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/ yamano-2004-2 Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2. in vitro In vitro pull down pull down predetermined predetermined participant predetermined Yamano H., Kominami K., Harrison C., Kitamura K., Katayama S., Dhut S., Hunt T., Toda T. toda@cancer.org.uk J. Biol. Chem. (0021-9258) 2004 yamano-2004-1 Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2. schpm Schizosaccharomyces pombe (fission yeast) Fission yeast anti tag coip anti tag coimmunoprecipitation anti tag coip predetermined predetermined participant predetermined Yamano H., Kominami K., Harrison C., Kitamura K., Katayama S., Dhut S., Hunt T., Toda T. toda@cancer.org.uk J. Biol. Chem. (0021-9258) 2004 cg22_schpo G2/mitotic-specific cyclin cig2 cig2 cyc17 SPAPB2B4.03 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MALYSISKPVGSKINKHSYQDENTLVGKQALSKGTEKTKLSTNFEINLPRRTVLSDVSNVGKNNADEKDTKKAKRSFDESNLSTNEEADKPVESKFVKKLKVYSKNADPSVETLQKDRVSNVDDHLSSNPLMAEEYAPEIFEYIRKLDLKCLPNPKYMDQQKELTWKMREILNEWLVEIHSNFCLMPETLYLAVNIIDRFLSRRSCSLSKFQLTGITALLIASKYEEVMCPSIQNFVYMTDGAFTVEDVCVAERYMLNVLNFDLSYPSPLNFLRKISQAEGYDAQTRTLGKYLTEIYLFDHDLLRYPMSKIAAAAMYLSRRLLRRGPWTPKLVESSGGYEEHELKEIAYIMLHYHNKPLEHKAFFQKYSSKRFLKASIFVHQLVRQRYSVNRTDDDDLQSEPSSSLTNDGH 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 cdc18_schpo Cell division control protein 18 cdc18 SPBC14C8.07c protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MCETPIGCHTPRRCNRFIDSAALIDCTNKTNQREHSPSFSIEIPTTPSRKRTLASSHFQTPTKRIKYELGELQEEKTDLYPNFPAQLKENKKPKLPTTPQTPKTPKRTIQIVTPKSLNRTCNPVPFATRLLQSTPHRQLFPPTPSTPSTPSYNSTAKLSLRKSYRSAGVVGRENEKSIVESFFRQHLDANAGGALYVSGAPGTGKTVLLHNVLDHVVSDYPKVNVCYINCMTINEPKAIFEKIHSKIVKEEILENEDHHINFQCELESHFTQSANELYNPVIIVLDEMDHLIAREQQVLYTLFEWPSRPTSRLILVGIANALDMTDRFLPRLRTKHITPKLLSFTPYTAQEISTIIKARLKTAATTSEKNNPFTPIKSISEVSDDSINVVSQHADETPFIHPAAIELCARKVAASSGDLRKALDICRHAIELAEREWKAQHDNTLSSVDIPRASIAHVVRATSAMSQSASARLKNLGLQQKAILCTLVVCEKTSLSVADVFEKYSSLCLRDRLIYPLTSSEFCDVANSLETLAIIRLRTKQRNGKPQDRIISLLVPEMDVITAVGDIGTLKRFFDRR rum1_schpo Cyclin-dependent kinase inhibitor rum1 rum1 SPBC32F12.09 p25-rum1 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MEPSTPPMRGLCTPSTPESPGSFKGVIDASLEGNSSIMIDEIPESDLPAPQVSTFPPTPAKTPKKQLLPNLMLQDRSNSLERCMEEDREHNPFLSSSDNQLLSRKKRKPTPPPSDGLYYVFRGKRIKKSFRPGTDLSTFKPKLLFADSAPSSSSDNPTSSVDLNDYSQIGILPPNLNSIGNKMFSLKSRVPSSSSGSFVAPPPQMRLPAYSSPQKSRSNTKDENRHNLLR pop1-cig2-1 Pull-down showing interaction between cig2 and pop1 596827 N/A 596829 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes N/A 596830 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) physical association physical association false false false Figure 4A. Pop1 was able to interact with Cig2 in-vitro. Mutation of the destruction box (AA 51-59)of cig2 (R51A, L54A) did not interfere with this interaction. pop1-cdc18 Pull-down showing interaction between cdc18 and pop1 596827 N/A 596831 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes N/A 596830 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) physical association physical association false false false Figure 4A, 5A, 5B. Pop1 was able to interact with Cdc18 in-vitro. pop1-rum1 Pull-down showing interaction between rum1 and pop1 596827 N/A 596832 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes N/A 596830 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) physical association physical association false false false Figure 4A, 5A, 5B. Pop1 was able to interact with Rum1 in-vitro. pop1-cig2-2 Pull-down showing interaction between phosphorylated cig2 and pop1 596827 N/A 596829 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes N/A 596830 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) physical association physical association false false false Figure 4B. It was observed that cig2 phosphorylation was a requirement for binding to pop1. pop1-cig2-3 Pull-down showing interaction between cig2 fragments and pop1 596827 N/A 596829 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes N/A 596830 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) physical association physical association false false false Figure 5A, 5B, 5C. It was discovered from deletion analysis, that an internal 93 amino acid region of Cig2 (AA 181-273) was necessary and sufficient for binding to Pop1. pop1-cig2 CoIP showing interaction between cig2 and pop1 596828 N/A 596829 unspecified role unspecified role prey prey schpm Schizosaccharomyces pombe (fission yeast) Fission yeast N/A 596830 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 false schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association false false false Figure 3A and 3B. Pop1 was able to interact with Cig2 in-vivo and this reaction was specific and able to take place even without the presence of Pop1's partner Pop2 in Pop2 depleted cells.