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/ Preprotein recognition by the Toc complex. Preprotein recognition by the Toc complex. EMBO J. (0261-4189) 2004 imex curation Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de 14-May-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro biochemical biochemical western blot western blot Immuno blot Immuno blot Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. It is not clear from the text how the recombinant Toc proteins were produced. The curator has assumed the proetins were expressed in ecoli and subsequently purified and used in the assay. The details of the method used to produce Toc protein containing liposomes was obtained from the reference provided Schleiff et al (2003) (PMID: 12665619) in the text. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de Accepted 2009-MAY-8 by JYOTI. 14-May-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2004 jyoti Only protein-protein interactions imex curation Preprotein recognition by the Toc complex. Preprotein recognition by the Toc complex. EMBO J. (0261-4189) 2004 imex curation Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de 14-May-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro pull down pull down immunostaining immunostaining Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. It is not clear from the text how the recombinant Toc159 protein was produced. The curator has assumed the proetins were expressed in ecoli and subsequently purified and used in the assay. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de Accepted 2009-MAY-8 by JYOTI. 14-May-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2004 jyoti Only protein-protein interactions imex curation Preprotein recognition by the Toc complex. Preprotein recognition by the Toc complex. EMBO J. (0261-4189) 2004 imex curation Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de 14-May-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati weight by staining molecular weight estimation by staining weight by staining Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de Accepted 2009-MAY-8 by JYOTI. 14-May-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2004 jyoti Only protein-protein interactions imex curation Preprotein recognition by the Toc complex. Preprotein recognition by the Toc complex. EMBO J. (0261-4189) 2004 imex curation Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de 14-May-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. It is not clear from the text how the recombinant Toc159 protein was produced. The curator has assumed the proetins were expressed in ecoli and subsequently purified and used in the assay. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de Accepted 2009-MAY-8 by JYOTI. 14-May-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2004 jyoti Only protein-protein interactions imex curation Preprotein recognition by the Toc complex. Preprotein recognition by the Toc complex. EMBO J. (0261-4189) 2004 imex curation Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de 14-May-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati western blot western blot Immuno blot Immuno blot Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de Accepted 2009-MAY-8 by JYOTI. 14-May-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2004 jyoti Only protein-protein interactions imex curation Preprotein recognition by the Toc complex. Preprotein recognition by the Toc complex. EMBO J. (0261-4189) 2004 imex curation Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de 14-May-2009: Contacted by JYOTI. jyoti Only protein-protein interactions arath-leaf Arabidopsis thaliana leaf leaf leaf far western blotting far western blotting Affinity blotting Affinity blotting western blot western blot Immuno blot Immuno blot Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de Accepted 2009-MAY-8 by JYOTI. 14-May-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2004 jyoti Only protein-protein interactions imex curation Preprotein recognition by the Toc complex. Preprotein recognition by the Toc complex. EMBO J. (0261-4189) 2004 imex curation Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de 14-May-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro gtpase assay gtpase assay GTPase gtp hydrolisis gtp hydrolisis GTPase autoradiography autoradiography Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. It is not clear from the text how the recombinant Toc159 protein was produced. The curator has assumed the proetins were expressed in ecoli and subsequently purified and used in the assay. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de Accepted 2009-MAY-8 by JYOTI. 14-May-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2004 jyoti Only protein-protein interactions imex curation Preprotein recognition by the Toc complex. Preprotein recognition by the Toc complex. EMBO J. (0261-4189) 2004 imex curation Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de 14-May-2009: Contacted by JYOTI. jyoti Only protein-protein interactions arath-leaf Arabidopsis thaliana leaf leaf leaf anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Becker T., Jelic M., Vojta A., Radunz A., Soll J., Schleiff E. Radio-labelled pSSU protein was detected by autoradiography whereas Toc159 or Toc34 proteins were detected by western blotting. schleiff@botanik.biologie.uni-muenchen.de Accepted 2009-MAY-8 by JYOTI. EMBO J. (0261-4189) 2004 jyoti Only protein-protein interactions imex curation ti110_arath Protein TIC110, chloroplastic TIC110 Translocon at the inner envelope membrane of chloroplasts 110 F4H5.1 F10K1.33 At1g06950 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MNPSLVTAINAPISPSPRSPLLSHFLPTLPHRFSKSECLSRRRYRVSFPRSSAASSDQLSVSTQAKNPGIHGNKKELTGLQPIVEKMTPPVRLATSAVVLAASLATGYGLGLRLAGSRNIAFGGAAVAGAAGGAVVYALNSAVPEVAAISLHNYVAEFEDPASVTKDDVEKIADRYGVNKGDEAFQAEICDIYCRYVTSVLPTEGQSLKGDEVAKIVKFKNALGIDEPDAAAMHMEIGRRIFRQRLETGEREGDAEQRRAFMRLVYVSALVFGDASSFLLPWKRVLKVTDAQVEIAIRENAKQLYAERLKLVGRDINVENLVDLRKSQLSFKLSDELAEDLFREHTRKVVVENISSALSILKSRTRAAKSLASVVEELEKVLEFNNLLVSLKSHSEADQFARGVGPISLIGDESDFERRMDDLKLLYRAYVTDALSGGRLEENKLVAMSQLRNILGLGKREAEAISVDVTSKSYRKRLANAVSSGDLEAQDSKAKYLQKLCEELHFDAQKAGAIHEEIYRQKLQQCVTDGELSDDNVAALLRLRVMLCIPQQTVDTAHAEICGTIFEKVVRDAISSGVDGYDAETRKSVRKAAHGLRLSRETAMSIASKAVRRVFTNYIRRARAAENRTDSAKELKKMIAFNTLVVTEMVADIKGESSDKAPEEDPVQEKEEDDEDEEWGSLESLRKTRPDKELAEKMGKPGQTEITLKDDLPDRDRIDLYKTYLLYCVTGEVTRIPFGAQITTKRDDSEYLLLNQLGGILGLSSKEIVNIHVGLAEQAFRQQAEVILADGQLTKARVEQLDELQKQVGLPQPQAEKVIKNITTTKMANAIETAVNQGRLNIKQIRELKEANVSLDSMIAVSLREKLFKKTVSDIFSSGTGEFDETEVYQTIPSDLSIDVEKAKRVVHDLAQSRLSNSLVQAVALLRQRNSKGVVLSLNDLLACDKAVPAEPMSWEVSEELSDLYAIYSKSDPKPAPEKVLRLQYLLGIDDSTATALREMEDGALSSAAEEGNFVF 814CCDE07EB5D0EA gtp guanosine triphosphate - GTP 5'-GTP GTP Guanosine 5'-triphosphate guanosine 5'-triphosphoric acid guanosine triphosphate GUANOSINE-5'-TRIPHOSPHATE H4gtp guanosine 5'-(tetrahydrogen triphosphate) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 oep37_arath Outer envelope pore protein 37, chloroplastic OEP37 Chloroplastic outer envelope pore protein of 37 kDa F6E13.8 At2g43950 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MADPSSQNPNLATPPPPSSPSPTHQIQSGTSELSPPSRPPCSTLSFLKTANRPKLRVTSEFDSDSLLFLNKVSCKLFDNLAKLKLSFQNNSQREISQPQVSFTSKHVSVLYDVEEKNTFIKSTLDVHPRLQLRALHNVKAQQGEVAMEANLTEPGYSLELSSPVPIGYPRATLKFPLGEISLQEKDEEEEEKQKRTLSVNGILKRQVMNGVCTALYTDEELRLRYAYKDDALSFIPSISLPSNAASFAFKRRFSPSDKLSYWYNFDSNMWSAVYKRTYGKDYKLKAGYDSDVRLGWASLWVGDEAGKVKTTPMKMKVQFMLQVPQDDIKSSVLMFRVKKRWDI FB26B8C037EF7169 tic40_arath Protein TIC 40, chloroplastic TIC40 PDE120 At5g16620 Protein PIGMENT DEFECTIVE EMBRYO 120 Translocon at the inner envelope membrane of chloroplasts 40 MTG13.6 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MENLTLVSCSASSPKLLIGCNFTSSLKNPTGFSRRTPNIVLRCSKISASAQSQSPSSRPENTGEIVVVKQRSKAFASIFSSSRDQQTTSVASPSVPVPPPSSSTIGSPLFWIGVGVGLSALFSYVTSNLKKYAMQTAMKTMMNQMNTQNSQFNNSGFPSGSPFPFPFPPQTSPASSPFQSQSQSSGATVDVTATKVETPPSTKPKPTPAKDIEVDKPSVVLEASKEKKEEKNYAFEDISPEETTKESPFSNYAEVSETNSPKETRLFEDVLQNGAGPANGATASEVFQSLGGGKGGPGLSVEALEKMMEDPTVQKMVYPYLPEEMRNPETFKWMLKNPQYRQQLQDMLNNMSGSGEWDKRMTDTLKNFDLNSPEVKQQFNQIGLTPEEVISKIMENPDVAMAFQNPRVQAALMECSENPMNIMKYQNDKEVMDVFNKISQLFPGMTG 52D3E7222AB461CE op24a_arath Outer envelope pore protein 24A, chloroplastic OEP24A Chloroplastic outer envelope pore protein of 24 kDa A F27F5.25 At1g45170 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MMKASFKGKFDVDKSGSVASLTFNAGNAKLRATMTDASFVAGPSFNGLSLAVEKPGFFIIDYNVPKKDVRFQFMNTIRIAEKPLNLTYIHMRGDNRTIVDGSFVIDPANKLSANYMVGTKNCKLKYTYVHGGIATFEPCYDVAKNMWDFAISHKLYGGDNLKATYQTSSKMLGLEWSNNSKSTGSFKVCASMNLAEELKPPKLTAETTWNLEL 693B6E72D8F362D0 rbs_tobac Ribulose bisphosphate carboxylase small subunit, chloroplastic RBCS TSSU3-8 protein protein tobac Nicotiana tabacum (Common tobacco) Common tobacco MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQCMQVWPPINKKKYETLSYLPDLSQEQLLSEVEYLLKNGWVPCLEFETEHGFVYRENNKSPGYYDGRYWTMWKLPMFGCTDATQVLAEVEEAKKAYPQAWIRIIGFDNVRQVQCISFIAYKPEGY DIP protein P69249 original sequence version: 38 26CF3F48EF0860AD tc159_arath Translocase of chloroplast 159, chloroplastic T14P8.24 At4g02510 TOC159 PPI2 TOC160 TOC86 Translocase of chloroplast 160, chloroplastic Translocase of chloroplast 86, chloroplastic 159 kDa chloroplast outer envelope protein Plastid protein import 2 T10P11.19 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDSKSVTPEPTNPFYASSGQSGKTYASVVAAAAAAAADKEDGGAVSSAKELDSSSEAVSGNSDKVGADDLSDSEKEKPNLVGDGKVSDEVDGSLKEDSTTPEATPKPEVVSGETIGVDDVSSLSPKPEAVSDGVGVVEENKKVKEDVEDIKDDGESKIENGSVDVDVKQASTDGESESKVKDVEEEDVGTKKDDEGESELGGKVDVDDKSDNVIEEEGVELTDKGDVIVNSSPVESVHVDVAKPGVVVVGDAEGSEELKINADAETLEVANKFDQIGDDDSGEFEPVSDKAIEEVEEKFTSESDSIADSSKLESVDTSAVEPEVVAAESGSEPKDVEKANGLEKGMTYAEVIKAASAVADNGTKEEESVLGGIVDDAEEGVKLNNKGDFVVDSSAIEAVNVDVAKPGVVVVGDVEVSEVLETDGNIPDVHNKFDPIGQGEGGEVELESDKATEEGGGKLVSEGDSMVDSSVVDSVDADINVAEPGVVVVGAAKEAVIKEDDKDDEVDKTISNIEEPDDLTAAYDGNFELAVKEISEAAKVEPDEPKVGVEVEELPVSESLKVGSVDAEEDSIPAAESQFEVRKVVEGDSAEEDENKLPVEDIVSSREFSFGGKEVDQEPSGEGVTRVDGSESEEETEEMIFGSSEAAKQFLAELEKASSGIEAHSDEANISNNMSDRIDGQIVTDSDEDVDTEDEGEEKMFDTAALAALLKAATGGGSSEGGNFTITSQDGTKLFSMDRPAGLSSSLRPLKPAAAPRANRSNIFSNSNVTMADETEINLSEEEKQKLEKLQSLRVKFLRLLQRLGHSAEDSIAAQVLYRLALLAGRQAGQLFSLDAAKKKAVESEAEGNEELIFSLNILVLGKAGVGKSATINSILGNQIASIDAFGLSTTSVREISGTVNGVKITFIDTPGLKSAAMDQSTNAKMLSSVKKVMKKCPPDIVLYVDRLDTQTRDLNNLPLLRTITASLGTSIWKNAIVTLTHAASAPPDGPSGTPLSYDVFVAQCSHIVQQSIGQAVGDLRLMNPSLMNPVSLVENHPLCRKNREGVKVLPNGQTWRSQLLLLCYSLKVLSETNSLLRPQEPLDHRKVFGFRVRSPPLPYLLSWLLQSRAHPKLPGDQGGDSVDSDIEIDDVSDSEQEDGEDDEYDQLPPFKPLRKTQLAKLSNEQRKAYFEEYDYRVKLLQKKQWREELKRMKEMKKNGKKLGESEFGYPGEEDDPENGAPAAVPVPLPDMVLPPSFDSDNSAYRYRYLEPTSQLLTRPVLDTHGWDHDCGYDGVNAEHSLALASRFPATATVQVTKDKKEFNIHLDSSVSAKHGENGSTMAGFDIQNVGKQLAYVVRGETKFKNLRKNKTTVGGSVTFLGENIATGVKLEDQIALGKRLVLVGSTGTMRSQGDSAYGANLEVRLREADFPIGQDQSSFGLSLVKWRGDLALGANLQSQVSVGRNSKIALRAGLNNKMSGQITVRTSSSDQLQIALTAILPIAMSIYKSIRPEATNDKYSMY 04AEDE84C1BED3F6 tc754_arath Protein TOC75-4, chloroplastic TOC75-4 F23J3.110 At4g09080 75 kDa translocon at the outer-envelope-membrane of chloroplasts 4 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEAVKEAVRKIKSLVIPHADEKDNGIVFEIKLNETDQRVEKWGLDPSLDFFEVTGNCNLGRPNSEGSNQSLMGSVTIRNIFNPKLDDLLSKIEYVRFLEAVKKPRNRTFKTSFFNSRKLSPVFTGGPGYEDLVPPMFVGRDCLKATITENLTRQRELTYGVMFEEIITRDENRRISENGLLLSPDGGISINGPPTTLSGTGIDHIATLQANITRDNTKLVNGAVVGEKNIFQVDQGLGIGNNFPLFNRHQLSLTSFIQLKQVEEGSDKPQPPVLVLHGRYGGCIGDLPSYDVFALGGPNSVRGYSMGELGAAKNILELGAEIRIPVKNTHVYAFAEHGNDLGSSKDVKGNPTGLYRKMGHGSSYGLGVKLGMVRAEYTVRHNRGTGALFLRFGERY F6606F1A031E9A6E toc34_arath Translocase of chloroplast 34, chloroplastic TOC34 OEP34 MUG13.14 At5g05000 PPI3 Plastid protein import 3 34 kDa chloroplast outer envelope protein GTP-binding protein OEP34 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAALQTLREWIGIQQFPPATQSKLLEILGKYKEEDVSSLTVLVMGKGGVGKSSTVNSVIGEKAAAVSTFQSEGLRPTLVSRTRSGFTLNIIDTPGLIEGGYVNDQAINIIKRFLLNMTIDVLLYVDRLDVYRVDDLDRQVVGAITDAFGKEIWKKSALVLTHAQFSPPDGLNYNHFVSKRSNALLKVIQTGAQLKKQDLQGFSIPVILVENSGRCHKNESDEKILPCGTSWIPNLFNKITEISFNGNKAIHVDKKLVEGPNPNERGKKLIPLMFAFQYLLVMKPLVRAIKSDVSRESKPAWELRDSGLASRRS DIP protein Q38906 original sequence version: 66 1C90E435B2EE0441 toc159-toc110 8 9 unspecified role unspecified role prey prey 16 unspecified role unspecified role prey prey 11 unspecified role unspecified role prey prey 15 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey association association Figure 1C Translocon components as well as Oep37 were coimmunoprecipitated by Toc159 antiserum toc34-rbcs 6 17 unspecified role unspecified role bait bait arath-leaf Arabidopsis thaliana leaf leaf leaf 14 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction Figure 3A Outer envelope proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane, followed by renaturation. Subsequently, the binding of a 35S-radiolabelled nonphosphorylated form of pSSU, as well as of 32P-radiolabelled phosphorylated pSSU, to outer envelope proteins was assayed (Figure 3A). The binding was analysed in the absence or presence of GMP-PNP. GMP-PNP was previously found to enhance the binding of pSSU to Toc34 (Schleiff et al, 2002), to Toc159 (Kouranov and Schnell, 1997) and to the Toc complex, respectively (Schleiff et al, 2003b). GMP-PNP strongly stimulated the binding of both pSSU forms to Toc34 (Figure 3A, lanes 1-4). Similarly, the interaction of the nonphosphorylated form of pSSU with Toc159 was stimulated by GMP-PNP (lanes 1, 2). However, no interaction of Toc159 with the phosphorylated pSSU was detected (lanes 3, 4). Furthermore, neither the phosphorylated nor the nonphosphorylated pSSU interacts with the outer envelope protein Oep37, which served as a negative control (upper part). GMP-PNP, a non-hydrolyzable analog of GTP. GMP-PNP is a compound in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. toc159-rbcs 6 14 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 15 unspecified role unspecified role bait bait arath-leaf Arabidopsis thaliana leaf leaf leaf direct interaction direct interaction Figure 3A Outer envelope proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane, followed by renaturation. Subsequently, the binding of a 35S-radiolabelled nonphosphorylated form of pSSU, as well as of 32P-radiolabelled phosphorylated pSSU, to outer envelope proteins was assayed (Figure 3A). The binding was analysed in the absence or presence of GMP-PNP. GMP-PNP was previously found to enhance the binding of pSSU to Toc34 (Schleiff et al, 2002), to Toc159 (Kouranov and Schnell, 1997) and to the Toc complex, respectively (Schleiff et al, 2003b). GMP-PNP strongly stimulated the binding of both pSSU forms to Toc34 (Figure 3A, lanes 1-4). Similarly, the interaction of the nonphosphorylated form of pSSU with Toc159 was stimulated by GMP-PNP (lanes 1, 2). However, no interaction of Toc159 with the phosphorylated pSSU was detected (lanes 3, 4). Furthermore, neither the phosphorylated nor the nonphosphorylated pSSU interacts with the outer envelope protein Oep37, which served as a negative control (upper part). GMP-PNP, a non-hydrolyzable analog of GTP. GMP-PNP is a compound, in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. toc34-rbcs-2 1 17 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction Figure 3B As before, Toc34 (lanes 5, 6) recognises pSSU with higher affinity in its GMP-PNP-bound stage. Furthermore, phosphorylation of the pSSU drastically enhances this interaction. GMP-PNP, a non-hydrolyzable analog of GTP, in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. Radiolabelling of P69249 was carried out using wheat germ lysate. toc34-rbcs-3 1 14 unspecified role unspecified role prey prey region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 17 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 3B As before, Toc34 (lanes 5, 6) recognises pSSU with higher affinity in its GMP-PNP-bound stage. Furthermore, phosphorylation of the pSSU drastically enhances this interaction. GMP-PNP, a non-hydrolyzable analog of GTP, in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. Phosphorylation of P69249 toc159-rbcs-1 1 14 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 15 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 3B As shown before, Toc159f interacts with the nonphosphorylated form of pSSU in a GMP-PNP-dependent manner GMP-PNP, a non-hydrolyzable analog of GTP, in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. Radiolabelling of P69249 was carried ot using reticulocyte lysate. toc34-rbcs-4 1 14 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 17 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 3B As before, Toc34 (lanes 5, 6) recognises pSSU with higher affinity in its GMP-PNP-bound stage. Furthermore, phosphorylation of the pSSU drastically enhances this interaction. GMP-PNP, a non-hydrolyzable analog of GTP, in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. Radiolabelling of P69249 was carried out using reticulocyte lysate. toc159-rbcs-2 2 14 unspecified role unspecified role bait bait in vitro In vitro 15 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 3C The observed nucleotide dependence of the interaction between Toc159 and the preprotein could also be assembled while incubation of immobilised pSSU with Toc159f in the absence or presence of different nucleotides. The strongest binding of Toc159f to pSSU was observed in the presence of GTP and GMP-PNP GMP-PNP, a non-hydrolyzable analog of GTP, in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. GTP GDP toc159-rbcs-3 4 14 unspecified role unspecified role bait bait n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 15 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 4 Toc159 recognises the N-terminal part of pSSU transit peptide with high affinity. GDP toc159-rbcs-4 4 14 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 15 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 4 Toc159 recognises the N-terminal part of pSSU transit peptide with high affinity. GDP GMP-PNP, a non-hydrolyzable analog of GTP. GMP-PNP is a compound, in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. toc34-rbcs-5 4 14 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 17 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 4 GMP-PNP, a non-hydrolyzable analog of GTP. no suitable source text GMP-PNP is a compound, in which the oxygen atom bridging the beta to the gamma phosphate is replaced by a nitrogen atom. It binds tightly to G-protein in the presence of Mg2+. The nucleotide is a potent stimulator of adenylate cyclase. toc159-rbcs-6 7 15 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) 10 enzyme target enzyme target substrate substrate neutral component neutral component gtpase reaction gtpase reaction Figure 5B Under the conditions used, pSSU was found to stimulate GTPase activity of Toc159f by about four-fold A chemically synthesised pSSU peptide (region 22 to 47 amino acid residues) was found to stimulate GTPase activity of Toc159f by about four-fold. toc34-rbcs-7 7 17 enzyme enzyme neutral component neutral component 10 enzyme target enzyme target substrate substrate neutral component neutral component gtpase reaction gtpase reaction Figure 5C In contrast, GTP hydrolysis by Toc34 was not affected by addition of the peptides (Figure 5C). toc159-toc75-1 8 13 unspecified role unspecified role prey prey 17 unspecified role unspecified role prey prey 16 unspecified role unspecified role prey prey 15 unspecified role unspecified role bait bait association association Figure 6A Therefore, solubilised OEVs were incubated with GDP or GMP-PNP. Subsequently, antisera against Toc34 or Toc159 were used for immunoprecipitation (Figure 6A). All the three Toc core components were coimmunoprecipitated with the same efficiency in the absence of nucleotides or in the presence of GMP-PNP by either Toc34 (Figure 6A, lanes 1, 3) or Toc159 antiserum (lanes 5, 7). In contrast, incubation with GDP leads to a reduced coimmunoprecipitation of Toc159 by Toc34 antiserum and vice versa The authors note that "incubation with GDP leads to a reduced coimmunoprecipitation of Toc159 by Toc34 antiserum and vice versa". The accession number for Oep24 (Q1H5C9) was derived from the closest amino acid sequence match for an Arabidopsis protein to the Oep24 Pisum sativaum (Pea) protein in the UniProt database. The Oep24 Pea protein was cited in a secondary reference from a reference provided by the authors. The authors concluded that "although small amounts of Oep24 were coimmunoprecipitated by the used Toc34 antiserum, it presents only a small portion of total Oep24 content in OEVs. Furthermore, Toc75, Toc159 and Toc34 were not coimmunoprecipitated by Oep24 antibodies regardless of the added nucleotides. Therefore, we consider the precipitation of Oep24 as an unspecific reaction". toc34-toc75-2 8 13 unspecified role unspecified role prey prey 16 unspecified role unspecified role prey prey 15 unspecified role unspecified role prey prey 17 unspecified role unspecified role bait bait association association Figure 6A Therefore, solubilised OEVs were incubated with GDP or GMP-PNP. Subsequently, antisera against Toc34 or Toc159 were used for immunoprecipitation (Figure 6A). All the three Toc core components were coimmunoprecipitated with the same efficiency in the absence of nucleotides or in the presence of GMP-PNP by either Toc34 (Figure 6A, lanes 1, 3) or Toc159 antiserum (lanes 5, 7). In contrast, incubation with GDP leads to a reduced coimmunoprecipitation of Toc159 by Toc34 antiserum and vice versa The authors note that "incubation with GDP leads to a reduced coimmunoprecipitation of Toc159 by Toc34 antiserum and vice versa". The accession number for Oep24 (Q1H5C9) was derived from the closest amino acid sequence match for an Arabidopsis protein to the Oep24 Pisum sativaum (Pea) protein in the UniProt database. The Oep24 Pea protein was cited in a secondary reference from a reference provided by the authors. The authors concluded that "although small amounts of Oep24 were coimmunoprecipitated by the used Toc34 antiserum, it presents only a small portion of total Oep24 content in OEVs. Furthermore, Toc75, Toc159 and Toc34 were not coimmunoprecipitated by Oep24 antibodies regardless of the added nucleotides. Therefore, we consider the precipitation of Oep24 as an unspecific reaction". toc159-toc75-2 3 15 unspecified role unspecified role neutral component neutral component 16 unspecified role unspecified role neutral component neutral component 17 unspecified role unspecified role neutral component neutral component physical association physical association Figure 6C To confirm our observation, the stability of the Toc complex was analysed during isolation by sucrose gradient centrifugation. OEVs were incubated with EDTA, GDP or GMP-PNP before solubilisation and subjected to linear sucrose gradient centrifugation. In the presence of GMP-PNP, the Toc components are enriched in higher density fractions as compared to the absence of nucleotide (Figure 6C, right and left parts). In the presence of GDP, Toc components migrate only to lower-density fractions The authors note that "in the presence of GMP-PNP, the Toc components are enriched in higher density fractions as compared to the absence of nucleotide". toc159-toc75-3 5 16 unspecified role unspecified role neutral component neutral component 15 unspecified role unspecified role neutral component neutral component 17 unspecified role unspecified role neutral component neutral component physical association physical association Figure 6D The samples were subjected to a sucrose step-gradient centrifugation. In the presence of GMP-PNP, the amount of Toc components, especially Toc34, is enriched in fractions of higher density in comparison to the amount in the absence of nucleotides The authors note that "in the presence of GMP-PNP, the Toc components are enriched in higher density fractions as compared to the absence of nucleotide" amnd that "In the presence of GDP, no Toc components were found in a fraction of high sucrose concentrations". The authors conclude that "GMP-PNP leads to a stabilisation of the Toc complex, whereas GDP weakens the interaction between the Toc components". toc34-toc159 2 17 unspecified role unspecified role bait bait c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 15 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 6E Toc159GMP-PNP binds to Toc34 to the same extent as nucleotide-free Toc159 (lanes 1, 3, 4, 6). In contrast, Toc159GDP shows reduced binding to Toc34 (lanes 2, 5) Presence of chemically synthesised peptides covering 1 to 21 and 22 to 47 amino acid residues of the pSSU protein (P69249). The authors note that "In contrast, Toc159GDP" Toc159 pretreated with GDP "shows reduced binding to Toc34".