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/ Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. EMBO J. (0261-4189) 2001 Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk 02-MAR-2006: Contacted by JYOTI. ecoli Escherichia coli (strain K12) pull down pull down western blot western blot Immuno blot Immuno blot Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk Accepted 2007-FEB-14 by DAVET. 02-MAR-2006: Contacted by JYOTI. EMBO J. (0261-4189) 2001 Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. EMBO J. (0261-4189) 2001 Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk 02-MAR-2006: Contacted by JYOTI. ecoli Escherichia coli (strain K12) bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk Accepted 2007-FEB-14 by DAVET. 02-MAR-2006: Contacted by JYOTI. EMBO J. (0261-4189) 2001 Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. EMBO J. (0261-4189) 2001 Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk 02-MAR-2006: Contacted by JYOTI. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk Accepted 2007-FEB-14 by DAVET. 02-MAR-2006: Contacted by JYOTI. EMBO J. (0261-4189) 2001 Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. EMBO J. (0261-4189) 2001 Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk 02-MAR-2006: Contacted by JYOTI. ecoli Escherichia coli (strain K12) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk Accepted 2007-FEB-14 by DAVET. 02-MAR-2006: Contacted by JYOTI. EMBO J. (0261-4189) 2001 Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. EMBO J. (0261-4189) 2001 Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk 02-MAR-2006: Contacted by JYOTI. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk Accepted 2007-FEB-14 by DAVET. 02-MAR-2006: Contacted by JYOTI. EMBO J. (0261-4189) 2001 Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. EMBO J. (0261-4189) 2001 Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk 02-MAR-2006: Contacted by JYOTI. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Luciano P., Vial S., Vergnolle MA., Dyall SD., Robinson DR., Tokatlidis K. The two genes were cloned as an operon in the same expression vector and co-expressed in E.coli. tokatlidis@man.ac.uk Accepted 2007-FEB-14 by DAVET. 02-MAR-2006: Contacted by JYOTI. EMBO J. (0261-4189) 2001 tim10_yeast Mitochondrial import inner membrane translocase subunit TIM10 MRS11 YHR005BC YHR005C-A TIM10 Mitochondrial intermembrane protein MRS11 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSFLGFGGGQPQLSSQQKIQAAEAELDLVTDMFNKLVNNCYKKCINTSYSEGELNKNESSCLDRCVAKYFETNVQVGENMQKMGQSFNAAGKF 70DD4764B944FF17 70DD4764B944FF17 tim9_yeast Mitochondrial import inner membrane translocase subunit TIM9 YEL020BW YEL020W-A TIM9 protein protein yeast Saccharomyces cerevisiae Baker's yeast MDALNSKEQQEFQKVVEQKQMKDFMRLYSNLVERCFTDCVNDFTTSKLTNKEQTCIMKCSEKFLKHSERVGQRFQEQNAALGQGLGR 5160E5DBA52065C7 tim9-tim10-1 1 7 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 8 unspecified role unspecified role prey prey physical association physical association To test for a direct interaction between Tim9 and Tim10, the soluble fraction, after disruption with French Press, was bound onto glutathione S�Sepharose beads. 1A Both proteins concomitantly released from the gluathione following cleavage of the GST tag. tim9-tim10-2 4 7 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component physical association physical association 1B Gel filtration analysis of this fraction after thrombin cleavage suggested that the complex of co-expressed Tim9 and Tim10 had the same size as the endogenous TIM10 complex from the IMS (Figure 1B). tim9-tim10-3 6 8 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component physical association physical association 1B Gel filtration analysis of this fraction after thrombin cleavage suggested that the complex of co-expressed Tim9 and Tim10 had the same size as the endogenous TIM10 complex from the IMS (Figure 1B). tim9-tim10-4 2 8 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component physical association physical association 1C BN-PAGE was performed on the recombinant TIM10 complex (Figure 1C) and we found that the recombinant TIM10 complex migrated as the endogenous, authentic complex from mitochondria tim9-tim10-5 5 7 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component physical association physical association 1C BN-PAGE was performed on the recombinant TIM10 complex (Figure 1C) and we found that the recombinant TIM10 complex migrated as the endogenous, authentic complex from mitochondria tim9-tim10-6 3 8 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait ecobb Escherichia coli (strain B / BL21) physical association physical association 3 The imported Tim10 and the endogenous Tim9 formed a complex and were co-immunoprecipitated (Figure 3, lanes IP10 and IP9). tim9-tim10-7 3 7 unspecified role unspecified role prey prey ecobb Escherichia coli (strain B / BL21) 8 unspecified role unspecified role bait bait physical association physical association 3 The imported Tim10 and the endogenous Tim9 formed a complex and were co-immunoprecipitated (Figure 3, lanes IP10 and IP9).