{"gene":"TIMM10","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1998,"finding":"Tim10 (with Tim12) localizes to the mitochondrial intermembrane space and interacts sequentially with carrier family precursor proteins to facilitate their translocation across the outer membrane in a membrane-potential-independent manner; Tim10 and Tim12 are found in a complex with Tim22, which mediates membrane-potential-dependent insertion into the inner membrane. Both Tim10 and Tim12 contain a zinc-finger-like motif with four cysteines and bind equimolar zinc ions; interaction with precursors depends on divalent metal ions.","method":"Biochemical fractionation, co-immunoprecipitation, reconstitution of import in yeast","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution of import pathway, co-IP, fractionation, replicated by multiple subsequent studies","pmids":["9495346"],"is_preprint":false},{"year":2001,"finding":"The TIM10 complex is composed exclusively of Tim9 and Tim10 (no other mitochondrial protein is required for complex formation); reconstituted recombinant Tim9-Tim10 complex restores ADP/ATP carrier import across the outer membrane and accurate inner membrane insertion in tim10-ts mitochondria lacking endogenous Tim10.","method":"Functional reconstitution using E. coli-expressed recombinant proteins, import assay in tim10-ts mitochondria","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus functional complementation assay with defined mutant background","pmids":["11483513"],"is_preprint":false},{"year":2004,"finding":"TIM10 assembly is redox-regulated: subunits are imported in a cysteine-reduced, unfolded state, then undergo intramolecular disulfide bonding (between their four conserved cysteines) to a zinc-devoid, assembly-competent structure, and finally assemble into the functional hexameric complex. Intramolecular disulfides form in vivo; intermolecular disulfides observed in vitro are abortive intermediates.","method":"Biochemical assays (redox trapping, import assays, mutagenesis of cysteines), in vivo labeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal biochemical methods plus mutagenesis in a single focused study","pmids":["14973127"],"is_preprint":false},{"year":2005,"finding":"Zinc binding stabilizes reduced Tim10 and slows oxidative folding by more than tenfold, maintaining it in an import-competent state in the cytosol; once imported, zinc must be released to permit oxidative folding and assembly. Oxidized (disulfide-bonded) Tim10 cannot be further reduced by glutathione, while reduced Tim10 is rapidly oxidized by oxidized glutathione at physiological concentrations. Protein disulfide isomerase can catalyze oxidative folding of Tim10 only after zinc removal.","method":"In vitro biochemical and biophysical assays (redox potential measurement, CD spectroscopy, fluorescence, glutathione redox assays)","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal in vitro methods (CD, fluorescence, redox potential measurement) in a single focused study","pmids":["16199054"],"is_preprint":false},{"year":2007,"finding":"Mia40 acts as a site-specific receptor for Tim10 biogenesis: only the most amino-terminal cysteine residue of Tim10 is critical for translocation across the outer membrane and interaction with Mia40, whereas all four cysteines are required for assembly of the Tim9-Tim10 chaperone complex.","method":"Systematic cysteine mutagenesis, in organello and in vitro import assays, co-immunoprecipitation with Mia40","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — systematic mutagenesis combined with in organello and in vitro assays in one study","pmids":["17553782"],"is_preprint":false},{"year":2007,"finding":"A conserved glutamate residue in the central core domain of Tim10 (within the CX3C motif region) is required for assembly of the hexameric TIM10 complex; mutations abolishing complex assembly are lethal on non-fermentable carbon sources but allow growth on glucose. The N-terminal substrate-binding region of Tim10 is essential for cell viability under all conditions.","method":"Site-directed mutagenesis, in vitro complex assembly assays, in vivo complementation using MET3-TIM10 strain","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis coupled with in vitro assembly and in vivo complementation in a focused study","pmids":["17618651"],"is_preprint":false},{"year":2008,"finding":"The crystal structure of the yeast Tim9-Tim10 hexameric complex was determined to 2.5 Å; each subunit contains a central loop flanked by disulfide bonds with N- and C-terminal tentacle-like helices. Buried salt bridges between conserved lysine and glutamate residues connect alternating subunits; mutation of these residues destabilizes the complex and causes defective precursor import. The N-terminal region of Tim9 is required for efficient trapping of incoming substrates into the IMS.","method":"X-ray crystallography (2.5 Å), site-directed mutagenesis, yeast growth assays, in vitro import assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and functional import assays in one study","pmids":["19037098"],"is_preprint":false},{"year":2007,"finding":"The Tim9-Tim10 complex assembles via a multi-step pathway with transient tetrameric intermediates before the final hexamer is formed; Tim9 forms a homodimer while Tim10 is a monomer. The N-terminal helices of subunits are assembled before the C-terminal helices during complex formation.","method":"Stopped-flow fluorescence with tryptophan mutagenesis, stopped-flow light scattering, analytical ultracentrifugation","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — multiple biophysical methods in vitro but single lab, no in vivo validation of intermediates","pmids":["18022191"],"is_preprint":false},{"year":2008,"finding":"Tim10 zinc binding proceeds via a two-step mechanism: an initial selective binding of Zn2+ to cysteine residues forming a structurally unfolded intermediate, followed by folding upon higher zinc concentrations. Zinc-binding affinity of Tim10 is ~8×10^-10 M. Oxidized (disulfide-bonded) Tim10 cannot bind zinc.","method":"Circular dichroism, fluorescence spectrometry, stopped-flow fluorescence, metal chelator competition assays","journal":"Proteins","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal biophysical methods in vitro, single lab","pmids":["17963238"],"is_preprint":false},{"year":2008,"finding":"Assembly of the Tim9-Tim10 complex is driven by electrostatic interactions (initial driving force, salt and pH dependence matching subunit isoelectric points) and is also regulated allosterically; Tim10 displays sigmoidal concentration dependence suggesting cooperativity, while Tim9 shows linear dependence.","method":"Stopped-flow kinetics with mutagenesis, pH and salt concentration variation","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — stopped-flow kinetics with mutagenesis, single lab","pmids":["18462749"],"is_preprint":false},{"year":2009,"finding":"A nine-amino-acid region within the Tim10 precursor (the IMS sorting signal) is sufficient for engagement with the Mia40 receptor and for transfer of proteins across the outer membrane to the IMS.","method":"Mutagenesis/deletion analysis, in organello and in vitro import assays, chimeric protein constructs","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — deletion and chimeric construct analysis with in organello and in vitro import assays in a focused study","pmids":["19297525"],"is_preprint":false},{"year":2011,"finding":"Dynamics of hydrophobic residues in the Tim9-Tim10 complex regulates its chaperone function; temperature-dependent conformational changes mimicking biological substrate-binding activity correspond to disruption of hydrophobic interactions, suggesting different functional conformational states exist at equilibrium.","method":"Temperature-dependent biochemical assays, substrate binding measurements, molecular dynamics simulation with energy decomposition analysis","journal":"Proteins","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, computational MD combined with limited biochemical assays, no mutagenesis validation","pmids":["22095685"],"is_preprint":false},{"year":2015,"finding":"Tim9 protects Tim10 from degradation by the mitochondrial i-AAA protease Yme1 by assembling into the Tim9-Tim10 complex; loss of Tim9's inner disulfide bond leads to degradation of both Tim9 and Tim10, and this is suppressed by deletion of YME1. Tim10 (rather than the hexameric complex) is proposed as the primary functional unit.","method":"Yeast genetics (temperature-sensitive mutants, YME1 deletion), biochemical and biophysical methods (complex stability, protein levels)","journal":"Bioscience reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (YME1 deletion suppression) plus biochemical validation, single lab","pmids":["26182355"],"is_preprint":false},{"year":2025,"finding":"Yme1 protease preferentially binds Tim10 over other small Tim proteins via a high-affinity interaction mediated primarily by Tim10's flexible N-terminal tentacle region, irrespective of disulfide bond status; substrate unfolding (disruption of disulfide bonds) exposes additional contact sites that enhance engagement and commit Tim10 to degradation. Yme1 also binds assembled Tim9-Tim10 complex independently of the Tim10 N-terminal tentacle.","method":"Biochemical and biophysical approaches (binding assays, degradation assays), analysis of disulfide bond variants","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical/biophysical methods in a focused preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.07.23.666395"],"is_preprint":true}],"current_model":"TIMM10 (Tim10) is an IMS-localized small Tim protein that is imported in a reduced, zinc-stabilized state via a 9-residue IMS-sorting signal recognized by Mia40 (which engages specifically the first cysteine of Tim10); after import, zinc is released, intramolecular disulfide bonds form between the four conserved CX3C cysteines, and Tim10 assembles with Tim9 through electrostatic and allosteric interactions into a heterohexameric (3×Tim9 + 3×Tim10) chaperone complex that chaperoning hydrophobic carrier precursors (including ADP/ATP carrier) across the IMS for Tim22-mediated insertion into the inner membrane, with Tim10's N-terminal tentacle being essential for substrate recognition and Tim9 serving to protect Tim10 from Yme1-mediated degradation."},"narrative":{"mechanistic_narrative":"TIMM10 (Tim10) is a small intermembrane-space (IMS) chaperone of mitochondria that, together with Tim9, escorts hydrophobic carrier-family precursor proteins across the IMS toward Tim22-mediated insertion into the inner membrane [PMID:9495346, PMID:11483513]. Tim10 and Tim9 assemble exclusively with one another into the functional chaperone; recombinant Tim9-Tim10 complex alone is sufficient to restore ADP/ATP carrier import and inner-membrane insertion in mitochondria lacking endogenous Tim10 [PMID:11483513]. The crystal structure reveals a heterohexamer in which each subunit contributes a central loop flanked by disulfide bonds and N- and C-terminal tentacle-like helices, with buried salt bridges between conserved lysine and glutamate residues stapling alternating subunits; mutation of these salt-bridge residues, or of a conserved core glutamate, destabilizes the complex and impairs precursor import [PMID:17618651, PMID:19037098]. Tim10's N-terminal substrate-binding tentacle is essential for cell viability and substrate recognition [PMID:17618651]. Biogenesis of Tim10 is redox- and metal-regulated: it is held in a reduced, zinc-stabilized, import-competent state that resists oxidative folding, and is delivered across the outer membrane through a nine-residue IMS sorting signal recognized by the Mia40 receptor, which engages specifically the most N-terminal cysteine [PMID:16199054, PMID:17553782, PMID:19297525]. Following import, zinc is released and intramolecular disulfide bonds form between the four conserved cysteines to generate the assembly-competent, oxidatively folded subunit [PMID:14973127, PMID:16199054]. Within the complex, Tim9 protects Tim10 from degradation by the i-AAA protease Yme1 [PMID:26182355].","teleology":[{"year":1998,"claim":"Established that Tim10 functions in the IMS to chaperone carrier precursors across the outer membrane and hand them to the Tim22 inner-membrane insertion machinery, defining the carrier import pathway.","evidence":"Biochemical fractionation, co-immunoprecipitation, and reconstitution of import in yeast mitochondria","pmids":["9495346"],"confidence":"High","gaps":["Did not define the minimal complement of subunits forming the functional chaperone","Structural basis of substrate recognition unknown"]},{"year":2001,"claim":"Resolved which subunits constitute the functional chaperone by showing the complex is composed exclusively of Tim9 and Tim10 and that recombinant Tim9-Tim10 alone restores carrier import.","evidence":"Functional reconstitution with E. coli-expressed proteins and import assay in tim10-ts mitochondria","pmids":["11483513"],"confidence":"High","gaps":["Stoichiometry and architecture of the complex not yet determined","Mechanism of substrate engagement not addressed"]},{"year":2004,"claim":"Showed Tim10 assembly is redox-controlled, proceeding from a reduced unfolded import substrate to an intramolecularly disulfide-bonded, zinc-devoid, assembly-competent form, distinguishing productive from abortive disulfide states.","evidence":"Redox trapping, import assays, cysteine mutagenesis, and in vivo labeling","pmids":["14973127"],"confidence":"High","gaps":["Catalyst driving in vivo oxidation not identified","Role of zinc in maintaining import competence not yet quantified"]},{"year":2005,"claim":"Defined the role of zinc as a kinetic brake that stabilizes reduced Tim10 and slows oxidative folding, coupling cytosolic metal binding to import competence and post-import folding upon zinc release.","evidence":"In vitro redox potential measurement, CD, fluorescence, and glutathione redox assays","pmids":["16199054"],"confidence":"High","gaps":["In vivo timing of zinc release not directly observed","Identity of the cellular zinc donor/acceptor not established"]},{"year":2007,"claim":"Identified Mia40 as a site-specific receptor that recognizes only the most N-terminal cysteine of Tim10 for outer-membrane translocation, separating the import-recognition determinant from the four-cysteine assembly requirement.","evidence":"Systematic cysteine mutagenesis with in organello and in vitro import and Mia40 co-immunoprecipitation","pmids":["17553782"],"confidence":"High","gaps":["Structure of the Mia40-Tim10 import intermediate not resolved","Sequence context beyond the cysteine not yet defined"]},{"year":2007,"claim":"Mapped functional determinants by showing a conserved core glutamate is required for hexamer assembly while the N-terminal substrate-binding region is essential for viability under all conditions.","evidence":"Site-directed mutagenesis, in vitro assembly assays, and in vivo complementation in a MET3-TIM10 strain","pmids":["17618651"],"confidence":"High","gaps":["Atomic basis of the glutamate contact not yet visualized","How the N-terminal region contacts substrate not defined"]},{"year":2007,"claim":"Dissected the assembly pathway, showing Tim9 dimerizes while Tim10 is monomeric and that the hexamer forms through tetrameric intermediates with N-terminal helices assembling before C-terminal helices.","evidence":"Stopped-flow fluorescence/light scattering with tryptophan mutagenesis and analytical ultracentrifugation","pmids":["18022191"],"confidence":"Medium","gaps":["Intermediates inferred in vitro and not validated in vivo","Single-lab biophysical observation"]},{"year":2008,"claim":"Provided the atomic architecture of the Tim9-Tim10 hexamer, revealing tentacle helices, disulfide-flanked central loops, and inter-subunit salt bridges whose mutation destabilizes the complex and blocks import.","evidence":"X-ray crystallography at 2.5 Å with mutagenesis and functional import assays","pmids":["19037098"],"confidence":"High","gaps":["No structure of a substrate-bound complex","Dynamics of the tentacles not captured by the static structure"]},{"year":2008,"claim":"Characterized the metal-binding mechanism, defining two-step zinc binding with sub-nanomolar affinity and showing oxidized Tim10 cannot bind zinc, linking redox state to metal occupancy.","evidence":"CD, fluorescence and stopped-flow spectrometry with chelator competition (idx 8); stopped-flow kinetics under varied pH and salt (idx 9)","pmids":["17963238","18462749"],"confidence":"Medium","gaps":["Cooperativity and allostery inferred from in vitro kinetics only","Physiological relevance of measured affinities not tested in vivo"]},{"year":2011,"claim":"Linked hydrophobic-residue dynamics within the complex to its chaperone function, suggesting multiple functional conformational states exist at equilibrium for substrate binding.","evidence":"Temperature-dependent biochemical assays and molecular dynamics simulation with energy decomposition","pmids":["22095685"],"confidence":"Low","gaps":["Computationally driven with no mutagenesis validation","Proposed conformational states not directly observed"]},{"year":2009,"claim":"Identified a transferable nine-residue IMS sorting signal in the Tim10 precursor sufficient for Mia40 engagement and outer-membrane transfer, defining the import targeting element.","evidence":"Deletion and chimeric-construct analysis with in organello and in vitro import assays","pmids":["19297525"],"confidence":"High","gaps":["Structural details of signal-Mia40 recognition not resolved","Generality of the signal across small Tim proteins not established"]},{"year":2015,"claim":"Revealed a quality-control axis in which Tim9 protects Tim10 from Yme1-mediated degradation through complex assembly, with loss of Tim9's inner disulfide triggering Yme1-dependent turnover of both subunits.","evidence":"Yeast genetics (ts mutants, YME1 deletion epistasis) with biochemical stability and abundance assays","pmids":["26182355"],"confidence":"Medium","gaps":["Molecular basis of Yme1 recognition not defined","Single-lab observation of degradation suppression"]},{"year":2025,"claim":"Defined the determinants of Tim10 turnover by showing Yme1 preferentially binds Tim10 via its N-terminal tentacle independent of disulfide status, with unfolding exposing additional contacts that commit it to degradation.","evidence":"Binding and degradation assays with disulfide-bond variants (preprint)","pmids":["bio_10.1101_2025.07.23.666395"],"confidence":"Medium","gaps":["Not yet peer-reviewed","In vivo relevance of tentacle-mediated Yme1 engagement not confirmed"]},{"year":null,"claim":"How the Tim9-Tim10 chaperone physically engages and releases diverse hydrophobic carrier substrates during transit, and how this is coordinated with handoff to Tim22, remains structurally undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No substrate-bound structure of the complex","Mechanism of substrate handoff to Tim22 not resolved","Human TIMM10 in vivo studies absent from this corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,1,6,11]}],"localization":[],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,4]}],"complexes":["TIM9-TIM10 hexameric chaperone complex","TIM22 carrier translocase"],"partners":["TIMM9","TIMM12","TIMM22","MIA40","YME1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P62072","full_name":"Mitochondrial import inner membrane translocase subunit Tim10","aliases":[],"length_aa":90,"mass_kda":10.3,"function":"Mitochondrial intermembrane chaperone that participates in the import and insertion of multi-pass transmembrane proteins into the mitochondrial inner membrane. May also be required for the transfer of beta-barrel precursors from the TOM complex to the sorting and assembly machinery (SAM complex) of the outer membrane. Acts as a chaperone-like protein that protects the hydrophobic precursors from aggregation and guide them through the mitochondrial intermembrane space","subcellular_location":"Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/P62072/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TIMM10","classification":"Common Essential","n_dependent_lines":1161,"n_total_lines":1208,"dependency_fraction":0.9610927152317881},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TIMM10","total_profiled":1310},"omim":[{"mim_id":"608109","title":"PSEUDOURIDINE SYNTHASE 1; PUS1","url":"https://www.omim.org/entry/608109"},{"mim_id":"602251","title":"TRANSLOCASE OF INNER MITOCHONDRIAL MEMBRANE 10; TIMM10","url":"https://www.omim.org/entry/602251"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Mitochondria","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TIMM10"},"hgnc":{"alias_symbol":["TIM10","TIM10A","TIMM10A"],"prev_symbol":[]},"alphafold":{"accession":"P62072","domains":[{"cath_id":"1.10.287.810","chopping":"2-36_45-70","consensus_level":"high","plddt":96.6238,"start":2,"end":70}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P62072","model_url":"https://alphafold.ebi.ac.uk/files/AF-P62072-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P62072-F1-predicted_aligned_error_v6.png","plddt_mean":93.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TIMM10","jax_strain_url":"https://www.jax.org/strain/search?query=TIMM10"},"sequence":{"accession":"P62072","fasta_url":"https://rest.uniprot.org/uniprotkb/P62072.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P62072/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P62072"}},"corpus_meta":[{"pmid":"9495346","id":"PMC_9495346","title":"Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5.","date":"1998","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/9495346","citation_count":250,"is_preprint":false},{"pmid":"19297525","id":"PMC_19297525","title":"Identification of the signal directing Tim9 and Tim10 into the intermembrane space of mitochondria.","date":"2009","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/19297525","citation_count":146,"is_preprint":false},{"pmid":"14973127","id":"PMC_14973127","title":"Functional TIM10 chaperone assembly is redox-regulated in vivo.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14973127","citation_count":105,"is_preprint":false},{"pmid":"17553782","id":"PMC_17553782","title":"Biogenesis of the essential Tim9-Tim10 chaperone complex of mitochondria: site-specific recognition of cysteine residues by the intermembrane space receptor Mia40.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17553782","citation_count":74,"is_preprint":false},{"pmid":"19037098","id":"PMC_19037098","title":"Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import.","date":"2008","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/19037098","citation_count":64,"is_preprint":false},{"pmid":"16199054","id":"PMC_16199054","title":"Zinc binding stabilizes mitochondrial Tim10 in a reduced and import-competent state kinetically.","date":"2005","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16199054","citation_count":50,"is_preprint":false},{"pmid":"11483513","id":"PMC_11483513","title":"Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex.","date":"2001","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11483513","citation_count":50,"is_preprint":false},{"pmid":"26182355","id":"PMC_26182355","title":"Mitochondrial Tim9 protects Tim10 from degradation by the protease Yme1.","date":"2015","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/26182355","citation_count":23,"is_preprint":false},{"pmid":"18022191","id":"PMC_18022191","title":"Assembly of the mitochondrial Tim9-Tim10 complex: a multi-step reaction with novel intermediates.","date":"2007","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18022191","citation_count":22,"is_preprint":false},{"pmid":"17618651","id":"PMC_17618651","title":"Mutation of conserved charged residues in mitochondrial TIM10 subunits precludes TIM10 complex assembly, but does not abolish growth of yeast cells.","date":"2007","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17618651","citation_count":16,"is_preprint":false},{"pmid":"17963238","id":"PMC_17963238","title":"Zinc binding of Tim10: evidence for existence of an unstructured binding intermediate for a zinc finger protein.","date":"2008","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/17963238","citation_count":15,"is_preprint":false},{"pmid":"18462749","id":"PMC_18462749","title":"Allosteric and electrostatic protein-protein interactions regulate the assembly of the heterohexameric Tim9-Tim10 complex.","date":"2008","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18462749","citation_count":10,"is_preprint":false},{"pmid":"22095685","id":"PMC_22095685","title":"Temperature-dependent study reveals that dynamics of hydrophobic residues plays an important functional role in the mitochondrial Tim9-Tim10 complex.","date":"2011","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/22095685","citation_count":4,"is_preprint":false},{"pmid":"10806421","id":"PMC_10806421","title":"Isolation and characterization of the TIM10 homologue from the yeast Pichia sorbitophila: a putative component of the mitochondrial protein import system.","date":"2000","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/10806421","citation_count":3,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.23.666395","title":"Recognition of small Tim chaperones by the mitochondrial Yme1 protease","date":"2025-07-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.23.666395","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8351,"output_tokens":3350,"usd":0.037651,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11023,"output_tokens":3949,"usd":0.07692,"stage2_stop_reason":"end_turn"},"total_usd":0.114571,"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\": 1998,\n      \"finding\": \"Tim10 (with Tim12) localizes to the mitochondrial intermembrane space and interacts sequentially with carrier family precursor proteins to facilitate their translocation across the outer membrane in a membrane-potential-independent manner; Tim10 and Tim12 are found in a complex with Tim22, which mediates membrane-potential-dependent insertion into the inner membrane. Both Tim10 and Tim12 contain a zinc-finger-like motif with four cysteines and bind equimolar zinc ions; interaction with precursors depends on divalent metal ions.\",\n      \"method\": \"Biochemical fractionation, co-immunoprecipitation, reconstitution of import in yeast\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution of import pathway, co-IP, fractionation, replicated by multiple subsequent studies\",\n      \"pmids\": [\"9495346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The TIM10 complex is composed exclusively of Tim9 and Tim10 (no other mitochondrial protein is required for complex formation); reconstituted recombinant Tim9-Tim10 complex restores ADP/ATP carrier import across the outer membrane and accurate inner membrane insertion in tim10-ts mitochondria lacking endogenous Tim10.\",\n      \"method\": \"Functional reconstitution using E. coli-expressed recombinant proteins, import assay in tim10-ts mitochondria\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus functional complementation assay with defined mutant background\",\n      \"pmids\": [\"11483513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TIM10 assembly is redox-regulated: subunits are imported in a cysteine-reduced, unfolded state, then undergo intramolecular disulfide bonding (between their four conserved cysteines) to a zinc-devoid, assembly-competent structure, and finally assemble into the functional hexameric complex. Intramolecular disulfides form in vivo; intermolecular disulfides observed in vitro are abortive intermediates.\",\n      \"method\": \"Biochemical assays (redox trapping, import assays, mutagenesis of cysteines), in vivo labeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal biochemical methods plus mutagenesis in a single focused study\",\n      \"pmids\": [\"14973127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Zinc binding stabilizes reduced Tim10 and slows oxidative folding by more than tenfold, maintaining it in an import-competent state in the cytosol; once imported, zinc must be released to permit oxidative folding and assembly. Oxidized (disulfide-bonded) Tim10 cannot be further reduced by glutathione, while reduced Tim10 is rapidly oxidized by oxidized glutathione at physiological concentrations. Protein disulfide isomerase can catalyze oxidative folding of Tim10 only after zinc removal.\",\n      \"method\": \"In vitro biochemical and biophysical assays (redox potential measurement, CD spectroscopy, fluorescence, glutathione redox assays)\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal in vitro methods (CD, fluorescence, redox potential measurement) in a single focused study\",\n      \"pmids\": [\"16199054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Mia40 acts as a site-specific receptor for Tim10 biogenesis: only the most amino-terminal cysteine residue of Tim10 is critical for translocation across the outer membrane and interaction with Mia40, whereas all four cysteines are required for assembly of the Tim9-Tim10 chaperone complex.\",\n      \"method\": \"Systematic cysteine mutagenesis, in organello and in vitro import assays, co-immunoprecipitation with Mia40\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — systematic mutagenesis combined with in organello and in vitro assays in one study\",\n      \"pmids\": [\"17553782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A conserved glutamate residue in the central core domain of Tim10 (within the CX3C motif region) is required for assembly of the hexameric TIM10 complex; mutations abolishing complex assembly are lethal on non-fermentable carbon sources but allow growth on glucose. The N-terminal substrate-binding region of Tim10 is essential for cell viability under all conditions.\",\n      \"method\": \"Site-directed mutagenesis, in vitro complex assembly assays, in vivo complementation using MET3-TIM10 strain\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis coupled with in vitro assembly and in vivo complementation in a focused study\",\n      \"pmids\": [\"17618651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The crystal structure of the yeast Tim9-Tim10 hexameric complex was determined to 2.5 Å; each subunit contains a central loop flanked by disulfide bonds with N- and C-terminal tentacle-like helices. Buried salt bridges between conserved lysine and glutamate residues connect alternating subunits; mutation of these residues destabilizes the complex and causes defective precursor import. The N-terminal region of Tim9 is required for efficient trapping of incoming substrates into the IMS.\",\n      \"method\": \"X-ray crystallography (2.5 Å), site-directed mutagenesis, yeast growth assays, in vitro import assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and functional import assays in one study\",\n      \"pmids\": [\"19037098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The Tim9-Tim10 complex assembles via a multi-step pathway with transient tetrameric intermediates before the final hexamer is formed; Tim9 forms a homodimer while Tim10 is a monomer. The N-terminal helices of subunits are assembled before the C-terminal helices during complex formation.\",\n      \"method\": \"Stopped-flow fluorescence with tryptophan mutagenesis, stopped-flow light scattering, analytical ultracentrifugation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple biophysical methods in vitro but single lab, no in vivo validation of intermediates\",\n      \"pmids\": [\"18022191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Tim10 zinc binding proceeds via a two-step mechanism: an initial selective binding of Zn2+ to cysteine residues forming a structurally unfolded intermediate, followed by folding upon higher zinc concentrations. Zinc-binding affinity of Tim10 is ~8×10^-10 M. Oxidized (disulfide-bonded) Tim10 cannot bind zinc.\",\n      \"method\": \"Circular dichroism, fluorescence spectrometry, stopped-flow fluorescence, metal chelator competition assays\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal biophysical methods in vitro, single lab\",\n      \"pmids\": [\"17963238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Assembly of the Tim9-Tim10 complex is driven by electrostatic interactions (initial driving force, salt and pH dependence matching subunit isoelectric points) and is also regulated allosterically; Tim10 displays sigmoidal concentration dependence suggesting cooperativity, while Tim9 shows linear dependence.\",\n      \"method\": \"Stopped-flow kinetics with mutagenesis, pH and salt concentration variation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — stopped-flow kinetics with mutagenesis, single lab\",\n      \"pmids\": [\"18462749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A nine-amino-acid region within the Tim10 precursor (the IMS sorting signal) is sufficient for engagement with the Mia40 receptor and for transfer of proteins across the outer membrane to the IMS.\",\n      \"method\": \"Mutagenesis/deletion analysis, in organello and in vitro import assays, chimeric protein constructs\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — deletion and chimeric construct analysis with in organello and in vitro import assays in a focused study\",\n      \"pmids\": [\"19297525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Dynamics of hydrophobic residues in the Tim9-Tim10 complex regulates its chaperone function; temperature-dependent conformational changes mimicking biological substrate-binding activity correspond to disruption of hydrophobic interactions, suggesting different functional conformational states exist at equilibrium.\",\n      \"method\": \"Temperature-dependent biochemical assays, substrate binding measurements, molecular dynamics simulation with energy decomposition analysis\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, computational MD combined with limited biochemical assays, no mutagenesis validation\",\n      \"pmids\": [\"22095685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Tim9 protects Tim10 from degradation by the mitochondrial i-AAA protease Yme1 by assembling into the Tim9-Tim10 complex; loss of Tim9's inner disulfide bond leads to degradation of both Tim9 and Tim10, and this is suppressed by deletion of YME1. Tim10 (rather than the hexameric complex) is proposed as the primary functional unit.\",\n      \"method\": \"Yeast genetics (temperature-sensitive mutants, YME1 deletion), biochemical and biophysical methods (complex stability, protein levels)\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (YME1 deletion suppression) plus biochemical validation, single lab\",\n      \"pmids\": [\"26182355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Yme1 protease preferentially binds Tim10 over other small Tim proteins via a high-affinity interaction mediated primarily by Tim10's flexible N-terminal tentacle region, irrespective of disulfide bond status; substrate unfolding (disruption of disulfide bonds) exposes additional contact sites that enhance engagement and commit Tim10 to degradation. Yme1 also binds assembled Tim9-Tim10 complex independently of the Tim10 N-terminal tentacle.\",\n      \"method\": \"Biochemical and biophysical approaches (binding assays, degradation assays), analysis of disulfide bond variants\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical/biophysical methods in a focused preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.07.23.666395\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TIMM10 (Tim10) is an IMS-localized small Tim protein that is imported in a reduced, zinc-stabilized state via a 9-residue IMS-sorting signal recognized by Mia40 (which engages specifically the first cysteine of Tim10); after import, zinc is released, intramolecular disulfide bonds form between the four conserved CX3C cysteines, and Tim10 assembles with Tim9 through electrostatic and allosteric interactions into a heterohexameric (3×Tim9 + 3×Tim10) chaperone complex that chaperoning hydrophobic carrier precursors (including ADP/ATP carrier) across the IMS for Tim22-mediated insertion into the inner membrane, with Tim10's N-terminal tentacle being essential for substrate recognition and Tim9 serving to protect Tim10 from Yme1-mediated degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TIMM10 (Tim10) is a small intermembrane-space (IMS) chaperone of mitochondria that, together with Tim9, escorts hydrophobic carrier-family precursor proteins across the IMS toward Tim22-mediated insertion into the inner membrane [#0, #1]. Tim10 and Tim9 assemble exclusively with one another into the functional chaperone; recombinant Tim9-Tim10 complex alone is sufficient to restore ADP/ATP carrier import and inner-membrane insertion in mitochondria lacking endogenous Tim10 [#1]. The crystal structure reveals a heterohexamer in which each subunit contributes a central loop flanked by disulfide bonds and N- and C-terminal tentacle-like helices, with buried salt bridges between conserved lysine and glutamate residues stapling alternating subunits; mutation of these salt-bridge residues, or of a conserved core glutamate, destabilizes the complex and impairs precursor import [#5, #6]. Tim10's N-terminal substrate-binding tentacle is essential for cell viability and substrate recognition [#5]. Biogenesis of Tim10 is redox- and metal-regulated: it is held in a reduced, zinc-stabilized, import-competent state that resists oxidative folding, and is delivered across the outer membrane through a nine-residue IMS sorting signal recognized by the Mia40 receptor, which engages specifically the most N-terminal cysteine [#3, #4, #10]. Following import, zinc is released and intramolecular disulfide bonds form between the four conserved cysteines to generate the assembly-competent, oxidatively folded subunit [#2, #3]. Within the complex, Tim9 protects Tim10 from degradation by the i-AAA protease Yme1 [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that Tim10 functions in the IMS to chaperone carrier precursors across the outer membrane and hand them to the Tim22 inner-membrane insertion machinery, defining the carrier import pathway.\",\n      \"evidence\": \"Biochemical fractionation, co-immunoprecipitation, and reconstitution of import in yeast mitochondria\",\n      \"pmids\": [\"9495346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the minimal complement of subunits forming the functional chaperone\", \"Structural basis of substrate recognition unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolved which subunits constitute the functional chaperone by showing the complex is composed exclusively of Tim9 and Tim10 and that recombinant Tim9-Tim10 alone restores carrier import.\",\n      \"evidence\": \"Functional reconstitution with E. coli-expressed proteins and import assay in tim10-ts mitochondria\",\n      \"pmids\": [\"11483513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the complex not yet determined\", \"Mechanism of substrate engagement not addressed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed Tim10 assembly is redox-controlled, proceeding from a reduced unfolded import substrate to an intramolecularly disulfide-bonded, zinc-devoid, assembly-competent form, distinguishing productive from abortive disulfide states.\",\n      \"evidence\": \"Redox trapping, import assays, cysteine mutagenesis, and in vivo labeling\",\n      \"pmids\": [\"14973127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalyst driving in vivo oxidation not identified\", \"Role of zinc in maintaining import competence not yet quantified\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined the role of zinc as a kinetic brake that stabilizes reduced Tim10 and slows oxidative folding, coupling cytosolic metal binding to import competence and post-import folding upon zinc release.\",\n      \"evidence\": \"In vitro redox potential measurement, CD, fluorescence, and glutathione redox assays\",\n      \"pmids\": [\"16199054\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo timing of zinc release not directly observed\", \"Identity of the cellular zinc donor/acceptor not established\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified Mia40 as a site-specific receptor that recognizes only the most N-terminal cysteine of Tim10 for outer-membrane translocation, separating the import-recognition determinant from the four-cysteine assembly requirement.\",\n      \"evidence\": \"Systematic cysteine mutagenesis with in organello and in vitro import and Mia40 co-immunoprecipitation\",\n      \"pmids\": [\"17553782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the Mia40-Tim10 import intermediate not resolved\", \"Sequence context beyond the cysteine not yet defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapped functional determinants by showing a conserved core glutamate is required for hexamer assembly while the N-terminal substrate-binding region is essential for viability under all conditions.\",\n      \"evidence\": \"Site-directed mutagenesis, in vitro assembly assays, and in vivo complementation in a MET3-TIM10 strain\",\n      \"pmids\": [\"17618651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic basis of the glutamate contact not yet visualized\", \"How the N-terminal region contacts substrate not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Dissected the assembly pathway, showing Tim9 dimerizes while Tim10 is monomeric and that the hexamer forms through tetrameric intermediates with N-terminal helices assembling before C-terminal helices.\",\n      \"evidence\": \"Stopped-flow fluorescence/light scattering with tryptophan mutagenesis and analytical ultracentrifugation\",\n      \"pmids\": [\"18022191\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Intermediates inferred in vitro and not validated in vivo\", \"Single-lab biophysical observation\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Provided the atomic architecture of the Tim9-Tim10 hexamer, revealing tentacle helices, disulfide-flanked central loops, and inter-subunit salt bridges whose mutation destabilizes the complex and blocks import.\",\n      \"evidence\": \"X-ray crystallography at 2.5 \\u00c5 with mutagenesis and functional import assays\",\n      \"pmids\": [\"19037098\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of a substrate-bound complex\", \"Dynamics of the tentacles not captured by the static structure\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Characterized the metal-binding mechanism, defining two-step zinc binding with sub-nanomolar affinity and showing oxidized Tim10 cannot bind zinc, linking redox state to metal occupancy.\",\n      \"evidence\": \"CD, fluorescence and stopped-flow spectrometry with chelator competition (idx 8); stopped-flow kinetics under varied pH and salt (idx 9)\",\n      \"pmids\": [\"17963238\", \"18462749\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cooperativity and allostery inferred from in vitro kinetics only\", \"Physiological relevance of measured affinities not tested in vivo\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linked hydrophobic-residue dynamics within the complex to its chaperone function, suggesting multiple functional conformational states exist at equilibrium for substrate binding.\",\n      \"evidence\": \"Temperature-dependent biochemical assays and molecular dynamics simulation with energy decomposition\",\n      \"pmids\": [\"22095685\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Computationally driven with no mutagenesis validation\", \"Proposed conformational states not directly observed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified a transferable nine-residue IMS sorting signal in the Tim10 precursor sufficient for Mia40 engagement and outer-membrane transfer, defining the import targeting element.\",\n      \"evidence\": \"Deletion and chimeric-construct analysis with in organello and in vitro import assays\",\n      \"pmids\": [\"19297525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural details of signal-Mia40 recognition not resolved\", \"Generality of the signal across small Tim proteins not established\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed a quality-control axis in which Tim9 protects Tim10 from Yme1-mediated degradation through complex assembly, with loss of Tim9's inner disulfide triggering Yme1-dependent turnover of both subunits.\",\n      \"evidence\": \"Yeast genetics (ts mutants, YME1 deletion epistasis) with biochemical stability and abundance assays\",\n      \"pmids\": [\"26182355\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of Yme1 recognition not defined\", \"Single-lab observation of degradation suppression\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined the determinants of Tim10 turnover by showing Yme1 preferentially binds Tim10 via its N-terminal tentacle independent of disulfide status, with unfolding exposing additional contacts that commit it to degradation.\",\n      \"evidence\": \"Binding and degradation assays with disulfide-bond variants (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.07.23.666395\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not yet peer-reviewed\", \"In vivo relevance of tentacle-mediated Yme1 engagement not confirmed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the Tim9-Tim10 chaperone physically engages and releases diverse hydrophobic carrier substrates during transit, and how this is coordinated with handoff to Tim22, remains structurally undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrate-bound structure of the complex\", \"Mechanism of substrate handoff to Tim22 not resolved\", \"Human TIMM10 in vivo studies absent from this corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 1, 6, 11]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": []}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005758\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"complexes\": [\"TIM9-TIM10 hexameric chaperone complex\", \"TIM22 carrier translocase\"],\n    \"partners\": [\"TIMM9\", \"TIMM12\", \"TIMM22\", \"MIA40\", \"YME1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}