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 Atp23 biogenesis reveals a chaperone-like folding activity of Mia40 in the IMS of mitochondria. Atp23 biogenesis reveals a chaperone-like folding activity of Mia40 in the IMS of mitochondria. EMBO J. (0261-4189) 2012 imex curation Weckbecker D., Longen S., Riemer J., Herrmann JM. Only protein-protein interactions in vitro In vitro crosslink cross-linking study crosslink weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Weckbecker D., Longen S., Riemer J., Herrmann JM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation Atp23 biogenesis reveals a chaperone-like folding activity of Mia40 in the IMS of mitochondria. Atp23 biogenesis reveals a chaperone-like folding activity of Mia40 in the IMS of mitochondria. EMBO J. (0261-4189) 2012 imex curation Weckbecker D., Longen S., Riemer J., Herrmann JM. Only protein-protein interactions in vitro In vitro light scattering light scattering predetermined predetermined participant predetermined Weckbecker D., Longen S., Riemer J., Herrmann JM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation Atp23 biogenesis reveals a chaperone-like folding activity of Mia40 in the IMS of mitochondria. Atp23 biogenesis reveals a chaperone-like folding activity of Mia40 in the IMS of mitochondria. EMBO J. (0261-4189) 2012 imex curation Weckbecker D., Longen S., Riemer J., Herrmann JM. Only protein-protein interactions in vitro In vitro peptide array peptide array experimental particp experimental participant identification experimental particp Weckbecker D., Longen S., Riemer J., Herrmann JM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation atp23_yeast Mitochondrial inner membrane protease ATP23 ATP23 N3212 YNR020C protein protein yeast Saccharomyces cerevisiae Baker's yeast MNSSGDNAGFEWWRRTMQYKTGIGLTPEEKTRYEDDSKARELKKECLKCYEYRDWMLKYSPTVRFMVQAITKLNKGSDSKFDDSKIICDYCPDWKGGGFHPELGILLCQNRLRDKWHLEDTLSHELIHYFDDLKWQIDWLNLKHHACSEIRASSLSGECRFWEEFKRRGFRTGFHVARGHQDCVRRRAIISVSGNPNCQSKEHAAKIVDEVWDSCFADTRPFDEIYR Has a dual role in the assembly of mitochondrial ATPase. Acts as a protease that removes the N-terminal 10 residues of mitochondrial ATPase CF(0) subunit 6 (ATP6) at the intermembrane space side. Also involved in the correct assembly of the membrane-embedded ATPase CF(0) particle, probably mediating association of ATP6 with the subunit 9 ring mint homomint 93232C45DB1DB10E tim9_yeast Mitochondrial import inner membrane translocase subunit TIM9 YEL020BW YEL020W-A TIM9 protein protein yeast Saccharomyces cerevisiae Baker's yeast MDALNSKEQQEFQKVVEQKQMKDFMRLYSNLVERCFTDCVNDFTTSKLTNKEQTCIMKCSEKFLKHSERVGQRFQEQNAALGQGLGR 5160E5DBA52065C7 mia40_yeast Mitochondrial intermembrane space import and assembly protein 40 YKL195W MIA40 TIM40 Mitochondrial import inner membrane translocase TIM40 protein protein yeast Saccharomyces cerevisiae Baker's yeast MLRNLVVRNACRNRPSIQVARGLCRHQTRRLMASSPQFGRNSNQEKTAGFIMGILSMAGALYFIAPNRKPLFASRKVESDKTAEEELSSGGEQSPENEDDNNSKSDENGDDNDSKNDETEAGPQLGGDKIGASKVAEDGELVVLAEEDNKSSEDKDTDESKVSTKDDEQSNEDNATANNQKDENISSENSEENTSDKTLDNNAGSSEKKDPEHSDDEKSQQGQSDDKTTTEDNNGEEESSKKTVSDSENSAKQSESSDEEKEELRKQEEKQMGPTEEEVQHEGAYNPDTGEINWDCPCLGGMAHGPCGEEFKSAFSCFVYSEAEPKGIDCVEKFQHMQDCFRKYPEHYAEQLKETSDDEEPQDKVKVNTIESAPNVSSAKENAAKKAEQSDVKKEPLNEESKP 5904E8D0213D6EFA 5904E8D0213D6EFA atp23-mia40 3 ATP23 4 peptide synthesis peptide synthesis experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain MIA40 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey purified purified P36046:p.Phe318Glu mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F E Tue Jul 12 10:19:58 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 16:54:07 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe318glu' Tue Jun 30 16:52:51 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe318Glu' P36046:p.Phe315Glu mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F E Tue Jul 12 10:19:57 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 19:17:23 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe315glu' Tue Jun 30 16:36:19 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe315Glu' direct interaction direct interaction f4a homomint mint atp23-mia40-1 3 ATP23 4 peptide synthesis peptide synthesis experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain MIA40 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey purified purified P36046:p.Phe315Glu mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F E Tue Jul 12 10:57:31 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 18:04:07 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe315glu' Tue Jun 30 17:31:17 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe315Glu' P36046:p.Phe318Glu mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F E Tue Jul 12 10:56:06 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 19:40:36 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe318glu' Tue Jun 30 16:42:31 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe318Glu' direct interaction direct interaction f4a homomint mint atp23-mia40-2 3 ATP23 4 peptide synthesis peptide synthesis experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain MIA40 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey purified purified P36046:p.Phe318Glu mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F E Tue Jul 12 10:19:04 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 15:08:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe318glu' Tue Jun 30 17:09:32 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe318Glu' P36046:p.Phe315Glu mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F E Tue Jul 12 10:19:05 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 16:31:13 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe315glu' Tue Jun 30 20:20:56 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe315Glu' direct interaction direct interaction f4a homomint mint atp23-mia40-3 3 ATP23 4 peptide synthesis peptide synthesis experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain MIA40 6 western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey purified purified P36046:p.Phe315Glu mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F E Tue Jul 12 10:55:57 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 19:23:04 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe315glu' Tue Jun 30 18:35:26 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe315Glu' P36046:p.Phe318Glu mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F E Tue Jul 12 10:18:39 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 17:02:28 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe318glu' Tue Jun 30 16:58:00 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe318Glu' direct interaction direct interaction f4a homomint mint atp23-atp23 2 ATP23 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified ATP23 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified physical association physical association homomint mint To study the influence of Mia40 on the aggregation of Atp23 in more detail we developed a light scattering approach. Dilution of denatured Atp23 or 10CS into GdmHClfree solution resulted in a rapid increase in light scattering intensity that was proportional to the protein concentration (Supplementary Figure S4A). Presence of Mia40, which itself did not show any light scattering signal (Supplementary Figure S4B), strongly reduced the aggregation of Atp23 (Figure 8A1). We estimated the concentration of Mia40 in isolated mitochondria to be about 5 mM based on Western blotting using purified Mia40 for comparison. Since the IMS is only a small subvolume of mitochondria, the Mia40 concentration in the IMS will be considerably higher The concentrations we used in the folding assay were 1.5 and 4.5 mM Mia40, thus they are presumably lower than the endogenous Mia40 concentrations in the IMS. Heatinactivated Mia40 which was used for control did not reduce aggregation of Atp23 (Supplementary Figure S4C). Likewise, Mia40F315E and Mia40F315,318E did not prevent aggregation of Atp23 (Figure 8A2,3) confirming that binding of Atp23 into the hydrophobic cleft of Mia40 is crucial for folding of Atp23. In contrast, the redox-active cysteine pair of Mia40 was not required for its activity in Atp23 stabilization since addition of purified Mia40SPS prevented aggregation at least to the same degree as wild type Mia40 (Figure 8A4). Nevertheless, the cysteine residues in Atp23 presumably play a critical role in the folding process since the 10CS mutant was only poorly stabilized by Mia40 or Mia40SPS (Figure 8A5 7). Physiological concentrations of glutathione (5mM GSH) did not prevent the stabilization of Atp23 by Mia40 (Figure 8A8). Addition of Mia40 had only a moderate effect on the solubility of the non-IMS protein citrate synthase (Figure 8A9) that is, for example, stabilized by members of the Hsp70 family (Silberg et al, 1998). In summary, these measurements indicate that Mia40 interacts with Atp23 and maintains the protein in a soluble, folding-competent conformation. In these reactions, the cysteine residues of Atp23 play a critical role and the 10CS mutant could not be efficiently stabilized by Mia40. f8a sf4 atp23-mia40-4 1 ATP23 4 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified MIA40 6 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f3a homomint mint Direct interaction of Mia40 with newly imported radiolabelled Atp23 was confirmed by immunoprecipitation under non-reducing conditions or after crosslinking with difluoro dinitrobenzene (DFDNB). Both in the presence and absence of DFDNB, Mia40-specific antibodies precipitated complexes of Mia40 and Atp23 (Figure 3A). Interestingly, unlike in experiments with conventional substrates of Mia40 (Figure 3B; Naoe et al, 2004; Mesecke et al, 2005; Terziyska et al, 2005), multiple complexes of different sizes were recovered suggesting the binding of several Mia40 proteins per Atp23 molecule (Figure 3, arrows). mia40-tim9 1 Tim9 5 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified MIA40 6 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f3b homomint mint Direct interaction of Mia40 with newly imported radiolabelled Atp23 was confirmed by immunoprecipitation under non-reducing conditions or after crosslinking with difluoro dinitrobenzene (DFDNB). Both in the presence and absence of DFDNB, Mia40-specific antibodies precipitated complexes of Mia40 and Atp23 (Figure 3A). Interestingly, unlike in experiments with conventional substrates of Mia40 (Figure 3B; Naoe et al, 2004; Mesecke et al, 2005; Terziyska et al, 2005), multiple complexes of different sizes were recovered suggesting the binding of several Mia40 proteins per Atp23 molecule (Figure 3, arrows).