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/ Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2. Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2. J. Biol. Chem. (0021-9258) 2004 Yamano H., Kominami K., Harrison C., Kitamura K., Katayama S., Dhut S., Hunt T., Toda T. toda@cancer.org.uk 10-APR-2007: Contacted by JYOTI. 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 Accepted 2007-APR-14 by JYOTI. 10-APR-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2004 Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2. Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2. J. Biol. Chem. (0021-9258) 2004 Yamano H., Kominami K., Harrison C., Kitamura K., Katayama S., Dhut S., Hunt T., Toda T. toda@cancer.org.uk 10-APR-2007: Contacted by JYOTI. 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 Accepted 2007-APR-14 by JYOTI. 10-APR-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2004 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 cdc18_schpo Cell division control protein 18 cdc18 SPBC14C8.07c protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MCETPIGCHTPRRCNRFIDSAALIDCTNKTNQREHSPSFSIEIPTTPSRKRTLASSHFQTPTKRIKYELGELQEEKTDLYPNFPAQLKENKKPKLPTTPQTPKTPKRTIQIVTPKSLNRTCNPVPFATRLLQSTPHRQLFPPTPSTPSTPSYNSTAKLSLRKSYRSAGVVGRENEKSIVESFFRQHLDANAGGALYVSGAPGTGKTVLLHNVLDHVVSDYPKVNVCYINCMTINEPKAIFEKIHSKIVKEEILENEDHHINFQCELESHFTQSANELYNPVIIVLDEMDHLIAREQQVLYTLFEWPSRPTSRLILVGIANALDMTDRFLPRLRTKHITPKLLSFTPYTAQEISTIIKARLKTAATTSEKNNPFTPIKSISEVSDDSINVVSQHADETPFIHPAAIELCARKVAASSGDLRKALDICRHAIELAEREWKAQHDNTLSSVDIPRASIAHVVRATSAMSQSASARLKNLGLQQKAILCTLVVCEKTSLSVADVFEKYSSLCLRDRLIYPLTSSEFCDVANSLETLAIIRLRTKQRNGKPQDRIISLLVPEMDVITAVGDIGTLKRFFDRR 74DBE9D5CF23504D rum1_schpo Cyclin-dependent kinase inhibitor rum1 rum1 SPBC32F12.09 p25-rum1 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MEPSTPPMRGLCTPSTPESPGSFKGVIDASLEGNSSIMIDEIPESDLPAPQVSTFPPTPAKTPKKQLLPNLMLQDRSNSLERCMEEDREHNPFLSSSDNQLLSRKKRKPTPPPSDGLYYVFRGKRIKKSFRPGTDLSTFKPKLLFADSAPSSSSDNPTSSVDLNDYSQIGILPPNLNSIGNKMFSLKSRVPSSSSGSFVAPPPQMRLPAYSSPQKSRSNTKDENRHNLLR C5199FE345F7484A cg22_schpo G2/mitotic-specific cyclin cig2 cig2 cyc17 SPAPB2B4.03 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MALYSISKPVGSKINKHSYQDENTLVGKQALSKGTEKTKLSTNFEINLPRRTVLSDVSNVGKNNADEKDTKKAKRSFDESNLSTNEEADKPVESKFVKKLKVYSKNADPSVETLQKDRVSNVDDHLSSNPLMAEEYAPEIFEYIRKLDLKCLPNPKYMDQQKELTWKMREILNEWLVEIHSNFCLMPETLYLAVNIIDRFLSRRSCSLSKFQLTGITALLIASKYEEVMCPSIQNFVYMTDGAFTVEDVCVAERYMLNVLNFDLSYPSPLNFLRKISQAEGYDAQTRTLGKYLTEIYLFDHDLLRYPMSKIAAAAMYLSRRLLRRGPWTPKLVESSGGYEEHELKEIAYIMLHYHNKPLEHKAFFQKYSSKRFLKASIFVHQLVRQRYSVNRTDDDDLQSEPSSSLTNDGH 6F255D2E26C6F8B3 pop1-cig2 1 6 unspecified role unspecified role prey prey 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 3A and 3B. As shown in Fig. 3A, Pop1-HA coimmunoprecipitated with Cig2 (lane 3). This interaction was specific, as Cig2 did not precipitate in the absence of primary anti-HA antibody (lanes 2 and 5), and in addition, Cdc13 was not pulled down in the precipitates under the same conditions (lanes 3 and 6). As Pop1 and Pop2 act in the same pathway by forming heterodimers as well as individual homodimers (26, 37), we then addressed whether Pop1 is still capable of interacting with Cig2 in the absence of Pop2. Pop1-HA was expressed in pop2 mutants, and immunoprecipitation was performed as described above. As shown in Fig. 3B, Pop1 coprecipitated with Cig2 in this mutant as efficiently as in wild type cells (lanes 4 and 7). Again, an interaction was specific, as Cig2 did not precipitate in the absence of primary antibody or with an excess amount of HA peptide competitors (lanes 3, 5, 6, and 8). These results show that Pop1 forms a complex with Cig2 in the cell, and for this interaction, its partner Pop2 is not required. 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. pop1-cig2-1 2 6 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 rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes 3 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 ecoli Escherichia coli (strain K12) physical association physical association Figure 4A. We next sought to examine binding characteristics between Cig2 and Pop1 using in vitro translation systems. At first, to assess binding capability and specificity, various full-length proteins were labeled with radioisotope, incubated with Cdc2 kinase, and then mixed with bacterially produced and purified GST-tagged Pop1 protein (GST-Pop1). The binding between radiolabeled proteins and GST-Pop1 was examined by pull-down with GSH-Sepharose beads. Proteins translated and labeled in these reactions included Cig2, Cig2/dm (Cig2 construct with a mutated destruction box), Cdc13, Cdc18, and Rum1 (Fig. 4A, lanes 1�6, and see �Experimental Procedures�). These pull-down experiments showed that GST-Pop1 bound Cig2 as efficiently as Cdc18 and Rum1 (lanes 7, 10, and 11), whereas GST-Pop1 did not bind Cdc13 (lane 9). Moreover, Cig2 was not pulled down by mock Sepharose beads (lane 12), indicating that the interaction depends on Pop1. Consistent with the independence of the APC/C in SCFPop1/Pop2-mediated Cig2 degradation, Cig2/dm was still capable of binding GST-Pop1 (lane 8). 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 2 4 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 rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes 3 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 ecoli Escherichia coli (strain K12) physical association physical association Figure 4A, 5A, 5B. We next sought to examine binding characteristics between Cig2 and Pop1 using in vitro translation systems. At first, to assess binding capability and specificity, various full-length proteins were labeled with radioisotope, incubated with Cdc2 kinase, and then mixed with bacterially produced and purified GST-tagged Pop1 protein (GST-Pop1). The binding between radiolabeled proteins and GST-Pop1 was examined by pull-down with GSH-Sepharose beads. Proteins translated and labeled in these reactions included Cig2, Cig2/dm (Cig2 construct with a mutated destruction box), Cdc13, Cdc18, and Rum1 (Fig. 4A, lanes 1�6, and see �Experimental Procedures�). These pull-down experiments showed that GST-Pop1 bound Cig2 as efficiently as Cdc18 and Rum1 (lanes 7, 10, and 11), whereas GST-Pop1 did not bind Cdc13 (lane 9). Moreover, Cig2 was not pulled down by mock Sepharose beads (lane 12), indicating that the interaction depends on Pop1. Consistent with the independence of the APC/C in SCFPop1/Pop2-mediated Cig2 degradation, Cig2/dm was still capable of binding GST-Pop1 (lane 8). Pop1 was able to interact with Cdc18 in-vitro. pop1-rum1 2 5 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 rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes 3 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 ecoli Escherichia coli (strain K12) physical association physical association Figure 4A, 5A, 5B. We next sought to examine binding characteristics between Cig2 and Pop1 using in vitro translation systems. At first, to assess binding capability and specificity, various full-length proteins were labeled with radioisotope, incubated with Cdc2 kinase, and then mixed with bacterially produced and purified GST-tagged Pop1 protein (GST-Pop1). The binding between radiolabeled proteins and GST-Pop1 was examined by pull-down with GSH-Sepharose beads. Proteins translated and labeled in these reactions included Cig2, Cig2/dm (Cig2 construct with a mutated destruction box), Cdc13, Cdc18, and Rum1 (Fig. 4A, lanes 1�6, and see �Experimental Procedures�). These pull-down experiments showed that GST-Pop1 bound Cig2 as efficiently as Cdc18 and Rum1 (lanes 7, 10, and 11), whereas GST-Pop1 did not bind Cdc13 (lane 9). Moreover, Cig2 was not pulled down by mock Sepharose beads (lane 12), indicating that the interaction depends on Pop1. Consistent with the independence of the APC/C in SCFPop1/Pop2-mediated Cig2 degradation, Cig2/dm was still capable of binding GST-Pop1 (lane 8). Pop1 was able to interact with Rum1 in-vitro. pop1-cig2-2 2 6 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 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 rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes 3 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 ecoli Escherichia coli (strain K12) physical association physical association Figure 4B. It was observed that cig2 phosphorylation was a requirement for binding to pop1. The requirement of Cig2 phosphorylation for binding to Pop1 was, therefore, examined in the above binding assays by treating Cig2 with lambda-phosphatase. As shown in Fig. 4B, it was evident that binding between Pop1 and Cig2 was abolished by phosphatase treatment (lane 5). Phosphatase activity was required for this inhibition, as simultaneous inclusion of phosphatase inhibitors blocked its inhibitory effect (lane 6). This result suggests that the interaction between Pop1 and Cig2 is dependent on phosphorylation of Cig2. pop1-cig2-3 2 6 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 region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes 3 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 ecoli Escherichia coli (strain K12) physical association physical association 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. we sought to identify the domain within Cig2 that is necessary for an interaction with Pop1. For this purpose, various subclones of Cig2 that lack either N-terminal or C-terminal regions were expressed in the rabbit reticulocyte lysate system (Fig. 5A, lanes 3-6). Binding between translated proteins and GST-Pop1 was then examined as before. In the case of N-terminal truncations, subclones lacking 180 amino acid residues (Cig2-N 180) as well as deletions of 60 and 121 residues were still capable of interacting with GST-Pop1 (Fig. 5A, lanes 12�15). Again, interaction was specific as Cig2 without GST-Pop1 or with GST alone did not pull down Cig2 (lanes 10 and 11), and only Cdc18 and Rum1, but not Cdc13, bound GST-Pop1 (lanes 16�18). This result shows that N-terminal 180 amino acid residues of Cig2 are dispensable for binding to Pop1. As for C-terminal truncations (Fig. 5B, lanes 1-5), two constructs (Cig2-C 53 and - 106, lanes 10 and 12) were still positive, although binding of Cig2-C 106 was somewhat weaker. In contrast, the other two deletions (Cig2- 85 and - 190, lanes 11 and 13) did not bind Pop1. We do not know at present the reason that Cig2- 85 was negative, whereas the shorter Cig2- 106 could bind Pop1. It is possible some structural abnormalities specifically created in Cig2- 85 may hinder an interaction with Pop1. To delineate further the minimal region of Cig2 for binding to Pop1, an internal fragment containing 181-358 was constructed and expressed (Fig. 5C, lane 4). As expected from the above results, this fragment was indeed capable of binding to Pop1 (lane 12). Then, this fragment was further divided into two regions, 181-283 and 284-385 (lanes 5 and 6, respectively), and binding of each fragment was examined. It was found that only the N-terminal region (181-283) was positive (lanes 13 and 14). Also, a shorter fragment consisting of 181-273 (lane 8) bound to Pop1 (lane 16). Taking these results together (see Fig. 5D for a summary of the binding assay), internal 93 amino acid residues of Cig2 (181-273) are necessary and sufficient for binding to Pop1.