) on Ni-agarose beads, but not on control beads (Figure 2a). In the reciprocal experiment, anti-Pop2p antisera, but not pre-immune sera, co-precipitated Myc-tagged Pop1p along with HA-Pop2p (Figure 2a). pop1-pop2-5 1 2 unspecified role unspecified role prey prey 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 3 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 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 schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Figure 2B The n-terminal region of pop2 (AA 2-241), even without the f-box is able to bind with pop1, however deletion of the pop2 n-terminal regions AA 171-703 and AA 345-703 prevents interaction with pop1 suggesting that there maybe an independent pop1-binding-domain in the region AA 2-171. To map the region in Pop2p required for the interaction with Pop1p, we analyzed various Pop2p truncation mutants (Figure 2b). Pop1p bound to full-length Pop2p as well as to amino-terminal Pop2p fragments containing residues 2-344 or 2-241(Figure 2b). In contrast, deletion of the amino-terminal 171 or 344 amino acids of Pop2p abolished the interaction with Pop1p (Figure 2b). These carboxy-terminal fragments were expressed at lower levels than the amino terminal fragments, however, so the carboxyl terminus of Pop2p may contain an independent Pop1p interaction motif not detected in this assay. cdc18-pop2 1 4 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 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 3 unspecified role unspecified role prey prey schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 2 unspecified role unspecified role ancillary ancillary schpm Schizosaccharomyces pombe (fission yeast) Fission yeast association association Figure 3D Interaction between Pop2p and GFP-Cdc18p was detected in lysates from wild-type cells, but was undetectable in lysates from pop1-deleted mutants, suggesting that Pop1p-Pop2p heterooligomers cooperate in substrate binding. Both Pop1p and Pop2p interact with their substrate Cdc18p in vivo [10,12]. To test whether the Cdc18p-Pop2p interaction depends on Pop1p, Mycepitope-tagged GFP-Cdc18p was expressed in wild-type cells, Dpop1 and Dpop2 mutant cells. Anti-Myc immunoprecipitates were tested for the presence of endogenous Pop2p with anti-Pop2p antisera. Although the interaction between Pop2p and GFP-Cdc18p was detected in lysates from wild-type cells, it was undetectable in lysates from Dpop1 mutants, suggesting that Pop1p-Pop2p heterooligomers cooperate in substrate binding.