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/ In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Plant Physiol. (0032-0889) 2007 imex curation Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl 01-April-2008: Contacted by JYOTI. orchard Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret predetermined predetermined participant predetermined Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl Accepted 2008-MAR-5 by ORCHARD. 01-April-2008: Contacted by JYOTI. Plant Physiol. (0032-0889) 2007 orchard Only protein-protein interactions imex curation In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Plant Physiol. (0032-0889) 2007 imex curation Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl 01-April-2008: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl Accepted 2008-MAR-5 by ORCHARD. 01-April-2008: Contacted by JYOTI. Plant Physiol. (0032-0889) 2007 orchard Only protein-protein interactions imex curation In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Plant Physiol. (0032-0889) 2007 imex curation Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl 01-April-2008: Contacted by JYOTI. orchard Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress fluorescence imaging fluorescence microscopy fluorescence imaging tag fluorescence tag visualisation by fluorescence tag fluorescence Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl Accepted 2008-MAR-5 by ORCHARD. 01-April-2008: Contacted by JYOTI. Plant Physiol. (0032-0889) 2007 orchard Only protein-protein interactions imex curation In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Plant Physiol. (0032-0889) 2007 imex curation Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl 01-April-2008: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro in gel kinase assay in-gel kinase assay in gel kinase assay fingerprinting peptide massfingerprinting fingerprinting Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. An in-vitro kinase assay was carried out using unlabelled ATP and the proteins isolated from a gel and analysed by liquid chromatography (LC)-tandem mass spectrometry. sacco.devries@wur.nl Accepted 2008-MAR-5 by ORCHARD. 01-April-2008: Contacted by JYOTI. Plant Physiol. (0032-0889) 2007 orchard Only protein-protein interactions imex curation In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Plant Physiol. (0032-0889) 2007 imex curation Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl 01-April-2008: Contacted by JYOTI. orchard Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress comigration in gel comigration in gel electrophoresis comigration in gel anti tag western anti tag western blot anti tag western Aker J., Hesselink R., Engel R., Karlova R., Borst JW., Visser AJ., de Vries SC. sacco.devries@wur.nl Accepted 2008-MAR-5 by ORCHARD. 01-April-2008: Contacted by JYOTI. Plant Physiol. (0032-0889) 2007 orchard Only protein-protein interactions imex curation serk1_arath Somatic embryogenesis receptor kinase 1 SERK1 Somatic embryogenesis receptor-like kinase 1 F14O23.21 F14O23_24 At1g71830 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MESSYVVFILLSLILLPNHSLWLASANLEGDALHTLRVTLVDPNNVLQSWDPTLVNPCTWFHVTCNNENSVIRVDLGNAELSGHLVPELGVLKNLQYLELYSNNITGPIPSNLGNLTNLVSLDLYLNSFSGPIPESLGKLSKLRFLRLNNNSLTGSIPMSLTNITTLQVLDLSNNRLSGSVPDNGSFSLFTPISFANNLDLCGPVTSHPCPGSPPFSPPPPFIQPPPVSTPSGYGITGAIAGGVAAGAALLFAAPAIAFAWWRRRKPLDIFFDVPAEEDPEVHLGQLKRFSLRELQVASDGFSNKNILGRGGFGKVYKGRLADGTLVAVKRLKEERTPGGELQFQTEVEMISMAVHRNLLRLRGFCMTPTERLLVYPYMANGSVASCLRERPPSQPPLDWPTRKRIALGSARGLSYLHDHCDPKIIHRDVKAANILLDEEFEAVVGDFGLAKLMDYKDTHVTTAVRGTIGHIAPEYLSTGKSSEKTDVFGYGIMLLELITGQRAFDLARLANDDDVMLLDWVKGLLKEKKLEMLVDPDLQTNYEERELEQVIQVALLCTQGSPMERPKMSEVVRMLEGDGLAEKWDEWQKVEILREEIDLSPNPNSDWILDSTYNLHAVELSGPR DIP protein Q94AG2 original sequence version: 112 DIP protein Q94AG2 original sequence version: 115 98F6B69126DB664A cd48c_arath Cell division control protein 48 homolog C EMB1354 Protein EMBRYO DEFECTIVE 1354 CDC48C F4P13.15 At3g01610 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MGRRGRGGGGGMGGGINRRYLSQVMDTCGKDLSTAEDIVDDLRSRYGNFARLTRQVLLLNVRQVLNVRNNKRVKDEDEDDNIGDEEGSASQRKKQRRVDEKEEKLQRAEQSHLRKRNMERSVSSSPSSSSSSEDSGDVSTSEDAVYGEKLSPPRFDLINDSLRDNYAKLNSSSKKPIGSPAEKNVEVETVSNKGRSKLATMGARKEAKVSLSLSGATGNGDLEVEGTKGPTFKDFGGIKKILDELEMNVLFPILNPEPFKKIGVKPPSGILFHGPPGCGKTKLANAIANEAGVPFYKISATEVISGVSGASEENIRELFSKAYRTAPSIVFIDEIDAIGSKRENQQREMEKRIVTQLLTCMDGPGNKGDKNAPDSSAGFVLVIGATNRPDALDPALRRSGRFETEIALTAPDEDARAEILSVVAQKLRLEGPFDKKRIARLTPGFVGADLESVAYLAGRKAIKRILDSRKSEQSGDGEDDKSWLRMPWPEEELEKLFVKMSDFEEAVNLVQASLTREGFSIVPDVKWDDVGGLDHLRLQFNRYIVRPIKKPDIYKAFGVDLETGFLLYGPPGCGKTLIAKAAANEAGANFMHIKGAELLNKYVGESELAIRTLFQRARTCAPCVIFFDEVDALTTSRGKEGAWVVERLLNQFLVELDGGERRNVYVIGATNRPDVVDPAFLRPGRFGNLLYVPLPNADERASILKAIARKKPIDPSVDLDGIAKNNCEGFSGADLAHLVQKATFQAVEEMIGSSESSEDDVTDITQCTIKTRHFEQALSLVSPSVNKQQRRHYDALSTKLQESVGRNTEQVTIGPSFTLE A9B46C26779DA8C9 cd48a_arath Cell division control protein 48 homolog A CDC48A CDC48 F8A24.11 At3g09840 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSTPAESSDSKSKKDFSTAILERKKSPNRLVVDEAINDDNSVVSLHPATMEKLQLFRGDTILIKGKKRKDTVCIALADETCEEPKIRMNKVVRSNLRVRLGDVISVHQCPDVKYGKRVHILPVDDTVEGVTGNLFDAYLKPYFLEAYRPVRKGDLFLVRGGMRSVEFKVIETDPAEYCVVAPDTEIFCEGEPVKREDEERLDDVGYDDVGGVRKQMAQIRELVELPLRHPQLFKSIGVKPPKGILLYGPPGSGKTLIARAVANETGAFFFCINGPEIMSKLAGESESNLRKAFEEAEKNAPSIIFIDEIDSIAPKREKTNGEVERRIVSQLLTLMDGLKSRAHVIVMGATNRPNSIDPALRRFGRFDREIDIGVPDEIGRLEVLRIHTKNMKLAEDVDLERISKDTHGYVGADLAALCTEAALQCIREKMDVIDLEDDSIDAEILNSMAVTNEHFHTALGNSNPSALRETVVEVPNVSWNDIGGLENVKRELQETVQYPVEHPEKFEKFGMSPSKGVLFYGPPGCGKTLLAKAIANECQANFISVKGPELLTMWFGESEANVREIFDKARQSAPCVLFFDELDSIATQRGGGSGGDGGGAADRVLNQLLTEMDGMNAKKTVFIIGATNRPDIIDSALLRPGRLDQLIYIPLPDEDSRLNIFKAALRKSPIAKDVDIGALAKYTQGFSGADITEICQRACKYAIRENIEKDIEKEKRRSENPEAMEEDGVDEVSEIKAAHFEESMKYARRSVSDADIRKYQAFAQTLQQSRGFGSEFRFENSAGSGATTGVADPFATSAAAAGDDDDLYN 93222D637EA193B0 cdc48a 5 8 unspecified role unspecified role neutral component neutral component c-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 1 Our data show that the CDC48ANdel protein lacking the completeNdomain (amino acids 1-188) is most likely in the monomeric form. The CDC48AN-D1 protein runs as a monomer, The complex of CDC48A proteins was identified as a hexamer on the basis of its calculated molecular weight. cdc48a-1 3 7 unspecified role unspecified role neutral component neutral component region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role neutral component neutral component c-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain colocalization colocalization 2 In Figure 2, images of the nucleus of CDC48A are shown in the focal plane. CDC48A colocalizes with CDC48C in the nucleus (Fig. 2, A�C) and also localizes to the PM and the cytoplasm. The PM is not in the focal plane and can therefore not be seen. The merge shows an overlay of the cells containing chloroplasts in red, CDC48A in yellow, and the nucleus in cyan. The CDC48AN-D1 protein (Fig. 2, D�F) is found in the cytoplasm but not in the nucleus The proteins colocalised to the nucleus. cdc48a-serk1 1 6 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role fluorescence donor fluorescence donor region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction 4 Table 1 As reported previously, coexpression of CDC48A-CrFP and SERK1-YFP resulted in a reduction of the fluorescence lifetime in a punctuate pattern at the membrane The proteins colocalised to the plasma membrane The authors contest that "in those membrane areas where interaction between SERK1 and CDC48A occurs, a substantial amount of CDC48A is in oligomeric form". cdc48c 1 7 unspecified role unspecified role fluorescence donor fluorescence donor n terminal cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 7 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept n terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range direct interaction direct interaction 4, 5, Table 1 The combination of CrFP and YFP-CDC48C showed a lifetime of 2.44 6 0.1 ns (Cr-C/Y-C; Fig. 4P; Table I), a significant reduction compared to the donor alone. cdc48a-serk1-1 2 6 enzyme enzyme neutral component neutral component 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 region binding region binding-associated region binding region range range range range ecoli Escherichia coli (strain K12) 8 enzyme target enzyme target substrate substrate neutral component neutral component 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) phosphorylation phosphorylation reaction phosphorylation 6 SERK1 Phosphorylates Ser-41 in the N Domain of CDC48A phosphorylation of CDC48A The coordinates of the SERK1 kinase domain did not appear in the paper and the coordinates of this domain are taken from the UniProt record of this protein. cdc48a-serk1-2 4 8 enzyme target enzyme target substrate substrate neutral component neutral component 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) 6 enzyme enzyme neutral component neutral component 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 region binding region binding-associated region binding region range range range range ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation 6 Only in the sample containing the SERK1 kinase, one phosphorylated Ser residue was found at position 41 of CDC48A, which is located in the N domain phosphorylation of CDC48A at Serine 41 The coordinates of the SERK1 kinase domain did not appear in the paper and the coordinates of this domain are taken from the UniProt record of this protein. The authors point out that they did " not have any peptide coverage in the C-terminal tail (from amino acid 775 onward; Fig. 6B), where three Sers, three Thrs, and one Tyr are present, so there may be additional targets for SERK1 phosphorylation". cdc48a-5 1 8 unspecified role unspecified role fluorescence donor fluorescence donor region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction 4 Table 1 Oligomerization of CDC48A at Locations with SERK1 Interaction serk1-5 2 6 putative self putative self putative self putative self 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 region binding region binding-associated region binding region range range range range ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation true 6 SK1-KD is autophosphorylated Autophosphorylation of Q94AG2 The coordinates of the SERK1 kinase domain did not appear in the paper and the coordinates of this domain are taken from the UniProt record of this protein.