{"gene":"TIMM50","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2002,"finding":"Tim50 is an essential subunit of the TIM23 presequence translocase that exposes its major domain to the intermembrane space, interacts with preproteins in transit, and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits import of matrix-targeting preproteins but does not strictly affect preproteins with additional inner membrane-sorting signals.","method":"Genetic depletion, co-immunoprecipitation, preprotein import assays in yeast","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional assays, two independent labs (PMID:12437924 and PMID:12437925) simultaneously identifying Tim50 with orthogonal methods","pmids":["12437924"],"is_preprint":false},{"year":2002,"finding":"Tim50 is anchored to the inner mitochondrial membrane with its C-terminal domain in the intermembrane space (IMS), interacts with the N-terminal IMS domain of Tim23, and facilitates transfer of translocating preproteins from the TOM complex to the TIM23 complex. A translocation intermediate was crosslinked to Tim50.","method":"Site-specific photocrosslinking of translocation intermediates, co-immunoprecipitation, anti-Tim50 antibody inhibition","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — site-specific photocrosslinking plus functional antibody inhibition, corroborated by independent lab same year","pmids":["12437925"],"is_preprint":false},{"year":2006,"finding":"The IMS domain of Tim50 closes the Tim23 channel to maintain the permeability barrier of the mitochondrial inner membrane; presequences overcome this effect and activate the channel for translocation, establishing an antagonistic regulatory mechanism.","method":"Reconstituted Tim23 channel electrophysiology, addition of recombinant Tim50 IMS domain, presequence peptide competition assay","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of channel gating with purified domains, single lab but clear functional assay","pmids":["16763150"],"is_preprint":false},{"year":2004,"finding":"Human TIMM50 possesses phosphatase activity and forms a complex with human TIM23. Knockdown of human TIMM50 by RNAi increases sensitivity to death stimuli by accelerating cytochrome c release from mitochondria.","method":"Phosphatase activity assay on purified human Tim50, co-immunoprecipitation with Tim23, RNAi knockdown with cytochrome c release measurement","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — enzymatic assay plus Co-IP plus RNAi phenotype, single lab","pmids":["15044455"],"is_preprint":false},{"year":2008,"finding":"The IMS domains of Tim50 and Tim23 directly interact in vitro; specific mutations in Tim23 that abolish this interaction in vitro also destabilize it in vivo and cause defective TIM23-dependent preprotein import and temperature-sensitive lethality.","method":"In vitro reconstitution with recombinant IMS domains, chemical cross-linking, surface plasmon resonance, in vivo mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted interaction in vitro with SPR + cross-linking + mutagenesis + in vivo functional validation, single lab","pmids":["19017642"],"is_preprint":false},{"year":2009,"finding":"Tim23-Tim50 IMS domain interactions facilitate two steps of protein import: transfer of precursors from TOM40 to TIM23, and a late step promoting motor functions of mitochondrial Hsp70 in the matrix.","method":"Genetic analysis of IMS domain mutants, import assays with isolated mitochondria, analysis of TOM40-TIM23 cooperation","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple import assay readouts with defined domain mutants, single lab","pmids":["19139266"],"is_preprint":false},{"year":2011,"finding":"Photo-affinity cross-linking with engineered presequence probes mapped a specific presequence-binding domain on Tim50, establishing Tim50 as the primary presequence receptor at the inner membrane. Targeting signals and Tim50 regulate the Tim23 channel antagonistically.","method":"Photo-affinity labeling with presequence probes, mass spectrometric mapping of cross-linking sites, import assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — photo-affinity labeling with mass spectrometric mapping plus functional channel assays, single lab but multiple orthogonal methods","pmids":["22065641"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of the yeast Tim50 IMS domain resolved to 1.83 Å reveals a protruding β-hairpin critical for interaction with Tim23, providing a structural basis for Tim50-Tim23 cooperation in preprotein translocation.","method":"X-ray crystallography at 1.83 Å, mutagenesis of the β-hairpin, functional import assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation of key structural element, single lab","pmids":["21704637"],"is_preprint":false},{"year":2011,"finding":"Human TIMM50 interacts with 3β-HSD2 primarily through the Tim50 N-terminus; Tim50 knockdown inhibited 3β-HSD2 enzymatic activity (conversion of DHEA to androstenedione and pregnenolone to progesterone) and reduced 3β-HSD2 expression, demonstrating a role for Tim50 in steroidogenesis beyond protein import.","method":"Mass spectrometry, co-immunoprecipitation, density-gradient ultracentrifugation, Tim50 siRNA knockdown with enzymatic activity assays, CD spectroscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus enzymatic assay readout plus KD, single lab, two orthogonal methods","pmids":["21930695"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of Tim50(164-361) at 2.67 Å reveals significant structural plasticity within the putative presequence-binding groove and in the β-hairpin; crystal packing shows helix A1 from a neighboring monomer docking into the groove, suggesting a hydrophobic mechanism for presequence recognition.","method":"X-ray crystallography at 2.67 Å, structural comparison with prior 1.83 Å structure","journal":"Acta crystallographica Section F","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure alone, no mutagenesis or functional validation in this paper","pmids":["26323300"],"is_preprint":false},{"year":2017,"finding":"Cardiolipin modulates the interaction between the soluble Tim50 receptor domain and the Tim23 channel. The Tim50 receptor domain interacts with membranes and specific sites on Tim23 in a cardiolipin-dependent manner; SAXS-based structure of the full soluble Tim50 receptor domain was obtained, and MD simulations confirmed cardiolipin-driven association with concomitant structural changes.","method":"In vivo assays, isolated mitochondria reconstitution, nanoscale model membrane systems, SAXS structure determination, molecular dynamics simulations, biophysical binding measurements","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal methods (SAXS structure, MD, in vivo, reconstitution in model membranes) in a single study","pmids":["28879236"],"is_preprint":false},{"year":2018,"finding":"Tim50 directly interacts with SCC (CYP11A1) amino acids 141-146 in the TIM23 complex IMS; absence of Tim50 or its mutation reduced SCC enzymatic activity. SCC is imported to the matrix, undergoes sequential N-terminal cleavage, then its C-terminus integrates into TIM23 and aligns with Tim50.","method":"Co-immunoprecipitation, Tim50 knockdown with enzymatic activity assay, domain mapping","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping plus KD functional readout, single lab","pmids":["30348838"],"is_preprint":false},{"year":2019,"finding":"Random mutagenesis of Tim50 identified two distinct surface patches whose mutation impairs TIM23-dependent precursor import and disrupts Tim50-Tim23 interaction; these patches map onto the Tim50 surface structure, suggesting two regions mediate Tim23 binding.","method":"Random mutagenesis, temperature-sensitive growth assays, import assays, co-immunoprecipitation","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with functional import assay and Co-IP, single lab","pmids":["30765764"],"is_preprint":false},{"year":2020,"finding":"Tim50 coordinates preprotein recognition with motor activation via three structural elements: (1) the matrix domain facilitates recruitment of the coupling factor Pam17; (2) the IMS domain promotes PAM recruitment to TIM23; (3) the transmembrane segment stimulates the matrix-directed import force by PAM. This establishes Tim50 as a transmembrane signal transducer coupling IMS presequence recognition to matrix motor activation.","method":"Genetic dissection of Tim50 domains, import assays, analysis of PAM/Pam17 recruitment in isolated mitochondria","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-specific functional dissection in multiple import assays, single lab","pmids":["32130909"],"is_preprint":false},{"year":2023,"finding":"The Tim50 IMS region contains two functionally distinct domains (core and PBD): the core domain carries the main presequence-binding site and is the main recruitment point to TIM23, while the PBD directly or indirectly facilitates cooperation between TOM and TIM23 complexes. The two domains can complement each other in trans.","method":"Domain-swap and trans-complementation experiments, import assays, co-immunoprecipitation","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — trans-complementation plus import assays plus Co-IP, single lab","pmids":["37748811"],"is_preprint":false},{"year":2024,"finding":"TIMM50 pathogenic mutations reduce levels of TIM23 core components (TIMM50, TIMM17A/B, TIMM23), decrease mitochondrial membrane potential, and impair TIM23-dependent protein import. Substrates imported via TIM23SORT (lateral release pathway) are most sensitive to TIMM50 loss. OXPHOS and mitochondrial ultrastructure proteins are enriched in the TIM23SORT substrate pool, explaining the specific clinical defects.","method":"Proteomics of patient fibroblasts and TIMM50 HEK293 CRISPR/Cas9 model, import assays, mitochondrial membrane potential measurement","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic mapping plus CRISPR model plus import assays, single lab","pmids":["38828998"],"is_preprint":false},{"year":2024,"finding":"TIMM50 deficiency in human fibroblasts and mouse neurons selectively reduces steady-state levels of OXPHOS and mitochondrial ribosome components, leading to declined respiration, reduced ATP, and defective mitochondrial trafficking in neuronal processes. TIMM50 knockdown in neurons correlates with reduced KCNJ10 and KCNA2 potassium channel levels and increased electrical activity.","method":"Patient fibroblast characterization, mouse neuron knockdown, respirometry, ATP measurement, live neuronal mitochondrial trafficking assay, proteomics, electrophysiology","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal assays (proteomics, respirometry, trafficking, electrophysiology) in patient and model cells, single lab","pmids":["39680434"],"is_preprint":false},{"year":2024,"finding":"eIF5A alleviates ribosome stalling at polyproline-encoding sequences in TIM50 mRNA at the mitochondrial surface. eIF5A depletion reduces Tim50 translation and protein levels, causing accumulation of mitoprotein precursors in the cytosol and triggering a mitochondrial import stress response; removal of polyprolines from Tim50 partially rescues this response.","method":"eIF5A depletion in yeast, ribosome profiling, Tim50 polyproline mutant rescue experiments, mitoprotein precursor accumulation assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ribosome profiling plus genetic rescue with polyproline mutant, single lab","pmids":["39509053"],"is_preprint":false},{"year":2005,"finding":"A nuclear isoform of Tim50, Tim50a, contains an N-terminal extension with a nuclear localization signal and localizes to nuclear speckles. Tim50a interacts with coilin, snRNPs, and SMN; coilin competes with Sm proteins and SMN for binding sites on Tim50a, suggesting a role in snRNP biogenesis distinct from the mitochondrial function of Tim50.","method":"Subcellular localization by immunofluorescence, co-immunoprecipitation, competition binding experiments","journal":"BMC cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus localization, single lab, no functional validation of the proposed snRNP role","pmids":["16008839"],"is_preprint":false},{"year":2011,"finding":"Mutant p53 (R175H and R273H) upregulates Tim50 expression by increasing histone acetylation and recruiting transcription factors Ets-1, CREB, and CBP to the Tim50 promoter. Reduction of Tim50 in cells harboring mutant p53 reduced growth rate and chemoresistance.","method":"Chromatin immunoprecipitation (ChIP), Tim50 promoter reporter assay, siRNA knockdown with proliferation and chemoresistance assays","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus promoter reporter assay plus functional KD, single lab","pmids":["21621504"],"is_preprint":false},{"year":2025,"finding":"TIMM50 downregulation in cellular senescence models is mediated by sirtuin1-dependent downregulation of the transcription factor CEBPα (a transcriptional activator of TIMM50). TIMM50 loss triggers all hallmarks of senescence via impaired mitochondrial function.","method":"Multiple senescence models, TIMM50 knockdown and overexpression, pathway analysis identifying sirtuin1-CEBPα axis, mitochondrial function assays","journal":"Advanced biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pathway analysis plus KD/OE phenotype, limited mechanistic detail in abstract","pmids":["40128440"],"is_preprint":false}],"current_model":"TIMM50 (Tim50) is a core subunit of the mitochondrial TIM23 translocase anchored in the inner membrane with a large IMS-exposed domain that serves as the primary presequence receptor, directing precursor proteins from the TOM complex to the Tim23 channel; its IMS domain closes the Tim23 channel to maintain the permeability barrier while presequences antagonistically open it, its matrix domain and transmembrane segment coordinate PAM motor recruitment and activation, cardiolipin modulates its receptor-channel interactions, and the two IMS sub-domains (core and PBD) perform distinct but complementary roles in TOM-TIM23 coupling—with loss-of-function causing impaired mitochondrial protein import, reduced OXPHOS assembly, decreased membrane potential, and severe neurological disease in humans."},"narrative":{"mechanistic_narrative":"TIMM50 (Tim50) is the principal presequence receptor of the mitochondrial TIM23 inner-membrane translocase, governing the import of matrix-targeted precursor proteins from the TOM complex into mitochondria [PMID:12437924, PMID:22065641]. It is anchored in the inner membrane with a large intermembrane-space (IMS) domain that engages translocating preproteins and hands them to the channel protein Tim23, an interaction mediated by a protruding β-hairpin and additional surface patches of the Tim50 IMS domain [PMID:12437925, PMID:21704637, PMID:30765764]. The Tim50 IMS domain enforces the inner-membrane permeability barrier by closing the Tim23 channel, while incoming presequences antagonistically open it, coupling cargo recognition to gated translocation [PMID:16763150, PMID:22065641]. Beyond receptor function, Tim50 acts as a transmembrane signal transducer: its matrix domain, IMS domain, and transmembrane segment cooperate to recruit and activate the PAM import motor (including Pam17), linking IMS presequence binding to matrix-directed motor force [PMID:19139266, PMID:32130909], and its IMS region resolves into a core presequence-binding/recruitment domain and a separable domain that promotes TOM–TIM23 cooperation [PMID:37748811]. Cardiolipin modulates the Tim50 receptor domain's association with membranes and Tim23 [PMID:28879236]. In humans, pathogenic TIMM50 mutations destabilize TIM23 core components, lower mitochondrial membrane potential, and impair import—most severely of laterally sorted OXPHOS and mitochondrial ribosome substrates—causing reduced respiration and ATP, defective neuronal mitochondrial trafficking, and a severe neurological disease phenotype [PMID:38828998, PMID:39680434]. Human TIMM50 additionally interacts with steroidogenic enzymes 3β-HSD2 and CYP11A1 within the TIM23 IMS to support their activity [PMID:21930695, PMID:30348838].","teleology":[{"year":2002,"claim":"Established Tim50 as an essential TIM23 subunit that exposes an IMS domain to receive preproteins and hand them to the Tim23 channel, defining the entry point for presequence import.","evidence":"Genetic depletion, co-IP, and preprotein import assays in yeast, with site-specific photocrosslinking of a translocation intermediate to Tim50","pmids":["12437924","12437925"],"confidence":"High","gaps":["Did not resolve the structural basis of presequence binding","Did not define how the channel is gated"]},{"year":2004,"claim":"Showed human TIMM50 forms a complex with human TIM23 and that its loss accelerates cytochrome c release, linking the import receptor to apoptotic sensitivity.","evidence":"Phosphatase activity assay on purified human Tim50, co-IP with Tim23, RNAi with cytochrome c release measurement","pmids":["15044455"],"confidence":"Medium","gaps":["Physiological substrate of the reported phosphatase activity unidentified","Mechanism connecting Tim50 loss to cytochrome c release unresolved"]},{"year":2006,"claim":"Defined the regulatory logic of import by showing the Tim50 IMS domain closes the Tim23 channel and presequences antagonistically open it.","evidence":"Reconstituted Tim23 channel electrophysiology with recombinant Tim50 IMS domain and presequence peptide competition","pmids":["16763150"],"confidence":"High","gaps":["Structural conformations of the open/closed channel not defined","Did not map the presequence-binding site on Tim50"]},{"year":2009,"claim":"Demonstrated that Tim23–Tim50 IMS contacts drive two import steps—TOM-to-TIM23 transfer and a late step promoting matrix Hsp70 motor function—extending Tim50's role beyond initial reception.","evidence":"Genetic IMS-domain mutants and import assays in isolated mitochondria; in vitro reconstitution with recombinant IMS domains, SPR, and cross-linking","pmids":["19139266","19017642"],"confidence":"Medium","gaps":["Molecular link between IMS-domain contacts and matrix motor activation unresolved","Did not define the structural element mediating the interaction"]},{"year":2011,"claim":"Mapped the presequence-binding site on Tim50 and resolved the IMS-domain crystal structure, establishing the structural basis for Tim50 as the primary inner-membrane presequence receptor and Tim23 partner.","evidence":"Photo-affinity presequence labeling with MS mapping plus import assays; X-ray crystallography at 1.83 Å with β-hairpin mutagenesis","pmids":["22065641","21704637"],"confidence":"High","gaps":["Conformational dynamics of the binding groove not captured in a single structure","Did not address how signal binding is transmitted across the membrane"]},{"year":2011,"claim":"Identified non-import functions of human TIMM50, including steroidogenic enzyme support and transcriptional upregulation by mutant p53 driving proliferation and chemoresistance.","evidence":"Co-IP and enzymatic assays with 3β-HSD2 after Tim50 knockdown; ChIP, promoter reporter, and siRNA proliferation/chemoresistance assays in mutant-p53 cells","pmids":["21930695","21621504"],"confidence":"Medium","gaps":["Whether steroidogenic support is separable from import function unresolved","Generality of mutant-p53 upregulation across tumor contexts untested"]},{"year":2017,"claim":"Showed cardiolipin modulates Tim50 receptor-domain association with membranes and Tim23, adding a lipid-dependent layer to receptor-channel coupling.","evidence":"In vivo assays, model-membrane reconstitution, SAXS structure of the soluble receptor domain, MD simulations, and biophysical binding measurements","pmids":["28879236"],"confidence":"High","gaps":["In situ cardiolipin dependence within intact translocase not directly imaged","Functional consequence for cargo throughput not quantified"]},{"year":2020,"claim":"Resolved how Tim50 transduces a signal across the membrane: distinct matrix, IMS, and transmembrane elements cooperate to recruit Pam17/PAM and stimulate import force, coupling IMS recognition to matrix motor activation.","evidence":"Genetic dissection of Tim50 domains, import assays, and PAM/Pam17 recruitment analysis in isolated mitochondria","pmids":["32130909"],"confidence":"Medium","gaps":["Atomic mechanism of transmembrane signal transmission undefined","Stoichiometry of Tim50-PAM coupling unresolved"]},{"year":2023,"claim":"Subdivided the Tim50 IMS region into a core presequence-binding/recruitment domain and a separable domain promoting TOM–TIM23 cooperation, refining the receptor's functional architecture.","evidence":"Domain-swap and trans-complementation experiments, import assays, and co-IP","pmids":["37748811"],"confidence":"Medium","gaps":["Direct versus indirect mechanism by which the PBD aids TOM-TIM23 coupling unresolved","Structural interface of the two subdomains not defined"]},{"year":2024,"claim":"Linked TIMM50 loss-of-function to human disease by showing pathogenic mutations destabilize TIM23 components, lower membrane potential, and selectively impair laterally sorted OXPHOS and mitoribosome substrate import, explaining the neurological phenotype.","evidence":"Proteomics of patient fibroblasts and a TIMM50 CRISPR HEK293 model, import assays, membrane potential measurements; patient and mouse neuron knockdown with respirometry, trafficking, and electrophysiology","pmids":["38828998","39680434"],"confidence":"Medium","gaps":["Causal chain from import defect to specific neuronal channel changes (KCNJ10/KCNA2) not fully mechanistic","Tissue-specific vulnerability not explained"]},{"year":2024,"claim":"Connected Tim50 biogenesis to translation control, showing eIF5A relieves ribosome stalling at TIM50 polyproline tracts and that Tim50 limitation triggers a mitochondrial import stress response.","evidence":"eIF5A depletion in yeast, ribosome profiling, and polyproline-mutant rescue with mitoprotein precursor accumulation assays","pmids":["39509053"],"confidence":"Medium","gaps":["Whether this regulation operates in human cells untested","Quantitative contribution of Tim50 stalling to the stress response unresolved"]},{"year":null,"claim":"How Tim50's distinct activities—presequence reception, channel gating, motor coupling, and its reported moonlighting nuclear/steroidogenic roles—are integrated and regulated in a single protein remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Nuclear Tim50a isoform role in snRNP biogenesis lacks functional validation (#18)","Senescence regulation via sirtuin1-CEBPα reported with limited mechanistic detail (#20)","No high-resolution structure of the assembled human translocase with Tim50 engaging cargo"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[19]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[13]},{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[0,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,6]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,16]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,6,15]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,5,13]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[15,16]}],"complexes":["TIM23 presequence translocase","PAM import motor"],"partners":["TIMM23","PAM17","TIMM17A","TIMM17B","HSPA9","HSD3B2","CYP11A1"],"other_free_text":[]}},"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":217,"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":211,"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":83,"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":54,"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":"17435247","id":"PMC_17435247","title":"Involvement of the mitochondrial protein translocator component tim50 in growth, cell proliferation and the modulation of respiration in 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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":18137,"output_tokens":4729,"usd":0.062673,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13114,"output_tokens":3996,"usd":0.082735,"stage2_stop_reason":"end_turn"},"total_usd":0.145408,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","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 presequence translocase that exposes its major domain to the intermembrane space, interacts with preproteins in transit, and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits import of matrix-targeting preproteins but does not strictly affect preproteins with additional inner membrane-sorting signals.\",\n      \"method\": \"Genetic depletion, co-immunoprecipitation, preprotein import assays in yeast\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional assays, two independent labs (PMID:12437924 and PMID:12437925) simultaneously identifying Tim50 with orthogonal methods\",\n      \"pmids\": [\"12437924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Tim50 is anchored to the inner mitochondrial membrane with its C-terminal domain in the intermembrane space (IMS), interacts with the N-terminal IMS domain of Tim23, and facilitates transfer of translocating preproteins from the TOM complex to the TIM23 complex. A translocation intermediate was crosslinked to Tim50.\",\n      \"method\": \"Site-specific photocrosslinking of translocation intermediates, co-immunoprecipitation, anti-Tim50 antibody inhibition\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — site-specific photocrosslinking plus functional antibody inhibition, corroborated by independent lab same year\",\n      \"pmids\": [\"12437925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The IMS domain of Tim50 closes the Tim23 channel to maintain the permeability barrier of the mitochondrial inner membrane; presequences overcome this effect and activate the channel for translocation, establishing an antagonistic regulatory mechanism.\",\n      \"method\": \"Reconstituted Tim23 channel electrophysiology, addition of recombinant Tim50 IMS domain, presequence peptide competition assay\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of channel gating with purified domains, single lab but clear functional assay\",\n      \"pmids\": [\"16763150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human TIMM50 possesses phosphatase activity and forms a complex with human TIM23. Knockdown of human TIMM50 by RNAi increases sensitivity to death stimuli by accelerating cytochrome c release from mitochondria.\",\n      \"method\": \"Phosphatase activity assay on purified human Tim50, co-immunoprecipitation with Tim23, RNAi knockdown with cytochrome c release measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzymatic assay plus Co-IP plus RNAi phenotype, single lab\",\n      \"pmids\": [\"15044455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The IMS domains of Tim50 and Tim23 directly interact in vitro; specific mutations in Tim23 that abolish this interaction in vitro also destabilize it in vivo and cause defective TIM23-dependent preprotein import and temperature-sensitive lethality.\",\n      \"method\": \"In vitro reconstitution with recombinant IMS domains, chemical cross-linking, surface plasmon resonance, in vivo mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted interaction in vitro with SPR + cross-linking + mutagenesis + in vivo functional validation, single lab\",\n      \"pmids\": [\"19017642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Tim23-Tim50 IMS domain interactions facilitate two steps of protein import: transfer of precursors from TOM40 to TIM23, and a late step promoting motor functions of mitochondrial Hsp70 in the matrix.\",\n      \"method\": \"Genetic analysis of IMS domain mutants, import assays with isolated mitochondria, analysis of TOM40-TIM23 cooperation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple import assay readouts with defined domain mutants, single lab\",\n      \"pmids\": [\"19139266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Photo-affinity cross-linking with engineered presequence probes mapped a specific presequence-binding domain on Tim50, establishing Tim50 as the primary presequence receptor at the inner membrane. Targeting signals and Tim50 regulate the Tim23 channel antagonistically.\",\n      \"method\": \"Photo-affinity labeling with presequence probes, mass spectrometric mapping of cross-linking sites, import assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — photo-affinity labeling with mass spectrometric mapping plus functional channel assays, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"22065641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of the yeast Tim50 IMS domain resolved to 1.83 Å reveals a protruding β-hairpin critical for interaction with Tim23, providing a structural basis for Tim50-Tim23 cooperation in preprotein translocation.\",\n      \"method\": \"X-ray crystallography at 1.83 Å, mutagenesis of the β-hairpin, functional import assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation of key structural element, single lab\",\n      \"pmids\": [\"21704637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Human TIMM50 interacts with 3β-HSD2 primarily through the Tim50 N-terminus; Tim50 knockdown inhibited 3β-HSD2 enzymatic activity (conversion of DHEA to androstenedione and pregnenolone to progesterone) and reduced 3β-HSD2 expression, demonstrating a role for Tim50 in steroidogenesis beyond protein import.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, density-gradient ultracentrifugation, Tim50 siRNA knockdown with enzymatic activity assays, CD spectroscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus enzymatic assay readout plus KD, single lab, two orthogonal methods\",\n      \"pmids\": [\"21930695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of Tim50(164-361) at 2.67 Å reveals significant structural plasticity within the putative presequence-binding groove and in the β-hairpin; crystal packing shows helix A1 from a neighboring monomer docking into the groove, suggesting a hydrophobic mechanism for presequence recognition.\",\n      \"method\": \"X-ray crystallography at 2.67 Å, structural comparison with prior 1.83 Å structure\",\n      \"journal\": \"Acta crystallographica Section F\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure alone, no mutagenesis or functional validation in this paper\",\n      \"pmids\": [\"26323300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cardiolipin modulates the interaction between the soluble Tim50 receptor domain and the Tim23 channel. The Tim50 receptor domain interacts with membranes and specific sites on Tim23 in a cardiolipin-dependent manner; SAXS-based structure of the full soluble Tim50 receptor domain was obtained, and MD simulations confirmed cardiolipin-driven association with concomitant structural changes.\",\n      \"method\": \"In vivo assays, isolated mitochondria reconstitution, nanoscale model membrane systems, SAXS structure determination, molecular dynamics simulations, biophysical binding measurements\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal methods (SAXS structure, MD, in vivo, reconstitution in model membranes) in a single study\",\n      \"pmids\": [\"28879236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Tim50 directly interacts with SCC (CYP11A1) amino acids 141-146 in the TIM23 complex IMS; absence of Tim50 or its mutation reduced SCC enzymatic activity. SCC is imported to the matrix, undergoes sequential N-terminal cleavage, then its C-terminus integrates into TIM23 and aligns with Tim50.\",\n      \"method\": \"Co-immunoprecipitation, Tim50 knockdown with enzymatic activity assay, domain mapping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping plus KD functional readout, single lab\",\n      \"pmids\": [\"30348838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Random mutagenesis of Tim50 identified two distinct surface patches whose mutation impairs TIM23-dependent precursor import and disrupts Tim50-Tim23 interaction; these patches map onto the Tim50 surface structure, suggesting two regions mediate Tim23 binding.\",\n      \"method\": \"Random mutagenesis, temperature-sensitive growth assays, import assays, co-immunoprecipitation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with functional import assay and Co-IP, single lab\",\n      \"pmids\": [\"30765764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Tim50 coordinates preprotein recognition with motor activation via three structural elements: (1) the matrix domain facilitates recruitment of the coupling factor Pam17; (2) the IMS domain promotes PAM recruitment to TIM23; (3) the transmembrane segment stimulates the matrix-directed import force by PAM. This establishes Tim50 as a transmembrane signal transducer coupling IMS presequence recognition to matrix motor activation.\",\n      \"method\": \"Genetic dissection of Tim50 domains, import assays, analysis of PAM/Pam17 recruitment in isolated mitochondria\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific functional dissection in multiple import assays, single lab\",\n      \"pmids\": [\"32130909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The Tim50 IMS region contains two functionally distinct domains (core and PBD): the core domain carries the main presequence-binding site and is the main recruitment point to TIM23, while the PBD directly or indirectly facilitates cooperation between TOM and TIM23 complexes. The two domains can complement each other in trans.\",\n      \"method\": \"Domain-swap and trans-complementation experiments, import assays, co-immunoprecipitation\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — trans-complementation plus import assays plus Co-IP, single lab\",\n      \"pmids\": [\"37748811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIMM50 pathogenic mutations reduce levels of TIM23 core components (TIMM50, TIMM17A/B, TIMM23), decrease mitochondrial membrane potential, and impair TIM23-dependent protein import. Substrates imported via TIM23SORT (lateral release pathway) are most sensitive to TIMM50 loss. OXPHOS and mitochondrial ultrastructure proteins are enriched in the TIM23SORT substrate pool, explaining the specific clinical defects.\",\n      \"method\": \"Proteomics of patient fibroblasts and TIMM50 HEK293 CRISPR/Cas9 model, import assays, mitochondrial membrane potential measurement\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic mapping plus CRISPR model plus import assays, single lab\",\n      \"pmids\": [\"38828998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIMM50 deficiency in human fibroblasts and mouse neurons selectively reduces steady-state levels of OXPHOS and mitochondrial ribosome components, leading to declined respiration, reduced ATP, and defective mitochondrial trafficking in neuronal processes. TIMM50 knockdown in neurons correlates with reduced KCNJ10 and KCNA2 potassium channel levels and increased electrical activity.\",\n      \"method\": \"Patient fibroblast characterization, mouse neuron knockdown, respirometry, ATP measurement, live neuronal mitochondrial trafficking assay, proteomics, electrophysiology\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal assays (proteomics, respirometry, trafficking, electrophysiology) in patient and model cells, single lab\",\n      \"pmids\": [\"39680434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"eIF5A alleviates ribosome stalling at polyproline-encoding sequences in TIM50 mRNA at the mitochondrial surface. eIF5A depletion reduces Tim50 translation and protein levels, causing accumulation of mitoprotein precursors in the cytosol and triggering a mitochondrial import stress response; removal of polyprolines from Tim50 partially rescues this response.\",\n      \"method\": \"eIF5A depletion in yeast, ribosome profiling, Tim50 polyproline mutant rescue experiments, mitoprotein precursor accumulation assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ribosome profiling plus genetic rescue with polyproline mutant, single lab\",\n      \"pmids\": [\"39509053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"A nuclear isoform of Tim50, Tim50a, contains an N-terminal extension with a nuclear localization signal and localizes to nuclear speckles. Tim50a interacts with coilin, snRNPs, and SMN; coilin competes with Sm proteins and SMN for binding sites on Tim50a, suggesting a role in snRNP biogenesis distinct from the mitochondrial function of Tim50.\",\n      \"method\": \"Subcellular localization by immunofluorescence, co-immunoprecipitation, competition binding experiments\",\n      \"journal\": \"BMC cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus localization, single lab, no functional validation of the proposed snRNP role\",\n      \"pmids\": [\"16008839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Mutant p53 (R175H and R273H) upregulates Tim50 expression by increasing histone acetylation and recruiting transcription factors Ets-1, CREB, and CBP to the Tim50 promoter. Reduction of Tim50 in cells harboring mutant p53 reduced growth rate and chemoresistance.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), Tim50 promoter reporter assay, siRNA knockdown with proliferation and chemoresistance assays\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus promoter reporter assay plus functional KD, single lab\",\n      \"pmids\": [\"21621504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TIMM50 downregulation in cellular senescence models is mediated by sirtuin1-dependent downregulation of the transcription factor CEBPα (a transcriptional activator of TIMM50). TIMM50 loss triggers all hallmarks of senescence via impaired mitochondrial function.\",\n      \"method\": \"Multiple senescence models, TIMM50 knockdown and overexpression, pathway analysis identifying sirtuin1-CEBPα axis, mitochondrial function assays\",\n      \"journal\": \"Advanced biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pathway analysis plus KD/OE phenotype, limited mechanistic detail in abstract\",\n      \"pmids\": [\"40128440\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TIMM50 (Tim50) is a core subunit of the mitochondrial TIM23 translocase anchored in the inner membrane with a large IMS-exposed domain that serves as the primary presequence receptor, directing precursor proteins from the TOM complex to the Tim23 channel; its IMS domain closes the Tim23 channel to maintain the permeability barrier while presequences antagonistically open it, its matrix domain and transmembrane segment coordinate PAM motor recruitment and activation, cardiolipin modulates its receptor-channel interactions, and the two IMS sub-domains (core and PBD) perform distinct but complementary roles in TOM-TIM23 coupling—with loss-of-function causing impaired mitochondrial protein import, reduced OXPHOS assembly, decreased membrane potential, and severe neurological disease in humans.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TIMM50 (Tim50) is the principal presequence receptor of the mitochondrial TIM23 inner-membrane translocase, governing the import of matrix-targeted precursor proteins from the TOM complex into mitochondria [#0, #6]. It is anchored in the inner membrane with a large intermembrane-space (IMS) domain that engages translocating preproteins and hands them to the channel protein Tim23, an interaction mediated by a protruding β-hairpin and additional surface patches of the Tim50 IMS domain [#1, #7, #12]. The Tim50 IMS domain enforces the inner-membrane permeability barrier by closing the Tim23 channel, while incoming presequences antagonistically open it, coupling cargo recognition to gated translocation [#2, #6]. Beyond receptor function, Tim50 acts as a transmembrane signal transducer: its matrix domain, IMS domain, and transmembrane segment cooperate to recruit and activate the PAM import motor (including Pam17), linking IMS presequence binding to matrix-directed motor force [#5, #13], and its IMS region resolves into a core presequence-binding/recruitment domain and a separable domain that promotes TOM–TIM23 cooperation [#14]. Cardiolipin modulates the Tim50 receptor domain's association with membranes and Tim23 [#10]. In humans, pathogenic TIMM50 mutations destabilize TIM23 core components, lower mitochondrial membrane potential, and impair import—most severely of laterally sorted OXPHOS and mitochondrial ribosome substrates—causing reduced respiration and ATP, defective neuronal mitochondrial trafficking, and a severe neurological disease phenotype [#15, #16]. Human TIMM50 additionally interacts with steroidogenic enzymes 3β-HSD2 and CYP11A1 within the TIM23 IMS to support their activity [#8, #11].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established Tim50 as an essential TIM23 subunit that exposes an IMS domain to receive preproteins and hand them to the Tim23 channel, defining the entry point for presequence import.\",\n      \"evidence\": \"Genetic depletion, co-IP, and preprotein import assays in yeast, with site-specific photocrosslinking of a translocation intermediate to Tim50\",\n      \"pmids\": [\"12437924\", \"12437925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of presequence binding\", \"Did not define how the channel is gated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed human TIMM50 forms a complex with human TIM23 and that its loss accelerates cytochrome c release, linking the import receptor to apoptotic sensitivity.\",\n      \"evidence\": \"Phosphatase activity assay on purified human Tim50, co-IP with Tim23, RNAi with cytochrome c release measurement\",\n      \"pmids\": [\"15044455\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological substrate of the reported phosphatase activity unidentified\", \"Mechanism connecting Tim50 loss to cytochrome c release unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the regulatory logic of import by showing the Tim50 IMS domain closes the Tim23 channel and presequences antagonistically open it.\",\n      \"evidence\": \"Reconstituted Tim23 channel electrophysiology with recombinant Tim50 IMS domain and presequence peptide competition\",\n      \"pmids\": [\"16763150\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural conformations of the open/closed channel not defined\", \"Did not map the presequence-binding site on Tim50\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated that Tim23–Tim50 IMS contacts drive two import steps—TOM-to-TIM23 transfer and a late step promoting matrix Hsp70 motor function—extending Tim50's role beyond initial reception.\",\n      \"evidence\": \"Genetic IMS-domain mutants and import assays in isolated mitochondria; in vitro reconstitution with recombinant IMS domains, SPR, and cross-linking\",\n      \"pmids\": [\"19139266\", \"19017642\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between IMS-domain contacts and matrix motor activation unresolved\", \"Did not define the structural element mediating the interaction\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapped the presequence-binding site on Tim50 and resolved the IMS-domain crystal structure, establishing the structural basis for Tim50 as the primary inner-membrane presequence receptor and Tim23 partner.\",\n      \"evidence\": \"Photo-affinity presequence labeling with MS mapping plus import assays; X-ray crystallography at 1.83 Å with β-hairpin mutagenesis\",\n      \"pmids\": [\"22065641\", \"21704637\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational dynamics of the binding groove not captured in a single structure\", \"Did not address how signal binding is transmitted across the membrane\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified non-import functions of human TIMM50, including steroidogenic enzyme support and transcriptional upregulation by mutant p53 driving proliferation and chemoresistance.\",\n      \"evidence\": \"Co-IP and enzymatic assays with 3β-HSD2 after Tim50 knockdown; ChIP, promoter reporter, and siRNA proliferation/chemoresistance assays in mutant-p53 cells\",\n      \"pmids\": [\"21930695\", \"21621504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether steroidogenic support is separable from import function unresolved\", \"Generality of mutant-p53 upregulation across tumor contexts untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed cardiolipin modulates Tim50 receptor-domain association with membranes and Tim23, adding a lipid-dependent layer to receptor-channel coupling.\",\n      \"evidence\": \"In vivo assays, model-membrane reconstitution, SAXS structure of the soluble receptor domain, MD simulations, and biophysical binding measurements\",\n      \"pmids\": [\"28879236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In situ cardiolipin dependence within intact translocase not directly imaged\", \"Functional consequence for cargo throughput not quantified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved how Tim50 transduces a signal across the membrane: distinct matrix, IMS, and transmembrane elements cooperate to recruit Pam17/PAM and stimulate import force, coupling IMS recognition to matrix motor activation.\",\n      \"evidence\": \"Genetic dissection of Tim50 domains, import assays, and PAM/Pam17 recruitment analysis in isolated mitochondria\",\n      \"pmids\": [\"32130909\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Atomic mechanism of transmembrane signal transmission undefined\", \"Stoichiometry of Tim50-PAM coupling unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Subdivided the Tim50 IMS region into a core presequence-binding/recruitment domain and a separable domain promoting TOM–TIM23 cooperation, refining the receptor's functional architecture.\",\n      \"evidence\": \"Domain-swap and trans-complementation experiments, import assays, and co-IP\",\n      \"pmids\": [\"37748811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct versus indirect mechanism by which the PBD aids TOM-TIM23 coupling unresolved\", \"Structural interface of the two subdomains not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked TIMM50 loss-of-function to human disease by showing pathogenic mutations destabilize TIM23 components, lower membrane potential, and selectively impair laterally sorted OXPHOS and mitoribosome substrate import, explaining the neurological phenotype.\",\n      \"evidence\": \"Proteomics of patient fibroblasts and a TIMM50 CRISPR HEK293 model, import assays, membrane potential measurements; patient and mouse neuron knockdown with respirometry, trafficking, and electrophysiology\",\n      \"pmids\": [\"38828998\", \"39680434\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from import defect to specific neuronal channel changes (KCNJ10/KCNA2) not fully mechanistic\", \"Tissue-specific vulnerability not explained\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected Tim50 biogenesis to translation control, showing eIF5A relieves ribosome stalling at TIM50 polyproline tracts and that Tim50 limitation triggers a mitochondrial import stress response.\",\n      \"evidence\": \"eIF5A depletion in yeast, ribosome profiling, and polyproline-mutant rescue with mitoprotein precursor accumulation assays\",\n      \"pmids\": [\"39509053\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this regulation operates in human cells untested\", \"Quantitative contribution of Tim50 stalling to the stress response unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Tim50's distinct activities—presequence reception, channel gating, motor coupling, and its reported moonlighting nuclear/steroidogenic roles—are integrated and regulated in a single protein remains unresolved.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Nuclear Tim50a isoform role in snRNP biogenesis lacks functional validation (#18)\", \"Senescence regulation via sirtuin1-CEBPα reported with limited mechanistic detail (#20)\", \"No high-resolution structure of the assembled human translocase with Tim50 engaging cargo\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 6, 15]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 5, 13]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [15, 16]}\n    ],\n    \"complexes\": [\"TIM23 presequence translocase\", \"PAM import motor\"],\n    \"partners\": [\"TIMM23\", \"PAM17\", \"TIMM17A\", \"TIMM17B\", \"HSPA9\", \"HSD3B2\", \"CYP11A1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}