MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it http://mint.bio.uniroma2.it/mint lytovchenko-2013-1 Signal recognition initiates reorganization of the presequence translocase during protein import. in vitro In vitro crosslink cross-linking study crosslink weight by comassie molecular weight estimation by coomasie staining weight by comassie Lytovchenko O., Melin J., Schulz C., Kilisch M., Hutu D.P., Rehling P. Peter.Rehling@medizin.uni-goettingen.de EMBO J. (0261-4189) 2013 Only protein-protein interactions imex curation lytovchenko-2013-2 Signal recognition initiates reorganization of the presequence translocase during protein import. in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Lytovchenko O., Melin J., Schulz C., Kilisch M., Hutu D.P., Rehling P. Peter.Rehling@medizin.uni-goettingen.de EMBO J. (0261-4189) 2013 Only protein-protein interactions imex curation lytovchenko-2013-5 Signal recognition initiates reorganization of the presequence translocase during protein import. yeast Saccharomyces cerevisiae Baker's yeast anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Lytovchenko O., Melin J., Schulz C., Kilisch M., Hutu D.P., Rehling P. Peter.Rehling@medizin.uni-goettingen.de EMBO J. (0261-4189) 2013 Only protein-protein interactions imex curation lytovchenko-2013-3 Signal recognition initiates reorganization of the presequence translocase during protein import. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Lytovchenko O., Melin J., Schulz C., Kilisch M., Hutu D.P., Rehling P. Peter.Rehling@medizin.uni-goettingen.de EMBO J. (0261-4189) 2013 Only protein-protein interactions imex curation lytovchenko-2013-4 Signal recognition initiates reorganization of the presequence translocase during protein import. yeast Saccharomyces cerevisiae Baker's yeast anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Lytovchenko O., Melin J., Schulz C., Kilisch M., Hutu D.P., Rehling P. Peter.Rehling@medizin.uni-goettingen.de EMBO J. (0261-4189) 2013 Only protein-protein interactions imex curation tim21_yeast Mitochondrial import inner membrane translocase subunit TIM21 TIM21 YGR033C protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSSLPRSLLRLGHRKPLFPRYNTFVNSSVITHTSLLRTRLYSNGTGATSGKKDDKTRNKPKPLWPQVKSASTFTFSGILVIGAVGISAIVIYLILSELFSPSGDTQLFNRAVSMVEKNKDIRSLLQCDDGITGKERLKAYGELITNDKWTRNRPIVSTKKLDKEGRTHHYMRFHVESKKKIALVHLEAKESKQNYQPDFINMYVDVPGEKRYYLIKPKLHPVSNSKGFLGIRWGPRKD FC140F3034E5B5BB tim50_yeast Mitochondrial import inner membrane translocase subunit TIM50 TIM50 YPL063W protein protein yeast Saccharomyces cerevisiae Baker's yeast MLSILRNSVRLNSRALRVVPSAANTLTSVQASRRLLTSYSSFLQKETKDDKPKSILTDDMLFKAGVDVDEKGQGKNEETSGEGGEDKNEPSSKSEKSRRKRQTSTDIKREKYANWFYIFSLSALTGTAIYMARDWEPQESEELKKDIDNGYTLSLMYKRFKARFNSMFTYFQEPPFPDLLPPPPPPPYQRPLTLVITLEDFLVHSEWSQKHGWRTAKRPGADYFLGYLSQYYEIVLFSSNYMMYSDKIAEKLDPIHAFVSYNLFKEHCVYKDGVHIKDLSKLNRDLSKVIIIDTDPNSYKLQPENAIPMEPWNGEADDKLVRLIPFLEYLATQQTKDVRPILNSFEDKKNLAEEFDHRVKKLKDKFYGDHKSGGNWAMTALGLGNSLGGSTKFPLDLIHEEGQKNYLMFMKMIEEEKEKIRIQQEQMGGQTFTLKDYVEGNLPSPEEQMKIQLEKQKEVDALFEEEKKKKKIAESK 778D54F5C4ED27C8 cox4_yeast Cytochrome c oxidase subunit 4, mitochondrial COX4 YGL187C G1362 Cytochrome c oxidase polypeptide IV protein protein yeast Saccharomyces cerevisiae Baker's yeast MLSLRQSIRFFKPATRTLCSSRYLLQQKPVVKTAQNLAEVNGPETLIGPGAKEGTVPTDLDQETGLARLELLGKLEGIDVFDTKPLDSSRKGTMKDPIIIESYDDYRYVGCTGSPAGSHTIMWLKPTVNEVARCWECGSVYKLNPVGVPNDDHHH 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 aldh2_rat Aldehyde dehydrogenase, mitochondrial ALDH class 2 ALDH1 ALDH-E2 Aldh2 protein protein rat Rattus norvegicus (Rat) Rat MLRAALSTARRGPRLSRLLSAAATSAVPAPNQQPEVFCNQIFINNEWHDAVSKKTFPTVNPSTGEVICQVAEGNKEDVDKAVKAAQAAFQLGSPWRRMDASDRGRLLYRLADLIERDRTYLAALETLDNGKPYVISYLVDLDMVLKCLRYYAGWADKYHGKTIPIDGDFFSYTRHEPVGVCGQIIPWNFPLLMQAWKLGPALATGNVVVMKVAEQTPLTALYVANLIKEAGFPPGVVNIVPGFGPTAGAAIASHEDVDKVAFTGSTEVGHLIQVAAGSSNLKRVTLELGGKSPNIIMSDADMDWAVEQAHFALFFNQGQCCCAGSRTFVQEDVYDEFVERSVARAKSRVVGNPFDSRTEQGPQVDETQFKKILGYIKSGQQEGAKLLCGGGAAADRGYFIQPTVFGDVKDGMTIAKEEIFGPVMQILKFKTIEEVVGRANNSKYGLAAAVFTKDLDKANYLSQALQAGTVWINCYDVFGAQSPFGGYKMSGSGRELGEYGLQAYTEVKTVTVKVPQKNS pam17_yeast Presequence translocated-associated motor subunit PAM17, mitochondrial YKR065C PAM17 FMP18 protein protein yeast Saccharomyces cerevisiae Baker's yeast MFTSAIRLSSQRLFASQPSVTAAALRSTATTLPLRSYSQPASLQDSSILTWSDFFKLRKQQRRINVGSSLFTALLGCNVSWAYLSTMEIDPTQMLFGFDPLTVISAGIIASGALGYLLGPIVGSQVFKLSHNQQLAQFNNKNKEFLKHIINNRVDASSQSFSNPVPDYYGEKIGSLKEYKQWLRDCHAYAKKAKEFL tim21-tim50 1497713 n/a 1497718 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497719 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false f2b f2c f2f Purified proteins were mixed in equimolar amounts and subjected to crosslinking prior to SDS–polyacrylamide gel electrophoresis (PAGE) analyses. Tim21IMS formed a dimer upon Cu2þ-induced crosslinking, while Tim21IMS-C128S did not, due to the absence of the cysteine residue (Figure 2B, lanes 4 versus 5). Similarly, Tim50IMS formed dimers upon crosslinking (Figure 2B, lane 6), tim21-tim50-1 1497713 n/a 1497718 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false P53220:p.Cys128Ser mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f2b f2c f2f Purified proteins were mixed in equimolar amounts and subjected to crosslinking prior to SDS–polyacrylamide gel electrophoresis (PAGE) analyses. Tim21IMS formed a dimer upon Cu2þ-induced crosslinking, while Tim21IMS-C128S did not, due to the absence of the cysteine residue (Figure 2B, lanes 4 versus 5). Similarly, Tim50IMS formed dimers upon crosslinking (Figure 2B, lane 6), tim21-tim50-2 1497713 n/a 1497719 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f2b Purified proteins were mixed in equimolar amounts and subjected to crosslinking prior to SDS–polyacrylamide gel electrophoresis (PAGE) analyses. Tim21IMS formed a dimer upon Cu2þ-induced crosslinking, while Tim21IMS-C128S did not, due to the absence of the cysteine residue (Figure 2B, lanes 4 versus 5). Similarly, Tim50IMS formed dimers upon crosslinking (Figure 2B, lane 6), tim21-tim50-3 1497713 n/a 1497718 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497719 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false f2f sf1a Purified proteins were mixed in equimolar amounts and subjected to crosslinking prior to SDS–polyacrylamide gel electrophoresis (PAGE) analyses. Tim21IMS formed a dimer upon Cu2þ-induced crosslinking, while Tim21IMS-C128S did not, due to the absence of the cysteine residue (Figure 2B, lanes 4 versus 5). Similarly, Tim50IMS formed dimers upon crosslinking (Figure 2B, lane 6), We purified Tim21IMS and Tim50C (amino acids 164–361), corresponding to the crystallized portion of Tim50, and addressed protein interactions in vitro. Similar to Tim50IMS, Tim50C was able to dimerize and to hetero-oligomerize with Tim21IMS (Supplementary Figure S1A). tim21-tim50-9 1497713 n/a 1497719 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497720 peptide synthesis peptide synthesis unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false sf1c Similarly, while Tim50IMS could be efficiently crosslinked to Cox4 presequence peptides with the homo-bifunctional chemical crosslinker 1,5-Difluoro-2,4-dinitrobenzene (DFDNB), Tim50DPBD and Tim50C displayed no significant presequence crosslinks (Supplementary Figure S1C). cox4-tim50 1497713 n/a 1497721 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified P32897:p.Tyr70_Leu71delinsAlaAla mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false n/a 1497719 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false sf2b Indeed, a crosslinking-adduct was formed between Tim50IMS and Tim23IMS, but not with Tim23IMS YL70AA in an in vitro chemical crosslinking experiment (Supplementary Figure S2B). tim21-tim50-4 1497714 n/a 1497718 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497719 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false 1497714 f2d t1 tim21-tim50-5 1497714 n/a 1497718 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497719 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false 1497714 f2g t1 tim21-tim50-6 1497714 n/a 1497722 peptide synthesis peptide synthesis unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497719 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false 1497714 t1 f3e aldh2-tim50 1497714 n/a 1497722 peptide synthesis peptide synthesis unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497719 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false 1497714 t1 f3a aldh2-tim50-1 1497714 n/a 1497719 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497722 peptide synthesis peptide synthesis unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false 1497714 t1 F3C tim21-tim50-7 1497714 n/a 1497721 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified n/a 1497718 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false 1497714 t1 sf2a sf2c Therefore, we analysed the binding affinity of Tim21IMS to immobilized Tim23IMS in an in vitro system via SPR (Supplementary Figure S2A). Our results confirmed their direct interaction with an KD of B1 mM (Table I), suggesting that Tim23 could exert its coordinating effect on Tim21– Tim50 complex formation via its direct interaction with both proteins. At the same time, SPR analysis showed that the mutant’s interaction with Tim21 was not affected (Supplementary Figure S2C). tim50-pam17 1497715 n/a 1497721 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level n/a 1497718 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level n/a 1497719 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level n/a 1497723 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association false false false f6a tim23-pam17 1497715 n/a 1497718 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level n/a 1497721 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level n/a 1497719 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level n/a 1497723 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association false false false f6a tim21-tim50-8 1497716 n/a 1497719 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1497718 predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false f5g To address the effect of Tim23IMS on Tim21–Tim50 by an alternative approach, we performed pull-down experiments. Therefore, purified Tim50IMS was added to Tim21IMS immobilized on Sepharose beads in the presence of increasing concentrations of Tim23IMS. Addition of wild-type Tim23IMS significantly increased the binding of Tim50IMS to the Tim21 beads, whereas Tim23IMS YL70AA did not promote this binding (Figure 5G and H). tim50-pam17-1 1497717 n/a 1497719 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait subcellular prep subcellular preparation subcellular prep over-expressed over expressed level over-expressed tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false range sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1497718 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association false false false sf2e