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/ SCF(Pof1)-ubiquitin and its target Zip1 transcription factor mediate cadmium response in fission yeast. SCF(Pof1)-ubiquitin and its target Zip1 transcription factor mediate cadmium response in fission yeast. EMBO J. (0261-4189) 2005 imex curation Harrison C., Katayama S., Dhut S., Chen D., Jones N., Bahler J., Toda T. takashi.toda@cancer.org.uk,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 Harrison C., Katayama S., Dhut S., Chen D., Jones N., Bahler J., Toda T. takashi.toda@cancer.org.uk,toda@cancer.org.uk Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. EMBO J. (0261-4189) 2005 Only protein-protein interactions imex curation zip1_schpo Transcription factor zip1 zip1 SPAC25G10.03 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MDFTPNSAINHLNLKFDDVPVSDDFSKDDLAEQLNVFTNPYFLDLEPSSMLSEGYYGFVSQPSGSSNSNKQEKNVQQQNPEKISTLQQVKEEEVSNTFSAPLNATGNFSSANPASIDLAYLDLQKLLTLPDHSKETQEKTSSQRELFEQKSSVASASKDNVSSSSILQGSASSKLLPDQSARQHQVLVGQTAIPTSEASSSINNTPLQAPVSSFADQNAFTNPLSTFASPDLASVSSPSLSSYKGAQSPNANSKRTKATSAIRTAAEEDKRRRNTAASARFRIKKKLKEQQLERTAKELTEKVAILETRVRELEMENNWLKGLIRPTSNF A681434C779DF960 pof1_schpo F-box/WD repeat-containing protein pof1 Skp1-binding protein 1 pof1 sbp1 SPAC57A10.05c protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MTTGYESVPTSEPSDNLAPRAELWQRHLLEKKEGDQSISVSAFNISSMHNELSGLSEKSRQRVEAVWAAFSEASCSERKLALQGILNNCSSSLLSFASSTLDSLVRLDFLSLLPVEISFRILSFLDARSLCQAAQVSKHWKELADDDVIWHRMCEQHINRKCEKCGWGLPLLERNTLYAAKASIQKRYERLTKRGVDQAHESSPVKKAKLDDYPTSSNEETISSVKPPSPNSDSKFFLPFKTRPWKEVYAERCRVECNWRHGRCRQVVLSGHSDGVMCLQLVRNILASGSYDATIRLWNLATFQQVALLEGHSSGVTCLQFDQCKLISGSMDKTIRIWNYRTSECISILHGHTDSVLCLTFDSTLLVSGSADCTVKLWHFSGGKRITLRGHTGPVNSVRIIRDRGLVLSGSDDSTIKIWSLETNTCLHTFSAHIGPVQSLALADSRLFSCSLDGTIKQWDIEKKKCVHTLFGHIEGVWEIAADHLRLISGAHDGVVKVWEACECVHTLKNHSEPVTSVALGDCEVVSGSEDGKIYLWLFNNAPNESPVSTQSVPISSLNGQRSNSSVQRALSSVPNYSSSLSNISTRNLNIPPSNANNDDVSIQS 7118C9379EC5C1F0 cul1_schpo Cullin-1 cul1 pcu1 SPAC17G6.12 Cell division control 53 homolog protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MTTLNTNDKDLPIVKKYDSLNGTWDFLKTGVSQIFERLDEGMTITKYMELYTAIHNYCADASKTITVDNFNDQTANVLGEALYNNLVLYLEEYLARLRKECISQTNHEEQLAAYAKYWTRFTTSARFINHLFGYLNRYWVKLKNRFTETLVYDIYTLCLVSWHHHVFSHIRDSLLQNLLYMFTKKRLYEPTDMKYVEVCVDSITSLSFDKTDMTKPNLSSYKTFFETNFIENTKNFYAKESSEYLASHSITDYLKKAEIRLAEEEELVRLYLHESTLKPLLEATEDVLIAQHEEVLHNDFARMLDQNCSEDIIRMYRLMSRTPNGLQPLRQTFEEFVKRSGFAAVAKIVPQVGGEADVDPKEYMEMLLSTYKASKELVNTAFHGDTDFTKSLDTAFRELVNRNVVCQRSSSRSPELLAKYADSILRKSNKNVDIDDVEDCLSSIIIIFRYVEDKDVFQNFYTKLLAKRLVNGTSNSQDAESSMLSKLKEVCGFEYTSKLQRMFQDISLSQEITEAFWQLPQSRAGNIDFSALVLGTSFWPLSPNNVNFHLPEELVPLYEGFQNYYYSCHNGRKLSWLFHLSKGEIKARINPQTNVTYVFQVSTYQMGVLLLYNHRDSYTYEELAKITGLSTDFLTGILNIFLKAKVLLLGDNDKLGDPNSTYKINENFRMKKIRVQLNLPIRSEQKQESLETHKTIEEDRKLLLQSAIVRIMKARRTLKHVVLVKETIDQIKSRFTPKVSDIKQCIDMLIEKEYLERQGRDEYIYLA 458CA31B5D97FAA9 pof1-cul1 1 4 unspecified role unspecified role prey prey c-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 3 unspecified role unspecified role bait bait c-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Coprecipitation of pof1 with cul1 showed that pof1 is a component of the fission yeast SCF ubiquitin ligase. Immunoblotting showed that Pof1-GST co-precipitated with Pcu1-13Myc (Figure 1A). Figure 1A pof1-zip1 1 3 unspecified role unspecified role bait bait c-terminal 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 c-term range C-terminal range c-term range c-term range C-terminal range c-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 2 unspecified role unspecified role prey prey c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range region phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) western blot western blot Immuno blot Immuno blot undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Coprecipitation of pof1 with phosphorylated zip1. Figure 4D, 4E If Zip1 is a substrate for the SCFPof1, Pof1 is expected to bind Zip1. Immunoprecipitation was performed in a strain containing Pof1-GFP and Zip1-HA. To prevent degradation of Zip1, experiments were again carried out in an mts3-1 background. Under this condition, Zip1 was detected as a doublet, the lower band of which corresponded to the band seen in wild-type cells (band 1 in Figure 4C) and the upper of which appeared to correlate to the faster migrating form (band 2 in Figure 4C) of the phosphorylated Zip1 bands (Figure 4D, lanes 2 and 4). Immunoprecipitation using anti-GFP antibody showed that Pof1 co-immunoprecipitated with Zip1, specifi- cally the upper band of the doublet (Figure 4D, lane 5). Reciprocal immunoprecipitation with anti-HA antibody also co-precipitated Pof1-GFP, albeit very faintly (lane 4). This is probably due to the existence of an excess amount of free Zip1-HA dissociated from Pof1-GFP in the mts3-1 mutant. In order to examine which of Zip1 forms binds Pof1 in the pof1-6 background, immunoprecipitation was performed using samples prepared from the pof1-6 mutant used in Figure 4A (doubly tagged with Pof1-6-GFP and Zip1-HA). For comparison, parallel immunoprecipitation was also performed in extracts from mts3-1 cells and precipitates were run side-by-side. As shown in Figure 4E, it is evident that two phosphorylated Zip1 forms interacted with Pof1-6 (lane 7, marked as bands 2 and 3). Taken together, these findings indicate that Zip1 is phosphorylated and this phosphorylation leads to an interaction with Pof1 and subsequent degradation.