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/ Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast. Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast. J. Biol. Chem. (0021-9258) 2003 imex curation Hermand D., Bamps S., Tafforeau L., Vandenhaute J., Makela TP. D.Hermand@cancer.org.uk 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 full dna sequence full identification by DNA sequencing full dna sequence Hermand D., Bamps S., Tafforeau L., Vandenhaute J., Makela TP. D.Hermand@cancer.org.uk Accepted 2007-JAN-23 by JYOTI. J. Biol. Chem. (0021-9258) 2003 Only protein-protein interactions imex curation Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast. Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast. J. Biol. Chem. (0021-9258) 2003 imex curation Hermand D., Bamps S., Tafforeau L., Vandenhaute J., Makela TP. D.Hermand@cancer.org.uk Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Hermand D., Bamps S., Tafforeau L., Vandenhaute J., Makela TP. D.Hermand@cancer.org.uk Accepted 2007-JAN-23 by JYOTI. J. Biol. Chem. (0021-9258) 2003 Only protein-protein interactions imex curation pof6_schpo F-box protein pof6 pof6 SPCC18.04 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MKPSEAREHAIRKIKEYLERRHKQQFKALFGCLTINIYLKIFTLISTPDLCNCRLVCRKFQQLCDYNSIYVKKLLLMNAWDVTLSRKLYYESQDGMSQANMSSNTSKGFHLQSSDKKYADSDRPKLSSSSLLSLPDQKIPFPVDYSTVQGKQVVLTVLDCVSQRVEFARLDYIKVWRALAPIYRNLAYASNTIDPIAFSAFRTPEEQSKVLKYLIRFGNSYPYGTYRQAMQNAILDMASLFEQACLDEFELGLHSRNLDLLRKFSHVLHDFSGPNAYVSMYLAKQTDFVRSFFHFDPYSLFISNNLEEIHINWNILESVVNDTIKLLESESKFSEATLPEPELVQVPYAKDILGNSLKDYVISICEHIGEEETELFLVFISGFYGLCKKFFSIPNGPALVDTIFQPQIDIFISQELHYFKTVGWSLVDQWDQKLEEKEDATECFFYKNVSQNTAKNNFLETFKNVMLLPVSLFTIPSENNSASNLAEKAIEQKEEEDPELSKLDAARFVPANIYVSKDRLKHLPTTELAAQAAVLDSKLEGISTMFSLELALKIVHLCKVSLARAKVFMGTSVPQDDDIKGLSKDLFVQLLRELGQGHLKHGFDRAIEHLSSFDPRRDFSSNTVEPVVKFLELINVGDMIQQMMDSFFNEEMSPICVKDDMFDPAIAEKKKFEQLLDERAAFGLHKGINVLIEHADFLLETKTPMNLFSDQTIGSITNTIEPTAAAKNVVQFLGFHMRILVGRADHEILDVFYKEVGMRLFDSLTRYIKSHKFSVDGGLKLLSDCNLYYEFIHSLHQSSLLPYFKTLKEIAHLFIIDGKNAEEIGKLATDTSRFSSAFHTEEVYEILHSRIDWLNIKYEVDKVIHGLACCIM 0F92F9824F579D6C 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 skp1-pof6-1 1 4 unspecified role unspecified role bait bait 3 unspecified role unspecified role prey prey physical association physical association Fig 3A Pof6 was identified through a twohybrid interaction with Skp1 (Fig. 3A). skp1-pof6-2 2 4 unspecified role unspecified role prey prey in vitro In vitro 3 unspecified role unspecified role bait bait region tap tagged calmodulin binding peptide plus protein a tag CBP-ProtA tagged CBP-ProtA tagged tap tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro physical association physical association Fig 4A To test if the Pof6 F-box protein is part of an SCF, a TAP fusion protein was expressed from the endogenous pof6 locus. After tandem affinity purification (45), the eluate was separated on lithium dodecyl sulfate-PAGE and silver-stained revealing several bands (Fig. 4A). The 19-kDa band represents Skp1 as demonstrated by Western blot analysis (Fig. 4A).