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/ Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell (0092-8674) 2005 imex curation Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Proteins isolated from yeast mitochondria lysed with digitonin. peter.rehling@biochemie.uni-freiburg.de Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast pull down pull down ms participant Identification by mass spectrometry ms participant Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Some data (Fig 5) could not be curated since identity of cytochrome B in a cytB/DHFR fusion protein could not be established. Proteins isolated from yeast mitochondria lysed with digitonin. Mass spectrometry identification confirmed by western blot peter.rehling@biochemie.uni-freiburg.de Accepted 2007-APR-8 by JYOTI. Cell (0092-8674) 2005 Only protein-protein interactions imex curation Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell (0092-8674) 2005 imex curation Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Proteins isolated from yeast mitochondria lysed with digitonin. peter.rehling@biochemie.uni-freiburg.de Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Proteins isolated from yeast mitochondria lysed with digitonin. peter.rehling@biochemie.uni-freiburg.de Accepted 2007-APR-8 by JYOTI. Cell (0092-8674) 2005 Only protein-protein interactions imex curation Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell (0092-8674) 2005 imex curation Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Proteins isolated from yeast mitochondria lysed with digitonin. peter.rehling@biochemie.uni-freiburg.de Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast pull down pull down western blot western blot Immuno blot Immuno blot Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Proteins isolated from yeast mitochondria lysed with digitonin. peter.rehling@biochemie.uni-freiburg.de Accepted 2007-APR-8 by JYOTI. Cell (0092-8674) 2005 Only protein-protein interactions imex curation Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell (0092-8674) 2005 imex curation Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Proteins isolated from yeast mitochondria lysed with digitonin. peter.rehling@biochemie.uni-freiburg.de Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Proteins isolated from yeast mitochondria lysed with digitonin. peter.rehling@biochemie.uni-freiburg.de Accepted 2007-APR-8 by JYOTI. Cell (0092-8674) 2005 Only protein-protein interactions imex curation Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell (0092-8674) 2005 imex curation Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Proteins isolated from yeast mitochondria lysed with digitonin. peter.rehling@biochemie.uni-freiburg.de Only protein-protein interactions in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Chacinska A., Lind M., Frazier AE., Dudek J., Meisinger C., Geissler A., Sickmann A., Meyer HE., Truscott KN., Guiard B., Pfanner N., Rehling P. Mitochondria from a strain expressing Tim21ProtA incubated with 250 uM crosslinker Dithiobis[succinimidyl propionate] for 30 minutes at 25C. peter.rehling@biochemie.uni-freiburg.de Accepted 2007-APR-8 by JYOTI. Cell (0092-8674) 2005 Only protein-protein interactions imex curation tim44_yeast Mitochondrial import inner membrane translocase subunit TIM44 ISP45 MIM44 MPI1 YIL022W TIM44 Mitochondrial protein import protein 1 Inner membrane import site protein 45 Membrane import machinery protein MIM44 protein protein yeast Saccharomyces cerevisiae Baker's yeast MHRSTFIRTSGTSSRTLTARYRSQYTGLLVARVLFSTSTTRAQGGNPRSPLQIFRDTFKKEWEKSQELQENIKTLQDASGKLGESEAYKKAREAYLKAQRGSTIVGKTLKKTGETMEHIATKAWESELGKNTRKAAAATAKKLDESFEPVRQTKIYKEVSEVIDDGESSRYGGFITKEQRRLKRERDLASGKRHRAVKSNEDAGTAVVATNIESKESFGKKVEDFKEKTVVGRSIQSLKNKLWDESENPLIVVMRKITNKVGGFFAETESSRVYSQFKLMDPTFSNESFTRHLREYIVPEILEAYVKGDVKVLKKWFSEAPFNVYAAQQKIFKEQDVYADGRILDIRGVEIVSAKLLAPQDIPVLVVGCRAQEINLYRKKKTGEIAAGDEANILMSSYAMVFTRDPEQIDDDETEGWKILEFVRGGSRQFT 8E98C738178B1E44 8E98C738178B1E44 tim23_yeast Mitochondrial import inner membrane translocase subunit TIM23 MIM23 MPI3 N3180 YNR017W TIM23 MAS6 Mitochondrial protein import protein 3 Mitochondrial protein import protein MAS6 Membrane import machinery protein MIM23 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSWLFGDKTPTDDANAAVGGQDTTKPKELSLKQSLGFEPNINNIISGPGGMHVDTARLHPLAGLDKGVEYLDLEEEQLSSLEGSQGLIPSRGWTDDLCYGTGAVYLLGLGIGGFSGMMQGLQNIPPNSPGKLQLNTVLNHITKRGPFLGNNAGILALSYNIINSTIDALRGKHDTAGSIGAGALTGALFKSSKGLKPMGYSSAMVAAACAVWCSVKKRLLEK 08E5AB4F400F675B 08E5AB4F400F675B hsp7e_yeast Heat shock protein SSC3, mitochondrial ECM10 SSC3 Extracellular mutant protein 10 YEL030W protein protein yeast Saccharomyces cerevisiae Baker's yeast MLPSWKAFKAHNILRILTRFQSTKIPDAVIGIDLGTTNSAVAIMEGKVPRIIENAEGSRTTPSVVAFTKDGERLVGEPAKRQSVINSENTLFATKRLIGRRFEDAEVQRDINQVPFKIVKHSNGDAWVEARNRTYSPAQIGGFILNKMKETAEAYLAKSVKNAVVTVPAYFNDAQRQATKDAGQIIGLNVLRVVNEPTAAALAYGLDKSEPKVIAVFDLGGGTFDISILDIDNGIFEVKSTNGDTHLGGEDFDIYLLQEIISHFKKETGIDLSNDRMAVQRIREAAEKAKIELSSTLSTEINLPFITADAAGPKHIRMPFSRVQLENITAPLIDRTVDPVKKALKDARITASDISDVLLVGGMSRMPKVADTVKKLFGKDASKAVNPDEAVALGAAIQAAVLSGEVTDVLLLDVTPLSLGIETLGGVFTKLIPRNSTIPNKKSQIFSTAASGQTSVEVKVFQGERELVKDNKLIGNFTLAGIPPAPKGTPQIEVTFDIDANGIINVSAKDLASHKDSSITVAGASGLSDTEIDRMVNEAERYKNQDRARRNAIETANKADQLANDTENSIKEFEGKLDKTDSQRLKDQISSLRELVSRSQAGDEVNDDDVGTKIDNLRTSSMKLFEQLYKNSDNPETKNGRENK 6A248DE208F513CB 6A248DE208F513CB tim14_yeast Mitochondrial import inner membrane translocase subunit TIM14 Presequence translocated-associated motor subunit PAM18 PAM18 TIM14 YLR008C protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSQSNTGNSIEAPQLPIPGQTNGSANVTVDGAGVNVGIQNGSQGQKTGMDLYFDQALNYMGEHPVITGFGAFLTLYFTAGAYKSISKGLNGGKSTTAFLKGGFDPKMNSKEALQILNLTENTLTKKKLKEVHRKIMLANHPDKGGSPFLATKINEAKDFLEKRGISK 9CBF71CA50A0F341 tom40_yeast Mitochondrial import receptor subunit TOM40 TOM40 ISP42 YMR203W YM8325.04 MOM38 Mitochondrial import site protein ISP42 Translocase of outer membrane 40 kDa subunit protein protein yeast Saccharomyces cerevisiae Baker's yeast MSAPTPLAEASQIPTIPALSPLTAKQSKGNFFSSNPISSFVVDTYKQLHSHRQSLELVNPGTVENLNKEVSRDVFLSQYFFTGLRADLNKAFSMNPAFQTSHTFSIGSQALPKYAFSALFANDNLFAQGNIDNDLSVSGRLNYGWDKKNISKVNLQISDGQPTMCQLEQDYQASDFSVNVKTLNPSFSEKGEFTGVAVASFLQSVTPQLALGLETLYSRTDGSAPGDAGVSYLTRYVSKKQDWIFSGQLQANGALIASLWRKVAQNVEAGIETTLQAGMVPITDPLMGTPIGIQPTVEGSTTIGAKYEYRQSVYRGTLDSNGKVACFLERKVLPTLSVLFCGEIDHFKNDTKIGCGLQFETAGNQELLMLQQGLDADGNPLQALPQL 6436FBBA4020464B tom22_yeast Mitochondrial import receptor subunit TOM22 MOM22 TOM22 MAS17 MAS22 YNL131W N1217 N1862 Mitochondrial 22 kDa outer membrane protein Translocase of outer membrane 22 kDa subunit Protein MAS17 Mitochondrial 17 kDa assembly protein protein protein yeast Saccharomyces cerevisiae Baker's yeast MVELTEIKDDVVQLDEPQFSRNQAIVEEKASATNNDVVDDEDDSDSDFEDEFDENETLLDRIVALKDIVPPGKRQTISNFFGFTSSFVRNAFTKSGNLAWTLTTTALLLGVPLSLSILAEQQLIEMEKTFDLQSDANNILAQGEKDAAATAN 6ACEC5D37D143CD3 6ACEC5D37D143CD3 tim21_yeast Mitochondrial import inner membrane translocase subunit TIM21 TIM21 YGR033C protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSSLPRSLLRLGHRKPLFPRYNTFVNSSVITHTSLLRTRLYSNGTGATSGKKDDKTRNKPKPLWPQVKSASTFTFSGILVIGAVGISAIVIYLILSELFSPSGDTQLFNRAVSMVEKNKDIRSLLQCDDGITGKERLKAYGELITNDKWTRNRPIVSTKKLDKEGRTHHYMRFHVESKKKIALVHLEAKESKQNYQPDFINMYVDVPGEKRYYLIKPKLHPVSNSKGFLGIRWGPRKD FC140F3034E5B5BB FC140F3034E5B5BB tim17_yeast Mitochondrial import inner membrane translocase subunit TIM17 MPI2 TIM17 MIM17 SMS1 YJL143W J0648 Mitochondrial protein import protein 2 Mitochondrial inner membrane protein MIM17 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSADHSRDPCPIVILNDFGGAFAMGAIGGVVWHGIKGFRNSPLGERGSGAMSAIKARAPVLGGNFGVWGGLFSTFDCAVKAVRKREDPWNAIIAGFFTGGALAVRGGWRHTRNSSITCACLLGVIEGVGLMFQRYAAWQAKPMAPPLPEAPSSQPLQA 1760B08F3B60D378 1760B08F3B60D378 tim16_yeast Mitochondrial import inner membrane translocase subunit TIM16 YJL104W J0822 PAM16 TIM16 Presequence translocated-associated motor subunit PAM16 protein protein yeast Saccharomyces cerevisiae Baker's yeast MAHRAFIQVIITGTQVFGKAFAEAYRQAASQSVKQGATNASRRGTGKGEYGGITLDESCKILNIEESKGDLNMDKINNRFNYLFEVNDKEKGGSFYLQSKVYRAAERLKWELAQREKNAKAKAGDASTAKPPPNSTNSSGADNSASSNQ 42BB69D746264B47 tim50_yeast Mitochondrial import inner membrane translocase subunit TIM50 TIM50 YPL063W protein protein yeast Saccharomyces cerevisiae Baker's yeast MLSILRNSVRLNSRALRVVPSAANTLTSVQASRRLLTSYSSFLQKETKDDKPKSILTDDMLFKAGVDVDEKGQGKNEETSGEGGEDKNEPSSKSEKSRRKRQTSTDIKREKYANWFYIFSLSALTGTAIYMARDWEPQESEELKKDIDNGYTLSLMYKRFKARFNSMFTYFQEPPFPDLLPPPPPPPYQRPLTLVITLEDFLVHSEWSQKHGWRTAKRPGADYFLGYLSQYYEIVLFSSNYMMYSDKIAEKLDPIHAFVSYNLFKEHCVYKDGVHIKDLSKLNRDLSKVIIIDTDPNSYKLQPENAIPMEPWNGEADDKLVRLIPFLEYLATQQTKDVRPILNSFEDKKNLAEEFDHRVKKLKDKFYGDHKSGGNWAMTALGLGNSLGGSTKFPLDLIHEEGQKNYLMFMKMIEEEKEKIRIQQEQMGGQTFTLKDYVEGNLPSPEEQMKIQLEKQKEVDALFEEEKKKKKIAESK 778D54F5C4ED27C8 778D54F5C4ED27C8 tim23-tim21-1 1 9 unspecified role unspecified role prey prey 12 unspecified role unspecified role prey prey 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait protein a tag region fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role prey prey 14 unspecified role unspecified role prey prey 8 unspecified role unspecified role prey prey association association 1A Isolation of protein A-Tim23 from wild-type (wt) and tim21 yeast mitochondria lysed with digitonin. tim23-tim50-1 2 15 unspecified role unspecified role bait bait 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 ims region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) 7 unspecified role unspecified role prey prey yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association 2A Ni-NTA agarose was left untreated (control) or loaded with purified Tim21IMS and Tim50IMS prior to incubation with Triton X-100 solubilized mitochondrial extracts. tim21-tom40-1 2 11 unspecified role unspecified role ancillary ancillary ims region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role prey prey yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 12 unspecified role unspecified role bait bait ims region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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) association association 2A & 2B Ni-NTA agarose was left untreated (control) or loaded with purified Tim21IMS and Tim50IMS prior to incubation with Triton X-100 solubilized mitochondrial extracts. tim21-tom22-1 2 11 unspecified role unspecified role prey prey ims region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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) 12 unspecified role unspecified role bait bait ims region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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 disulfide bond disulfide bond disulfide bridge SS-bond 2C GST-Tom22IMS but not GST itself bound to immobilized Tim21IMS (Figure 2C). Thus, Tim21IMS interacts with the TOM complex via direct binding to Tom22IMS. tim23-tim21-2 4 13 unspecified role unspecified role neutral component neutral component 12 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component physical association physical association Figure 3A Upon separation of digitonin-lysed mitochondria by BN-PAGE, Tim23 and Tim17 are found in a 90 kDa core complex (TIM23core) and additionally in larger TIM23* complexes (Dekker et al., 1997 and Chacinska et al., 2003) (Figure 3A, lanes 1 and 5). 90kDa core complex tim23-tim17-1 4 7 unspecified role unspecified role neutral component neutral component 13 unspecified role unspecified role neutral component neutral component physical association physical association Figure 3A Upon separation of digitonin-lysed mitochondria by BN-PAGE, Tim23 and Tim17 are found in a 90 kDa core complex (TIM23core) and additionally in larger TIM23* complexes (Dekker et al., 1997 and Chacinska et al., 2003) (Figure 3A, lanes 1 and 5). 90kDa core complex tim23-tim21-3 3 12 unspecified role unspecified role bait bait protein a tag region fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey physical association physical association Figure 3B While the purification via tagged Tim23 yielded both TIM23core and TIM23*, the purification via tagged Tim21 yielded only TIM23* (Figure 3B), tim23-tim21-4 3 7 unspecified role unspecified role bait bait protein a tag region fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role prey prey 12 unspecified role unspecified role prey prey 6 unspecified role unspecified role prey prey 13 unspecified role unspecified role prey prey 14 unspecified role unspecified role prey prey 8 unspecified role unspecified role prey prey 15 unspecified role unspecified role prey prey association association 3F & 6D We thus directly compared which components of TIM23-PAM were copurified with either tagged Tim23 or tagged Tim21 (Figure 3F). In cells defficient in Tim17, only Tim44 (Q01852), Tim21 ( P53220) and Tom50 (Q02776) were pulled down with Tom23 tim23-tim21-5 3 12 unspecified role unspecified role bait bait protein a tag region fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey 13 unspecified role unspecified role prey prey 15 unspecified role unspecified role prey prey association association 3F We thus directly compared which components of TIM23-PAM were copurified with either tagged Tim23 or tagged Tim21 (Figure 3F). pam18-tim17-1 2 9 unspecified role unspecified role bait bait n-terminal region osulftyr O4'-sulfo-L-tyrosine (S)-2-amino-3-(4-sulfooxyphenyl)propanoic acid MOD_RES Sulfotyrosine O4-sulfotyrosine tyrosine sulfate tyrosine-O-sulfonic acid tyrosine-O-sulphonic acid 2-amino-3-(4-hydroxyphenyl)propanoic acid 4'-sulfate O4'-sulfo-L-tyrosine O-Sulfonation OSulfTyr Sulfo Sulphation (of O of Tyrosine) Tyrosinyl Sulphate sulfationy deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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) 13 unspecified role unspecified role prey prey yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association We expressed and purified Pam18N and incubated it with lysed mitochondria. 6F & 6G tim23-tim21-6 5 7 unspecified role unspecified role neutral component neutral component yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 12 unspecified role unspecified role neutral component neutral component protein a tag region tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast direct interaction direct interaction S1 Tim21 can be crosslinked to Tim23.