UniProt UniProt http://www.uniprot.org Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast. Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast. J. Biol. Chem. (0021-9258) 2001 imex curation Traverso EE., Baskerville C., Liu Y., Shou W., James P., Deshaies RJ., Charbonneau H. charb@purdue.edu 03-Feb-2009: Contacted by JYOTI. orchard 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 Traverso EE., Baskerville C., Liu Y., Shou W., James P., Deshaies RJ., Charbonneau H. charb@purdue.edu Accepted 2009-FEB-13 by ORCHARD. 03-Mar-2009: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2001 orchard Only protein-protein interactions imex curation Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast. Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast. J. Biol. Chem. (0021-9258) 2001 imex curation Traverso EE., Baskerville C., Liu Y., Shou W., James P., Deshaies RJ., Charbonneau H. charb@purdue.edu 03-Feb-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro phosphatase assay phosphatase assay predetermined predetermined participant predetermined Traverso EE., Baskerville C., Liu Y., Shou W., James P., Deshaies RJ., Charbonneau H. charb@purdue.edu Accepted 2009-FEB-13 by ORCHARD. 03-Feb-2009: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2001 orchard Only protein-protein interactions imex curation Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast. Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast. J. Biol. Chem. (0021-9258) 2001 imex curation Traverso EE., Baskerville C., Liu Y., Shou W., James P., Deshaies RJ., Charbonneau H. charb@purdue.edu 03-Feb-2009: Contacted by JYOTI. orchard 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Traverso EE., Baskerville C., Liu Y., Shou W., James P., Deshaies RJ., Charbonneau H. charb@purdue.edu Accepted 2009-FEB-13 by ORCHARD. 03-Feb-2009: Contacted by JYOTI. 3 distinct genomic libraries were used. PubMed 8978031. J. Biol. Chem. (0021-9258) 2001 orchard Only protein-protein interactions imex curation Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast. Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast. J. Biol. Chem. (0021-9258) 2001 imex curation Traverso EE., Baskerville C., Liu Y., Shou W., James P., Deshaies RJ., Charbonneau H. charb@purdue.edu 03-Feb-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Traverso EE., Baskerville C., Liu Y., Shou W., James P., Deshaies RJ., Charbonneau H. charb@purdue.edu Accepted 2009-FEB-13 by ORCHARD. 03-Feb-2009: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2001 orchard Only protein-protein interactions imex curation sic1_yeast Protein SIC1 SIC1 SDB25 YLR079W L9449.8 CDK inhibitor p40 protein protein yeast Saccharomyces cerevisiae Baker's yeast MTPSTPPRSRGTRYLAQPSGNTSSSALMQGQKTPQKPSQNLVPVTPSTTKSFKNAPLLAPPNSNMGMTSPFNGLTSPQRSPFPKSSVKRTLFQFESHDNGTVREEQEPLGRVNRILFPTQQNVDIDAAEEEEEGEVLLPPSRPTSARQLHLSLERDEFDQTHRKKIIKDVPGTPSDKVITFELAKNWNNNSPKNDARSQESEDEEDIIINPVRVGKNPFASDELVTQEIRNERKRAMLRENPDIEDVITYVNKKGEVVEKRRLTDEEKRRFKPKALFQSRDQEH 7913636340678F78 7913636340678F78 cdc14_yeast Tyrosine-protein phosphatase CDC14 CDC14 OAF3 YFR028C protein protein yeast Saccharomyces cerevisiae Baker's yeast MRRSVYLDNTIEFLRGRVYLGAYDYTPEDTDELVFFTVEDAIFYNSFHLDFGPMNIGHLYRFAVIFHEILNDPENANKAVVFYSSASTRQRANAACMLCCYMILVQAWTPHQVLQPLAQVDPPFMPFRDAGYSNADFEITIQDVVYGVWRAKEKGLIDLHSFNLESYEKYEHVEFGDFNVLTPDFIAFASPQEDHPKGYLATKSSHLNQPFKSVLNFFANNNVQLVVRLNSHLYNKKHFEDIGIQHLDLIFEDGTCPDLSIVKNFVGAAETIIKRGGKIAVHCKAGLGRTGCLIGAHLIYTYGFTANECIGFLRFIRPGMVVGPQQHWLYLHQNDFREWKYTTRISLKPSEAIGGLYPLISLEEYRLQKKKLKDDKRVAQNNIEGELRDLTMTPPSNGHGALSARNSSQPSTANNGSNSFKSSAVPQTSPGQPRKGQNGSNTIEDINNNRNPTSHANRKVVIESNNSDDESMQDTNGTSNHYPKVSRKKNDISSASSSRMEDNEPSATNINNAADDTILRQLLPKNRRVTSGRRTTSAAGGIRKISGSIKK 4EB3985DFA3FD823 4EB3985DFA3FD823 swi5_yeast Transcriptional factor SWI5 SWI5 YD8358.03C YDR146C protein protein yeast Saccharomyces cerevisiae Baker's yeast MDTSNSWFDASKVQSLNFDLQTNSYYSNARGSDPSSYAIEGEYKTLATDDLGNILNLNYGETNEVIMNEINDLNLPLGPLSDEKSVKVSTFSELIGNDWQSMNFDLENNSREVTLNATSLLNENRLNQDSGMTVYQKTMSDKPHDEKKISMADNLLSTINKSEINKGFDRNLGELLLQQQQELREQLRAQQEANKKLELELKQTQYKQQQLQATLENSDGPQFLSPKRKISPASENVEDVYANSLSPMISPPMSNTSFTGSPSRRNNRQKYCLQRKNSSGTVGPLCFQELNEGFNDSLISPKKIRSNPNENLSSKTKFITPFTPKSRVSSATSNSANITPNNLRLDFKINVEDQESEYSEKPLGLGIELLGKPGPSPTKSVSLKSASVDIMPTIPGSVNNTPSVNKVSLSSSYIDQYTPRGKQLHFSSISENALGINAATPHLKPPSQQARHREGVFNDLDPNVLTKNTDNEGDDNEENEPESRFVISETPSPVLKSQSKYEGRSPQFGTHIKEINTYTTNSPSKITRKLTTLPRGSIDKYVKEMPDKTFECLFPGCTKTFKRRYNIRSHIQTHLEDRPYSCDHPGCDKAFVRNHDLIRHKKSHQEKAYACPCGKKFNREDALVVHRSRMICSGGKKYENVVIKRSPRKRGRPRKDGTSSVSSSPIKENINKDHNGQLMFKLEDQLRRERSYDGNGTGIMVSPMKTNQR BEF5ED5BFB6E30F6 BEF5ED5BFB6E30F6 net1_yeast Nucleolar protein NET1 YJL076W J1038 NET1 CFI1 ESC5 SRM8 protein protein yeast Saccharomyces cerevisiae Baker's yeast MYKLQVVLVPPSLQATMPIQFGYGPTIAESSQLLPNRTNMAQSAGDASLQYANLRSANVSFTPSYFNQSRFRKFLLFTKPTNTLLNLSDEIIDKCEKMYPSLQEDIEILSLQDNSGCDLDPDFLVKDVFNVNNIVRVILKNEIDLDDSAPVSLYKSVKRSKLNNGSPQSVQPQQQIPSSSGVLRIAKKRPPTGTTTTTTIRSATNGSMRVSTPLARQIYPPPSSKIVSNNSDDEDEDIGERSFLPPPTQPQSPPIRISSGIDAGKKIKSSIVEEDIVSRSATVDPDKTKQQRLLSGTPIMSTMTPNRVTLTGQRVVSEHAHKNELVFSASASSSSFANGGTAAVTAQDINRKPPVTTPRITSGMLKIPEPRISEIEKELKEGPSSPASILPAKAAKIPMKKPYLENGENYESDDSSSSENQETPETEPHSKASLQRSQSSIADNNGSPVKNSPLGDAMPHNVHLAELPKASNTSITKSSNGESWGKQQEHQPPRKSSLETIVEKKSQAEPSGIVEPKRMTNFLDDNQVREKEDTNDKLLEKEILPTIPHNDQPILASSDKSNGTLKSLAGKVSSNNNASKEDGTIINGTIEDDGNDNDEVDTTVRIVPQDSDSSSFPKSDLFKMIEGDDTDLPQWFKGKNSRTSGNSKNSKPYTTVLNKDIDNSKPDPRNILPQRTPRSAAKRAAQLLAGAKKNEVPQKSTEDSSSAASTDDESESGIETDFSSDDDFKRKNMSVPNNGPKDISLHSLKGSVVPVKDSKIINKEVDEERNDKRDSQKKSAVSESSVTNSKISEQMAKSFYPNSNKKQNEATKVETKPATQASSFPVVGGSPSVATKGTTSFNEEGNRKNVKTKAKNESAQIDRQQKETTSRVADLKSANIGGEDLNKKAEGSKEPEKASANIQDANDKNNSKEKEDSKSKQVSQKKLKMTDHLKEGNVQLPKPSANDKLKDLKAKFTNSKTLVPPGIISNEKNNSSANDDDSSSSGSSTEDESSSSSSSSDEETSTSRKARRVVVNTPREPVRSSSKIEAPSPSVNKKINATPDKIPVTQLMDMSSPPSVKSKTTSNPSSILHDLPRKVRPSLSSLSDLVSRGIPDVKEKTSKSNEKSQTKAPSSSDDESSSDSDSNSSSDSVSDSSSDSKSESDSDDSGDSSDDGKSFISAKSASAALGKKKKPSGGFASLIKDFKKK 80EDF9A8065EB5A2 80EDF9A8065EB5A2 cdc14-net1-2 1 8 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role bait bait physical association physical association Table 1 A two-hybrid assay confirmed that a fragment of Net1 containing residues 1-601 could interact with Cdc14 cdc14-net1-3 3 8 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait physical association physical association In addition to a genetic screen described by Shou et al. (18), we and others (19) also identified Net1 in a yeast two-hybrid screen for regulators or substrates of Cdc14 cdc14-net1-4 4 8 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecobb Escherichia coli (strain B / BL21) 6 unspecified role unspecified role bait bait n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Fig. 1, 3, 4, 5A, 5B. By sodium tungstate. purified Net1(1-341)-His6 (1 nmol) was incubated for 30 min at 4 C with GST and GST-Cdc14 affinity matrices containing 0.25 nmol of each protein. cdc14-swi5-1 2 6 enzyme enzyme neutral component neutral component n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobb Escherichia coli (strain B / BL21) 7 enzyme target enzyme target substrate substrate neutral component neutral component c-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range residue 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 ecobb Escherichia coli (strain B / BL21) dephosphorylation dephosphorylation reaction dephosphorylation Fig. 2A Interaction is inhibited by NET1. cdc14-sic1-1 2 5 enzyme target enzyme target substrate substrate neutral component neutral component c-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range residue 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 ecobb Escherichia coli (strain B / BL21) 6 enzyme enzyme neutral component neutral component n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobb Escherichia coli (strain B / BL21) dephosphorylation dephosphorylation reaction dephosphorylation Fig. 2A Interaction is inhibited by NET1.