{"gene":"TIMM23","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1996,"finding":"Tim23 forms dimers promoted by membrane potential (ΔΨ); binding of a matrix targeting sequence to Tim23 triggers dimer dissociation. Dimerization depends on a heptad leucine repeat motif in the N-terminal hydrophilic domain (IMS-exposed). Monomeric Tim23 is present when a preprotein is in transit, suggesting Tim23 dimers act as receptors for matrix targeting sequences and dimer dissociation triggers TIM channel opening.","method":"Biochemical dimerization assays, membrane potential manipulation, preprotein binding experiments, mutational analysis of leucine repeat motif","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal biochemical methods, foundational study replicated conceptually by many subsequent papers","pmids":["8858146"],"is_preprint":false},{"year":2000,"finding":"Tim23 has a two-membrane-spanning topology: its C-terminal domain is anchored in the inner membrane, an intermediate domain is exposed in the IMS as a presequence receptor, and the N-terminal domain is exposed on the surface of the outer mitochondrial membrane. This simultaneous integration into two membranes forms contact sites between outer and inner membranes, facilitating transfer of precursors from TOM to TIM23.","method":"Protease accessibility assays, GFP fusion topology mapping, import competition, electron microscopy","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods in a single rigorous study, replicated by subsequent work","pmids":["10830167"],"is_preprint":false},{"year":2000,"finding":"The TIM8-Tim13 complex in the IMS interacts with translocation intermediates of Tim23 (partially translocated across the outer membrane), binding to the N-terminal or intermediate domain of Tim23 to prevent retrograde translocation. The TIM8-13 complex is required for Tim23 import under low membrane potential conditions.","method":"Co-immunoprecipitation, import assays with TIM8-13 deletion/depletion strains, crosslinking of translocation intermediates","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding, functional import assays, loss-of-function with defined phenotype, replicated by crystal structure paper","pmids":["11101512"],"is_preprint":false},{"year":1997,"finding":"Tim23 is required for normal activity of the multiple conductance channel (MCC) of the mitochondrial inner membrane. Anti-Tim23 antibodies that inhibit protein import also inhibit MCC activity; the tim23-1 mutation alters MCC conductance and abolishes presequence-peptide blockage of MCC, identifying Tim23 as essential for the protein-conducting pore activity.","method":"Electrophysiology (patch clamp), antibody inhibition, yeast tim23-1 mutant analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — electrophysiology with mutagenesis and antibody blocking, single lab but multiple orthogonal methods","pmids":["9128249"],"is_preprint":false},{"year":2002,"finding":"Tim50 is a subunit of the TIM23 complex anchored to the inner membrane with its C-terminal domain exposed to the IMS. Tim50 interacts with the N-terminal IMS domain of Tim23. Tim50 depletion or antibody addition blocks protein translocation across the inner membrane. A translocation intermediate at TOM is crosslinked to Tim50, establishing Tim50 as a component that mediates transfer of translocating proteins from TOM to TIM23.","method":"Site-specific photocrosslinking, co-immunoprecipitation, protein depletion, antibody inhibition assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — site-specific crosslinking, reciprocal co-IP, depletion + antibody phenotypes, replicated by multiple subsequent studies","pmids":["12437925"],"is_preprint":false},{"year":2003,"finding":"Tim14 (Pam18) is an integral inner membrane protein with a J-domain exposed to the matrix. It is an essential component of the TIM23 import motor, interacting with Tim44 and mtHsp70 in an ATP-dependent manner. A mutation in the HPD motif of the J-domain is lethal, establishing Tim14 as the J-protein co-chaperone that activates mtHsp70 ATPase activity within the TIM23 motor.","method":"Gene depletion, import assays, co-immunoprecipitation, HPD motif mutagenesis, ATP-dependence studies","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis of catalytic motif, co-IP with motor components, loss-of-function phenotype, independently replicated","pmids":["14517234"],"is_preprint":false},{"year":2004,"finding":"Tim16 (Pam16) is a novel J-domain-related cochaperone that forms a stable subcomplex with Tim14 within the TIM23 translocase. Depletion of Tim16 markedly impairs matrix protein import and disrupts Tim14 interaction with the TIM23 complex, leading to severe structural changes of the import motor.","method":"Protein identification, co-immunoprecipitation, gene depletion, import assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, depletion phenotype, replicated by subsequent association studies","pmids":["14981506"],"is_preprint":false},{"year":1998,"finding":"Tim23 contains two independent import signals: one in the first 62 residues of the N-terminal hydrophilic domain mediating ΔΨ-independent outer membrane translocation, and a second in the C-terminal membrane-integrated domain mediating ΔΨ-dependent translocation and inner membrane insertion. Import of Tim23 into the inner membrane requires Tim22 (not functional Tim23), establishing that TIM23 biogenesis depends on the TIM22 complex.","method":"Deletion mutagenesis, import assays with ΔΨ manipulation, genetic epistasis with Tim22/Tim23 depletion strains","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic deletion mutagenesis, epistasis, multiple orthogonal import assays","pmids":["9501078"],"is_preprint":false},{"year":2006,"finding":"Tim17p regulates twin-pore structure and voltage gating of TIM23. Patch clamp of reconstituted inner membranes showed TIM23 has twin pores that cooperatively gate. Tim17p depletion collapses the twin pores into a single pore; N-terminal deletion or C-terminal truncation of Tim17p produces variable-sized pores. Tim17p N-terminus is vital for voltage sensing and protein sorting, while Tim23p is the main structural unit of the pore.","method":"Patch clamp electrophysiology of reconstituted inner membranes, Tim17p deletion and truncation mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro electrophysiology reconstitution with systematic mutagenesis, single lab","pmids":["17148445"],"is_preprint":false},{"year":2008,"finding":"Fluorescence mapping using environment-sensitive probes positioned along transmembrane segment 2 (TMS2) of Tim23 revealed an amphipathic alpha-helix in the protein-conducting channel region. Probes on the aqueous-facing helical surface underwent spectral changes during active protein import, and their accessibility to hydrophilic quenchers changes with channel gating, defining the channel-lining face of Tim23 TMS2.","method":"Site-directed fluorescence labeling, multiple spectral analyses (polarization, intensity, lifetime), hydrophilic quenching in functional intact mitochondria","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in situ fluorescence mapping with multiple spectral techniques in functional mitochondria, functionally validated","pmids":["18692467"],"is_preprint":false},{"year":2008,"finding":"The Tim8-Tim13 complex assembles as a hexamer and binds Tim23 cooperatively with ~6 binding sites, exhibiting positive cooperativity. The crystal structure (2.6 Å) reveals tentacle-like helices with hydrophobic pockets that interact with Tim23 transmembrane helices, shielding them during chaperoned translocation across the IMS.","method":"X-ray crystallography (2.6 Å), surface plasmon resonance binding kinetics","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with quantitative binding measurements, single lab","pmids":["18706423"],"is_preprint":false},{"year":2008,"finding":"Tim23 cross-links to Tim17 at its first transmembrane segment, to Tim50 at the C-terminal end of its hydrophilic IMS region, and Tim23 IMS domains cross-link to each other (dimerization). These proximity relationships reversibly change upon membrane potential alterations and when a translocating substrate is trapped, demonstrating dynamic conformational rearrangements of the TIM23 quaternary structure.","method":"Site-specific chemical cross-linking of radiolabeled Tim23 monocysteine mutants imported into functional mitochondria","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — systematic cross-linking at defined positions with functional correlation, single lab","pmids":["17959826"],"is_preprint":false},{"year":2008,"finding":"Yeast Aac2 (ADP/ATP carrier) physically associates with both the cytochrome bc1-COX supercomplex and the TIM23 machinery. Affinity purification with His-tagged Aac2 co-purifies TIM23 components. This association can occur independently of the fully assembled cytochrome bc1-COX supercomplex.","method":"Affinity purification (His-tag), co-immunoprecipitation, blue native gel electrophoresis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — affinity purification and co-IP from single lab, functional consequence of TIM23-Aac2 association not directly tested","pmids":["18614795"],"is_preprint":false},{"year":2009,"finding":"Tim23-Tim50 interactions in the IMS facilitate both transfer of precursor proteins from TOM40 to TIM23 and a late step of translocation across the inner membrane by promoting mtHsp70 motor functions. The Tim23-Tim50 pair thus coordinates actions of TOM40 and TIM23 complexes together with the import motor.","method":"Genetic analysis of Tim23/Tim50 IMS domain mutants, co-immunoprecipitation, import assays, crosslinking","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (genetics, co-IP, crosslinking, import assays) in single lab","pmids":["19139266"],"is_preprint":false},{"year":2008,"finding":"In vitro reconstitution of the Tim23-Tim50 IMS domain interaction using purified recombinant proteins. Chemical cross-linking and surface plasmon resonance defined the interaction. Mutations in Tim23 that abolish Tim50 binding in vitro also destabilize the interaction in vivo, causing defective protein import and temperature-sensitive lethality.","method":"In vitro reconstitution with purified recombinant proteins, chemical cross-linking, surface plasmon resonance, in vivo import assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution plus mutagenesis plus in vivo validation, single lab","pmids":["19017642"],"is_preprint":false},{"year":2010,"finding":"The IMS domain of Tim23 (residues 1-96, Tim23N) is an intrinsically disordered protein. NMR titrations with two presequences identified a distinct presequence-binding region formed by residues 71-84, making Tim23N the only IMS-exposed domain of TOM/TIM23 predicted to be intrinsically disordered.","method":"NMR spectroscopy, NMR titrations with presequence peptides, charge-hydropathy analysis","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure determination with functional binding mapping, single lab","pmids":["20718036"],"is_preprint":false},{"year":2011,"finding":"Tim50's presequence-binding domain was mapped by photo-affinity labeling with engineered presequence probes and mass spectrometric identification of cross-linking sites. Tim50 is established as the primary presequence receptor at the inner membrane. Targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner: presequences promote channel opening while Tim50 keeps the channel closed.","method":"Photo-affinity labeling, mass spectrometric mapping, in vitro import assays, electrophysiology","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — photo-crosslinking with MS mapping combined with electrophysiology, single lab","pmids":["22065641"],"is_preprint":false},{"year":2011,"finding":"Purified Tim50IMS and Tim44 directly bind presequence peptides (Hsp60, mHsp70, cytochrome P450SCC presequences), demonstrated by chemical cross-linking and surface plasmon resonance with defined KD values. Tim23IMS binding to presequences was also characterized. These stronger interactions on the trans side of the channel support directional import.","method":"Chemical cross-linking of purified recombinant proteins, surface plasmon resonance with KD determination","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified components, quantitative binding measurements, single lab","pmids":["21969381"],"is_preprint":false},{"year":2011,"finding":"TIM23-mediated membrane insertion of transmembrane alpha-helices into the mitochondrial inner membrane depends strongly on hydrophobicity and position of polar/aromatic residues, paralleling the ER system, but shows striking differences in the effect of flanking charged residues compared to the ER translocon.","method":"Systematic mutagenesis of model transmembrane segments, in vitro import assays in yeast mitochondria","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with quantitative insertion assays, single lab","pmids":["21326212"],"is_preprint":false},{"year":2013,"finding":"High-resolution fluorescence mapping of TMS2 of Tim23 in energized versus depolarized membranes showed that TMS2 forms a continuous alpha-helix inaccessible to the IMS aqueous phase in energized membranes. Upon depolarization, helical periodicity is disrupted and the channel becomes exposed to the IMS. Kinetic measurements confirmed that TMS2 conformational changes coincide with depolarization.","method":"Site-directed fluorescence labeling in intact yeast membranes, multiple spectroscopic methods, kinetic measurements","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution fluorescence mapping in functional membranes with multiple orthogonal spectral methods, single lab","pmids":["23832274"],"is_preprint":false},{"year":2012,"finding":"Sym1 (yeast ortholog of MPV17), a multispanning inner membrane protein, is imported via TIM23 in a presequence-independent manner using internal targeting signals, rather than via TIM22 as expected for carrier-type proteins. This defines a novel transport mechanism for a polytopic inner membrane protein through TIM23.","method":"Import assays with TIM23/TIM22 depletion, translocation intermediate trapping, protease protection","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — import pathway determination using multiple depletion strains with defined intermediates, single lab","pmids":["23045398"],"is_preprint":false},{"year":2014,"finding":"Molecular basis of TIM23 architecture: Tim23 loop 1 (between TM1 and TM2, matrix-exposed) cross-links to Tim44; alterations in this loop destabilize Tim44 interaction with the translocon. Tim17 loop 1 cross-links to Pam17; alterations destabilize Pam17-translocon interaction. This positions Tim44 and Pam17 as regulatory subunits at distinct translocon interaction sites.","method":"Site-specific in vivo photocross-linking, co-immunoprecipitation, genetic analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — site-specific in vivo photocross-linking with genetic and biochemical validation, single lab","pmids":["25157107"],"is_preprint":false},{"year":2014,"finding":"The IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N-terminus. The membrane-bound Tim23 IMS domain remains highly dynamic. Cardiolipin enhances Tim23 membrane attachment, suggesting cardiolipin influences preprotein import.","method":"NMR spectroscopy with membrane model systems, cardiolipin-containing liposomes","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — NMR with model membranes, single lab, no in vivo functional validation of cardiolipin effect","pmids":["25349212"],"is_preprint":false},{"year":2014,"finding":"NMR characterization of the dynamic interaction network within the TIM23 IMS: Tim23 IMS domain contains multiple sites for interaction with Tim21 IMS domain, and also interacts with Tim50 and Tom22. The atomic details of the Tim23IMS-Tim21IMS complex were determined, establishing Tim23 IMS as a hub for TIM23 complex organization.","method":"NMR spectroscopy, chemical shift perturbation mapping at single-residue level","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR with single-residue resolution mapping of multiple interactions, single lab","pmids":["25263020"],"is_preprint":false},{"year":2015,"finding":"GxxxG motifs in transmembrane segments 1 and 2 of Tim23 are necessary for structural integrity of the TIM23 complex. Mutations of specific glycines in TM1 and TM2 caused lethal or temperature-sensitive phenotypes correlated with TIM23 complex destabilization, without impairing Tim23 import or membrane integration itself.","method":"Systematic mutagenesis of GxxxG motifs, yeast growth assays, blue native gel electrophoresis","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with complex stability readout, single lab","pmids":["25765297"],"is_preprint":false},{"year":2016,"finding":"Tim17 contains a pair of highly conserved cysteine residues forming a structural disulfide bond exposed to the IMS. This disulfide bond is critical for efficient protein translocation through TIM23 and for dynamic gating of the preprotein-conducting channel. The disulfide is formed during Tim17 insertion, dependent on Mia40 binding but not its oxidoreductase activity; direct oxidation by Erv1 is suggested.","method":"Cysteine mutagenesis, electrophysiology (channel gating assays), import assays, redox biochemistry","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with electrophysiology and import assays, single lab","pmids":["27502485"],"is_preprint":false},{"year":2017,"finding":"Both domains of Tim44 interact with the major matrix-exposed loop of Tim23: the C-terminal domain (CTD) anchors Tim44 to the translocon (also binding Tim17), while the N-terminal domain (NTD) is intrinsically disordered and dynamically interacts with presequences near the region important for Hsp70 and Tim23 interaction.","method":"Site-specific in vivo crosslinking, genetic analysis, in vitro binding assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — site-specific in vivo crosslinking with in vitro validation and genetic support, single lab","pmids":["28440746"],"is_preprint":false},{"year":2017,"finding":"Cardiolipin directly modulates interactions between the Tim50 soluble receptor domain and the Tim23 channel. The Tim50 receptor domain interacts with membranes and with specific sites on Tim23 in a cardiolipin-dependent manner. SAXS-based structure of the full soluble Tim50 receptor was determined. Molecular dynamics simulations confirmed cardiolipin-driven Tim50 association with lipid bilayers with concomitant structural changes.","method":"SAXS structure determination, molecular dynamics simulations, biophysical measurements in liposomes, in vivo and isolated mitochondria assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — SAXS structure combined with MD simulations and multiple biophysical validations in reductionist and cellular systems, single lab","pmids":["28879236"],"is_preprint":false},{"year":2017,"finding":"Cation selectivity of the Tim23 channel is essential for protein import. Structure-based mutations of highly conserved pore-lining amino acids reduce selectivity, reduce protein import capacity, and render the Tim23 channel insensitive to substrates, demonstrating that cation selectivity is a key feature for substrate recognition.","method":"Structure-based mutagenesis, electrophysiology (cation selectivity measurements), in vitro import assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structure-guided mutagenesis with electrophysiology and import assays, single lab","pmids":["28857742"],"is_preprint":false},{"year":2018,"finding":"The import motor J-protein Pam18 controls lateral protein release into the lipid bilayer through the TIM23 lateral gate. Constitutively translocase-associated Pam18 obstructs lateral precursor transport and displaces Mgr2 from the translocase, demonstrating that during motor-dependent matrix import, the Pam18 transmembrane segment closes the lateral gate to promote anterograde translocation.","method":"Genetic and biochemical analysis, import assays of matrix-targeted vs stop-transfer substrates, co-immunoprecipitation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic manipulation with defined substrate-specific import phenotypes, single lab","pmids":["30279421"],"is_preprint":false},{"year":2019,"finding":"Mutant huntingtin (mHTT) specifically binds the TIM23 subunit (of the four TIM23 complex proteins) and resides in the mitochondrial intermembrane space. mHTT binding inhibits mitochondrial protein import specifically through TIM23, reducing levels of TIM23-imported soluble matrix proteins in mHTT-expressing cells and HD patient brain tissue.","method":"Co-immunoprecipitation, mitochondrial fractionation, quantitative proteomics, quantitative immunoblotting in cell lines and human HD brain tissue","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, fractionation, quantitative proteomics in multiple model systems plus human tissue","pmids":["31346086"],"is_preprint":false},{"year":2019,"finding":"In the IMS, the Tim23-Tim50 interaction surface is larger than previously thought; an unexpected interaction of Tim23 with Pam17 in the IMS was identified. Mutations of two conserved negatively charged residues of Tim23 near the inner membrane prevented Tim23 dimerization and increased Tim23 surface exposure, while membrane potential dissipation decreased surface exposure.","method":"In vivo mutagenesis, crosslinking, dimerization assays, surface accessibility experiments","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo crosslinking and mutagenesis with multiple readouts, single lab","pmids":["32277989"],"is_preprint":false},{"year":2021,"finding":"Chemical cross-linking combined with mass spectrometry of a stalled TOM-TIM23 supercomplex mapped protein-protein interactions at the IMS interface between TOM and TIM23 at amino acid resolution, including contacts involving Tim23 and Tim50. The import motor interactions were also mapped in this context.","method":"Chemical cross-linking with mass spectrometric analysis, structural modeling, purification of stalled translocation intermediate","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — cross-linking MS with structural modeling of captured translocation intermediate, single lab","pmids":["34588454"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structure of the core TIM23 complex (Tim17-Tim23-Tim44 heterotrimer) from S. cerevisiae was determined. Contrary to the prevailing model, Tim23 and Tim17 do not form a shared water-filled channel; instead, they have separate lipid-exposed concave cavities facing opposite directions. The cavity of Tim17 (not Tim23) forms the protein translocation path; Tim23 has primarily a structural role. Mgr2 seals the lateral opening of the Tim17 cavity to facilitate translocation.","method":"Cryo-electron microscopy structure determination, biochemical validation","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with biochemical validation, revises prevailing model, published in high-impact journal","pmids":["37344598"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of the active TOM-TIM23 supercomplex with translocating substrate revealed that TIM23 contains a heterotrimer of Tim23, Tim17, and Mgr2. Polypeptide substrates are shielded from lipids by Mgr2 and Tim17, with a translocation pathway characterized by a negatively charged entrance and central hydrophobic region. In TOM, the substrate passes through the center of Tom40.","method":"Cryo-electron microscopy of active supercomplex with translocating substrate, biochemical analysis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM of active translocation complex with substrate, single lab","pmids":["37696957"],"is_preprint":false},{"year":2023,"finding":"TIM23 interacts with PINK1 and facilitates PINK1 accumulation upon mitochondrial depolarization by protecting PINK1 from degradation by the OMA1 protease. TIM23 knockdown decreases PINK1 levels and delays autophosphorylation; OMA1 inactivation rescues PINK1 levels when TIM23 is depleted. Pathogenic PINK1 mutants that fail to interact with TIM23 have deficiencies partially restored by OMA1 inactivation.","method":"Mass spectrometric identification of PINK1 co-immunoprecipitates, co-immunoprecipitation, TIM23 knockdown, OMA1 inactivation, PINK1 autophosphorylation assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction validated by co-IP with multiple genetic perturbations, single lab","pmids":["37160114"],"is_preprint":false},{"year":2024,"finding":"PINK1 forms a high-molecular-weight PINK1-TOM-TIM23 supercomplex upon mitochondrial stress in human cultured cell lines, dopamine neurons, and midbrain organoids. PINK1 is required to stably tether TOM to TIM23 complexes in response to stress. This tethering depends on an interaction between the PINK1 N-terminal C-terminal extension module and the cytosolic domain of Tom20; disruption by designer or PD-associated PINK1 mutations inhibits downstream mitophagy.","method":"Co-immunoprecipitation, blue native gel electrophoresis, PINK1 mutagenesis, mitophagy assays in multiple cell models including organoids","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, multiple cell models, PINK1 mutagenesis with functional mitophagy readout","pmids":["38416681"],"is_preprint":false},{"year":2024,"finding":"OCIAD1 and the prohibitin complex regulate the stability of the human TIM23 translocase. Prohibitins stabilize both TIMM17A- and TIMM17B-containing TIM23 variants. OCIAD1 assembles with the prohibitin complex to specifically protect the TIMM17A variant from degradation by the YME1L protease. OCIAD1 expression is regulated by TIM23 complex status.","method":"Co-immunoprecipitation, protease inhibition, siRNA knockdown, quantitative proteomics","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and genetic perturbations with protease identification, single lab","pmids":["39630581"],"is_preprint":false},{"year":2024,"finding":"TIMM50 pathogenic variants reduce levels and activity of the TIM23 complex, and proteomic analysis reveals that laterally released substrates imported via the TIM23SORT pathway are most sensitive to TIMM50 loss. Proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for TIMM50-associated disease.","method":"Quantitative proteomics of patient fibroblasts and HEK293 cell model, import assays, blue native gel electrophoresis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative proteomics with import assays in patient and engineered cells, single lab","pmids":["38828998"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structures of the active TOM-TIM23 supercomplex captured with translocating substrate show that the Tim17-Mgr2 pathway creates the translocation channel with a central restriction formed by four conserved hydrophobic residues. Substrate hydrophobicity modulates Mgr2 association with Tim17, enabling dynamic regulation of protein sorting toward matrix or membrane.","method":"Cryo-electron microscopy of active translocating supercomplex, biochemical validation","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structures with translocating substrate in multiple conformations, biochemical validation, single lab","pmids":["40877479"],"is_preprint":false},{"year":2020,"finding":"Tim23 knockdown in mouse muscle (40% reduction) caused a 32% decrease in matrix-destined protein import and activated the CHOP branch of the UPRmt (increased ClpP and cpn10) without activating the ATF5 arm, demonstrating that reduced TIM23 import capacity triggers a specific branch of the mitochondrial unfolded protein response.","method":"In vivo antisense oligonucleotide knockdown, import assays, UPRmt marker measurements","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockdown with defined import and UPRmt phenotype, single lab","pmids":["29949403"],"is_preprint":false},{"year":2008,"finding":"Tim23 undergoes intra-mitochondrial proteolytic degradation following mitochondrial outer membrane permeabilization (MOMP) in a caspase-independent but Bax/Bak-dependent manner. This degradation is mitochondrion-autonomous (occurs in isolated mitochondria undergoing permeability transition), selective for Tim23, and depletion of Tim23 by siRNA inhibits cell proliferation and prevents long-term survival.","method":"Caspase inhibition, Bax/Bak knockout, isolated mitochondria assays, siRNA knockdown, Tim23-GFP complementation","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic perturbations with complementation, single lab","pmids":["18174902"],"is_preprint":false},{"year":2023,"finding":"NOX4 localizes to the mitochondrial matrix following asbestos exposure in lung macrophages via direct interaction with TIM23. TIM23 augments NOX4-induced mitochondrial ROS and metabolic reprogramming to oxidative phosphorylation. Silencing TIM23 decreased mitochondrial ROS and oxidative phosphorylation. The interaction was localized to the proximal transmembrane region of NOX4.","method":"Co-immunoprecipitation, TIM23 silencing, mitochondrial fractionation, ROS measurement, OXPHOS analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with domain mapping and functional knockdown readouts, single lab","pmids":["37044213"],"is_preprint":false},{"year":2020,"finding":"Mitochondrial ROS induces rapid translocation of APE1 from the IMS into the matrix through the TIM23/PAM machinery complex. The IMS serves as a storage site for APE1 under basal conditions.","method":"Mitochondrial fractionation, import assays, TIM23 knockdown, oxidative stress induction","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — fractionation and TIM23 knockdown with functional import readout, single lab","pmids":["33197464"],"is_preprint":false},{"year":2013,"finding":"TM1 of Tim23 is required for homodimerization; TM1 and TM2 together are involved in preprotein binding within the channel, and TM2 recruits Tim21 and the PAM subcomplex to Tim23. The matrix-exposed loop L1 generates specificity in PAM subcomplex association. The C-terminal sequence of Tim23 functions as an inhibitor of Tim21 binding.","method":"Mutagenesis, co-immunoprecipitation, genetic complementation, import assays in yeast","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with biochemical and functional readouts, single lab","pmids":["24061477"],"is_preprint":false},{"year":2019,"finding":"Deletion of Mgr2 decreases normal TIM23 channel gating frequency and produces a residual channel activity lacking gating transitions but remaining sensitive to import signal peptides. A G145L mutation in Tim23 displaces Mgr2 from the complex leading to the same gating impairment, establishing that Mgr2 regulates TIM23 channel gating behavior.","method":"Patch clamp electrophysiology of yeast inner membranes, Mgr2 deletion and Tim23 point mutation","journal":"Frontiers in physiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — electrophysiology with genetic perturbations, single lab","pmids":["30697167"],"is_preprint":false},{"year":2019,"finding":"Mgr2 controls the threshold hydrophobicity and flanking charge requirements for TIM23-mediated membrane insertion via the stop-transfer mechanism. Deletion of Mgr2 reduces the hydrophobicity threshold required for membrane insertion and reduces the requirement for matrix-facing positive charges, establishing Mgr2 as a lateral gatekeeper that discriminates stop-transfer signal features.","method":"Systematic mutagenesis of model transmembrane segments, membrane insertion assays in Mgr2-deletion and overexpression yeast strains","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with quantitative insertion assays in defined genetic backgrounds, single lab","pmids":["31764998"],"is_preprint":false},{"year":2026,"finding":"PGC-1α promotes binding of NRF1 to the Tim23 promoter, upregulating Tim23 expression, which in turn reduces DRP1 transcription and ACSL4 mitochondrial translocation, thereby inhibiting ferroptosis and MASH. This establishes a PGC-1α-Tim23-DRP1-ACSL4 axis in hepatocyte ferroptosis.","method":"Western blot, RT-qPCR, immunofluorescence, luciferase reporter assays, co-immunoprecipitation, mouse MASH models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and reporter assays with in vivo models, single lab, mechanistic chain not fully reconstituted","pmids":["41724762"],"is_preprint":false},{"year":2025,"finding":"In the absence of TIM23, PINK1 is stabilized in the TOM complex. The MMP (not the PAM import motor) is the primary driving force for PINK1 import through TIM23. Loss of TIM23 is sufficient to activate Parkin, establishing TIM23 as the essential inner membrane translocase that separates PINK1 from TOM as the key damage-sensing switch in the PINK1-Parkin mitophagy pathway.","method":"Genome-wide screens, PINK1 import assays, TIM23 knockout, Parkin activation assays, membrane potential manipulation","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen with genetic validation and import assays, preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"OMA1-mediated degradation of DNAJC15 reduces protein import of OXPHOS-related proteins via the TIMM23-TIMM17A translocase under mitochondrial stress, limiting OXPHOS biogenesis. Loss of DNAJC15 specifically reduces TIMM17A-containing TIM23 complex-dependent import.","method":"Proteomic analysis, import assays, OMA1 and DNAJC15 genetic perturbations, blue native gel electrophoresis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics with genetic perturbations and import assays, preprint not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"TIMM23 (Tim23) is the central scaffold subunit of the mitochondrial TIM23 presequence translocase in the inner membrane, where structural studies reveal it has primarily a structural role while its homolog Tim17 (with Mgr2) forms the actual protein-conducting channel; Tim23 functions as a dynamic IMS-exposed hub that dimerizes in a ΔΨ-dependent manner to act as a presequence receptor, interacts with Tim50 (the primary presequence receptor) and other TOM/TIM23 subunits to coordinate precursor handover from TOM, bridges the outer and inner membranes through its two-membrane-spanning topology, and recruits the mtHsp70-based PAM motor (via Tim44, Tim14/Pam18, and Tim16/Pam16) to drive ATP-dependent translocation into the matrix, while also regulating lateral sorting of stop-transfer substrates into the inner membrane through its association with Mgr2; additionally, Tim23 participates in PINK1 stabilization for mitophagy initiation and is targeted by pathological proteins such as mutant huntingtin that inhibit its import function."},"narrative":{"mechanistic_narrative":"TIMM23 (Tim23) is the central inner-membrane subunit of the mitochondrial TIM23 presequence translocase that imports presequence-bearing precursor proteins from the cytosol into the matrix and inner membrane [PMID:9128249, PMID:37696957]. It adopts a two-membrane-spanning topology in which its C-terminal domain is integrated in the inner membrane while an intrinsically disordered N-terminal/intermembrane-space (IMS) domain reaches across to the outer membrane, forming contact sites that channel precursors from TOM to TIM23 [PMID:10830167, PMID:20718036]. The IMS domain serves as a dynamic organizing hub: it dimerizes in a membrane-potential-dependent manner to act as a presequence receptor, and dimer dissociation upon presequence binding is coupled to channel opening [PMID:8858146, PMID:32277989]. This domain coordinates the translocase by binding Tim50, the primary presequence receptor, as well as Tim21 and Tom22, integrating substrate handover with channel gating in which presequences and Tim50 act antagonistically on the channel [PMID:12437925, PMID:22065641, PMID:25263020]. Conformational rearrangements of the transmembrane segments accompany membrane potential changes and substrate trapping, and pore properties such as cation selectivity are essential for import [PMID:23832274, PMID:28857742]. Tim23 recruits the matrix mtHsp70-based PAM import motor through its matrix-exposed loop, binding Tim44 and the Tim14(Pam18)/Tim16(Pam16) J-protein subcomplex that drives ATP-dependent translocation [PMID:14517234, PMID:14981506, PMID:28440746, PMID:24061477]. Recent cryo-EM structures revise the long-standing channel model: Tim23 plays a primarily structural role within a Tim17–Tim23–Tim44/Mgr2 core, while the protein-conducting path is formed by the Tim17 cavity sealed laterally by Mgr2, which gates lateral release of hydrophobic stop-transfer substrates into the inner membrane [PMID:37344598, PMID:37696957, PMID:40877479]. Beyond constitutive import, TIM23 interacts with PINK1 and protects it from OMA1-mediated degradation, positioning the translocase as a damage-sensing switch in PINK1–Parkin mitophagy [PMID:37160114, PMID:38416681]. The translocase is a target of pathology: mutant huntingtin binds Tim23 and inhibits matrix import [PMID:31346086], and reduced TIM23 import capacity activates the CHOP branch of the mitochondrial unfolded protein response [PMID:29949403].","teleology":[{"year":1996,"claim":"Established that Tim23 is not a static channel but a membrane-potential-regulated presequence receptor, defining the first dynamic gating principle of the translocase.","evidence":"Biochemical dimerization assays with membrane potential manipulation and leucine-repeat mutagenesis in yeast mitochondria","pmids":["8858146"],"confidence":"High","gaps":["Did not resolve the structural basis of dimerization","Coupling between dimer dissociation and physical channel opening inferred indirectly"]},{"year":1997,"claim":"Linked Tim23 directly to the protein-conducting pore activity of the inner membrane, identifying it as essential for the multiple conductance channel.","evidence":"Patch-clamp electrophysiology with antibody inhibition and the tim23-1 mutant","pmids":["9128249"],"confidence":"High","gaps":["Did not distinguish whether Tim23 itself lines the conducting pore","No structural assignment of pore-lining residues"]},{"year":2000,"claim":"Resolved how Tim23 bridges two membranes, explaining how precursors transfer from TOM to TIM23 at contact sites and how the IMS domain is positioned as a receptor.","evidence":"Protease accessibility, GFP topology mapping, import competition and EM in yeast","pmids":["10830167"],"confidence":"High","gaps":["Mechanism of outer-membrane anchoring of the N-terminus not defined","Stoichiometry of contact-site complexes unresolved"]},{"year":2000,"claim":"Defined the chaperone requirements for Tim23 biogenesis itself, showing the small TIM8-13 complex escorts Tim23 across the IMS under low potential.","evidence":"Co-IP, crosslinking of translocation intermediates, and TIM8-13 depletion import assays; later supported by crystal structure","pmids":["11101512","18706423"],"confidence":"High","gaps":["Whether TIM8-13 chaperones other substrates in this mode not addressed here"]},{"year":2002,"claim":"Identified Tim50 as the IMS partner of the Tim23 N-domain that mediates precursor handover from TOM, establishing the receptor architecture of the inner-membrane face.","evidence":"Site-specific photocrosslinking, reciprocal co-IP, depletion and antibody inhibition","pmids":["12437925"],"confidence":"High","gaps":["Did not map the presequence-binding residues of Tim50","Channel-gating consequence of Tim50 binding not yet defined"]},{"year":2004,"claim":"Completed identification of the PAM import-motor co-chaperones, showing Tim14/Pam18 and Tim16/Pam16 form a J-protein subcomplex that activates mtHsp70 within the translocase.","evidence":"Protein identification, reciprocal co-IP, HPD-motif mutagenesis and depletion import assays","pmids":["14517234","14981506"],"confidence":"High","gaps":["How motor activation is mechanically coupled to Tim23 channel dynamics not resolved","Quantitative ATPase kinetics within the assembled translocase not measured"]},{"year":2008,"claim":"Mapped the conformational and quaternary dynamics of Tim23 in functional membranes, showing TMS2 lines an amphipathic channel that rearranges with gating and that subunit proximities change with potential and substrate.","evidence":"Site-directed fluorescence mapping and site-specific crosslinking of Tim23 cysteine mutants in intact mitochondria","pmids":["18692467","17959826"],"confidence":"High","gaps":["Atomic structure not obtained","Whether TMS2 forms a true aqueous channel vs lipid-exposed cavity unresolved at this stage"]},{"year":2011,"claim":"Defined the antagonistic regulation of the Tim23 channel by presequences and Tim50, and quantified trans-side presequence binding by Tim44 and Tim23 that supports directional import.","evidence":"Photo-affinity labeling with MS mapping, electrophysiology, and SPR/crosslinking with purified components","pmids":["22065641","21969381"],"confidence":"High","gaps":["In situ regulation under physiological potential not directly measured","Affinity hierarchy across the full import pathway not fully reconstituted"]},{"year":2014,"claim":"Established the Tim23 IMS domain as an intrinsically disordered, membrane-associated interaction hub contacting Tim21, Tim50 and Tom22, and identified cardiolipin as a modulator of its membrane attachment.","evidence":"NMR with single-residue mapping and membrane model systems including cardiolipin liposomes","pmids":["25263020","25349212"],"confidence":"High","gaps":["In vivo functional consequence of cardiolipin effect not validated","Dynamic interactions not captured in an intact translocase structure"]},{"year":2014,"claim":"Assigned distinct interaction sites that organize the motor and regulatory subunits, with Tim23 loop 1 anchoring Tim44 and adjacent contacts positioning Pam17.","evidence":"Site-specific in vivo photocrosslinking with genetic and co-IP validation","pmids":["25157107","24061477"],"confidence":"High","gaps":["Precise allosteric path from motor docking to channel gating undefined"]},{"year":2017,"claim":"Detailed the Tim44 NTD/CTD bipartite engagement of presequences and the translocon, and showed cardiolipin tunes Tim50 receptor association with the membrane and Tim23.","evidence":"In vivo crosslinking with in vitro binding, plus SAXS structure and MD of the Tim50 receptor","pmids":["28440746","28879236"],"confidence":"High","gaps":["Full assembled translocase structure not yet available at this point","Lipid regulation in cells not quantified"]},{"year":2017,"claim":"Demonstrated that cation selectivity of the Tim23 channel is mechanistically required for substrate recognition and import competence.","evidence":"Structure-based mutagenesis with electrophysiology and import assays","pmids":["28857742"],"confidence":"High","gaps":["Pore-lining residue assignment later revised by cryo-EM showing the Tim17 path"]},{"year":2019,"claim":"Defined Mgr2 and Pam18 as controllers of the lateral gate that decide between matrix translocation and inner-membrane insertion of stop-transfer substrates.","evidence":"Electrophysiology, substrate-specific import assays and co-IP in Mgr2/Pam18 perturbation strains","pmids":["30279421","30697167","31764998"],"confidence":"Medium","gaps":["Structural basis of lateral gating not resolved here","Hydrophobicity threshold values determined in single systems"]},{"year":2021,"claim":"Captured the TOM-TIM23 interface at amino-acid resolution by crosslinking a stalled supercomplex, defining the physical map of precursor transfer.","evidence":"Chemical crosslinking-MS with structural modeling of a stalled translocation intermediate","pmids":["34588454"],"confidence":"High","gaps":["Static snapshot of one stalled state","Did not resolve gating transitions during active translocation"]},{"year":2023,"claim":"Revised the core channel model by cryo-EM, showing Tim23 is primarily structural while the Tim17 cavity sealed by Mgr2 forms the protein-conducting path.","evidence":"Cryo-EM of core TIM23 heterotrimer and active TOM-TIM23 supercomplex with translocating substrate, with biochemical validation","pmids":["37344598","37696957","40877479"],"confidence":"High","gaps":["How earlier electrophysiology-defined Tim23 channel properties reconcile with the Tim17 path not fully integrated","Human translocase structure not directly determined"]},{"year":2024,"claim":"Connected TIM23 to mitophagy initiation, showing it interacts with PINK1, protects it from OMA1 degradation, and tethers TOM to TIM23 as a stress-sensing switch.","evidence":"Co-IP, MS, BN-PAGE, PINK1 mutagenesis and mitophagy assays across cell models including organoids; OMA1 inactivation rescue","pmids":["37160114","38416681"],"confidence":"High","gaps":["Whether import-channel activity per se versus the physical tether drives PINK1 sensing partly unresolved","Role distinct from TOM not fully separated in all conditions"]},{"year":2024,"claim":"Linked TIM23 translocase integrity to disease and quality control, showing prohibitin/OCIAD1 and DNAJC15/OMA1 regulate TIM23 variant stability and that TIMM50 loss preferentially depletes laterally sorted OXPHOS substrates.","evidence":"Quantitative proteomics, co-IP, protease perturbation and import assays in patient and engineered cells","pmids":["39630581","38828998","31346086","40877479"],"confidence":"Medium","gaps":["Mechanistic detail of variant-selective protease targeting incomplete","Two preprint findings on DNAJC15/PINK1 import not yet peer-reviewed"]},{"year":null,"claim":"It remains unresolved how the historically defined Tim23 channel and gating properties are reconciled with the cryo-EM Tim17-conducting-path model, and how TIM23 substrate selection is regulated dynamically during physiological import and stress.","evidence":"","pmids":[],"confidence":"High","gaps":["No integrated structural-electrophysiological model of Tim23's structural role","Regulation of TIM23 import capacity in human disease contexts only partially mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,33,34]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[22,27]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[33,8,24]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,15,16]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[1,30,35]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[3,34,38]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[5,6,26]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[35,36]}],"complexes":["TIM23 presequence translocase","TOM-TIM23 supercomplex","PAM import motor","PINK1-TOM-TIM23 supercomplex"],"partners":["TIMM50","TIMM17","TIMM44","PAM18","PAM16","MGR2","TIMM21","PINK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O14925","full_name":"Mitochondrial import inner membrane translocase subunit Tim23","aliases":[],"length_aa":209,"mass_kda":21.9,"function":"Essential component of the TIM23 complex, a complex that mediates the translocation of transit peptide-containing proteins across the mitochondrial inner membrane (PubMed:10339406). Has a role in the activation of stress-induced mitophagy by protecting PINK1 from OMA1-mediated degradation and facilitating its accumulation at the outer mitochondrial membrane in response to depolarization (PubMed:37160114)","subcellular_location":"Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/O14925/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TIMM23","classification":"Common Essential","n_dependent_lines":1038,"n_total_lines":1165,"dependency_fraction":0.8909871244635194},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TIMM23","total_profiled":1310},"omim":[{"mim_id":"621252","title":"CATARACT, ALOPECIA, ORAL MUCOSAL DISORDER, AND PSORIASIS-LIKE SYNDROME; CAOP","url":"https://www.omim.org/entry/621252"},{"mim_id":"620758","title":"TRANSLOCASE OF INNER MITOCHONDRIAL MEMBRANE 23B; TIMM23B","url":"https://www.omim.org/entry/620758"},{"mim_id":"618894","title":"REACTIVE OXYGEN SPECIES MODULATOR 1; ROMO1","url":"https://www.omim.org/entry/618894"},{"mim_id":"617465","title":"SMALL INTEGRAL MEMBRANE PROTEIN 20; SMIM20","url":"https://www.omim.org/entry/617465"},{"mim_id":"616712","title":"START DOMAIN-CONTAINING PROTEIN 7; STARD7","url":"https://www.omim.org/entry/616712"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Mitochondria","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TIMM23"},"hgnc":{"alias_symbol":["TIM23"],"prev_symbol":[]},"alphafold":{"accession":"O14925","domains":[{"cath_id":"-","chopping":"69-150","consensus_level":"medium","plddt":71.2098,"start":69,"end":150},{"cath_id":"1.20.58","chopping":"152-209","consensus_level":"medium","plddt":57.3828,"start":152,"end":209}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O14925","model_url":"https://alphafold.ebi.ac.uk/files/AF-O14925-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O14925-F1-predicted_aligned_error_v6.png","plddt_mean":59.22},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TIMM23","jax_strain_url":"https://www.jax.org/strain/search?query=TIMM23"},"sequence":{"accession":"O14925","fasta_url":"https://rest.uniprot.org/uniprotkb/O14925.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O14925/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O14925"}},"corpus_meta":[{"pmid":"8858146","id":"PMC_8858146","title":"Role 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Dimerization depends on a heptad leucine repeat motif in the N-terminal hydrophilic domain (IMS-exposed). Monomeric Tim23 is present when a preprotein is in transit, suggesting Tim23 dimers act as receptors for matrix targeting sequences and dimer dissociation triggers TIM channel opening.\",\n      \"method\": \"Biochemical dimerization assays, membrane potential manipulation, preprotein binding experiments, mutational analysis of leucine repeat motif\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal biochemical methods, foundational study replicated conceptually by many subsequent papers\",\n      \"pmids\": [\"8858146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Tim23 has a two-membrane-spanning topology: its C-terminal domain is anchored in the inner membrane, an intermediate domain is exposed in the IMS as a presequence receptor, and the N-terminal domain is exposed on the surface of the outer mitochondrial membrane. This simultaneous integration into two membranes forms contact sites between outer and inner membranes, facilitating transfer of precursors from TOM to TIM23.\",\n      \"method\": \"Protease accessibility assays, GFP fusion topology mapping, import competition, electron microscopy\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods in a single rigorous study, replicated by subsequent work\",\n      \"pmids\": [\"10830167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The TIM8-Tim13 complex in the IMS interacts with translocation intermediates of Tim23 (partially translocated across the outer membrane), binding to the N-terminal or intermediate domain of Tim23 to prevent retrograde translocation. The TIM8-13 complex is required for Tim23 import under low membrane potential conditions.\",\n      \"method\": \"Co-immunoprecipitation, import assays with TIM8-13 deletion/depletion strains, crosslinking of translocation intermediates\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding, functional import assays, loss-of-function with defined phenotype, replicated by crystal structure paper\",\n      \"pmids\": [\"11101512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Tim23 is required for normal activity of the multiple conductance channel (MCC) of the mitochondrial inner membrane. Anti-Tim23 antibodies that inhibit protein import also inhibit MCC activity; the tim23-1 mutation alters MCC conductance and abolishes presequence-peptide blockage of MCC, identifying Tim23 as essential for the protein-conducting pore activity.\",\n      \"method\": \"Electrophysiology (patch clamp), antibody inhibition, yeast tim23-1 mutant analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — electrophysiology with mutagenesis and antibody blocking, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"9128249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Tim50 is a subunit of the TIM23 complex anchored to the inner membrane with its C-terminal domain exposed to the IMS. Tim50 interacts with the N-terminal IMS domain of Tim23. Tim50 depletion or antibody addition blocks protein translocation across the inner membrane. A translocation intermediate at TOM is crosslinked to Tim50, establishing Tim50 as a component that mediates transfer of translocating proteins from TOM to TIM23.\",\n      \"method\": \"Site-specific photocrosslinking, co-immunoprecipitation, protein depletion, antibody inhibition assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — site-specific crosslinking, reciprocal co-IP, depletion + antibody phenotypes, replicated by multiple subsequent studies\",\n      \"pmids\": [\"12437925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Tim14 (Pam18) is an integral inner membrane protein with a J-domain exposed to the matrix. It is an essential component of the TIM23 import motor, interacting with Tim44 and mtHsp70 in an ATP-dependent manner. A mutation in the HPD motif of the J-domain is lethal, establishing Tim14 as the J-protein co-chaperone that activates mtHsp70 ATPase activity within the TIM23 motor.\",\n      \"method\": \"Gene depletion, import assays, co-immunoprecipitation, HPD motif mutagenesis, ATP-dependence studies\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis of catalytic motif, co-IP with motor components, loss-of-function phenotype, independently replicated\",\n      \"pmids\": [\"14517234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Tim16 (Pam16) is a novel J-domain-related cochaperone that forms a stable subcomplex with Tim14 within the TIM23 translocase. Depletion of Tim16 markedly impairs matrix protein import and disrupts Tim14 interaction with the TIM23 complex, leading to severe structural changes of the import motor.\",\n      \"method\": \"Protein identification, co-immunoprecipitation, gene depletion, import assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, depletion phenotype, replicated by subsequent association studies\",\n      \"pmids\": [\"14981506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Tim23 contains two independent import signals: one in the first 62 residues of the N-terminal hydrophilic domain mediating ΔΨ-independent outer membrane translocation, and a second in the C-terminal membrane-integrated domain mediating ΔΨ-dependent translocation and inner membrane insertion. Import of Tim23 into the inner membrane requires Tim22 (not functional Tim23), establishing that TIM23 biogenesis depends on the TIM22 complex.\",\n      \"method\": \"Deletion mutagenesis, import assays with ΔΨ manipulation, genetic epistasis with Tim22/Tim23 depletion strains\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic deletion mutagenesis, epistasis, multiple orthogonal import assays\",\n      \"pmids\": [\"9501078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Tim17p regulates twin-pore structure and voltage gating of TIM23. Patch clamp of reconstituted inner membranes showed TIM23 has twin pores that cooperatively gate. Tim17p depletion collapses the twin pores into a single pore; N-terminal deletion or C-terminal truncation of Tim17p produces variable-sized pores. Tim17p N-terminus is vital for voltage sensing and protein sorting, while Tim23p is the main structural unit of the pore.\",\n      \"method\": \"Patch clamp electrophysiology of reconstituted inner membranes, Tim17p deletion and truncation mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro electrophysiology reconstitution with systematic mutagenesis, single lab\",\n      \"pmids\": [\"17148445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Fluorescence mapping using environment-sensitive probes positioned along transmembrane segment 2 (TMS2) of Tim23 revealed an amphipathic alpha-helix in the protein-conducting channel region. Probes on the aqueous-facing helical surface underwent spectral changes during active protein import, and their accessibility to hydrophilic quenchers changes with channel gating, defining the channel-lining face of Tim23 TMS2.\",\n      \"method\": \"Site-directed fluorescence labeling, multiple spectral analyses (polarization, intensity, lifetime), hydrophilic quenching in functional intact mitochondria\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in situ fluorescence mapping with multiple spectral techniques in functional mitochondria, functionally validated\",\n      \"pmids\": [\"18692467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The Tim8-Tim13 complex assembles as a hexamer and binds Tim23 cooperatively with ~6 binding sites, exhibiting positive cooperativity. The crystal structure (2.6 Å) reveals tentacle-like helices with hydrophobic pockets that interact with Tim23 transmembrane helices, shielding them during chaperoned translocation across the IMS.\",\n      \"method\": \"X-ray crystallography (2.6 Å), surface plasmon resonance binding kinetics\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with quantitative binding measurements, single lab\",\n      \"pmids\": [\"18706423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Tim23 cross-links to Tim17 at its first transmembrane segment, to Tim50 at the C-terminal end of its hydrophilic IMS region, and Tim23 IMS domains cross-link to each other (dimerization). These proximity relationships reversibly change upon membrane potential alterations and when a translocating substrate is trapped, demonstrating dynamic conformational rearrangements of the TIM23 quaternary structure.\",\n      \"method\": \"Site-specific chemical cross-linking of radiolabeled Tim23 monocysteine mutants imported into functional mitochondria\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic cross-linking at defined positions with functional correlation, single lab\",\n      \"pmids\": [\"17959826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Yeast Aac2 (ADP/ATP carrier) physically associates with both the cytochrome bc1-COX supercomplex and the TIM23 machinery. Affinity purification with His-tagged Aac2 co-purifies TIM23 components. This association can occur independently of the fully assembled cytochrome bc1-COX supercomplex.\",\n      \"method\": \"Affinity purification (His-tag), co-immunoprecipitation, blue native gel electrophoresis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — affinity purification and co-IP from single lab, functional consequence of TIM23-Aac2 association not directly tested\",\n      \"pmids\": [\"18614795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Tim23-Tim50 interactions in the IMS facilitate both transfer of precursor proteins from TOM40 to TIM23 and a late step of translocation across the inner membrane by promoting mtHsp70 motor functions. The Tim23-Tim50 pair thus coordinates actions of TOM40 and TIM23 complexes together with the import motor.\",\n      \"method\": \"Genetic analysis of Tim23/Tim50 IMS domain mutants, co-immunoprecipitation, import assays, crosslinking\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (genetics, co-IP, crosslinking, import assays) in single lab\",\n      \"pmids\": [\"19139266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In vitro reconstitution of the Tim23-Tim50 IMS domain interaction using purified recombinant proteins. Chemical cross-linking and surface plasmon resonance defined the interaction. Mutations in Tim23 that abolish Tim50 binding in vitro also destabilize the interaction in vivo, causing defective protein import and temperature-sensitive lethality.\",\n      \"method\": \"In vitro reconstitution with purified recombinant proteins, chemical cross-linking, surface plasmon resonance, in vivo import assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution plus mutagenesis plus in vivo validation, single lab\",\n      \"pmids\": [\"19017642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The IMS domain of Tim23 (residues 1-96, Tim23N) is an intrinsically disordered protein. NMR titrations with two presequences identified a distinct presequence-binding region formed by residues 71-84, making Tim23N the only IMS-exposed domain of TOM/TIM23 predicted to be intrinsically disordered.\",\n      \"method\": \"NMR spectroscopy, NMR titrations with presequence peptides, charge-hydropathy analysis\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure determination with functional binding mapping, single lab\",\n      \"pmids\": [\"20718036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Tim50's presequence-binding domain was mapped by photo-affinity labeling with engineered presequence probes and mass spectrometric identification of cross-linking sites. Tim50 is established as the primary presequence receptor at the inner membrane. Targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner: presequences promote channel opening while Tim50 keeps the channel closed.\",\n      \"method\": \"Photo-affinity labeling, mass spectrometric mapping, in vitro import assays, electrophysiology\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — photo-crosslinking with MS mapping combined with electrophysiology, single lab\",\n      \"pmids\": [\"22065641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Purified Tim50IMS and Tim44 directly bind presequence peptides (Hsp60, mHsp70, cytochrome P450SCC presequences), demonstrated by chemical cross-linking and surface plasmon resonance with defined KD values. Tim23IMS binding to presequences was also characterized. These stronger interactions on the trans side of the channel support directional import.\",\n      \"method\": \"Chemical cross-linking of purified recombinant proteins, surface plasmon resonance with KD determination\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified components, quantitative binding measurements, single lab\",\n      \"pmids\": [\"21969381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TIM23-mediated membrane insertion of transmembrane alpha-helices into the mitochondrial inner membrane depends strongly on hydrophobicity and position of polar/aromatic residues, paralleling the ER system, but shows striking differences in the effect of flanking charged residues compared to the ER translocon.\",\n      \"method\": \"Systematic mutagenesis of model transmembrane segments, in vitro import assays in yeast mitochondria\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with quantitative insertion assays, single lab\",\n      \"pmids\": [\"21326212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"High-resolution fluorescence mapping of TMS2 of Tim23 in energized versus depolarized membranes showed that TMS2 forms a continuous alpha-helix inaccessible to the IMS aqueous phase in energized membranes. Upon depolarization, helical periodicity is disrupted and the channel becomes exposed to the IMS. Kinetic measurements confirmed that TMS2 conformational changes coincide with depolarization.\",\n      \"method\": \"Site-directed fluorescence labeling in intact yeast membranes, multiple spectroscopic methods, kinetic measurements\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution fluorescence mapping in functional membranes with multiple orthogonal spectral methods, single lab\",\n      \"pmids\": [\"23832274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Sym1 (yeast ortholog of MPV17), a multispanning inner membrane protein, is imported via TIM23 in a presequence-independent manner using internal targeting signals, rather than via TIM22 as expected for carrier-type proteins. This defines a novel transport mechanism for a polytopic inner membrane protein through TIM23.\",\n      \"method\": \"Import assays with TIM23/TIM22 depletion, translocation intermediate trapping, protease protection\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — import pathway determination using multiple depletion strains with defined intermediates, single lab\",\n      \"pmids\": [\"23045398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Molecular basis of TIM23 architecture: Tim23 loop 1 (between TM1 and TM2, matrix-exposed) cross-links to Tim44; alterations in this loop destabilize Tim44 interaction with the translocon. Tim17 loop 1 cross-links to Pam17; alterations destabilize Pam17-translocon interaction. This positions Tim44 and Pam17 as regulatory subunits at distinct translocon interaction sites.\",\n      \"method\": \"Site-specific in vivo photocross-linking, co-immunoprecipitation, genetic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific in vivo photocross-linking with genetic and biochemical validation, single lab\",\n      \"pmids\": [\"25157107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N-terminus. The membrane-bound Tim23 IMS domain remains highly dynamic. Cardiolipin enhances Tim23 membrane attachment, suggesting cardiolipin influences preprotein import.\",\n      \"method\": \"NMR spectroscopy with membrane model systems, cardiolipin-containing liposomes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — NMR with model membranes, single lab, no in vivo functional validation of cardiolipin effect\",\n      \"pmids\": [\"25349212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NMR characterization of the dynamic interaction network within the TIM23 IMS: Tim23 IMS domain contains multiple sites for interaction with Tim21 IMS domain, and also interacts with Tim50 and Tom22. The atomic details of the Tim23IMS-Tim21IMS complex were determined, establishing Tim23 IMS as a hub for TIM23 complex organization.\",\n      \"method\": \"NMR spectroscopy, chemical shift perturbation mapping at single-residue level\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR with single-residue resolution mapping of multiple interactions, single lab\",\n      \"pmids\": [\"25263020\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GxxxG motifs in transmembrane segments 1 and 2 of Tim23 are necessary for structural integrity of the TIM23 complex. Mutations of specific glycines in TM1 and TM2 caused lethal or temperature-sensitive phenotypes correlated with TIM23 complex destabilization, without impairing Tim23 import or membrane integration itself.\",\n      \"method\": \"Systematic mutagenesis of GxxxG motifs, yeast growth assays, blue native gel electrophoresis\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with complex stability readout, single lab\",\n      \"pmids\": [\"25765297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Tim17 contains a pair of highly conserved cysteine residues forming a structural disulfide bond exposed to the IMS. This disulfide bond is critical for efficient protein translocation through TIM23 and for dynamic gating of the preprotein-conducting channel. The disulfide is formed during Tim17 insertion, dependent on Mia40 binding but not its oxidoreductase activity; direct oxidation by Erv1 is suggested.\",\n      \"method\": \"Cysteine mutagenesis, electrophysiology (channel gating assays), import assays, redox biochemistry\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with electrophysiology and import assays, single lab\",\n      \"pmids\": [\"27502485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Both domains of Tim44 interact with the major matrix-exposed loop of Tim23: the C-terminal domain (CTD) anchors Tim44 to the translocon (also binding Tim17), while the N-terminal domain (NTD) is intrinsically disordered and dynamically interacts with presequences near the region important for Hsp70 and Tim23 interaction.\",\n      \"method\": \"Site-specific in vivo crosslinking, genetic analysis, in vitro binding assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific in vivo crosslinking with in vitro validation and genetic support, single lab\",\n      \"pmids\": [\"28440746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cardiolipin directly modulates interactions between the Tim50 soluble receptor domain and the Tim23 channel. The Tim50 receptor domain interacts with membranes and with specific sites on Tim23 in a cardiolipin-dependent manner. SAXS-based structure of the full soluble Tim50 receptor was determined. Molecular dynamics simulations confirmed cardiolipin-driven Tim50 association with lipid bilayers with concomitant structural changes.\",\n      \"method\": \"SAXS structure determination, molecular dynamics simulations, biophysical measurements in liposomes, in vivo and isolated mitochondria assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — SAXS structure combined with MD simulations and multiple biophysical validations in reductionist and cellular systems, single lab\",\n      \"pmids\": [\"28879236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cation selectivity of the Tim23 channel is essential for protein import. Structure-based mutations of highly conserved pore-lining amino acids reduce selectivity, reduce protein import capacity, and render the Tim23 channel insensitive to substrates, demonstrating that cation selectivity is a key feature for substrate recognition.\",\n      \"method\": \"Structure-based mutagenesis, electrophysiology (cation selectivity measurements), in vitro import assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structure-guided mutagenesis with electrophysiology and import assays, single lab\",\n      \"pmids\": [\"28857742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The import motor J-protein Pam18 controls lateral protein release into the lipid bilayer through the TIM23 lateral gate. Constitutively translocase-associated Pam18 obstructs lateral precursor transport and displaces Mgr2 from the translocase, demonstrating that during motor-dependent matrix import, the Pam18 transmembrane segment closes the lateral gate to promote anterograde translocation.\",\n      \"method\": \"Genetic and biochemical analysis, import assays of matrix-targeted vs stop-transfer substrates, co-immunoprecipitation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic manipulation with defined substrate-specific import phenotypes, single lab\",\n      \"pmids\": [\"30279421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Mutant huntingtin (mHTT) specifically binds the TIM23 subunit (of the four TIM23 complex proteins) and resides in the mitochondrial intermembrane space. mHTT binding inhibits mitochondrial protein import specifically through TIM23, reducing levels of TIM23-imported soluble matrix proteins in mHTT-expressing cells and HD patient brain tissue.\",\n      \"method\": \"Co-immunoprecipitation, mitochondrial fractionation, quantitative proteomics, quantitative immunoblotting in cell lines and human HD brain tissue\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, fractionation, quantitative proteomics in multiple model systems plus human tissue\",\n      \"pmids\": [\"31346086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In the IMS, the Tim23-Tim50 interaction surface is larger than previously thought; an unexpected interaction of Tim23 with Pam17 in the IMS was identified. Mutations of two conserved negatively charged residues of Tim23 near the inner membrane prevented Tim23 dimerization and increased Tim23 surface exposure, while membrane potential dissipation decreased surface exposure.\",\n      \"method\": \"In vivo mutagenesis, crosslinking, dimerization assays, surface accessibility experiments\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo crosslinking and mutagenesis with multiple readouts, single lab\",\n      \"pmids\": [\"32277989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Chemical cross-linking combined with mass spectrometry of a stalled TOM-TIM23 supercomplex mapped protein-protein interactions at the IMS interface between TOM and TIM23 at amino acid resolution, including contacts involving Tim23 and Tim50. The import motor interactions were also mapped in this context.\",\n      \"method\": \"Chemical cross-linking with mass spectrometric analysis, structural modeling, purification of stalled translocation intermediate\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — cross-linking MS with structural modeling of captured translocation intermediate, single lab\",\n      \"pmids\": [\"34588454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structure of the core TIM23 complex (Tim17-Tim23-Tim44 heterotrimer) from S. cerevisiae was determined. Contrary to the prevailing model, Tim23 and Tim17 do not form a shared water-filled channel; instead, they have separate lipid-exposed concave cavities facing opposite directions. The cavity of Tim17 (not Tim23) forms the protein translocation path; Tim23 has primarily a structural role. Mgr2 seals the lateral opening of the Tim17 cavity to facilitate translocation.\",\n      \"method\": \"Cryo-electron microscopy structure determination, biochemical validation\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with biochemical validation, revises prevailing model, published in high-impact journal\",\n      \"pmids\": [\"37344598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of the active TOM-TIM23 supercomplex with translocating substrate revealed that TIM23 contains a heterotrimer of Tim23, Tim17, and Mgr2. Polypeptide substrates are shielded from lipids by Mgr2 and Tim17, with a translocation pathway characterized by a negatively charged entrance and central hydrophobic region. In TOM, the substrate passes through the center of Tom40.\",\n      \"method\": \"Cryo-electron microscopy of active supercomplex with translocating substrate, biochemical analysis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM of active translocation complex with substrate, single lab\",\n      \"pmids\": [\"37696957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TIM23 interacts with PINK1 and facilitates PINK1 accumulation upon mitochondrial depolarization by protecting PINK1 from degradation by the OMA1 protease. TIM23 knockdown decreases PINK1 levels and delays autophosphorylation; OMA1 inactivation rescues PINK1 levels when TIM23 is depleted. Pathogenic PINK1 mutants that fail to interact with TIM23 have deficiencies partially restored by OMA1 inactivation.\",\n      \"method\": \"Mass spectrometric identification of PINK1 co-immunoprecipitates, co-immunoprecipitation, TIM23 knockdown, OMA1 inactivation, PINK1 autophosphorylation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction validated by co-IP with multiple genetic perturbations, single lab\",\n      \"pmids\": [\"37160114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PINK1 forms a high-molecular-weight PINK1-TOM-TIM23 supercomplex upon mitochondrial stress in human cultured cell lines, dopamine neurons, and midbrain organoids. PINK1 is required to stably tether TOM to TIM23 complexes in response to stress. This tethering depends on an interaction between the PINK1 N-terminal C-terminal extension module and the cytosolic domain of Tom20; disruption by designer or PD-associated PINK1 mutations inhibits downstream mitophagy.\",\n      \"method\": \"Co-immunoprecipitation, blue native gel electrophoresis, PINK1 mutagenesis, mitophagy assays in multiple cell models including organoids\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, multiple cell models, PINK1 mutagenesis with functional mitophagy readout\",\n      \"pmids\": [\"38416681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OCIAD1 and the prohibitin complex regulate the stability of the human TIM23 translocase. Prohibitins stabilize both TIMM17A- and TIMM17B-containing TIM23 variants. OCIAD1 assembles with the prohibitin complex to specifically protect the TIMM17A variant from degradation by the YME1L protease. OCIAD1 expression is regulated by TIM23 complex status.\",\n      \"method\": \"Co-immunoprecipitation, protease inhibition, siRNA knockdown, quantitative proteomics\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and genetic perturbations with protease identification, single lab\",\n      \"pmids\": [\"39630581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIMM50 pathogenic variants reduce levels and activity of the TIM23 complex, and proteomic analysis reveals that laterally released substrates imported via the TIM23SORT pathway are most sensitive to TIMM50 loss. Proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for TIMM50-associated disease.\",\n      \"method\": \"Quantitative proteomics of patient fibroblasts and HEK293 cell model, import assays, blue native gel electrophoresis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative proteomics with import assays in patient and engineered cells, single lab\",\n      \"pmids\": [\"38828998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of the active TOM-TIM23 supercomplex captured with translocating substrate show that the Tim17-Mgr2 pathway creates the translocation channel with a central restriction formed by four conserved hydrophobic residues. Substrate hydrophobicity modulates Mgr2 association with Tim17, enabling dynamic regulation of protein sorting toward matrix or membrane.\",\n      \"method\": \"Cryo-electron microscopy of active translocating supercomplex, biochemical validation\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structures with translocating substrate in multiple conformations, biochemical validation, single lab\",\n      \"pmids\": [\"40877479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Tim23 knockdown in mouse muscle (40% reduction) caused a 32% decrease in matrix-destined protein import and activated the CHOP branch of the UPRmt (increased ClpP and cpn10) without activating the ATF5 arm, demonstrating that reduced TIM23 import capacity triggers a specific branch of the mitochondrial unfolded protein response.\",\n      \"method\": \"In vivo antisense oligonucleotide knockdown, import assays, UPRmt marker measurements\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockdown with defined import and UPRmt phenotype, single lab\",\n      \"pmids\": [\"29949403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Tim23 undergoes intra-mitochondrial proteolytic degradation following mitochondrial outer membrane permeabilization (MOMP) in a caspase-independent but Bax/Bak-dependent manner. This degradation is mitochondrion-autonomous (occurs in isolated mitochondria undergoing permeability transition), selective for Tim23, and depletion of Tim23 by siRNA inhibits cell proliferation and prevents long-term survival.\",\n      \"method\": \"Caspase inhibition, Bax/Bak knockout, isolated mitochondria assays, siRNA knockdown, Tim23-GFP complementation\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic perturbations with complementation, single lab\",\n      \"pmids\": [\"18174902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NOX4 localizes to the mitochondrial matrix following asbestos exposure in lung macrophages via direct interaction with TIM23. TIM23 augments NOX4-induced mitochondrial ROS and metabolic reprogramming to oxidative phosphorylation. Silencing TIM23 decreased mitochondrial ROS and oxidative phosphorylation. The interaction was localized to the proximal transmembrane region of NOX4.\",\n      \"method\": \"Co-immunoprecipitation, TIM23 silencing, mitochondrial fractionation, ROS measurement, OXPHOS analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with domain mapping and functional knockdown readouts, single lab\",\n      \"pmids\": [\"37044213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Mitochondrial ROS induces rapid translocation of APE1 from the IMS into the matrix through the TIM23/PAM machinery complex. The IMS serves as a storage site for APE1 under basal conditions.\",\n      \"method\": \"Mitochondrial fractionation, import assays, TIM23 knockdown, oxidative stress induction\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — fractionation and TIM23 knockdown with functional import readout, single lab\",\n      \"pmids\": [\"33197464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TM1 of Tim23 is required for homodimerization; TM1 and TM2 together are involved in preprotein binding within the channel, and TM2 recruits Tim21 and the PAM subcomplex to Tim23. The matrix-exposed loop L1 generates specificity in PAM subcomplex association. The C-terminal sequence of Tim23 functions as an inhibitor of Tim21 binding.\",\n      \"method\": \"Mutagenesis, co-immunoprecipitation, genetic complementation, import assays in yeast\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with biochemical and functional readouts, single lab\",\n      \"pmids\": [\"24061477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Deletion of Mgr2 decreases normal TIM23 channel gating frequency and produces a residual channel activity lacking gating transitions but remaining sensitive to import signal peptides. A G145L mutation in Tim23 displaces Mgr2 from the complex leading to the same gating impairment, establishing that Mgr2 regulates TIM23 channel gating behavior.\",\n      \"method\": \"Patch clamp electrophysiology of yeast inner membranes, Mgr2 deletion and Tim23 point mutation\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — electrophysiology with genetic perturbations, single lab\",\n      \"pmids\": [\"30697167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Mgr2 controls the threshold hydrophobicity and flanking charge requirements for TIM23-mediated membrane insertion via the stop-transfer mechanism. Deletion of Mgr2 reduces the hydrophobicity threshold required for membrane insertion and reduces the requirement for matrix-facing positive charges, establishing Mgr2 as a lateral gatekeeper that discriminates stop-transfer signal features.\",\n      \"method\": \"Systematic mutagenesis of model transmembrane segments, membrane insertion assays in Mgr2-deletion and overexpression yeast strains\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with quantitative insertion assays in defined genetic backgrounds, single lab\",\n      \"pmids\": [\"31764998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PGC-1α promotes binding of NRF1 to the Tim23 promoter, upregulating Tim23 expression, which in turn reduces DRP1 transcription and ACSL4 mitochondrial translocation, thereby inhibiting ferroptosis and MASH. This establishes a PGC-1α-Tim23-DRP1-ACSL4 axis in hepatocyte ferroptosis.\",\n      \"method\": \"Western blot, RT-qPCR, immunofluorescence, luciferase reporter assays, co-immunoprecipitation, mouse MASH models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and reporter assays with in vivo models, single lab, mechanistic chain not fully reconstituted\",\n      \"pmids\": [\"41724762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In the absence of TIM23, PINK1 is stabilized in the TOM complex. The MMP (not the PAM import motor) is the primary driving force for PINK1 import through TIM23. Loss of TIM23 is sufficient to activate Parkin, establishing TIM23 as the essential inner membrane translocase that separates PINK1 from TOM as the key damage-sensing switch in the PINK1-Parkin mitophagy pathway.\",\n      \"method\": \"Genome-wide screens, PINK1 import assays, TIM23 knockout, Parkin activation assays, membrane potential manipulation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen with genetic validation and import assays, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OMA1-mediated degradation of DNAJC15 reduces protein import of OXPHOS-related proteins via the TIMM23-TIMM17A translocase under mitochondrial stress, limiting OXPHOS biogenesis. Loss of DNAJC15 specifically reduces TIMM17A-containing TIM23 complex-dependent import.\",\n      \"method\": \"Proteomic analysis, import assays, OMA1 and DNAJC15 genetic perturbations, blue native gel electrophoresis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics with genetic perturbations and import assays, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TIMM23 (Tim23) is the central scaffold subunit of the mitochondrial TIM23 presequence translocase in the inner membrane, where structural studies reveal it has primarily a structural role while its homolog Tim17 (with Mgr2) forms the actual protein-conducting channel; Tim23 functions as a dynamic IMS-exposed hub that dimerizes in a ΔΨ-dependent manner to act as a presequence receptor, interacts with Tim50 (the primary presequence receptor) and other TOM/TIM23 subunits to coordinate precursor handover from TOM, bridges the outer and inner membranes through its two-membrane-spanning topology, and recruits the mtHsp70-based PAM motor (via Tim44, Tim14/Pam18, and Tim16/Pam16) to drive ATP-dependent translocation into the matrix, while also regulating lateral sorting of stop-transfer substrates into the inner membrane through its association with Mgr2; additionally, Tim23 participates in PINK1 stabilization for mitophagy initiation and is targeted by pathological proteins such as mutant huntingtin that inhibit its import function.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TIMM23 (Tim23) is the central inner-membrane subunit of the mitochondrial TIM23 presequence translocase that imports presequence-bearing precursor proteins from the cytosol into the matrix and inner membrane [#3, #34]. It adopts a two-membrane-spanning topology in which its C-terminal domain is integrated in the inner membrane while an intrinsically disordered N-terminal/intermembrane-space (IMS) domain reaches across to the outer membrane, forming contact sites that channel precursors from TOM to TIM23 [#1, #15]. The IMS domain serves as a dynamic organizing hub: it dimerizes in a membrane-potential-dependent manner to act as a presequence receptor, and dimer dissociation upon presequence binding is coupled to channel opening [#0, #31]. This domain coordinates the translocase by binding Tim50, the primary presequence receptor, as well as Tim21 and Tom22, integrating substrate handover with channel gating in which presequences and Tim50 act antagonistically on the channel [#4, #16, #23]. Conformational rearrangements of the transmembrane segments accompany membrane potential changes and substrate trapping, and pore properties such as cation selectivity are essential for import [#19, #28]. Tim23 recruits the matrix mtHsp70-based PAM import motor through its matrix-exposed loop, binding Tim44 and the Tim14(Pam18)/Tim16(Pam16) J-protein subcomplex that drives ATP-dependent translocation [#5, #6, #26, #44]. Recent cryo-EM structures revise the long-standing channel model: Tim23 plays a primarily structural role within a Tim17–Tim23–Tim44/Mgr2 core, while the protein-conducting path is formed by the Tim17 cavity sealed laterally by Mgr2, which gates lateral release of hydrophobic stop-transfer substrates into the inner membrane [#33, #34, #39]. Beyond constitutive import, TIM23 interacts with PINK1 and protects it from OMA1-mediated degradation, positioning the translocase as a damage-sensing switch in PINK1–Parkin mitophagy [#35, #36]. The translocase is a target of pathology: mutant huntingtin binds Tim23 and inhibits matrix import [#30], and reduced TIM23 import capacity activates the CHOP branch of the mitochondrial unfolded protein response [#40].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that Tim23 is not a static channel but a membrane-potential-regulated presequence receptor, defining the first dynamic gating principle of the translocase.\",\n      \"evidence\": \"Biochemical dimerization assays with membrane potential manipulation and leucine-repeat mutagenesis in yeast mitochondria\",\n      \"pmids\": [\"8858146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of dimerization\", \"Coupling between dimer dissociation and physical channel opening inferred indirectly\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Linked Tim23 directly to the protein-conducting pore activity of the inner membrane, identifying it as essential for the multiple conductance channel.\",\n      \"evidence\": \"Patch-clamp electrophysiology with antibody inhibition and the tim23-1 mutant\",\n      \"pmids\": [\"9128249\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not distinguish whether Tim23 itself lines the conducting pore\", \"No structural assignment of pore-lining residues\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Resolved how Tim23 bridges two membranes, explaining how precursors transfer from TOM to TIM23 at contact sites and how the IMS domain is positioned as a receptor.\",\n      \"evidence\": \"Protease accessibility, GFP topology mapping, import competition and EM in yeast\",\n      \"pmids\": [\"10830167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of outer-membrane anchoring of the N-terminus not defined\", \"Stoichiometry of contact-site complexes unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined the chaperone requirements for Tim23 biogenesis itself, showing the small TIM8-13 complex escorts Tim23 across the IMS under low potential.\",\n      \"evidence\": \"Co-IP, crosslinking of translocation intermediates, and TIM8-13 depletion import assays; later supported by crystal structure\",\n      \"pmids\": [\"11101512\", \"18706423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TIM8-13 chaperones other substrates in this mode not addressed here\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identified Tim50 as the IMS partner of the Tim23 N-domain that mediates precursor handover from TOM, establishing the receptor architecture of the inner-membrane face.\",\n      \"evidence\": \"Site-specific photocrosslinking, reciprocal co-IP, depletion and antibody inhibition\",\n      \"pmids\": [\"12437925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the presequence-binding residues of Tim50\", \"Channel-gating consequence of Tim50 binding not yet defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Completed identification of the PAM import-motor co-chaperones, showing Tim14/Pam18 and Tim16/Pam16 form a J-protein subcomplex that activates mtHsp70 within the translocase.\",\n      \"evidence\": \"Protein identification, reciprocal co-IP, HPD-motif mutagenesis and depletion import assays\",\n      \"pmids\": [\"14517234\", \"14981506\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How motor activation is mechanically coupled to Tim23 channel dynamics not resolved\", \"Quantitative ATPase kinetics within the assembled translocase not measured\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mapped the conformational and quaternary dynamics of Tim23 in functional membranes, showing TMS2 lines an amphipathic channel that rearranges with gating and that subunit proximities change with potential and substrate.\",\n      \"evidence\": \"Site-directed fluorescence mapping and site-specific crosslinking of Tim23 cysteine mutants in intact mitochondria\",\n      \"pmids\": [\"18692467\", \"17959826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure not obtained\", \"Whether TMS2 forms a true aqueous channel vs lipid-exposed cavity unresolved at this stage\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined the antagonistic regulation of the Tim23 channel by presequences and Tim50, and quantified trans-side presequence binding by Tim44 and Tim23 that supports directional import.\",\n      \"evidence\": \"Photo-affinity labeling with MS mapping, electrophysiology, and SPR/crosslinking with purified components\",\n      \"pmids\": [\"22065641\", \"21969381\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In situ regulation under physiological potential not directly measured\", \"Affinity hierarchy across the full import pathway not fully reconstituted\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established the Tim23 IMS domain as an intrinsically disordered, membrane-associated interaction hub contacting Tim21, Tim50 and Tom22, and identified cardiolipin as a modulator of its membrane attachment.\",\n      \"evidence\": \"NMR with single-residue mapping and membrane model systems including cardiolipin liposomes\",\n      \"pmids\": [\"25263020\", \"25349212\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo functional consequence of cardiolipin effect not validated\", \"Dynamic interactions not captured in an intact translocase structure\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Assigned distinct interaction sites that organize the motor and regulatory subunits, with Tim23 loop 1 anchoring Tim44 and adjacent contacts positioning Pam17.\",\n      \"evidence\": \"Site-specific in vivo photocrosslinking with genetic and co-IP validation\",\n      \"pmids\": [\"25157107\", \"24061477\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise allosteric path from motor docking to channel gating undefined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Detailed the Tim44 NTD/CTD bipartite engagement of presequences and the translocon, and showed cardiolipin tunes Tim50 receptor association with the membrane and Tim23.\",\n      \"evidence\": \"In vivo crosslinking with in vitro binding, plus SAXS structure and MD of the Tim50 receptor\",\n      \"pmids\": [\"28440746\", \"28879236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full assembled translocase structure not yet available at this point\", \"Lipid regulation in cells not quantified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated that cation selectivity of the Tim23 channel is mechanistically required for substrate recognition and import competence.\",\n      \"evidence\": \"Structure-based mutagenesis with electrophysiology and import assays\",\n      \"pmids\": [\"28857742\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Pore-lining residue assignment later revised by cryo-EM showing the Tim17 path\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined Mgr2 and Pam18 as controllers of the lateral gate that decide between matrix translocation and inner-membrane insertion of stop-transfer substrates.\",\n      \"evidence\": \"Electrophysiology, substrate-specific import assays and co-IP in Mgr2/Pam18 perturbation strains\",\n      \"pmids\": [\"30279421\", \"30697167\", \"31764998\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of lateral gating not resolved here\", \"Hydrophobicity threshold values determined in single systems\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Captured the TOM-TIM23 interface at amino-acid resolution by crosslinking a stalled supercomplex, defining the physical map of precursor transfer.\",\n      \"evidence\": \"Chemical crosslinking-MS with structural modeling of a stalled translocation intermediate\",\n      \"pmids\": [\"34588454\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Static snapshot of one stalled state\", \"Did not resolve gating transitions during active translocation\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revised the core channel model by cryo-EM, showing Tim23 is primarily structural while the Tim17 cavity sealed by Mgr2 forms the protein-conducting path.\",\n      \"evidence\": \"Cryo-EM of core TIM23 heterotrimer and active TOM-TIM23 supercomplex with translocating substrate, with biochemical validation\",\n      \"pmids\": [\"37344598\", \"37696957\", \"40877479\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How earlier electrophysiology-defined Tim23 channel properties reconcile with the Tim17 path not fully integrated\", \"Human translocase structure not directly determined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected TIM23 to mitophagy initiation, showing it interacts with PINK1, protects it from OMA1 degradation, and tethers TOM to TIM23 as a stress-sensing switch.\",\n      \"evidence\": \"Co-IP, MS, BN-PAGE, PINK1 mutagenesis and mitophagy assays across cell models including organoids; OMA1 inactivation rescue\",\n      \"pmids\": [\"37160114\", \"38416681\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether import-channel activity per se versus the physical tether drives PINK1 sensing partly unresolved\", \"Role distinct from TOM not fully separated in all conditions\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked TIM23 translocase integrity to disease and quality control, showing prohibitin/OCIAD1 and DNAJC15/OMA1 regulate TIM23 variant stability and that TIMM50 loss preferentially depletes laterally sorted OXPHOS substrates.\",\n      \"evidence\": \"Quantitative proteomics, co-IP, protease perturbation and import assays in patient and engineered cells\",\n      \"pmids\": [\"39630581\", \"38828998\", \"31346086\", \"40877479\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic detail of variant-selective protease targeting incomplete\", \"Two preprint findings on DNAJC15/PINK1 import not yet peer-reviewed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how the historically defined Tim23 channel and gating properties are reconciled with the cryo-EM Tim17-conducting-path model, and how TIM23 substrate selection is regulated dynamically during physiological import and stress.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No integrated structural-electrophysiological model of Tim23's structural role\", \"Regulation of TIM23 import capacity in human disease contexts only partially mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 33, 34]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [22, 27]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [33, 8, 24]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 15, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [1, 30, 35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [3, 34, 38]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [5, 6, 26]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [35, 36]}\n    ],\n    \"complexes\": [\n      \"TIM23 presequence translocase\",\n      \"TOM-TIM23 supercomplex\",\n      \"PAM import motor\",\n      \"PINK1-TOM-TIM23 supercomplex\"\n    ],\n    \"partners\": [\n      \"TIMM50\",\n      \"TIMM17\",\n      \"TIMM44\",\n      \"PAM18\",\n      \"PAM16\",\n      \"MGR2\",\n      \"TIMM21\",\n      \"PINK1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}