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/ The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell (1040-4651) 2007 imex curation Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu 01-April-2008: 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu Accepted 2008-MAR-1 by JYOTI. 01-April-2008: Contacted by JYOTI. A cDNA library from etiolated Arabidopsis (ecotype Columbia) seedlings. Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell (1040-4651) 2007 imex curation Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu 01-April-2008: Contacted by JYOTI. Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy tag visualisation tag visualisation Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu Accepted 2008-MAR-1 by JYOTI. 01-April-2008: Contacted by JYOTI. Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell (1040-4651) 2007 imex curation Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu 01-April-2008: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu Accepted 2008-MAR-1 by JYOTI. 01-April-2008: Contacted by JYOTI. Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell (1040-4651) 2007 imex curation Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu 01-April-2008: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro atpase assay atpase assay predetermined predetermined participant predetermined Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu Accepted 2008-MAR-1 by JYOTI. 01-April-2008: Contacted by JYOTI. Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell (1040-4651) 2007 imex curation Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu 01-April-2008: Contacted by JYOTI. Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Haas TJ., Sliwinski MK., Martinez DE., Preuss M., Ebine K., Ueda T., Nielsen E., Odorizzi G., Otegui MS. Transgenic arabidopsis plants (ecotype Columbia) expressing tagged proteins were used in this study. otegui@wisc.edu Accepted 2008-MAR-1 by JYOTI. 01-April-2008: Contacted by JYOTI. Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation raf2b_arath Ras-related protein RABF2b RABF2B ARA-7 RAB5B At4g19640 Ras-related protein Ara-7 Ras-related protein Rab5B F24J7.190 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAAAGNKSINAKLVLLGDVGAGKSSLVLRFVKDQFVEFQESTIGAAFFSQTLAVNDATVKFEIWDTAGQERYHSLAPMYYRGAAAAIIVFDVTNQASFERAKKWVQELQAQGNPNMVMALAGNKSDLLDARKVTAEDAQTYAQENGLFFMETSAKTATNVKEIFYEIARRLPRVQPTENPTGMVLPDRAMDRAVSSSCCA 769F75CFC708C6D0 atp ATP Adenosine 5'-triphosphate Adenosine triphosphate ADENOSINE-5'-TRIPHOSPHATE Adephos Adetol Adynol Atipi ATP Atriphos Cardenosine Fosfobion Glucobasin H4atp Myotriphos Triadenyl Triphosphaden adenosine 5'-(tetrahydrogen triphosphate) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) rabf1_arath Ras-related protein RABF1 RABF1 At3g54840 ARA-6 RAB5C Ras-related protein Ara-6 Ras-related protein Rab5C F28P10.180 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MGCASSLPDRNSGTLSGLSNSENAVPADAKNLRVKLVLLGDSGVGKSCIVLRFVRGQFDATSKVTVGASFLSQTIALQDSTTVKFEIWDTAGQERYSALAPLYYRGAGVAVIVYDITSPESFKKAQYWVKELQKHGSPDIVMALVGNKADLHEKREVPTEDGMELAEKNGMFFIETSAKTADNINQLFEEIGKRLPRPAPSS 528D7DAD2AB58398 raf2a_arath Ras-related protein RABF2a RABF2A K17O22.15 At5g45130 RAB5A RHA1 Ras-related protein Rab5A Ras-related protein Rha1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MASSGNKNINAKLVLLGDVGAGKSSLVLRFVKDQFVEFQESTIGAAFFSQTLAVNDATVKFEIWDTAGQERYHSLAPMYYRGAAAAIIVFDITNQASFERAKKWVQELQAQGNPNMVMALAGNKADLLDARKVSAEEAEIYAQENSLFFMETSAKTATNVKDIFYEIAKRLPRVQPAENPTGMVLPNGPGATAVSSSCCA 98E2ED559A59DE35 vps4_arath Protein SUPPRESSOR OF K(+) TRANSPORT GROWTH DEFECT 1 SKD1 Protein VACUOLAR PROTEIN SORTING 4 VPS4 F10A12.27 F15K20 At2g27600 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MYSNFKEQAIEYVKQAVHEDNAGNYNKAFPLYMNALEYFKTHLKYEKNPKIREAITQKFTEYLRRAEEIRAVLDEGGSGPGSNGDAAVATRPKTKPKDGEGGGKDGEDPEQSKLRAGLNSAIVREKPNIKWSDVAGLESAKQALQEAVILPVKFPQFFTGKRRPWRAFLLYGPPGTGKSYLAKAVATEADSTFFSVSSSDLVSKWMGESEKLVSNLFEMARESAPSIIFVDEIDSLCGTRGEGNESEASRRIKTELLVQMQGVGHNDEKVLVLAATNTPYALDQAIRRRFDKRIYIPLPEAKARQHMFKVHLGDTPHNLTEPDFEYLGQKTEGFSGSDVSVCVKDVLFEPVRKTQDAMFFFKSPDGTWMPCGPRHPGAIQTTMQDLATKGLAEKIIPPPITRTDFEKVLARQRPTVSKSDLDVHERFTQEFGEEG B56F8B39419C20AA lip5_arath Protein HOMOLOG OF MAMMALIAN LYST-INTERACTING PROTEIN 5 LIP5 Protein EXTENSIN-LIKE VTA1 F10M23.90 At4g26750 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSNPNEPARALLPYLQRADELQKHEPLVAYYCRLYAMERGLKIPQSERTKTTNSILMSLINQLEKDKKSLTLSPDDNMHVEGFALSVFAKADKQDRAGRADLGTAKTFYAASIFFEILSQFGPVPPDIEQKHKYAAWKAADIRKAIKEGRKPTPGDPVDDDTDLSIPSSGPSGSYDHSASDTNTTSHHRTELDPPHDSNDDSSHHQFPEVPQHPLPPRFYDNPTNDYPADVPPPPPSSYPSNDHLPPPTGPSDSPYPHPYSHQPYHQDPPKHMPPPQNYSSHEPSPNSLPNFQSYPSFSESSLPSTSPHYPSHYQNPEPYYSSPHSAPAPSSTSFSSAPPPPPYSSNGRINIAPVLDPAPSSAQKYHYDSSYQPGPEKVAEALKAARFAVGALAFDEVSTAVEHLKKSLELLTNPSAGAGH 1BD515B10FA4FA77 at2g27600-ara6 5 10 unspecified role unspecified role neutral component neutral component region green fluorescent protein gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role neutral component neutral component c-terminal monomeric red fluorescent protein tag rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range colocalization colocalization Figure 4A Partial colocalization of ARA6/RabF1-mRFP and GFP-SKD1 in Arabidopsis root epidermal cells. Partial colocalisation was observed in epidermal root cells. at2g27600-rha1 5 9 unspecified role unspecified role neutral component neutral component n-terminal enhanced yellow fluorescent protein 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 10 unspecified role unspecified role neutral component neutral component region green fluorescent protein gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Figure 4C Partial colocalization of EYFP-RHA1/RabF2a and GFP-SKD1 in Arabidopsis root epidermal cells. Partial colocalisation was observed in epidermal root cells. at2g27600-at4g19640 5 6 unspecified role unspecified role neutral component neutral component c-terminal monomeric red fluorescent protein rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 10 unspecified role unspecified role neutral component neutral component region green fluorescent protein gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Figure 4B Partial colocalization of mRFP-ARA7/RabF2b and GFP-SKD1 in Arabidopsis root epidermal cells. Partial colocalisation was observed in epidermal root cells. at2g27600-ara6-1 2 8 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component colocalization colocalization Figure 6D We then performed double immunogold labeling with antibodies against Arabidopsis SKD1 and against the three Rab5-type GTPases (Figures 6D to 6F). In all cases, the immunogold results support the partial colocalization of SKD1 and the three endosomal Rab GTPases on MVBs shown in the confocal analysis of the fluorescent proteins. at2g27600-at4g19640- 2 6 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component colocalization colocalization Figure 6F We then performed double immunogold labeling with antibodies against Arabidopsis SKD1 and against the three Rab5-type GTPases (Figures 6D to 6F). In all cases, the immunogold results support the partial colocalization of SKD1 and the three endosomal Rab GTPases on MVBs shown in the confocal analysis of the fluorescent proteins. A weak cross reaction of the anti-body to Rab2a (P31582) was observed in this study. at2g27600-rha1-1 2 10 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component colocalization colocalization Figure 6E We then performed double immunogold labeling with antibodies against Arabidopsis SKD1 and against the three Rab5-type GTPases (Figures 6D to 6F). In all cases, the immunogold results support the partial colocalization of SKD1 and the three endosomal Rab GTPases on MVBs shown in the confocal analysis of the fluorescent proteins. A weak cross reaction of the anti-body was observed with protein Rab2b (Q9SN68) at2g27600-at4g26750 1 10 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey physical association physical association These isolated prey plasmids were retransformed into the alpha mating type of PJ694 and validated in a mating and selection assay with the Arabidopsis SKD1 bait, the empty bait vector, and unrelated baits. Out of seven confirmed positive clones, three were found to encode a predicted protein of 421 amino acids (At4g26750). at2g27600-at4g26750- 3 11 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role bait bait 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) direct interaction direct interaction In vitro pull-down assay to confirm interactions between LIP5 and GST-SKD1 but not between LIP5 and GST alone. Figure 9 at2g27600-atp 4 7 enzyme target enzyme target substrate substrate neutral component neutral component 10 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 ecoli Escherichia coli (strain K12) atpase reaction atpase reaction In vitro ATPase activity of recombinant SKD1, Figure 10A Lip5 proten (Q9SZ15) stimulated ATPase activity In the absence of LIP5, SKD1 hydrolyzes approximately 106 micro Moles of ATP per min/per mg,in the presence of recombinant LIP5, the hydrolysis rate shows a fourfold to fivefold increase.