{"gene":"TIMM50","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2002,"finding":"Tim50 is an essential subunit of the TIM23 complex, anchored to the inner mitochondrial membrane with its major domain exposed to the intermembrane space, where it interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits import of preproteins with classical matrix-targeting signals.","method":"Genetic depletion, co-immunoprecipitation, in vivo import assays, fractionation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — independently replicated in two simultaneous Cell papers by different labs with multiple orthogonal methods","pmids":["12437924","12437925"],"is_preprint":false},{"year":2002,"finding":"Tim50 interacts with the N-terminal intermembrane space domain of Tim23, and a translocation intermediate accumulated at the TOM complex is crosslinked to Tim50, indicating Tim50 facilitates transfer of translocating proteins from the TOM complex to the TIM23 complex.","method":"Site-specific photocrosslinking of translocation intermediates, co-immunoprecipitation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — photocrosslinking establishes direct contact, replicated across two independent labs","pmids":["12437925","12437924"],"is_preprint":false},{"year":2006,"finding":"The intermembrane space domain of Tim50 induces the Tim23 channel to close, maintaining the permeability barrier of the mitochondrial inner membrane. Presequences overcome this effect and activate the channel for translocation, establishing a presequence-regulated gating mechanism.","method":"Reconstituted channel electrophysiology, in vitro assay with purified IMS domain of Tim50","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of Tim23 channel regulation by Tim50 IMS domain","pmids":["16763150"],"is_preprint":false},{"year":2008,"finding":"The IMS domains of Tim50 and Tim23 interact directly; specific mutations in Tim23 that abolish Tim50 binding in vitro also destabilize the interaction in vivo and cause defective preprotein import and cell death at elevated temperatures.","method":"In vitro reconstitution with purified recombinant IMS domains, chemical cross-linking, surface plasmon resonance, in vivo import assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis and SPR, validated in vivo","pmids":["19017642"],"is_preprint":false},{"year":2009,"finding":"Tim23-Tim50 interactions in the IMS facilitate transfer of precursor proteins from the TOM40 complex to the TIM23 complex, and also facilitate a late step of translocation by promoting motor functions of mitochondrial Hsp70 in the matrix.","method":"Genetic epistasis, co-immunoprecipitation, in vivo import assays with IMS domain mutants","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, coordinates both TOM-TIM transfer and motor activation","pmids":["19139266"],"is_preprint":false},{"year":2011,"finding":"Tim50 is the primary presequence receptor at the inner membrane; photo-affinity labeling and mass spectrometric mapping identified a presequence-binding domain in Tim50, and targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.","method":"Photo-affinity labeling with engineered presequence probes, mass spectrometric mapping of crosslink sites","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — photocrosslinking with mass spectrometry identifies binding domain, functional channel regulation shown","pmids":["22065641"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of the IMS domain of yeast Tim50 resolved to 1.83 Å; a protruding β-hairpin is crucial for interaction with Tim23, providing structural basis for Tim50-Tim23 cooperation in preprotein translocation.","method":"X-ray crystallography at 1.83 Å resolution, functional mutagenesis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional mutagenesis validation","pmids":["21704637"],"is_preprint":false},{"year":2004,"finding":"Human TIMM50 is present in a complex with human Tim23, possesses phosphatase activity, and its knockdown by RNAi increases sensitivity to death stimuli by accelerating cytochrome c release from mitochondria.","method":"Co-immunoprecipitation, RNAi knockdown, cytochrome c release assay, phosphatase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods; first characterization of human TIMM50 with enzymatic and complex data","pmids":["15044455"],"is_preprint":false},{"year":2011,"finding":"Human TIMM50 interacts with 3β-HSD2 primarily through Tim50's intermembrane space domain binding the N-terminus of 3β-HSD2; this interaction contributes to 3β-HSD2 enzymatic activity and conformational change, and Tim50 knockdown inhibits steroidogenic catalysis without rescuing activity by restoring protein levels alone.","method":"Co-immunoprecipitation, mass spectrometry of mitochondrial complexes, density gradient ultracentrifugation, Tim50 knockdown, CD spectroscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods but single lab; novel non-canonical Tim50 function in steroidogenesis","pmids":["21930695"],"is_preprint":false},{"year":2015,"finding":"A second crystal structure of the Tim50 IMS domain [Tim50(164-361)] at 2.67 Å reveals significant conformational plasticity in the β-hairpin and helix A2, and crystal packing shows helix A1 from a neighboring monomer docking into the presequence-binding groove, suggesting Tim50 recognizes presequences via hydrophobic interactions within the β-hairpin.","method":"X-ray crystallography at 2.67 Å resolution, structural analysis of crystal packing","journal":"Acta crystallographica Section F","confidence":"Medium","confidence_rationale":"Tier 1 method (crystallography) but structural inference without direct mutagenesis validation of binding mechanism","pmids":["26323300"],"is_preprint":false},{"year":2017,"finding":"Cardiolipin directly modulates the interaction between the soluble receptor domain of Tim50 and the Tim23 channel; Tim50 binds membranes and specific sites on Tim23 in a cardiolipin-dependent manner, and SAXS-based structural analysis of the Tim50 receptor domain combined with molecular dynamics identified structural elements mediating this interaction.","method":"In vivo and in vitro interaction assays, nanoscale model membranes, small-angle X-ray scattering (SAXS), molecular dynamics simulations, biophysical measurements","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal methods including SAXS structure and in vitro reconstitution in model membranes","pmids":["28879236"],"is_preprint":false},{"year":2019,"finding":"Random mutagenesis of Tim50 identified two distinct surface patches whose mutation impairs interaction with Tim23 and causes defective TIM23-dependent preprotein import, establishing that two regions of Tim50 are required for Tim23 binding.","method":"Random mutagenesis, temperature-sensitive mutant analysis, co-immunoprecipitation, in vivo import assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis with functional validation; single lab","pmids":["30765764"],"is_preprint":false},{"year":2020,"finding":"Tim50 transmits presequence recognition signals across the inner membrane: the Tim50 matrix domain facilitates recruitment of the PAM coupling factor Pam17, the IMS domain of Tim50 promotes PAM recruitment to TIM23, and the Tim50 transmembrane segment stimulates the matrix-directed import-driving force by PAM, coordinating preprotein recognition with motor activation.","method":"Genetic analysis, in vivo import assays, domain dissection with functional complementation","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — domain-level dissection with functional readouts; single lab","pmids":["32130909"],"is_preprint":false},{"year":2023,"finding":"The two IMS domains of Tim50 (core and PBD) have distinct essential roles: the core domain contains the main presequence-binding site and is the main recruitment point to TIM23, while the PBD plays a critical role in cooperation between TOM and TIM23 complexes; the two domains can complement each other in trans.","method":"Trans-complementation experiments, in vivo import assays, domain deletion analysis","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 — trans-complementation establishes distinct domain functions; single lab","pmids":["37748811"],"is_preprint":false},{"year":2024,"finding":"Pathogenic variants in TIMM50 specifically reduce laterally released substrates imported via the TIM23SORT pathway; proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated disease.","method":"Quantitative proteomics of patient fibroblasts and CRISPR TIMM50 HEK293 model, pathway analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — proteomic mapping in disease model with CRISPR validation; single lab","pmids":["38828998"],"is_preprint":false},{"year":2024,"finding":"eIF5A controls mitochondrial protein import by alleviating ribosome stalling at polyproline sequences in Tim50 mRNA; eIF5A depletion reduces Tim50 levels and causes mitoprotein precursor accumulation and mitochondrial import stress; removal of polyprolines from Tim50 rescues the import stress response.","method":"eIF5A depletion in yeast, ribosome profiling, mitochondrial import stress assay, polyproline deletion mutagenesis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — ribosome profiling plus mutagenesis rescue; published in peer-reviewed journal","pmids":["39509053"],"is_preprint":false},{"year":2024,"finding":"TIMM50 deficiency in neurons reduces levels of OXPHOS and mitochondrial ribosome complex components, decreases respiration and ATP, causes defective mitochondrial trafficking in neuronal processes, and increases electrical activity correlated with reduced KCNJ10 and KCNA2 potassium channels.","method":"TIMM50 knockdown in mouse neurons, proteomics, respirometry, ATP measurement, neuronal imaging, electrophysiology","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods in neuronal model; single lab","pmids":["39680434"],"is_preprint":false},{"year":2005,"finding":"A nuclear isoform of Tim50, Tim50a, localizes strictly to the nucleus (enriched in speckles with snRNPs) due to an N-terminal nuclear localization signal, interacts with coilin, snRNPs, and SMN, and competition binding shows coilin competes with Sm proteins and SMN for Tim50a binding sites, suggesting a role in snRNP biogenesis.","method":"Subcellular fractionation, co-immunoprecipitation, competition binding assay, fluorescence microscopy","journal":"BMC cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple binding methods and localization; single lab, isoform-specific","pmids":["16008839"],"is_preprint":false},{"year":2018,"finding":"Tim50 directly interacts with cytochrome P450 SCC (CYP11A1) via SCC amino acids 141-146 after SCC is imported into the matrix and partially processed, forming a large complex at the TIM23 translocase; Tim50 knockdown or mutation of the SCC-Tim50 interaction site reduces SCC enzymatic activity.","method":"Fractionation, mass spectrometry, co-immunoprecipitation, Tim50 knockdown, mutagenesis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods; single lab, novel non-canonical function","pmids":["30348838"],"is_preprint":false},{"year":2025,"finding":"TIMM50 downregulation is sufficient to trigger cellular senescence through impaired mitochondrial function; TIMM50 expression is regulated by sirtuin1-dependent downregulation of CEBPα (a transcriptional activator of TIMM50); overexpression of TIMM50 slows senescence onset.","method":"Multiple senescence models, pathway analysis, overexpression/knockdown, mitochondrial function assays","journal":"Advanced biology","confidence":"Low","confidence_rationale":"Tier 3 — pathway inference from knockdown/overexpression; single lab, no direct mechanistic reconstitution","pmids":["40128440"],"is_preprint":false}],"current_model":"TIMM50 (Tim50) is an essential subunit of the mitochondrial TIM23 complex, anchored to the inner membrane with its large IMS domain acting as the primary presequence receptor that captures incoming preproteins from the TOM complex, directs them to the Tim23 channel (via a direct IMS domain–IMS domain interaction mediated by a β-hairpin), closes the Tim23 pore in the absence of substrate to maintain the inner membrane permeability barrier, and transmits presequence-recognition signals across the membrane through its matrix domain to coordinate recruitment and activation of the PAM motor, while cardiolipin modulates Tim50-Tim23 channel interactions and human TIMM50 additionally harbors phosphatase activity and regulates steroidogenic enzyme activity."},"narrative":{"teleology":[{"year":2002,"claim":"The discovery that Tim50 is an essential TIM23 subunit that contacts preproteins in the IMS and directs them to Tim23 established Tim50 as a previously unknown central component of the preprotein import pathway.","evidence":"Genetic depletion, co-immunoprecipitation, photocrosslinking of translocation intermediates, and in vivo import assays in yeast, reported independently by two labs","pmids":["12437924","12437925"],"confidence":"High","gaps":["Mechanism of presequence recognition by Tim50 not yet identified","No structural information on Tim50","How Tim50 regulates the Tim23 channel was unknown"]},{"year":2004,"claim":"Characterization of human TIMM50 showed conservation of the Tim23 interaction and revealed an intrinsic phosphatase activity, while establishing that TIMM50 loss sensitizes cells to apoptosis by facilitating cytochrome c release.","evidence":"Co-immunoprecipitation, RNAi knockdown, phosphatase activity assay, cytochrome c release assay in human cells","pmids":["15044455"],"confidence":"High","gaps":["Physiological substrate of phosphatase activity unidentified","Whether apoptosis sensitization reflects import defects or a direct barrier-maintenance function was unclear"]},{"year":2005,"claim":"Identification of a nuclear isoform (Tim50a) that localizes to nuclear speckles and interacts with coilin, snRNPs, and SMN raised the possibility of a non-mitochondrial role in snRNP biogenesis.","evidence":"Subcellular fractionation, co-immunoprecipitation, competition binding, fluorescence microscopy in human cells","pmids":["16008839"],"confidence":"Medium","gaps":["Functional consequence of Tim50a on snRNP assembly not demonstrated","Not independently replicated by other labs","Relationship to mitochondrial isoform unclear"]},{"year":2006,"claim":"Reconstituted electrophysiology revealed that the Tim50 IMS domain closes the Tim23 channel in the absence of substrate and that presequences antagonize this closure, establishing Tim50 as a gatekeeper that maintains the inner membrane permeability barrier while enabling presequence-regulated channel opening.","evidence":"Purified Tim50 IMS domain applied to reconstituted Tim23 channels; electrophysiology with presequence peptides","pmids":["16763150"],"confidence":"High","gaps":["Structural basis for Tim50-mediated channel closure unknown","In vivo relevance of reconstituted gating not yet confirmed"]},{"year":2008,"claim":"Direct IMS domain–IMS domain binding between Tim50 and Tim23 was quantified and specific mutations that disrupt this interaction were shown to be lethal, establishing this interaction as essential for import.","evidence":"SPR with purified recombinant IMS domains, chemical cross-linking, mutagenesis with in vivo growth and import assays in yeast","pmids":["19017642"],"confidence":"High","gaps":["Structural determinants at atomic resolution not yet resolved","Whether multiple binding surfaces exist was unknown"]},{"year":2009,"claim":"Tim50–Tim23 IMS interactions were shown to facilitate not only TOM-to-TIM23 transfer but also a late import step by promoting matrix Hsp70 motor function, revealing that Tim50 coordinates both early and late phases of translocation.","evidence":"Genetic epistasis, co-immunoprecipitation, in vivo import assays with IMS domain mutants in yeast","pmids":["19139266"],"confidence":"High","gaps":["Mechanism by which IMS signals reach the matrix motor was unknown","Role of Tim50 transmembrane and matrix domains not addressed"]},{"year":2011,"claim":"Two landmark studies established that Tim50 is the primary presequence receptor at the inner membrane—photo-affinity labeling mapped its presequence-binding domain—and the 1.83 Å crystal structure of the IMS domain identified a protruding β-hairpin essential for Tim23 binding.","evidence":"Photo-affinity labeling with mass spectrometric mapping; X-ray crystallography at 1.83 Å with functional mutagenesis in yeast","pmids":["22065641","21704637"],"confidence":"High","gaps":["Atomic-level view of presequence bound to Tim50 not obtained","Conformational dynamics of the β-hairpin during translocation unknown"]},{"year":2015,"claim":"A second crystal form of Tim50 IMS domain revealed conformational plasticity in the β-hairpin and helix A2 regions, with crystal packing suggesting that presequence recognition involves hydrophobic interactions within the β-hairpin groove.","evidence":"X-ray crystallography at 2.67 Å; structural analysis of crystal contacts","pmids":["26323300"],"confidence":"Medium","gaps":["Presequence-binding mode inferred from crystal packing rather than a co-crystal structure","No mutagenesis validation of the proposed binding groove"]},{"year":2017,"claim":"Cardiolipin was identified as a direct modulator of Tim50–Tim23 interaction, with Tim50 binding membranes and specific Tim23 sites in a cardiolipin-dependent manner, adding a lipid regulatory layer to translocase function.","evidence":"In vivo/in vitro interaction assays, nanoscale model membranes, SAXS, molecular dynamics in yeast system","pmids":["28879236"],"confidence":"High","gaps":["Physiological consequence of cardiolipin depletion on Tim50 gating in vivo not fully resolved","Structural details of the cardiolipin binding site on Tim50 at atomic resolution unknown"]},{"year":2018,"claim":"Human TIMM50 was shown to interact directly with cytochrome P450scc (CYP11A1) at the TIM23 translocase, with this interaction required for full SCC enzymatic activity, extending Tim50 function to regulation of steroidogenesis beyond canonical import.","evidence":"Co-immunoprecipitation, mass spectrometry, mutagenesis, Tim50 knockdown in steroidogenic cells","pmids":["30348838"],"confidence":"Medium","gaps":["Whether this reflects a chaperoning role during import or a stable post-import complex is unclear","Not replicated independently"]},{"year":2019,"claim":"Random mutagenesis identified two distinct surface patches on Tim50 required for Tim23 binding, resolving earlier ambiguity about whether a single or multiple contact sites mediate the interaction.","evidence":"Random mutagenesis, temperature-sensitive mutant analysis, co-immunoprecipitation, in vivo import in yeast","pmids":["30765764"],"confidence":"Medium","gaps":["No co-crystal of Tim50–Tim23 complex to assign patches at atomic resolution","How the two patches coordinate during translocation is unclear"]},{"year":2020,"claim":"Domain dissection established that Tim50 transmits presequence recognition across the inner membrane: the matrix domain recruits PAM coupling factor Pam17, the IMS domain promotes PAM recruitment to TIM23, and the transmembrane segment stimulates import-driving force, resolving how IMS presequence sensing is mechanistically coupled to matrix motor activation.","evidence":"Domain dissection with functional complementation, genetic analysis, in vivo import in yeast","pmids":["32130909"],"confidence":"Medium","gaps":["Molecular mechanism of signal transduction across the transmembrane segment unresolved","No direct biochemical reconstitution of the Tim50–Pam17 interaction"]},{"year":2023,"claim":"The two IMS sub-domains of Tim50 (core and PBD) were shown to have distinct essential functions—core domain for presequence binding and TIM23 recruitment, PBD for TOM–TIM23 cooperation—and can complement each other in trans, revealing a modular architecture.","evidence":"Trans-complementation, domain deletion, in vivo import assays in yeast","pmids":["37748811"],"confidence":"Medium","gaps":["Structural basis for independent function of PBD at TOM–TIM23 interface not determined","Whether trans-complementation reflects physiological sub-complex dynamics is unknown"]},{"year":2024,"claim":"Three concurrent studies revealed that TIMM50 pathogenic variants specifically impair the TIM23SORT lateral release pathway (reducing OXPHOS and mitoribosomal components), that TIMM50 deficiency in neurons decreases respiration/ATP and disrupts mitochondrial trafficking and electrical activity, and that eIF5A controls Tim50 levels by alleviating ribosome stalling at polyproline stretches in Tim50 mRNA.","evidence":"Quantitative proteomics in patient fibroblasts and CRISPR models; neuronal knockdown with respirometry, imaging, and electrophysiology; ribosome profiling with polyproline deletion rescue in yeast","pmids":["38828998","39680434","39509053"],"confidence":"High","gaps":["Relative contribution of TIM23SORT vs. TIM23MOTOR pathway impairment to disease phenotype unknown","Whether eIF5A-dependent Tim50 regulation operates in mammalian neurons not tested","No therapeutic strategy to restore TIM23SORT function in TIMM50 disease"]},{"year":null,"claim":"A high-resolution structure of the full-length Tim50 in complex with Tim23, a co-crystal with bound presequence, and a detailed mechanism for how presequence recognition is transduced across the membrane to activate the PAM motor remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of full-length Tim50 or Tim50–Tim23 complex","No co-crystal or cryo-EM structure with bound presequence","Transmembrane signal transduction mechanism from IMS to matrix unresolved at molecular level","Physiological role and substrate of phosphatase activity remain unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,5,12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,5,12]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,2,3,4,5,6,7,10,12]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,1,4,5,12,13,14]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[14,16]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,12,15]}],"complexes":["TIM23 complex"],"partners":["TIM23","PAM17","CYP11A1","HSD3B2","COILIN","SMN"],"other_free_text":[]},"mechanistic_narrative":"TIMM50 (Tim50) is an essential subunit of the mitochondrial TIM23 translocase that serves as the primary presequence receptor at the inner membrane, coupling preprotein recognition in the intermembrane space (IMS) with channel gating and import motor activation in the matrix. Its large IMS domain captures preproteins emerging from the TOM complex and directs them to the Tim23 channel via a structurally characterized β-hairpin-mediated interaction, while simultaneously closing the Tim23 pore in the absence of substrate to maintain the inner membrane permeability barrier; presequences antagonize this closure to activate translocation [PMID:12437924, PMID:16763150, PMID:21704637]. Tim50 transmits presequence recognition across the membrane through its transmembrane and matrix domains, recruiting the PAM coupling factor Pam17 and stimulating Hsp70-driven import, and cardiolipin modulates the Tim50–Tim23 channel interaction [PMID:32130909, PMID:28879236]. Pathogenic TIMM50 variants preferentially impair the TIM23^SORT lateral release pathway, reducing OXPHOS and mitochondrial ribosomal components, and TIMM50 deficiency in neurons decreases respiration, ATP production, and mitochondrial trafficking while increasing aberrant electrical activity [PMID:38828998, PMID:39680434]."},"prefetch_data":{"uniprot":{"accession":"Q3ZCQ8","full_name":"Mitochondrial import inner membrane translocase subunit TIM50","aliases":[],"length_aa":353,"mass_kda":39.6,"function":"Essential component of the TIM23 complex, a complex that mediates the translocation of transit peptide-containing proteins across the mitochondrial inner membrane (PubMed:30190335, PubMed:38828998). Has some phosphatase activity in vitro; however such activity may not be relevant in vivo May participate in the release of snRNPs and SMN from the Cajal body","subcellular_location":"Nucleus speckle","url":"https://www.uniprot.org/uniprotkb/Q3ZCQ8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TIMM50","classification":"Not Classified","n_dependent_lines":148,"n_total_lines":383,"dependency_fraction":0.38642297650130547},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"TRIM28","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TIMM50","total_profiled":1310},"omim":[{"mim_id":"617698","title":"3-@METHYLGLUTACONIC ACIDURIA, TYPE IX; MGCA9","url":"https://www.omim.org/entry/617698"},{"mim_id":"615339","title":"DNAJ/HSP40 HOMOLOG, SUBFAMILY C, MEMBER 15; DNAJC15","url":"https://www.omim.org/entry/615339"},{"mim_id":"607381","title":"TRANSLOCASE OF INNER MITOCHONDRIAL MEMBRANE 50; TIMM50","url":"https://www.omim.org/entry/607381"},{"mim_id":"250950","title":"3-@METHYLGLUTACONIC ACIDURIA, TYPE I; MGCA1","url":"https://www.omim.org/entry/250950"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Mitochondria","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TIMM50"},"hgnc":{"alias_symbol":["TIM50L","TIM50"],"prev_symbol":[]},"alphafold":{"accession":"Q3ZCQ8","domains":[{"cath_id":"-","chopping":"82-128","consensus_level":"medium","plddt":83.4126,"start":82,"end":128},{"cath_id":"3.40.50.1000","chopping":"149-304","consensus_level":"high","plddt":93.9197,"start":149,"end":304}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q3ZCQ8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q3ZCQ8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q3ZCQ8-F1-predicted_aligned_error_v6.png","plddt_mean":79.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TIMM50","jax_strain_url":"https://www.jax.org/strain/search?query=TIMM50"},"sequence":{"accession":"Q3ZCQ8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q3ZCQ8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q3ZCQ8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q3ZCQ8"}},"corpus_meta":[{"pmid":"12437924","id":"PMC_12437924","title":"The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel.","date":"2002","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/12437924","citation_count":216,"is_preprint":false},{"pmid":"12437925","id":"PMC_12437925","title":"Tim50 is a subunit of the TIM23 complex that links protein translocation across the outer and inner mitochondrial membranes.","date":"2002","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/12437925","citation_count":210,"is_preprint":false},{"pmid":"16763150","id":"PMC_16763150","title":"Tim50 maintains the permeability barrier of the mitochondrial inner membrane.","date":"2006","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/16763150","citation_count":152,"is_preprint":false},{"pmid":"19139266","id":"PMC_19139266","title":"Tim23-Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import.","date":"2009","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/19139266","citation_count":117,"is_preprint":false},{"pmid":"15044455","id":"PMC_15044455","title":"Tim50, a component of the mitochondrial translocator, regulates mitochondrial integrity and cell death.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15044455","citation_count":82,"is_preprint":false},{"pmid":"22065641","id":"PMC_22065641","title":"Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex.","date":"2011","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/22065641","citation_count":79,"is_preprint":false},{"pmid":"19017642","id":"PMC_19017642","title":"Interaction of Tim23 with Tim50 Is essential for protein translocation by the mitochondrial TIM23 complex.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19017642","citation_count":55,"is_preprint":false},{"pmid":"28879236","id":"PMC_28879236","title":"Cardiolipin mediates membrane and channel interactions of the mitochondrial TIM23 protein import complex receptor Tim50.","date":"2017","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/28879236","citation_count":53,"is_preprint":false},{"pmid":"21704637","id":"PMC_21704637","title":"Structural basis for the function of Tim50 in the mitochondrial presequence translocase.","date":"2011","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21704637","citation_count":41,"is_preprint":false},{"pmid":"21930695","id":"PMC_21930695","title":"Inner mitochondrial translocase Tim50 interacts with 3β-hydroxysteroid dehydrogenase type 2 to regulate adrenal and gonadal steroidogenesis.","date":"2011","source":"The Journal of biological 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Diverse Functions of Tim50, a Component of the Mitochondrial Inner Membrane Protein Translocase. Int. J. Mol. Sci. 2021, 22, 7779.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35887402","citation_count":1,"is_preprint":false},{"pmid":"40128440","id":"PMC_40128440","title":"Ameliorating TIMM50 Loss Slows Senescence by Improving Mitochondrial Structure and Function.","date":"2025","source":"Advanced biology","url":"https://pubmed.ncbi.nlm.nih.gov/40128440","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17675,"output_tokens":4145,"usd":0.0576},"stage2":{"model":"claude-opus-4-6","input_tokens":7684,"output_tokens":3592,"usd":0.19233},"total_usd":0.24993,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"Tim50 is an essential subunit of the TIM23 complex, anchored to the inner mitochondrial membrane with its major domain exposed to the intermembrane space, where it interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits import of preproteins with classical matrix-targeting signals.\",\n      \"method\": \"Genetic depletion, co-immunoprecipitation, in vivo import assays, fractionation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — independently replicated in two simultaneous Cell papers by different labs with multiple orthogonal methods\",\n      \"pmids\": [\"12437924\", \"12437925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Tim50 interacts with the N-terminal intermembrane space domain of Tim23, and a translocation intermediate accumulated at the TOM complex is crosslinked to Tim50, indicating Tim50 facilitates transfer of translocating proteins from the TOM complex to the TIM23 complex.\",\n      \"method\": \"Site-specific photocrosslinking of translocation intermediates, co-immunoprecipitation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — photocrosslinking establishes direct contact, replicated across two independent labs\",\n      \"pmids\": [\"12437925\", \"12437924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The intermembrane space domain of Tim50 induces the Tim23 channel to close, maintaining the permeability barrier of the mitochondrial inner membrane. Presequences overcome this effect and activate the channel for translocation, establishing a presequence-regulated gating mechanism.\",\n      \"method\": \"Reconstituted channel electrophysiology, in vitro assay with purified IMS domain of Tim50\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of Tim23 channel regulation by Tim50 IMS domain\",\n      \"pmids\": [\"16763150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The IMS domains of Tim50 and Tim23 interact directly; specific mutations in Tim23 that abolish Tim50 binding in vitro also destabilize the interaction in vivo and cause defective preprotein import and cell death at elevated temperatures.\",\n      \"method\": \"In vitro reconstitution with purified recombinant IMS domains, chemical cross-linking, surface plasmon resonance, in vivo import assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis and SPR, validated in vivo\",\n      \"pmids\": [\"19017642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Tim23-Tim50 interactions in the IMS facilitate transfer of precursor proteins from the TOM40 complex to the TIM23 complex, and also facilitate a late step of translocation by promoting motor functions of mitochondrial Hsp70 in the matrix.\",\n      \"method\": \"Genetic epistasis, co-immunoprecipitation, in vivo import assays with IMS domain mutants\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, coordinates both TOM-TIM transfer and motor activation\",\n      \"pmids\": [\"19139266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Tim50 is the primary presequence receptor at the inner membrane; photo-affinity labeling and mass spectrometric mapping identified a presequence-binding domain in Tim50, and targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.\",\n      \"method\": \"Photo-affinity labeling with engineered presequence probes, mass spectrometric mapping of crosslink sites\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — photocrosslinking with mass spectrometry identifies binding domain, functional channel regulation shown\",\n      \"pmids\": [\"22065641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of the IMS domain of yeast Tim50 resolved to 1.83 Å; a protruding β-hairpin is crucial for interaction with Tim23, providing structural basis for Tim50-Tim23 cooperation in preprotein translocation.\",\n      \"method\": \"X-ray crystallography at 1.83 Å resolution, functional mutagenesis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional mutagenesis validation\",\n      \"pmids\": [\"21704637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human TIMM50 is present in a complex with human Tim23, possesses phosphatase activity, and its knockdown by RNAi increases sensitivity to death stimuli by accelerating cytochrome c release from mitochondria.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, cytochrome c release assay, phosphatase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods; first characterization of human TIMM50 with enzymatic and complex data\",\n      \"pmids\": [\"15044455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Human TIMM50 interacts with 3β-HSD2 primarily through Tim50's intermembrane space domain binding the N-terminus of 3β-HSD2; this interaction contributes to 3β-HSD2 enzymatic activity and conformational change, and Tim50 knockdown inhibits steroidogenic catalysis without rescuing activity by restoring protein levels alone.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry of mitochondrial complexes, density gradient ultracentrifugation, Tim50 knockdown, CD spectroscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods but single lab; novel non-canonical Tim50 function in steroidogenesis\",\n      \"pmids\": [\"21930695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A second crystal structure of the Tim50 IMS domain [Tim50(164-361)] at 2.67 Å reveals significant conformational plasticity in the β-hairpin and helix A2, and crystal packing shows helix A1 from a neighboring monomer docking into the presequence-binding groove, suggesting Tim50 recognizes presequences via hydrophobic interactions within the β-hairpin.\",\n      \"method\": \"X-ray crystallography at 2.67 Å resolution, structural analysis of crystal packing\",\n      \"journal\": \"Acta crystallographica Section F\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 method (crystallography) but structural inference without direct mutagenesis validation of binding mechanism\",\n      \"pmids\": [\"26323300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cardiolipin directly modulates the interaction between the soluble receptor domain of Tim50 and the Tim23 channel; Tim50 binds membranes and specific sites on Tim23 in a cardiolipin-dependent manner, and SAXS-based structural analysis of the Tim50 receptor domain combined with molecular dynamics identified structural elements mediating this interaction.\",\n      \"method\": \"In vivo and in vitro interaction assays, nanoscale model membranes, small-angle X-ray scattering (SAXS), molecular dynamics simulations, biophysical measurements\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal methods including SAXS structure and in vitro reconstitution in model membranes\",\n      \"pmids\": [\"28879236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Random mutagenesis of Tim50 identified two distinct surface patches whose mutation impairs interaction with Tim23 and causes defective TIM23-dependent preprotein import, establishing that two regions of Tim50 are required for Tim23 binding.\",\n      \"method\": \"Random mutagenesis, temperature-sensitive mutant analysis, co-immunoprecipitation, in vivo import assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with functional validation; single lab\",\n      \"pmids\": [\"30765764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Tim50 transmits presequence recognition signals across the inner membrane: the Tim50 matrix domain facilitates recruitment of the PAM coupling factor Pam17, the IMS domain of Tim50 promotes PAM recruitment to TIM23, and the Tim50 transmembrane segment stimulates the matrix-directed import-driving force by PAM, coordinating preprotein recognition with motor activation.\",\n      \"method\": \"Genetic analysis, in vivo import assays, domain dissection with functional complementation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain-level dissection with functional readouts; single lab\",\n      \"pmids\": [\"32130909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The two IMS domains of Tim50 (core and PBD) have distinct essential roles: the core domain contains the main presequence-binding site and is the main recruitment point to TIM23, while the PBD plays a critical role in cooperation between TOM and TIM23 complexes; the two domains can complement each other in trans.\",\n      \"method\": \"Trans-complementation experiments, in vivo import assays, domain deletion analysis\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — trans-complementation establishes distinct domain functions; single lab\",\n      \"pmids\": [\"37748811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Pathogenic variants in TIMM50 specifically reduce laterally released substrates imported via the TIM23SORT pathway; proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated disease.\",\n      \"method\": \"Quantitative proteomics of patient fibroblasts and CRISPR TIMM50 HEK293 model, pathway analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proteomic mapping in disease model with CRISPR validation; single lab\",\n      \"pmids\": [\"38828998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"eIF5A controls mitochondrial protein import by alleviating ribosome stalling at polyproline sequences in Tim50 mRNA; eIF5A depletion reduces Tim50 levels and causes mitoprotein precursor accumulation and mitochondrial import stress; removal of polyprolines from Tim50 rescues the import stress response.\",\n      \"method\": \"eIF5A depletion in yeast, ribosome profiling, mitochondrial import stress assay, polyproline deletion mutagenesis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — ribosome profiling plus mutagenesis rescue; published in peer-reviewed journal\",\n      \"pmids\": [\"39509053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIMM50 deficiency in neurons reduces levels of OXPHOS and mitochondrial ribosome complex components, decreases respiration and ATP, causes defective mitochondrial trafficking in neuronal processes, and increases electrical activity correlated with reduced KCNJ10 and KCNA2 potassium channels.\",\n      \"method\": \"TIMM50 knockdown in mouse neurons, proteomics, respirometry, ATP measurement, neuronal imaging, electrophysiology\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in neuronal model; single lab\",\n      \"pmids\": [\"39680434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"A nuclear isoform of Tim50, Tim50a, localizes strictly to the nucleus (enriched in speckles with snRNPs) due to an N-terminal nuclear localization signal, interacts with coilin, snRNPs, and SMN, and competition binding shows coilin competes with Sm proteins and SMN for Tim50a binding sites, suggesting a role in snRNP biogenesis.\",\n      \"method\": \"Subcellular fractionation, co-immunoprecipitation, competition binding assay, fluorescence microscopy\",\n      \"journal\": \"BMC cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple binding methods and localization; single lab, isoform-specific\",\n      \"pmids\": [\"16008839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Tim50 directly interacts with cytochrome P450 SCC (CYP11A1) via SCC amino acids 141-146 after SCC is imported into the matrix and partially processed, forming a large complex at the TIM23 translocase; Tim50 knockdown or mutation of the SCC-Tim50 interaction site reduces SCC enzymatic activity.\",\n      \"method\": \"Fractionation, mass spectrometry, co-immunoprecipitation, Tim50 knockdown, mutagenesis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods; single lab, novel non-canonical function\",\n      \"pmids\": [\"30348838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TIMM50 downregulation is sufficient to trigger cellular senescence through impaired mitochondrial function; TIMM50 expression is regulated by sirtuin1-dependent downregulation of CEBPα (a transcriptional activator of TIMM50); overexpression of TIMM50 slows senescence onset.\",\n      \"method\": \"Multiple senescence models, pathway analysis, overexpression/knockdown, mitochondrial function assays\",\n      \"journal\": \"Advanced biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — pathway inference from knockdown/overexpression; single lab, no direct mechanistic reconstitution\",\n      \"pmids\": [\"40128440\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TIMM50 (Tim50) is an essential subunit of the mitochondrial TIM23 complex, anchored to the inner membrane with its large IMS domain acting as the primary presequence receptor that captures incoming preproteins from the TOM complex, directs them to the Tim23 channel (via a direct IMS domain–IMS domain interaction mediated by a β-hairpin), closes the Tim23 pore in the absence of substrate to maintain the inner membrane permeability barrier, and transmits presequence-recognition signals across the membrane through its matrix domain to coordinate recruitment and activation of the PAM motor, while cardiolipin modulates Tim50-Tim23 channel interactions and human TIMM50 additionally harbors phosphatase activity and regulates steroidogenic enzyme activity.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TIMM50 (Tim50) is an essential subunit of the mitochondrial TIM23 translocase that serves as the primary presequence receptor at the inner membrane, coupling preprotein recognition in the intermembrane space (IMS) with channel gating and import motor activation in the matrix. Its large IMS domain captures preproteins emerging from the TOM complex and directs them to the Tim23 channel via a structurally characterized β-hairpin-mediated interaction, while simultaneously closing the Tim23 pore in the absence of substrate to maintain the inner membrane permeability barrier; presequences antagonize this closure to activate translocation [PMID:12437924, PMID:16763150, PMID:21704637]. Tim50 transmits presequence recognition across the membrane through its transmembrane and matrix domains, recruiting the PAM coupling factor Pam17 and stimulating Hsp70-driven import, and cardiolipin modulates the Tim50–Tim23 channel interaction [PMID:32130909, PMID:28879236]. Pathogenic TIMM50 variants preferentially impair the TIM23^SORT lateral release pathway, reducing OXPHOS and mitochondrial ribosomal components, and TIMM50 deficiency in neurons decreases respiration, ATP production, and mitochondrial trafficking while increasing aberrant electrical activity [PMID:38828998, PMID:39680434].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"The discovery that Tim50 is an essential TIM23 subunit that contacts preproteins in the IMS and directs them to Tim23 established Tim50 as a previously unknown central component of the preprotein import pathway.\",\n      \"evidence\": \"Genetic depletion, co-immunoprecipitation, photocrosslinking of translocation intermediates, and in vivo import assays in yeast, reported independently by two labs\",\n      \"pmids\": [\"12437924\", \"12437925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism of presequence recognition by Tim50 not yet identified\",\n        \"No structural information on Tim50\",\n        \"How Tim50 regulates the Tim23 channel was unknown\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Characterization of human TIMM50 showed conservation of the Tim23 interaction and revealed an intrinsic phosphatase activity, while establishing that TIMM50 loss sensitizes cells to apoptosis by facilitating cytochrome c release.\",\n      \"evidence\": \"Co-immunoprecipitation, RNAi knockdown, phosphatase activity assay, cytochrome c release assay in human cells\",\n      \"pmids\": [\"15044455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological substrate of phosphatase activity unidentified\",\n        \"Whether apoptosis sensitization reflects import defects or a direct barrier-maintenance function was unclear\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of a nuclear isoform (Tim50a) that localizes to nuclear speckles and interacts with coilin, snRNPs, and SMN raised the possibility of a non-mitochondrial role in snRNP biogenesis.\",\n      \"evidence\": \"Subcellular fractionation, co-immunoprecipitation, competition binding, fluorescence microscopy in human cells\",\n      \"pmids\": [\"16008839\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional consequence of Tim50a on snRNP assembly not demonstrated\",\n        \"Not independently replicated by other labs\",\n        \"Relationship to mitochondrial isoform unclear\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Reconstituted electrophysiology revealed that the Tim50 IMS domain closes the Tim23 channel in the absence of substrate and that presequences antagonize this closure, establishing Tim50 as a gatekeeper that maintains the inner membrane permeability barrier while enabling presequence-regulated channel opening.\",\n      \"evidence\": \"Purified Tim50 IMS domain applied to reconstituted Tim23 channels; electrophysiology with presequence peptides\",\n      \"pmids\": [\"16763150\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for Tim50-mediated channel closure unknown\",\n        \"In vivo relevance of reconstituted gating not yet confirmed\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Direct IMS domain–IMS domain binding between Tim50 and Tim23 was quantified and specific mutations that disrupt this interaction were shown to be lethal, establishing this interaction as essential for import.\",\n      \"evidence\": \"SPR with purified recombinant IMS domains, chemical cross-linking, mutagenesis with in vivo growth and import assays in yeast\",\n      \"pmids\": [\"19017642\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural determinants at atomic resolution not yet resolved\",\n        \"Whether multiple binding surfaces exist was unknown\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Tim50–Tim23 IMS interactions were shown to facilitate not only TOM-to-TIM23 transfer but also a late import step by promoting matrix Hsp70 motor function, revealing that Tim50 coordinates both early and late phases of translocation.\",\n      \"evidence\": \"Genetic epistasis, co-immunoprecipitation, in vivo import assays with IMS domain mutants in yeast\",\n      \"pmids\": [\"19139266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which IMS signals reach the matrix motor was unknown\",\n        \"Role of Tim50 transmembrane and matrix domains not addressed\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Two landmark studies established that Tim50 is the primary presequence receptor at the inner membrane—photo-affinity labeling mapped its presequence-binding domain—and the 1.83 Å crystal structure of the IMS domain identified a protruding β-hairpin essential for Tim23 binding.\",\n      \"evidence\": \"Photo-affinity labeling with mass spectrometric mapping; X-ray crystallography at 1.83 Å with functional mutagenesis in yeast\",\n      \"pmids\": [\"22065641\", \"21704637\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Atomic-level view of presequence bound to Tim50 not obtained\",\n        \"Conformational dynamics of the β-hairpin during translocation unknown\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"A second crystal form of Tim50 IMS domain revealed conformational plasticity in the β-hairpin and helix A2 regions, with crystal packing suggesting that presequence recognition involves hydrophobic interactions within the β-hairpin groove.\",\n      \"evidence\": \"X-ray crystallography at 2.67 Å; structural analysis of crystal contacts\",\n      \"pmids\": [\"26323300\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Presequence-binding mode inferred from crystal packing rather than a co-crystal structure\",\n        \"No mutagenesis validation of the proposed binding groove\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Cardiolipin was identified as a direct modulator of Tim50–Tim23 interaction, with Tim50 binding membranes and specific Tim23 sites in a cardiolipin-dependent manner, adding a lipid regulatory layer to translocase function.\",\n      \"evidence\": \"In vivo/in vitro interaction assays, nanoscale model membranes, SAXS, molecular dynamics in yeast system\",\n      \"pmids\": [\"28879236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological consequence of cardiolipin depletion on Tim50 gating in vivo not fully resolved\",\n        \"Structural details of the cardiolipin binding site on Tim50 at atomic resolution unknown\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Human TIMM50 was shown to interact directly with cytochrome P450scc (CYP11A1) at the TIM23 translocase, with this interaction required for full SCC enzymatic activity, extending Tim50 function to regulation of steroidogenesis beyond canonical import.\",\n      \"evidence\": \"Co-immunoprecipitation, mass spectrometry, mutagenesis, Tim50 knockdown in steroidogenic cells\",\n      \"pmids\": [\"30348838\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether this reflects a chaperoning role during import or a stable post-import complex is unclear\",\n        \"Not replicated independently\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Random mutagenesis identified two distinct surface patches on Tim50 required for Tim23 binding, resolving earlier ambiguity about whether a single or multiple contact sites mediate the interaction.\",\n      \"evidence\": \"Random mutagenesis, temperature-sensitive mutant analysis, co-immunoprecipitation, in vivo import in yeast\",\n      \"pmids\": [\"30765764\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No co-crystal of Tim50–Tim23 complex to assign patches at atomic resolution\",\n        \"How the two patches coordinate during translocation is unclear\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Domain dissection established that Tim50 transmits presequence recognition across the inner membrane: the matrix domain recruits PAM coupling factor Pam17, the IMS domain promotes PAM recruitment to TIM23, and the transmembrane segment stimulates import-driving force, resolving how IMS presequence sensing is mechanistically coupled to matrix motor activation.\",\n      \"evidence\": \"Domain dissection with functional complementation, genetic analysis, in vivo import in yeast\",\n      \"pmids\": [\"32130909\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular mechanism of signal transduction across the transmembrane segment unresolved\",\n        \"No direct biochemical reconstitution of the Tim50–Pam17 interaction\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The two IMS sub-domains of Tim50 (core and PBD) were shown to have distinct essential functions—core domain for presequence binding and TIM23 recruitment, PBD for TOM–TIM23 cooperation—and can complement each other in trans, revealing a modular architecture.\",\n      \"evidence\": \"Trans-complementation, domain deletion, in vivo import assays in yeast\",\n      \"pmids\": [\"37748811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis for independent function of PBD at TOM–TIM23 interface not determined\",\n        \"Whether trans-complementation reflects physiological sub-complex dynamics is unknown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Three concurrent studies revealed that TIMM50 pathogenic variants specifically impair the TIM23SORT lateral release pathway (reducing OXPHOS and mitoribosomal components), that TIMM50 deficiency in neurons decreases respiration/ATP and disrupts mitochondrial trafficking and electrical activity, and that eIF5A controls Tim50 levels by alleviating ribosome stalling at polyproline stretches in Tim50 mRNA.\",\n      \"evidence\": \"Quantitative proteomics in patient fibroblasts and CRISPR models; neuronal knockdown with respirometry, imaging, and electrophysiology; ribosome profiling with polyproline deletion rescue in yeast\",\n      \"pmids\": [\"38828998\", \"39680434\", \"39509053\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contribution of TIM23SORT vs. TIM23MOTOR pathway impairment to disease phenotype unknown\",\n        \"Whether eIF5A-dependent Tim50 regulation operates in mammalian neurons not tested\",\n        \"No therapeutic strategy to restore TIM23SORT function in TIMM50 disease\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of the full-length Tim50 in complex with Tim23, a co-crystal with bound presequence, and a detailed mechanism for how presequence recognition is transduced across the membrane to activate the PAM motor remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structure of full-length Tim50 or Tim50–Tim23 complex\",\n        \"No co-crystal or cryo-EM structure with bound presequence\",\n        \"Transmembrane signal transduction mechanism from IMS to matrix unresolved at molecular level\",\n        \"Physiological role and substrate of phosphatase activity remain unknown\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 5, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 5, 12]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6, 7, 10, 12]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 1, 4, 5, 12, 13, 14]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [14, 16]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 12, 15]}\n    ],\n    \"complexes\": [\n      \"TIM23 complex\"\n    ],\n    \"partners\": [\n      \"Tim23\",\n      \"Pam17\",\n      \"CYP11A1\",\n      \"HSD3B2\",\n      \"coilin\",\n      \"SMN\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}