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/ http://www.ebi.ac.uk/ espinosa-2009-2 RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. 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 Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. cell membrane fraction enriched in Golgi and endosomes was prepared by sucrose gradient flotation. Crosslinking agent 1mMDithiobis(sulfosuccinimidylproprionate), proteins separated on blue native gel electrophoresis Detail on constructs supplied by the author. pfeffer@stanford.edu Human placental cDNA Cell (0092-8674) 2009 Only protein-protein interactions 8 imex curation espinosa-2009-4 RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. in vitro In vitro atpase assay atpase assay predetermined predetermined participant predetermined Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. cell membrane fraction enriched in Golgi and endosomes was prepared by sucrose gradient flotation. Crosslinking agent 1mMDithiobis(sulfosuccinimidylproprionate), proteins separated on blue native gel electrophoresis Detail on constructs supplied by the author. pfeffer@stanford.edu Human placental cDNA Cell (0092-8674) 2009 Only protein-protein interactions 8 imex curation espinosa-2009-1 RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. 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 Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. cell membrane fraction enriched in Golgi and endosomes was prepared by sucrose gradient flotation. Crosslinking agent 1mMDithiobis(sulfosuccinimidylproprionate), proteins separated on blue native gel electrophoresis Detail on constructs supplied by the author. pfeffer@stanford.edu Human placental cDNA Cell (0092-8674) 2009 Only protein-protein interactions 8 imex curation espinosa-2009-3 RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. in vitro In vitro pull down pull down autoradiography autoradiography Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. cell membrane fraction enriched in Golgi and endosomes was prepared by sucrose gradient flotation. Crosslinking agent 1mMDithiobis(sulfosuccinimidylproprionate), proteins separated on blue native gel electrophoresis Detail on constructs supplied by the author. pfeffer@stanford.edu Human placental cDNA Cell (0092-8674) 2009 Only protein-protein interactions 8 imex curation espinosa-2009-5 RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. human-k_562 Homo sapiens myeloid leukemia cells. k562 Myelogenous leukemia K562 cells crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. cell membrane fraction enriched in Golgi and endosomes was prepared by sucrose gradient flotation. Crosslinking agent 1mMDithiobis(sulfosuccinimidylproprionate), proteins separated on blue native gel electrophoresis Detail on constructs supplied by the author. pfeffer@stanford.edu Human placental cDNA Cell (0092-8674) 2009 Only protein-protein interactions 8 imex curation espinosa-2009-6 RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. human-k_562 Homo sapiens myeloid leukemia cells. k562 Myelogenous leukemia K562 cells bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. cell membrane fraction enriched in Golgi and endosomes was prepared by sucrose gradient flotation. Crosslinking agent 1mMDithiobis(sulfosuccinimidylproprionate), proteins separated on blue native gel electrophoresis Detail on constructs supplied by the author. pfeffer@stanford.edu Human placental cDNA Cell (0092-8674) 2009 Only protein-protein interactions 8 imex curation rab9a_canlf Ras-related protein Rab-9A RAB9A RAB9 protein protein canlf Canis familiaris (dog) Canis lupus familiaris Dog Canis familiaris MAGKSSLFKVILLGDGGVGKSSLMNRYVTNKFDTQLFHTIGVEFLNKDLEVDGHFVTMQIWDTAGQERFRSLRTPFYRGSDCCLLTFSVDDSQSFQNLSNWKKEFIYYADVKEPESFPFVILGNKIDISERQVSTEEAQAWCRDNGDYPYFETSAKDATNVAAAFEEAVRRVLATEDRSDHLIQTDTVSLHRKPKPSSSCC rhbt3_human Rho-related BTB domain-containing protein 3 RHOBTB3 KIAA0878 protein protein human Homo sapiens Human MSIHIVALGNEGDTFHQDNRPSGLIRTYLGRSPLVSGDESSLLLNAASTVARPVFTEYQASAFGNVKLVVHDCPVWDIFDSDWYTSRNLIGGADIIVIKYNVNDKFSFHEVKDNYIPVIKRALNSVPVIIAAVGTRQNEELPCTCPLCTSDRGSCVSTTEGIQLAKELGATYLELHSLDDFYIGKYFGGVLEYFMIQALNQKTSEKMKKRKMSNSFHGIRPPQLEQPEKMPVLKAEASHYNSDLNNLLFCCQCVDVVFYNPNLKKVVEAHKIVLCAVSHVFMLLFNVKSPTDIQDSSIIRTTQDLFAINRDTAFPGASHESSGNPPLRVIVKDALFCSCLSDILRFIYSGAFQWEELEEDIRKKLKDSGDVSNVIEKVKCILKTPGKINCLRNCKTYQARKPLWFYNTSLKFFLNKPMLADVVFEIQGTTVPAHRAILVARCEVMAAMFNGNYMEAKSVLIPVYGVSKETFLSFLEYLYTDSCCPAGIFQAMCLLICAEMYQVSRLQHICELFIITQLQSMPSRELASMNLDIVDLLKKAKFHHSDCLSTWLLHFIATNYLIFSQKPEFQDLSVEERSFVEKHRWPSNMYLKQLAEYRKYIHSRKCRCLVM rab9b_human Ras-related protein Rab-9B RAB9B RAB9L Rab-9-like protein protein protein human Homo sapiens Human MSGKSLLLKVILLGDGGVGKSSLMNRYVTNKFDSQAFHTIGVEFLNRDLEVDGRFVTLQIWDTAGQERFKSLRTPFYRGADCCLLTFSVDDRQSFENLGNWQKEFIYYADVKDPEHFPFVVLGNKVDKEDRQVTTEEAQTWCMENGDYPYLETSAKDDTNVTVAFEEAVRQVLAVEEQLEHCMLGHTIDLNSGSKAGSSCC 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) plin3_human Perilipin-3 Cargo selection protein TIP47 47 kDa mannose 6-phosphate receptor-binding protein Placental protein 17 PLIN3 TIP47 M6PRBP1 Mannose-6-phosphate receptor-binding protein 1 protein protein human Homo sapiens Human MSADGAEADGSTQVTVEEPVQQPSVVDRVASMPLISSTCDMVSAAYASTKESYPHIKTVCDAAEKGVRTLTAAAVSGAQPILSKLEPQIASASEYAHRGLDKLEENLPILQQPTEKVLADTKELVSSKVSGAQEMVSSAKDTVATQLSEAVDATRGAVQSGVDKTKSVVTGGVQSVMGSRLGQMVLSGVDTVLGKSEEWADNHLPLTDAELARIATSLDGFDVASVQQQRQEQSYFVRLGSLSERLRQHAYEHSLGKLRATKQRAQEALLQLSQVLSLMETVKQGVDQKLVEGQEKLHQMWLSWNQKQLQGPEKEPPKPEQVESRALTMFRDIAQQLQATCTSLGSSIQGLPTNVKDQVQQARRQVEDLQATFSSIHSFQDLSSSILAQSRERVASAREALDHMVEYVAQNTPVTWLVGPFAPGITEKAPEEKK rab9a-rhobtb3-2 Interaction between RAB9A and RHOBTB3 identified by Y2H 833810 n/a 833816 unspecified role unspecified role bait bait P24408:p.Gln66Leu GTP-binding mutation mutation certain certain sequence position certain certain certain sequence position certain false P24408:p.Ser21Asn GDP binding mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false n/a 833817 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 false physical association physical association false false false Fig. 2A RhoBTB3 interacted with a mutant of Rab9 (Rab9Q66L) that binds but does not hydrolyse GTP, but not a mutant Rab9 that binds GDP with 50-fold preference to GTP (Rab9S21N). Interaction requires RAB9A to be GTP-bound. rab9a-rhobtb3-3 Interaction between RAB9A and RHOBTB3 identified by Y2H 833810 n/a 833817 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 false n/a 833816 unspecified role unspecified role bait bait P24408:p.Gln66Leu GTP-binding mutation mutation certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 2B Three point mutation - ala498thr, asp532glu and iso533lys were reported to inhibit the interaction - data not shown. rab9b-rhobtb3-1 Interaction between RAB9B and RHOBTB3 identified by Y2H 833810 n/a 833818 unspecified role unspecified role unspecified role unspecified role n/a 833817 unspecified role unspecified role unspecified role unspecified role physical association physical association false false false Supp. Fig. 1 It is probable that Gln66 is of RAB9B is mutated to prevent GTPase activity but this information is not given in the text. rab9a-rhobtb3-5 Interaction between RAB9A and RHOBTB3 identified by Y2H 833810 n/a 833816 unspecified role unspecified role unspecified role unspecified role n/a 833817 unspecified role unspecified role unspecified role unspecified role physical association physical association false false false Supp. Fig. 1 It is probable that Gln66 is of RAB9A is mutated to prevent GTPase activity but this information is not given in the text. rhobtb3-atp-1 ATPase activity of RHOBTB3 833811 n/a 833819 enzyme target enzyme target substrate substrate neutral component neutral component n/a 833817 enzyme enzyme neutral component neutral component rho region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false O94955:p.Asn138Asp Alters kinetics of interaction mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false 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 false ecoli Escherichia coli (strain K12) atpase reaction atpase reaction false false false Kcat/KM = 93,750 /M-/min Figs. 6F, 6G rhobtb3-atp-2 ATPase activity of RHOBTB3 833811 n/a 833817 enzyme enzyme neutral component neutral component 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 false ecoli Escherichia coli (strain K12) n/a 833819 enzyme target enzyme target substrate substrate neutral component neutral component atpase reaction atpase reaction false false false Fig. 7A Full-length RhoBTB3 protein displayed approximately 50% the rate of ATP hydrolysis seen for the Rho domain alone rhobtb3-atp-3 ATPase activity of RHOBTB3 - inhibition by RAB9A 833811 n/a 833816 inhibitor inhibitor neutral component neutral component n/a 833819 enzyme target enzyme target substrate substrate neutral component neutral component n/a 833817 enzyme enzyme neutral component neutral component 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 false ecoli Escherichia coli (strain K12) atpase reaction atpase reaction false false false Fig. 7A rab9a-rhobtb3-1 Interaction between RAB9A and RHOBTB3 identified by Y2H 833812 n/a 833817 unspecified role unspecified role prey prey n/a 833816 unspecified role unspecified role bait bait physical association physical association false false false rab9a-rhobtb3-4 Interaction between RAB9A and RHOBTB3 identified by pulldown 833813 n/a 833816 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 833817 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 false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Figs. 2C, 2D Binding showed strong preference for Rab9-GTP. rhobtb3-m6prbp1-1 Interaction between RHOBTB3 abd M6PRBP1 demonstrated by crosslinking 833814 n/a 833817 unspecified role unspecified role neutral component neutral component n/a 833820 unspecified role unspecified role neutral component neutral component physical association physical association false false false Rab9 and ATP stimulate release of TIP47 from this complex Figs. 7B, 7C, 7D m6rbp1-1 Homodimerisation of M6PRBP1 833815 n/a 833820 unspecified role unspecified role neutral component neutral component Stoichiometry: 2.0 physical association physical association false false false Fig. 7B