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/ Fission yeast Mcl1 interacts with SCF(Pof3) and is required for centromere formation. Fission yeast Mcl1 interacts with SCF(Pof3) and is required for centromere formation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Mamnun YM., Katayama S., Toda T. toda@cancer.org.uk 12-MAR-2007: Contacted by JYOTI. 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 Mamnun YM., Katayama S., Toda T. toda@cancer.org.uk Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation Fission yeast Mcl1 interacts with SCF(Pof3) and is required for centromere formation. Fission yeast Mcl1 interacts with SCF(Pof3) and is required for centromere formation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Mamnun YM., Katayama S., 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 Mamnun YM., Katayama S., Toda T. toda@cancer.org.uk Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation skp1_schpo Suppressor of kinetochore protein 1 skp1 psh1 sph1 SPBC409.05 P19/Skp1 homolog protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MSKIKLISSDNEEFVVDQLIAERSMLIKNMLEDVGEINVPIPLPNVSSNVLRKVLEWCEHHKNDLYSGTEEESDIRLKKSTDIDEWDRKFMAVDQEMLFEIVLASNYLDIKPLLDTGCKTVANMIRGKSPEDIRKTFNIPNDFTPEEEEQIRKENEWAEDR 07EA1E97A3556E78 mcl1_schpo Minichromosome loss protein 1 mcl1 SPAPB1E7.02c slr3 DNA polymerase alpha accessory factor Mcl1 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MAGNRLVPRYAHTDGLTRLAYTRDGKFLLTVGSNQVIRKFQVGSDEEPDSIDNHQDPITGIAVAENYFCTCSEDATVCVYPIDSPTEHTLLARTTLPIRDVAYSVDGNWIAIASDETAVKVVSSTDSSQIFSLRPAKASNKHVTYSPNGNFLAVSSCNGILYFYDTQTRELIKFLTNTIASLEAESEICSKAAWHPKNGTFAVASTDHFVSVISPDDWLPLYKLLPKENHSGVTDISWSSNGMYIAASFKKGGILIWDTQSHEVVVELPYSTVVALAWQPFENVLSFTTNQGILYSCPDVIPKSILKEENDPTKPLTSSKSKNRTSKELDDLFGSDDEQSQNVNDLDGNSANEENEFINHDGLDSSLDLDGDSYMVDENDLNLAKKRKQKALIDRTTTIENGSSKRRLLQASIHKPVHTGSTPWQGNRRYLCLNLVGFIWTVQQDAEHNTITVEFHDETTHRKYHFVDDQKFEMACLDHEGALYASPATESSPGVIYYKAHVDWSRKSEWAMALPMENESPVTISLSSSVVLVCTSAGYVRVFSRQGFPISIHRSKHLPFVACSSFQDTIITIANDGLSSDGNSRLVYSIEDISRDEMLQTGDGVALPPQGTLESVFFSDVGDPYIYDSTGVLLVLMHWRIPGQAKWIPVLDTNELERRKSRQESYWPVTVADNQFHCILLKGASRYPYFPRPMFTEFDFRIPCNTNNPDASTSVPVLEELQLRNKLFLTLLEDSIGDGDVTEDEKISIARLEANIDKALLQLIQKACLEERIERVYELTKTLRRTTSIAAAQKIALHHSLTNVAEKIGNLLSNV D3AD3EF2D7997C54 pof3_schpo F-box/TPR repeat protein pof3 pof3 SPCC338.16 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MNNYQVKAIKEKTQQYLSKRKFEDALTFITKTIEQEPNPTIDLFELRAQVYEKSGQYSQAELDAKRMIHLNARNARGYLRLGKLLQLDGFDKKADQLYTQGLRMVHKMDPLRPVLKKVSQRLNERILRTRPVLDLFRILPREVLLCILQQLNFKSIVQCMQVCKHWRDCIKKEPSLFCCLDFSCASPRSVNSRDRNVMAVARYSVYSKDNIQEVIGLEKLGILTPTKALLRSVKSLKVYKTISPLHTQSTDKLYTIWTPFSELHYFYCATPITFSIASKILSCCKKLKQVELVDLIPDLIFDSMDWDKLFNAESVPLALKSLTFIRNQKFPFHHKEQQFLKDLLSASPYLEYLEASYQSDLVAAIKKYKINLRSLIIIDEGVSNTVKDLAFLPQSLTTLIVKPCNPASTILCPYLFPTNVRMESLINLELFLYLRLSQNDIDNVVKFLTSCYKLKKLVLHDSLALAPHFFEIFASLPELEHLEIPDNVALQNKHAIHITDCCPNLKYVNFSNSISLDGSGFIAVLRGLKELKRIDIINCDSVSRDAIDWARSKGMQVTVASSLPNSQPLGTKKIRLI 507D7901C1980D3F pof3-skp1-1 1 5 unspecified role unspecified role bait bait 3 unspecified role unspecified role prey prey physical association physical association To gain further insight into the function of Pof3, we performed a yeast two-hybrid screen to identify Pof3-interacting proteins. Using the full-length Pof3 as bait, we screened 2.7x10.6 colonies and isolated 16 candidate plasmids, which were able to confer growth on plates lacking histidine specifically in the presence of Pof3. Sequencing revealed that 12 of these plasmids contained the known Pof3-binding partner Skp1 and therefore served as positive controls for the screening conditions. Two of the plasmids encoded a previously not described open reading frame and two corresponded to an N-terminal truncated version of Mcl1. Cloning full-length Mcl1 into the pGAN6 vector confirmed that Pof3 and Mcl1 interact, as cells could only grow in the absence of histidine when both plasmids were present (Fig. 1A). Figure 1A. pof3-mcl1 1 5 unspecified role unspecified role bait bait 4 unspecified role unspecified role prey prey physical association physical association To gain further insight into the function of Pof3, we performed a yeast two-hybrid screen to identify Pof3-interacting proteins. Using the full-length Pof3 as bait, we screened 2.7x10.6 colonies and isolated 16 candidate plasmids, which were able to confer growth on plates lacking histidine specifically in the presence of Pof3. Sequencing revealed that 12 of these plasmids contained the known Pof3-binding partner Skp1 and therefore served as positive controls for the screening conditions. Two of the plasmids encoded a previously not described open reading frame and two corresponded to an N-terminal truncated version of Mcl1. Cloning full-length Mcl1 into the pGAN6 vector confirmed that Pof3 and Mcl1 interact, as cells could only grow in the absence of histidine when both plasmids were present (Fig. 1A). Figure 1A. pof3-skp1-2 2 5 unspecified role unspecified role bait bait 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 prey prey schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Figure 1B A substantial amount of Skp1 also copurified with Pof3-13myc (Fig. 1B), again serving as an internal control. An anti-skp1 antibody was used to detect the presence of skp1 and the anti-myc antibody showed the presence of pof3-myc with skp1. pof3-mcl1-1 2 5 unspecified role unspecified role bait bait 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 4 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association To confirm this interaction in Schizosaccharomyces pombe cells we performed immunoprecipitation experiments (Fig. 1B and C). Strains carrying either Pof3-13myc or GFP-Mcl1, the latter under the control of the thiamine-repressible nmt1 promoter, or both were grown in YE5S medium, harvested, and immunoprecipitation was performed from cell extracts using either anti-myc (Fig. 1B) or anti-GFP antibody (Fig. 1C). Western blot analysis of the precipitates showed that the band corresponding to GFP-Mcl1 could only be detected in the samples containing Pof3-13myc, demonstrating that purification of GFPMcl1 is dependent on its interaction with the F-box protein. The same was true for the immunoprecipitation of GFPMcl1 (Fig. 1C), in which Pof3-13myc could only be precipitated with GFP antibodies in the presence of GFP-Mcl1 but not in its absence. We therefore concluded that Mcl1 is indeed a binding partner of Pof3. Figure 1B and 1C. Pof3 is shown to interact with Mcl1.