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/ RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. Cell (0092-8674) 2009 imex curation Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. This interaction is not reported in PMID: 16885419 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 27-Oct-2007: Contacted by JYOTI. Human placental cDNA davet Only protein-protein interactions 8 in vitro In vitro pull down pull down autoradiography autoradiography Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. This interaction is not reported in PMID: 16885419 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 Accepted 2009-AUG-24 by DAVET. 27-Oct-2007: Contacted by JYOTI. Human placental cDNA Cell (0092-8674) 2009 davet Only protein-protein interactions 8 imex curation RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. Cell (0092-8674) 2009 imex curation Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. This interaction is not reported in PMID: 16885419 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 27-Oct-2007: Contacted by JYOTI. Human placental cDNA davet Only protein-protein interactions 8 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. This interaction is not reported in PMID: 16885419 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 Accepted 2009-AUG-24 by DAVET. 27-Oct-2007: Contacted by JYOTI. Human placental cDNA Cell (0092-8674) 2009 davet Only protein-protein interactions 8 imex curation RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. Cell (0092-8674) 2009 imex curation Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. This interaction is not reported in PMID: 16885419 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 27-Oct-2007: Contacted by JYOTI. Human placental cDNA davet Only protein-protein interactions 8 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. This interaction is not reported in PMID: 16885419 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 Accepted 2009-AUG-24 by DAVET. 27-Oct-2007: Contacted by JYOTI. Human placental cDNA Cell (0092-8674) 2009 davet Only protein-protein interactions 8 imex curation RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. Cell (0092-8674) 2009 imex curation Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. This interaction is not reported in PMID: 16885419 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 27-Oct-2007: Contacted by JYOTI. Human placental cDNA davet Only protein-protein interactions 8 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. This interaction is not reported in PMID: 16885419 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 Accepted 2009-AUG-24 by DAVET. Human placental cDNA Cell (0092-8674) 2009 davet Only protein-protein interactions 8 imex curation RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. Cell (0092-8674) 2009 imex curation Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. This interaction is not reported in PMID: 16885419 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 27-Oct-2007: Contacted by JYOTI. Human placental cDNA davet Only protein-protein interactions 8 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. This interaction is not reported in PMID: 16885419 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 Accepted 2009-AUG-24 by DAVET. 27-Oct-2007: Contacted by JYOTI. Human placental cDNA Cell (0092-8674) 2009 davet Only protein-protein interactions 8 imex curation RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. Cell (0092-8674) 2009 imex curation Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. This interaction is not reported in PMID: 16885419 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 27-Oct-2007: Contacted by JYOTI. Human placental cDNA davet Only protein-protein interactions 8 in vitro In vitro atpase assay atpase assay predetermined predetermined participant predetermined Espinosa EJ., Calero M., Sridevi K., Pfeffer SR. This interaction is not reported in PMID: 16885419 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 Accepted 2009-AUG-24 by DAVET. 27-Oct-2007: Contacted by JYOTI. Human placental cDNA Cell (0092-8674) 2009 davet Only protein-protein interactions 8 imex curation 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 67B2B9CDBC523043 rab9a_canlf Ras-related protein Rab-9A RAB9A RAB9 protein protein canlf Canis familiaris (dog) Canis lupus familiaris Dog Canis familiaris MAGKSSLFKVILLGDGGVGKSSLMNRYVTNKFDTQLFHTIGVEFLNKDLEVDGHFVTMQIWDTAGQERFRSLRTPFYRGSDCCLLTFSVDDSQSFQNLSNWKKEFIYYADVKEPESFPFVILGNKIDISERQVSTEEAQAWCRDNGDYPYFETSAKDATNVAAAFEEAVRRVLATEDRSDHLIQTDTVSLHRKPKPSSSCC 65B502C204BEDB72 rab9b_human Ras-related protein Rab-9B RAB9B RAB9L Rab-9-like protein protein protein human Homo sapiens Human MSGKSLLLKVILLGDGGVGKSSLMNRYVTNKFDSQAFHTIGVEFLNRDLEVDGRFVTLQIWDTAGQERFKSLRTPFYRGADCCLLTFSVDDRQSFENLGNWQKEFIYYADVKDPEHFPFVVLGNKVDKEDRQVTTEEAQTWCMENGDYPYLETSAKDDTNVTVAFEEAVRQVLAVEEQLEHCMLGHTIDLNSGSKAGSSCC B35EDA8E8358C49C 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) rhbt3_human Rho-related BTB domain-containing protein 3 RHOBTB3 KIAA0878 protein protein human Homo sapiens Human MSIHIVALGNEGDTFHQDNRPSGLIRTYLGRSPLVSGDESSLLLNAASTVARPVFTEYQASAFGNVKLVVHDCPVWDIFDSDWYTSRNLIGGADIIVIKYNVNDKFSFHEVKDNYIPVIKRALNSVPVIIAAVGTRQNEELPCTCPLCTSDRGSCVSTTEGIQLAKELGATYLELHSLDDFYIGKYFGGVLEYFMIQALNQKTSEKMKKRKMSNSFHGIRPPQLEQPEKMPVLKAEASHYNSDLNNLLFCCQCVDVVFYNPNLKKVVEAHKIVLCAVSHVFMLLFNVKSPTDIQDSSIIRTTQDLFAINRDTAFPGASHESSGNPPLRVIVKDALFCSCLSDILRFIYSGAFQWEELEEDIRKKLKDSGDVSNVIEKVKCILKTPGKINCLRNCKTYQARKPLWFYNTSLKFFLNKPMLADVVFEIQGTTVPAHRAILVARCEVMAAMFNGNYMEAKSVLIPVYGVSKETFLSFLEYLYTDSCCPAGIFQAMCLLICAEMYQVSRLQHICELFIITQLQSMPSRELASMNLDIVDLLKKAKFHHSDCLSTWLLHFIATNYLIFSQKPEFQDLSVEERSFVEKHRWPSNMYLKQLAEYRKYIHSRKCRCLVM 430ED42327AF08B6 rab9a-rhobtb3-1 4 11 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association RhoBTB3 was identified in a yeast two-hybrid screen for Rab9-GTP-interacting proteins. rab9a-rhobtb3-2 2 8 unspecified role unspecified role bait bait P24408:p.Gln66Leu GTP-binding mutation mutation certain certain sequence position certain certain certain sequence position certain Q L Wed May 02 17:50:58 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln66leu' Tue Jun 30 18:03:27 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln66Leu' 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 S N Wed May 02 15:47:48 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser21asn' Tue Jun 30 18:28:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser21Asn' 11 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 physical association physical association Binding was quantified by liquid culture analysis: full-length RhoBTB3 interacted with the GTP conformation of Rab9 (Rab9Q66L) but not a mutant Rab9 that binds GDP with 50-fold preference to GTP (Rab9S21N; Riederer et al., 1994) (Figure 2A). 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 2 11 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 8 unspecified role unspecified role bait bait P24408:p.Gln66Leu GTP-binding mutation mutation certain certain sequence position certain certain certain sequence position certain Q L Wed May 02 16:23:32 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln66leu' Tue Jun 30 19:55:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln66Leu' physical association physical association Liquid culture of b-galactosidase activity of yeast strains coexpressing the indicated RhoBTB3 fragments with the GAL4-DNA binding domain of Rab9Q66L. Fig. 2B Three point mutation - ala498thr, asp532glu and iso533lys were reported to inhibit the interaction - data not shown. rab9b-rhobtb3-1 2 9 unspecified role unspecified role unspecified role unspecified role 11 unspecified role unspecified role unspecified role unspecified role physical association physical association Subsequent analysis of 54 different Rab GTPases confirmed binding specificity: only Rab9A and, to a lesser extent, Rab9B showed interaction with RhoBTB3 (Figure S1available online). 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-rhobtb3-4 1 8 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 11 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction To verify that the binding observed in yeast was due to a direct protein:protein interaction, we tested the ability of purified GSTRhoBTB3 C-terminal residues 420�611 to bind purified Rab proteins. Figs. 2C, 2D Binding showed strong preference for Rab9-GTP. rhobtb3-atp-1 6 10 enzyme target enzyme target substrate substrate neutral component neutral component 11 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 O94955:p.Asn138Asp Alters kinetics of interaction mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain N D Wed May 02 16:51:25 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asn138asp' Tue Jun 30 20:15:07 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asn138Asp' 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 Kcat/KM = 93,750 /M-/min RhoBTB3 Binds and Hydrolyzes ATP Figs. 6F, 6G rhobtb3-atp-2 6 11 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 ecoli Escherichia coli (strain K12) 10 enzyme target enzyme target substrate substrate neutral component neutral component atpase reaction atpase reaction RhoBTB3 Binds and Hydrolyzes ATP Fig. 7A Full-length RhoBTB3 protein displayed approximately 50% the rate of ATP hydrolysis seen for the Rho domain alone rhobtb3-atp-3 6 8 inhibitor inhibitor neutral component neutral component 10 enzyme target enzyme target substrate substrate neutral component neutral component 11 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 ecoli Escherichia coli (strain K12) atpase reaction atpase reaction Satisfyingly, addition of Rab9 GTPase (closed circles) but not Rab5 GTPase (closed diamonds) brought the hydrolysis rate of full-length RhoBTB3 to that of the Rho domain alone. Fig. 7A rhobtb3-m6prbp1-1 3 11 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component physical association physical association Rab9 and ATP stimulate release of TIP47 from this complex Figs. 7B, 7C, 7D m6rbp1-1 5 7 unspecified role unspecified role neutral component neutral component physical association physical association migrated as a dimer upon blue native gel electrophoresis in dodecyl maltoside Fig. 7B rab9a-rhobtb3-5 2 8 unspecified role unspecified role unspecified role unspecified role 11 unspecified role unspecified role unspecified role unspecified role physical association physical association Subsequent analysis of 54 different Rab GTPases confirmed binding specificity: only Rab9A and, to a lesser extent, Rab9B showed interaction with RhoBTB3 (Figure S1available online). 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.