{"gene":"TIMM22","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1996,"finding":"Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane. Tim22 is part of a high-molecular-mass assembly distinct from the Tim23-Tim17 complex, and import via Tim22 is independent of Tim23, establishing two separate TIM pathways.","method":"Genetic depletion of Tim22 in yeast, in organello import assays, native gel fractionation","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function with specific import phenotype, independently replicated in subsequent work across multiple labs","pmids":["8955274"],"is_preprint":false},{"year":2002,"finding":"Tim22 is the only essential membrane-integrated subunit of the protein insertion complex. Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters that is voltage-activated and specifically gated by internal targeting signals (not presequences), combining signal recognition, channel formation, and energy transduction in one component.","method":"Reconstitution of purified Tim22 into lipid bilayers; electrophysiology; genetic complementation showing Tim22 is the sole essential membrane subunit","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with electrophysiological characterization, multiple orthogonal methods, replicated by subsequent electrophysiology study","pmids":["11864609"],"is_preprint":false},{"year":1999,"finding":"Tim9 is a component of the TIM22.54 translocase. Tim9 forms two distinct hetero-oligomeric complexes in the intermembrane space: one with Tim10, and one with Tim9, Tim10, and Tim12 that is tightly associated with Tim22 in the inner membrane. The Tim9-Tim10 complex mediates partial translocation of carrier proteins across the outer membrane, while the Tim9.10.12 complex assists further translocation into the inner membrane via TIM22.54.","method":"Protein purification, native gel electrophoresis, co-immunoprecipitation, in organello import assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, native gel, import assays; replicated across multiple subsequent studies","pmids":["9889188"],"is_preprint":false},{"year":2011,"finding":"Sdh3, subunit 3 of respiratory complex II (succinate dehydrogenase), is also a subunit of the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex, demonstrating dual function of Sdh3 in both respiratory complex II and the TIM22 translocase.","method":"Genetic and biochemical approaches including co-immunoprecipitation, native gel electrophoresis, blue native PAGE, and yeast mutant growth analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, genetic epistasis, biochemical fractionation, multiple orthogonal methods in one study","pmids":["22152483"],"is_preprint":false},{"year":2017,"finding":"Acylglycerol kinase (AGK) is a constituent of the TIM22 complex in the mitochondrial inner membrane, assembling with TIMM22 and TIMM29 to support import of a subset of multi-spanning membrane proteins. AGK's function as a TIM22 subunit does not depend on its kinase activity, but enzymatically active AGK is separately required for mitochondrial cristae morphogenesis and apoptotic resistance.","method":"Mitochondrial interactome determination (mass spectrometry), co-immunoprecipitation, BN-PAGE, import assays in AGK-deficient cells and patient tissues","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — independently replicated in two simultaneous papers from different labs (PMIDs 28712724 and 28712726), reciprocal co-IP, import assays, patient-derived cells","pmids":["28712724","28712726"],"is_preprint":false},{"year":2017,"finding":"AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex and facilitate the import and assembly of mitochondrial carrier proteins. Mitochondria from Sengers syndrome patient cells show destabilized TIM22 complex and defects in carrier biogenesis, with downstream perturbations in the TCA cycle.","method":"BN-PAGE of patient mitochondria, import assays, metabolic profiling (TCA cycle), co-immunoprecipitation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient-derived cells, multiple orthogonal methods, replicated in companion paper (PMID 28712724)","pmids":["28712726"],"is_preprint":false},{"year":2016,"finding":"Tim29 (C19orf52) is a novel metazoan-specific subunit of the human TIM22 complex, integrated into the mitochondrial inner membrane with C-terminus in the intermembrane space. Tim29 is required for TIM22 complex stability and hTim22 assembly, and contacts the TOM complex, enabling transport of hydrophobic carrier substrates across the aqueous intermembrane space.","method":"Co-immunoprecipitation, BN-PAGE, siRNA knockdown, in vitro import assays, protease-protection assay for topology","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, reciprocal co-IP, import assays, topology determination, replicated in AGK papers","pmids":["27554484"],"is_preprint":false},{"year":2000,"finding":"Tim18p is an integral membrane subunit of the yeast TIM22 complex. It comigrates and co-immunoprecipitates with Tim54p and Tim12p. Deletion of Tim18p impairs import of several precursor proteins and lowers the apparent mass of the TIM22 complex, suggesting Tim18p functions in assembly and stabilization of the TIM22 complex.","method":"Co-immunoprecipitation, native gel electrophoresis, import assays in tim18Δ yeast, synthetic lethality with tim9/tim10 mutants","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, import assays, genetic epistasis in single lab with multiple methods","pmids":["10648604"],"is_preprint":false},{"year":2013,"finding":"Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria, and Mia40 also binds the Tim22 precursor via noncovalent interactions, assisting Tim22 integration into the inner membrane — extending the MIA pathway beyond IMS proteins.","method":"In organello import assays, disulfide trapping, co-immunoprecipitation, non-reducing SDS-PAGE","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — disulfide trapping and co-IP in single lab, single study","pmids":["23283984"],"is_preprint":false},{"year":2014,"finding":"Conserved cysteine residues of Tim22 form an intramolecular disulfide bond that stabilizes Tim22, particularly at elevated temperatures, through interactions with Tim18. The disulfide bond is required for TIM22 complex integrity and for efficient assembly of TIM22 pathway substrates into the inner membrane under excess substrate conditions.","method":"Cys→Ser mutagenesis, non-reducing SDS-PAGE, import assays in yeast mutants, temperature-sensitive growth analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — mutagenesis with functional readout in single lab, single study","pmids":["24385427"],"is_preprint":false},{"year":2004,"finding":"Hot13p is the first identified component of a pathway mediating assembly of the small TIM complexes in the intermembrane space. The small Tim proteins require Hot13p for assembly into the 70-kDa complex. Oxidizing conditions arrest the ADP/ATP carrier bound to the Tim9-Tim10 complex in the intermembrane space, and this intermediate can be chased into the inner membrane by reductant, indicating that redox state of the small TIMs regulates translocation of substrates to the TIM22 complex.","method":"In organello import assays with oxidant/reductant treatment, native gel electrophoresis, genetic analysis (ΔHot13 mitochondria)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in organello import with chemical manipulation, native gel, single lab","pmids":["15294910"],"is_preprint":false},{"year":2007,"finding":"TIM22 channel activity can be detected in intact mitoplasts (in organello) when an internal signal peptide is present in the intermembrane space; without signal peptide the channel is silent. The channel shows high-conductance (~1000 pS) slightly cationic activity, with low membrane potential keeping it fully open when signal peptide is present, portraying TIM22 as a dynamic, ligand-gated channel.","method":"Electrophysiology of intact mitoplasts, internal signal peptide titration","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — electrophysiological reconstitution in organelle, single lab, single study","pmids":["17462993"],"is_preprint":false},{"year":2010,"finding":"The Tim9-Tim10 complex mediates the import of Tim22 and Tafazzin, but not Tim23, indicating that the Tim9-Tim10 complex selectively handles a subset of inner membrane proteins routed through the TIM22 pathway. MitoBloCK-1, a small molecule, blocks binding of Tim9-Tim10 to substrate during early translocation across the outer membrane, impairing import of carrier proteins (ADP/ATP and phosphate carriers) through the TIM22 but not the TIM23 or Mia40/Erv1 pathways.","method":"Chemical-genetic screen, in vitro import assays, chemical inhibitor (MitoBloCK-1), yeast genetics","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemical inhibitor with specific import pathway readout, multiple substrates tested, validated in mammalian cells","pmids":["20457929"],"is_preprint":false},{"year":2019,"finding":"The human TIM22 complex associates with the MICOS (mitochondrial contact site and cristae organizing system) complex. This association is required for efficient import of carrier proteins into the human mitochondrial inner membrane, suggesting that positioning of the carrier translocase at crista junctions and in proximity to the TOM complex coordinates carrier transport across the intermembrane space.","method":"Proteomic approaches (co-IP/MS), BN-PAGE, MIC10 knockout in HEK293T cells, import assays","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP/MS plus KO cell line with import phenotype, but association not yet structurally defined","pmids":["31103774"],"is_preprint":false},{"year":2020,"finding":"Using a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum, pyruvate carrier (MPC) subunits were identified as unconventional TIM22 cargos with atypical topology (not typical 4 or 6 TM carrier proteins), broadening the known substrate repertoire of the TIM22 pathway.","method":"Temperature-conditional tim22 yeast mutant, quantitative proteomics, import assays, patient cell analysis","journal":"Current biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional mutant with proteomic substrate mapping plus patient validation, two orthogonal approaches","pmids":["32142709"],"is_preprint":false},{"year":2021,"finding":"Sideroflexin (SFXN) proteins, which contain five transmembrane domains, are novel substrates of the human TIM22 complex. Loss of TIM22 function via AGK knockout reduces SFXN protein biogenesis and impairs one-carbon metabolism (serine-dependent cell proliferation).","method":"Quantitative proteomics of AGK KO cells, import assays, serine auxotrophy proliferation assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative proteomics plus import assays, functional metabolic readout, single lab","pmids":["33476211"],"is_preprint":false},{"year":2018,"finding":"Compound heterozygous mutations in human TIMM22 (p.Tyr25Ter and p.Val33Leu in the IMS region) cause early-onset mitochondrial myopathy, reducing TIM22 protein levels and complex assembly, and impairing carrier protein amounts in the inner mitochondrial membrane, establishing that the pore-forming subunit TIMM22 is required for carrier protein biogenesis in humans.","method":"Patient fibroblast biochemistry, BN-PAGE, import assays, cybrid cell lines (mtDNA exclusion), whole-exome sequencing","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with biochemical import phenotype, multiple orthogonal methods, single case","pmids":["30452684"],"is_preprint":false},{"year":2020,"finding":"The IMS and TM4 regions of Tim22 are critically required for interactions with membrane-embedded subunits Tim54, Tim18, and Sdh3, maintaining TIM22 complex architecture. TM1 and TM2 are important for association with Tim18, while TM3 is exclusively required for interaction with Sdh3. Impairment of TIM22 complex assembly reduces translocase activity, alters the mitochondrial network, and affects viability of rho0 cells.","method":"Tim22 region mutagenesis, co-immunoprecipitation, BN-PAGE, import assays, yeast genetics","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis with co-IP and functional import readout, single lab","pmids":["32591483"],"is_preprint":false},{"year":2017,"finding":"Granzyme B breaches the mitochondrial inner membrane through Tim22 (the metabolite carrier translocase pore) in a mitochondrial Hsp70 (mtHsp70)-dependent manner. This noncanonical import pathway (requiring Sam50 for outer membrane entry and Tim22 for inner membrane translocation) is used by granzyme B, granzyme A, and caspase-3 to induce mitochondrial dysfunction and cell death.","method":"Tim22 siRNA knockdown, Sam50 depletion, Tim22 K243/R244 mutagenesis, cell death assays, ROS measurements","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with functional cell death readout, mutagenesis, multiple cytotoxic substrates tested, single lab","pmids":["28338658"],"is_preprint":false},{"year":2023,"finding":"Genetic epistasis in S. cerevisiae shows that impairment of the TIM22 complex rescues respiratory growth defects of yme1Δ cells, and that Yme1 metalloprotease is required for the stability of the TIM22 complex and regulates proteostasis of TIM22 pathway substrates. Excessive TIM22 pathway substrate accumulation is a contributor to the respiratory growth defects caused by loss of Yme1.","method":"Yeast genetic epistasis (double mutants), growth assays, BN-PAGE, import/degradation assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic epistasis with biochemical follow-up, single lab, single study","pmids":["36601773"],"is_preprint":false},{"year":2025,"finding":"The TIM22 complex is selectively required for Fe-S biogenesis in mammalian cells. Loss of TIM22 function reduces iron transporter (mitoferrin) presence on mitochondria, impairing iron uptake from the cytosol; reconstituting mitochondrial iron levels rescues Fe-S biogenesis and cell proliferation in TIMM29-deficient cells and embryonic development in timm29-deficient zebrafish.","method":"Mitochondria-focused CRISPR screening, DepMap co-essentiality analysis, iron sensor (fluorescent), mitoferrin import/localization assays, zebrafish timm29 KO rescue experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR screen plus co-essentiality convergence, direct iron measurement, in vivo rescue in zebrafish, multiple orthogonal methods","pmids":["41418755"],"is_preprint":false},{"year":2025,"finding":"TIMM22 knockdown selectively reduces OCT3 levels on mitochondria without impairing mitochondrial morphology or membrane potential, and this reduction in mitochondrial OCT3 decreases mitochondrial MPP+ uptake, rescuing MPP+-induced mitochondrial fragmentation, complex I inhibition, membrane potential reduction, and caspase activation in SH-SY5Y neuronal cells.","method":"TIMM22 siRNA knockdown, mitochondrial fractionation, OCT3 localization, MPP+ uptake assays, mitochondrial functional assays","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — knockdown with multiple mitochondrial functional readouts, single lab, single study","pmids":["40660178"],"is_preprint":false},{"year":2024,"finding":"Overexpression of TIM22 (and TIM29) reduces intracellular HBV DNA/RNA levels and secreted HBV antigens by reducing HBV core promoter activity through increased expression of SRSF1, which acts as a suppressor of HBV replication.","method":"Overexpression in HBV-infected cells, HBV DNA/RNA quantification, core promoter reporter assay, SRSF1 expression analysis","journal":"Journal of medical virology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — overexpression with reporter assay, single lab, single method per mechanistic step, indirect pathway connection","pmids":["38294104"],"is_preprint":false},{"year":2020,"finding":"Chemical crosslinking mass spectrometry (XL-MS) of the isolated human TIM22 complex defined the molecular arrangement of its subunits, including unexpected crosslinks between the small TIM chaperone complex and core TIM22 subunits, revealing proximity relationships not previously characterized.","method":"Chemical crosslinking (BS3) coupled with mass spectrometry of purified human TIM22 complex","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — structural proximity mapping by XL-MS on purified complex, single lab, no mutagenesis validation","pmids":["33125709"],"is_preprint":false}],"current_model":"TIMM22 is the pore-forming core subunit of the mitochondrial inner membrane TIM22 carrier translocase, forming a voltage-activated, signal-gated aqueous channel that mediates the membrane insertion of multi-spanning hydrophobic proteins (carrier family proteins, TIM subunits, pyruvate carrier subunits, sideroflexins) bearing internal targeting signals; the complex is assembled with multiple accessory subunits (Tim9, Tim10, Tim12, Tim18/Tim54 in yeast; AGK/Tim29/small TIMs in humans) and associates with MICOS at crista junctions, with Tim22's conserved cysteines forming an intramolecular disulfide bond (assisted by Mia40) that stabilizes the complex, while its primary anabolic function includes facilitating transporter-mediated mitochondrial iron uptake required for Fe-S cluster biogenesis."},"narrative":{"mechanistic_narrative":"TIMM22 is the pore-forming core subunit of the mitochondrial inner-membrane TIM22 translocase, which mediates membrane insertion of multi-spanning hydrophobic proteins bearing internal targeting signals along a pathway functionally distinct from the presequence-driven TIM23 route [PMID:8955274]. As the sole essential membrane-integral subunit, purified Tim22 reconstitutes a voltage-activated, high-conductance aqueous channel that is specifically gated open by internal targeting signals rather than presequences, thereby combining signal recognition, channel formation, and energy transduction in one component [PMID:11864609, PMID:17462993]. The active complex is built around accessory subunits that stabilize Tim22 and deliver substrates: the intermembrane-space small TIM chaperones (Tim9-Tim10-Tim12) hand off hydrophobic carriers from the TOM complex to the inner-membrane translocase [PMID:9889188, PMID:20457929], while membrane-embedded partners Tim18 and the dual-function respiratory subunit Sdh3 support assembly and architecture of the complex through distinct contacts mapped to Tim22's transmembrane and IMS regions [PMID:22152483, PMID:10648604, PMID:32591483]. In humans the complex incorporates the metazoan-specific subunit TIM29 and acylglycerol kinase (AGK), the latter acting kinase-independently to maintain complex integrity and carrier import [PMID:28712724, PMID:28712726, PMID:27554484], and it docks at crista junctions via association with the MICOS complex to coordinate transport across the intermembrane space [PMID:31103774]. The translocase imports a broad substrate repertoire including carrier-family proteins, pyruvate carrier (MPC) subunits, and five-transmembrane sideroflexins, linking it to one-carbon metabolism [PMID:32142709, PMID:33476211], and its loss reduces mitoferrin levels and mitochondrial iron uptake required for Fe-S cluster biogenesis [PMID:41418755]. Compound heterozygous TIMM22 mutations cause early-onset mitochondrial myopathy by destabilizing the complex and impairing carrier biogenesis, while AGK mutations cause Sengers syndrome through the same import defect [PMID:30452684, PMID:28712726].","teleology":[{"year":1996,"claim":"Established that carrier-family proteins use an import route separate from the presequence translocase, defining TIM22 as the founding component of a distinct inner-membrane insertion pathway.","evidence":"Genetic depletion of Tim22 in yeast with in organello import assays and native gel fractionation","pmids":["8955274"],"confidence":"High","gaps":["Did not define the channel mechanism or the accessory subunit composition","Substrate spectrum beyond AAC family unknown"]},{"year":1999,"claim":"Resolved how hydrophobic carriers traverse the aqueous intermembrane space by identifying small TIM chaperone complexes that escort substrates to TIM22.","evidence":"Protein purification, native gels, reciprocal co-IP, and import assays of Tim9/Tim10/Tim12 complexes in yeast","pmids":["9889188"],"confidence":"High","gaps":["Structural basis of substrate handoff not defined","Regulation of chaperone-translocase docking unresolved"]},{"year":2000,"claim":"Identified Tim18 as a membrane-integral subunit functioning in assembly and stabilization of the TIM22 complex.","evidence":"Co-IP, native gels, import assays in tim18Δ yeast, and synthetic lethality with small TIM mutants","pmids":["10648604"],"confidence":"High","gaps":["Mechanism by which Tim18 stabilizes the complex not defined","Human ortholog/counterpart not addressed"]},{"year":2002,"claim":"Demonstrated that Tim22 alone forms the import channel, unifying signal recognition, pore formation, and energy transduction in the single essential membrane subunit.","evidence":"Reconstitution of purified Tim22 in lipid bilayers with electrophysiology plus genetic complementation in yeast","pmids":["11864609"],"confidence":"High","gaps":["Atomic structure of the open/closed channel not resolved","How internal signals physically gate the pore unknown"]},{"year":2004,"claim":"Showed that redox state of the small TIM chaperones regulates substrate translocation, with Hot13p mediating their oxidative assembly.","evidence":"In organello import with oxidant/reductant manipulation and native gels in ΔHot13 mitochondria","pmids":["15294910"],"confidence":"Medium","gaps":["Single-lab study without structural validation","Physiological redox triggers in vivo not defined"]},{"year":2007,"claim":"Confirmed in intact organelles that TIM22 is a ligand-gated channel, silent without an internal signal peptide and opened by signal presentation in the IMS.","evidence":"Electrophysiology of intact mitoplasts with internal signal peptide titration","pmids":["17462993"],"confidence":"Medium","gaps":["Single-lab in organello measurement","Quantitative coupling between gating and substrate flux not established"]},{"year":2010,"claim":"Defined substrate selectivity of the small TIM chaperones and provided a chemical tool (MitoBloCK-1) that blocks early TIM22-pathway translocation without affecting other import routes.","evidence":"Chemical-genetic screen, in vitro import assays with inhibitor, validated in mammalian cells","pmids":["20457929"],"confidence":"Medium","gaps":["Molecular target site of MitoBloCK-1 on chaperones not mapped","Full substrate selectivity rules incomplete"]},{"year":2011,"claim":"Revealed an unexpected moonlighting role of the respiratory complex II subunit Sdh3 as a TIM22 assembly factor, linking translocase biogenesis to respiratory subunit pools.","evidence":"Co-IP, native/blue-native PAGE, and genetic growth analysis in yeast","pmids":["22152483"],"confidence":"High","gaps":["How Sdh3 partitions between complex II and TIM22 not defined","Human counterpart for this dual function not established"]},{"year":2013,"claim":"Extended the MIA disulfide-relay pathway beyond IMS proteins by showing Mia40 assists Tim22 integration into the inner membrane.","evidence":"In organello import, disulfide trapping, co-IP, and non-reducing SDS-PAGE","pmids":["23283984"],"confidence":"Medium","gaps":["Single-study disulfide trapping without reconstitution","Kinetics of the Mia40-Tim22 intermediate not quantified"]},{"year":2014,"claim":"Showed that an intramolecular disulfide bond in Tim22, supported by Tim18, stabilizes the protein and is required for complex integrity under substrate or thermal stress.","evidence":"Cys→Ser mutagenesis, non-reducing SDS-PAGE, import assays, and temperature-sensitive growth in yeast","pmids":["24385427"],"confidence":"Medium","gaps":["Single-lab study","Conservation and role of the disulfide in the human complex not directly tested here"]},{"year":2016,"claim":"Identified the metazoan-specific subunit Tim29, defining how the human complex is stabilized and physically bridges to the TOM complex for substrate transfer.","evidence":"Co-IP, BN-PAGE, siRNA knockdown, in vitro import, and protease-protection topology in human cells","pmids":["27554484"],"confidence":"High","gaps":["Structural details of the TIM22-TOM contact not resolved","Whether Tim29 directly gates substrate entry unknown"]},{"year":2017,"claim":"Established AGK as a kinase-independent TIM22 subunit required for complex integrity and carrier import, mechanistically explaining the carrier-biogenesis defect in Sengers syndrome.","evidence":"Mitochondrial interactome MS, co-IP, BN-PAGE, and import/metabolic assays in patient-derived cells (two companion studies)","pmids":["28712724","28712726"],"confidence":"High","gaps":["How a lipid kinase doubles as a structural subunit not fully defined","Separation of AGK's kinase and assembly roles in vivo incomplete"]},{"year":2017,"claim":"Uncovered a noncanonical cytotoxic role: granzymes and caspase-3 exploit Tim22 to breach the inner membrane and induce cell death in an mtHsp70-dependent manner.","evidence":"Tim22 siRNA knockdown, Sam50 depletion, Tim22 residue mutagenesis, and cell-death/ROS assays","pmids":["28338658"],"confidence":"Medium","gaps":["Single-lab study","Whether the death substrates use the canonical signal-gated channel mode unknown"]},{"year":2018,"claim":"Provided direct human genetic proof that TIMM22 is essential for carrier biogenesis, with loss-of-function mutations causing early-onset mitochondrial myopathy.","evidence":"Patient fibroblast biochemistry, BN-PAGE, import assays, cybrids, and whole-exome sequencing","pmids":["30452684"],"confidence":"Medium","gaps":["Single case","Genotype-phenotype relationship across the mutation spectrum not established"]},{"year":2020,"claim":"Broadened the TIM22 substrate repertoire to include atypical-topology cargos (pyruvate carrier subunits) beyond classical 4/6-TM carriers.","evidence":"Temperature-conditional tim22 yeast mutant, quantitative proteomics, import assays, patient cell analysis","pmids":["32142709"],"confidence":"Medium","gaps":["Recognition determinants for atypical cargos not defined","Full substrate census incomplete"]},{"year":2020,"claim":"Mapped the Tim22 regions governing contacts with membrane-embedded subunits, linking complex architecture to translocase activity and mitochondrial network integrity.","evidence":"Tim22 region mutagenesis, co-IP, BN-PAGE, and import assays in yeast","pmids":["32591483"],"confidence":"Medium","gaps":["Single-lab domain-mapping without structure","Human subunit contacts not directly tested"]},{"year":2020,"claim":"Provided proximity-based architecture of the human complex, including unexpected small-TIM-to-core crosslinks.","evidence":"Chemical crosslinking (BS3) mass spectrometry of the purified human TIM22 complex","pmids":["33125709"],"confidence":"Medium","gaps":["No mutagenesis validation of crosslinks","No high-resolution structure"]},{"year":2021,"claim":"Connected TIM22 import to one-carbon metabolism by identifying five-TM sideroflexins as human substrates whose loss impairs serine-dependent proliferation.","evidence":"Quantitative proteomics of AGK KO cells, import assays, and serine auxotrophy proliferation assays","pmids":["33476211"],"confidence":"Medium","gaps":["Direct demonstration of SFXN insertion mechanism limited","Single-lab study"]},{"year":2023,"claim":"Revealed reciprocal proteostatic control: Yme1 metalloprotease stabilizes the TIM22 complex and limits accumulation of TIM22-pathway substrates that otherwise impair respiration.","evidence":"Yeast genetic epistasis, growth assays, BN-PAGE, and import/degradation assays","pmids":["36601773"],"confidence":"Medium","gaps":["Mechanism of Yme1-mediated TIM22 stabilization not defined","Single-lab, yeast-only study"]},{"year":2025,"claim":"Defined a primary anabolic role for TIM22 in mitochondrial iron homeostasis: it sustains mitoferrin levels and iron uptake required for Fe-S cluster biogenesis and growth.","evidence":"Mitochondria-focused CRISPR screen, DepMap co-essentiality, iron sensors, mitoferrin localization, and zebrafish timm29 KO rescue","pmids":["41418755"],"confidence":"High","gaps":["Whether all iron-dependent phenotypes trace to mitoferrin import alone unresolved","Structural basis of mitoferrin insertion not defined"]},{"year":2025,"claim":"Showed TIM22 controls neuronal toxin susceptibility by setting mitochondrial OCT3 levels and thereby MPP+ uptake.","evidence":"TIMM22 siRNA knockdown, mitochondrial fractionation, OCT3 localization, and MPP+ uptake/functional assays in SH-SY5Y cells","pmids":["40660178"],"confidence":"Medium","gaps":["Single-lab study","Whether OCT3 is a direct TIM22 substrate not established"]},{"year":null,"claim":"A high-resolution structure of the assembled human TIM22 complex and a mechanistic account of how internal signals gate the channel during substrate insertion remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No atomic structure of the gated channel","Physical mechanism coupling signal recognition to membrane insertion undefined","Determinants distinguishing canonical carriers from atypical and cytotoxic cargos unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,1,14,15,20]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,11,17]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,4,6,16]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,1,6,14,15]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,16]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[3,6,13,17]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[14,15,20]}],"complexes":["TIM22 carrier translocase","MICOS","TIM9-TIM10-TIM12 small TIM complex"],"partners":["TIMM29","AGK","TIM9","TIM10","TIM12","TIM18","SDH3","MIA40"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y584","full_name":"Mitochondrial import inner membrane translocase subunit Tim22","aliases":["Testis-expressed protein 4"],"length_aa":194,"mass_kda":20.0,"function":"Essential core component of the TIM22 complex, a complex that mediates the import and insertion of multi-pass transmembrane proteins into the mitochondrial inner membrane. In the TIM22 complex, it constitutes the voltage-activated and signal-gated channel. Forms a twin-pore translocase that uses the membrane potential as external driving force in 2 voltage-dependent steps (By similarity)","subcellular_location":"Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y584/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TIMM22","classification":"Common Essential","n_dependent_lines":732,"n_total_lines":1208,"dependency_fraction":0.6059602649006622},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TIMM22","total_profiled":1310},"omim":[{"mim_id":"619465","title":"VISCERAL NEUROPATHY, FAMILIAL, 2, AUTOSOMAL RECESSIVE; VSCN2","url":"https://www.omim.org/entry/619465"},{"mim_id":"618851","title":"COMBINED OXIDATIVE PHOSPHORYLATION DEFICIENCY 43; COXPD43","url":"https://www.omim.org/entry/618851"},{"mim_id":"617380","title":"TRANSLOCASE OF INNER MITOCHONDRIAL MEMBRANE 29; TIMM29","url":"https://www.omim.org/entry/617380"},{"mim_id":"614948","title":"TAM41 MITOCHONDRIAL TRANSLOCATOR ASSEMBLY AND MAINTENANCE HOMOLOG; TAMM41","url":"https://www.omim.org/entry/614948"},{"mim_id":"613659","title":"GASTRIC CANCER","url":"https://www.omim.org/entry/613659"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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of carrier proteins into the mitochondrial inner membrane mediated by Tim22.","date":"1996","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8955274","citation_count":266,"is_preprint":false},{"pmid":"11864609","id":"PMC_11864609","title":"Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel.","date":"2002","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/11864609","citation_count":135,"is_preprint":false},{"pmid":"9889188","id":"PMC_9889188","title":"Tim9, a new component of the TIM22.54 translocase in mitochondria.","date":"1999","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9889188","citation_count":124,"is_preprint":false},{"pmid":"22152483","id":"PMC_22152483","title":"Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane.","date":"2011","source":"Molecular 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mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis.","date":"2017","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/28338658","citation_count":40,"is_preprint":false},{"pmid":"32142709","id":"PMC_32142709","title":"Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos.","date":"2020","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/32142709","citation_count":36,"is_preprint":false},{"pmid":"20457929","id":"PMC_20457929","title":"Substrate specificity of the TIM22 mitochondrial import pathway revealed with small molecule inhibitor of protein translocation.","date":"2010","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/20457929","citation_count":36,"is_preprint":false},{"pmid":"30452684","id":"PMC_30452684","title":"Mutations of the mitochondrial carrier translocase channel subunit TIM22 cause early-onset mitochondrial myopathy.","date":"2018","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30452684","citation_count":33,"is_preprint":false},{"pmid":"17462993","id":"PMC_17462993","title":"Awaking TIM22, a dynamic ligand-gated channel for protein insertion in the mitochondrial inner membrane.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17462993","citation_count":33,"is_preprint":false},{"pmid":"24385427","id":"PMC_24385427","title":"Intramolecular disulfide bond of Tim22 protein maintains integrity of the TIM22 complex in the mitochondrial inner membrane.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24385427","citation_count":28,"is_preprint":false},{"pmid":"11509656","id":"PMC_11509656","title":"The essential function of the small Tim proteins in the TIM22 import pathway does not depend on formation of the soluble 70-kilodalton complex.","date":"2001","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11509656","citation_count":28,"is_preprint":false},{"pmid":"33476211","id":"PMC_33476211","title":"The TIM22 complex mediates the import of sideroflexins and is required for efficient mitochondrial one-carbon metabolism.","date":"2021","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/33476211","citation_count":27,"is_preprint":false},{"pmid":"12656987","id":"PMC_12656987","title":"The role of Tim9p in the assembly of the TIM22 import complexes.","date":"2003","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/12656987","citation_count":21,"is_preprint":false},{"pmid":"15644337","id":"PMC_15644337","title":"The phosphate carrier has an ability to be sorted to either the TIM22 pathway or the TIM23 pathway for its import into yeast mitochondria.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15644337","citation_count":15,"is_preprint":false},{"pmid":"33125709","id":"PMC_33125709","title":"Defining the architecture of the human TIM22 complex by chemical crosslinking.","date":"2020","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/33125709","citation_count":15,"is_preprint":false},{"pmid":"15320873","id":"PMC_15320873","title":"A cryptic matrix targeting signal of the yeast ADP/ATP carrier normally inserted by the TIM22 complex is recognized by the TIM23 machinery.","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15320873","citation_count":9,"is_preprint":false},{"pmid":"36601773","id":"PMC_36601773","title":"Functional crosstalk between the TIM22 complex and YME1 machinery maintains mitochondrial proteostasis and integrity.","date":"2023","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/36601773","citation_count":8,"is_preprint":false},{"pmid":"33014348","id":"PMC_33014348","title":"Independent accretion of TIM22 complex subunits in the animal and fungal lineages.","date":"2020","source":"F1000Research","url":"https://pubmed.ncbi.nlm.nih.gov/33014348","citation_count":8,"is_preprint":false},{"pmid":"32591483","id":"PMC_32591483","title":"Conserved regions of budding yeast Tim22 have a role in structural organization of the carrier translocase.","date":"2020","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/32591483","citation_count":6,"is_preprint":false},{"pmid":"38294104","id":"PMC_38294104","title":"TIM22 and TIM29 inhibit HBV replication by up-regulating SRSF1 expression.","date":"2024","source":"Journal of medical virology","url":"https://pubmed.ncbi.nlm.nih.gov/38294104","citation_count":4,"is_preprint":false},{"pmid":"41418755","id":"PMC_41418755","title":"The TIM22 carrier translocase supports cell proliferation by facilitating mitochondrial iron uptake for Fe-S biogenesis.","date":"2025","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/41418755","citation_count":1,"is_preprint":false},{"pmid":"40660178","id":"PMC_40660178","title":"Organic cation transporter 3 on neuronal mitochondria mediates MPP+-induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner.","date":"2025","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/40660178","citation_count":0,"is_preprint":false},{"pmid":"41418750","id":"PMC_41418750","title":"Transporting the transporter: TIM22 translocates mitoferrins to enable mitochondrial iron-sulfur cluster synthesis.","date":"2025","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/41418750","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.13.682092","title":"The Mitochondrial F-box protein 1 and DJ-1 homolog HSP31 support cellular proteostasis during mitochondrial protein import clogging","date":"2025-10-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.13.682092","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15364,"output_tokens":5788,"usd":0.066456,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14554,"output_tokens":5730,"usd":0.10801,"stage2_stop_reason":"end_turn"},"total_usd":0.174466,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane. Tim22 is part of a high-molecular-mass assembly distinct from the Tim23-Tim17 complex, and import via Tim22 is independent of Tim23, establishing two separate TIM pathways.\",\n      \"method\": \"Genetic depletion of Tim22 in yeast, in organello import assays, native gel fractionation\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss-of-function with specific import phenotype, independently replicated in subsequent work across multiple labs\",\n      \"pmids\": [\"8955274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Tim22 is the only essential membrane-integrated subunit of the protein insertion complex. Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters that is voltage-activated and specifically gated by internal targeting signals (not presequences), combining signal recognition, channel formation, and energy transduction in one component.\",\n      \"method\": \"Reconstitution of purified Tim22 into lipid bilayers; electrophysiology; genetic complementation showing Tim22 is the sole essential membrane subunit\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with electrophysiological characterization, multiple orthogonal methods, replicated by subsequent electrophysiology study\",\n      \"pmids\": [\"11864609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Tim9 is a component of the TIM22.54 translocase. Tim9 forms two distinct hetero-oligomeric complexes in the intermembrane space: one with Tim10, and one with Tim9, Tim10, and Tim12 that is tightly associated with Tim22 in the inner membrane. The Tim9-Tim10 complex mediates partial translocation of carrier proteins across the outer membrane, while the Tim9.10.12 complex assists further translocation into the inner membrane via TIM22.54.\",\n      \"method\": \"Protein purification, native gel electrophoresis, co-immunoprecipitation, in organello import assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, native gel, import assays; replicated across multiple subsequent studies\",\n      \"pmids\": [\"9889188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Sdh3, subunit 3 of respiratory complex II (succinate dehydrogenase), is also a subunit of the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex, demonstrating dual function of Sdh3 in both respiratory complex II and the TIM22 translocase.\",\n      \"method\": \"Genetic and biochemical approaches including co-immunoprecipitation, native gel electrophoresis, blue native PAGE, and yeast mutant growth analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, genetic epistasis, biochemical fractionation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"22152483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Acylglycerol kinase (AGK) is a constituent of the TIM22 complex in the mitochondrial inner membrane, assembling with TIMM22 and TIMM29 to support import of a subset of multi-spanning membrane proteins. AGK's function as a TIM22 subunit does not depend on its kinase activity, but enzymatically active AGK is separately required for mitochondrial cristae morphogenesis and apoptotic resistance.\",\n      \"method\": \"Mitochondrial interactome determination (mass spectrometry), co-immunoprecipitation, BN-PAGE, import assays in AGK-deficient cells and patient tissues\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independently replicated in two simultaneous papers from different labs (PMIDs 28712724 and 28712726), reciprocal co-IP, import assays, patient-derived cells\",\n      \"pmids\": [\"28712724\", \"28712726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex and facilitate the import and assembly of mitochondrial carrier proteins. Mitochondria from Sengers syndrome patient cells show destabilized TIM22 complex and defects in carrier biogenesis, with downstream perturbations in the TCA cycle.\",\n      \"method\": \"BN-PAGE of patient mitochondria, import assays, metabolic profiling (TCA cycle), co-immunoprecipitation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient-derived cells, multiple orthogonal methods, replicated in companion paper (PMID 28712724)\",\n      \"pmids\": [\"28712726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Tim29 (C19orf52) is a novel metazoan-specific subunit of the human TIM22 complex, integrated into the mitochondrial inner membrane with C-terminus in the intermembrane space. Tim29 is required for TIM22 complex stability and hTim22 assembly, and contacts the TOM complex, enabling transport of hydrophobic carrier substrates across the aqueous intermembrane space.\",\n      \"method\": \"Co-immunoprecipitation, BN-PAGE, siRNA knockdown, in vitro import assays, protease-protection assay for topology\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, reciprocal co-IP, import assays, topology determination, replicated in AGK papers\",\n      \"pmids\": [\"27554484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Tim18p is an integral membrane subunit of the yeast TIM22 complex. It comigrates and co-immunoprecipitates with Tim54p and Tim12p. Deletion of Tim18p impairs import of several precursor proteins and lowers the apparent mass of the TIM22 complex, suggesting Tim18p functions in assembly and stabilization of the TIM22 complex.\",\n      \"method\": \"Co-immunoprecipitation, native gel electrophoresis, import assays in tim18Δ yeast, synthetic lethality with tim9/tim10 mutants\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, import assays, genetic epistasis in single lab with multiple methods\",\n      \"pmids\": [\"10648604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria, and Mia40 also binds the Tim22 precursor via noncovalent interactions, assisting Tim22 integration into the inner membrane — extending the MIA pathway beyond IMS proteins.\",\n      \"method\": \"In organello import assays, disulfide trapping, co-immunoprecipitation, non-reducing SDS-PAGE\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — disulfide trapping and co-IP in single lab, single study\",\n      \"pmids\": [\"23283984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Conserved cysteine residues of Tim22 form an intramolecular disulfide bond that stabilizes Tim22, particularly at elevated temperatures, through interactions with Tim18. The disulfide bond is required for TIM22 complex integrity and for efficient assembly of TIM22 pathway substrates into the inner membrane under excess substrate conditions.\",\n      \"method\": \"Cys→Ser mutagenesis, non-reducing SDS-PAGE, import assays in yeast mutants, temperature-sensitive growth analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — mutagenesis with functional readout in single lab, single study\",\n      \"pmids\": [\"24385427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Hot13p is the first identified component of a pathway mediating assembly of the small TIM complexes in the intermembrane space. The small Tim proteins require Hot13p for assembly into the 70-kDa complex. Oxidizing conditions arrest the ADP/ATP carrier bound to the Tim9-Tim10 complex in the intermembrane space, and this intermediate can be chased into the inner membrane by reductant, indicating that redox state of the small TIMs regulates translocation of substrates to the TIM22 complex.\",\n      \"method\": \"In organello import assays with oxidant/reductant treatment, native gel electrophoresis, genetic analysis (ΔHot13 mitochondria)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in organello import with chemical manipulation, native gel, single lab\",\n      \"pmids\": [\"15294910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TIM22 channel activity can be detected in intact mitoplasts (in organello) when an internal signal peptide is present in the intermembrane space; without signal peptide the channel is silent. The channel shows high-conductance (~1000 pS) slightly cationic activity, with low membrane potential keeping it fully open when signal peptide is present, portraying TIM22 as a dynamic, ligand-gated channel.\",\n      \"method\": \"Electrophysiology of intact mitoplasts, internal signal peptide titration\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — electrophysiological reconstitution in organelle, single lab, single study\",\n      \"pmids\": [\"17462993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The Tim9-Tim10 complex mediates the import of Tim22 and Tafazzin, but not Tim23, indicating that the Tim9-Tim10 complex selectively handles a subset of inner membrane proteins routed through the TIM22 pathway. MitoBloCK-1, a small molecule, blocks binding of Tim9-Tim10 to substrate during early translocation across the outer membrane, impairing import of carrier proteins (ADP/ATP and phosphate carriers) through the TIM22 but not the TIM23 or Mia40/Erv1 pathways.\",\n      \"method\": \"Chemical-genetic screen, in vitro import assays, chemical inhibitor (MitoBloCK-1), yeast genetics\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemical inhibitor with specific import pathway readout, multiple substrates tested, validated in mammalian cells\",\n      \"pmids\": [\"20457929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The human TIM22 complex associates with the MICOS (mitochondrial contact site and cristae organizing system) complex. This association is required for efficient import of carrier proteins into the human mitochondrial inner membrane, suggesting that positioning of the carrier translocase at crista junctions and in proximity to the TOM complex coordinates carrier transport across the intermembrane space.\",\n      \"method\": \"Proteomic approaches (co-IP/MS), BN-PAGE, MIC10 knockout in HEK293T cells, import assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP/MS plus KO cell line with import phenotype, but association not yet structurally defined\",\n      \"pmids\": [\"31103774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Using a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum, pyruvate carrier (MPC) subunits were identified as unconventional TIM22 cargos with atypical topology (not typical 4 or 6 TM carrier proteins), broadening the known substrate repertoire of the TIM22 pathway.\",\n      \"method\": \"Temperature-conditional tim22 yeast mutant, quantitative proteomics, import assays, patient cell analysis\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional mutant with proteomic substrate mapping plus patient validation, two orthogonal approaches\",\n      \"pmids\": [\"32142709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Sideroflexin (SFXN) proteins, which contain five transmembrane domains, are novel substrates of the human TIM22 complex. Loss of TIM22 function via AGK knockout reduces SFXN protein biogenesis and impairs one-carbon metabolism (serine-dependent cell proliferation).\",\n      \"method\": \"Quantitative proteomics of AGK KO cells, import assays, serine auxotrophy proliferation assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative proteomics plus import assays, functional metabolic readout, single lab\",\n      \"pmids\": [\"33476211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Compound heterozygous mutations in human TIMM22 (p.Tyr25Ter and p.Val33Leu in the IMS region) cause early-onset mitochondrial myopathy, reducing TIM22 protein levels and complex assembly, and impairing carrier protein amounts in the inner mitochondrial membrane, establishing that the pore-forming subunit TIMM22 is required for carrier protein biogenesis in humans.\",\n      \"method\": \"Patient fibroblast biochemistry, BN-PAGE, import assays, cybrid cell lines (mtDNA exclusion), whole-exome sequencing\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with biochemical import phenotype, multiple orthogonal methods, single case\",\n      \"pmids\": [\"30452684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The IMS and TM4 regions of Tim22 are critically required for interactions with membrane-embedded subunits Tim54, Tim18, and Sdh3, maintaining TIM22 complex architecture. TM1 and TM2 are important for association with Tim18, while TM3 is exclusively required for interaction with Sdh3. Impairment of TIM22 complex assembly reduces translocase activity, alters the mitochondrial network, and affects viability of rho0 cells.\",\n      \"method\": \"Tim22 region mutagenesis, co-immunoprecipitation, BN-PAGE, import assays, yeast genetics\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis with co-IP and functional import readout, single lab\",\n      \"pmids\": [\"32591483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Granzyme B breaches the mitochondrial inner membrane through Tim22 (the metabolite carrier translocase pore) in a mitochondrial Hsp70 (mtHsp70)-dependent manner. This noncanonical import pathway (requiring Sam50 for outer membrane entry and Tim22 for inner membrane translocation) is used by granzyme B, granzyme A, and caspase-3 to induce mitochondrial dysfunction and cell death.\",\n      \"method\": \"Tim22 siRNA knockdown, Sam50 depletion, Tim22 K243/R244 mutagenesis, cell death assays, ROS measurements\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with functional cell death readout, mutagenesis, multiple cytotoxic substrates tested, single lab\",\n      \"pmids\": [\"28338658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Genetic epistasis in S. cerevisiae shows that impairment of the TIM22 complex rescues respiratory growth defects of yme1Δ cells, and that Yme1 metalloprotease is required for the stability of the TIM22 complex and regulates proteostasis of TIM22 pathway substrates. Excessive TIM22 pathway substrate accumulation is a contributor to the respiratory growth defects caused by loss of Yme1.\",\n      \"method\": \"Yeast genetic epistasis (double mutants), growth assays, BN-PAGE, import/degradation assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic epistasis with biochemical follow-up, single lab, single study\",\n      \"pmids\": [\"36601773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The TIM22 complex is selectively required for Fe-S biogenesis in mammalian cells. Loss of TIM22 function reduces iron transporter (mitoferrin) presence on mitochondria, impairing iron uptake from the cytosol; reconstituting mitochondrial iron levels rescues Fe-S biogenesis and cell proliferation in TIMM29-deficient cells and embryonic development in timm29-deficient zebrafish.\",\n      \"method\": \"Mitochondria-focused CRISPR screening, DepMap co-essentiality analysis, iron sensor (fluorescent), mitoferrin import/localization assays, zebrafish timm29 KO rescue experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR screen plus co-essentiality convergence, direct iron measurement, in vivo rescue in zebrafish, multiple orthogonal methods\",\n      \"pmids\": [\"41418755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TIMM22 knockdown selectively reduces OCT3 levels on mitochondria without impairing mitochondrial morphology or membrane potential, and this reduction in mitochondrial OCT3 decreases mitochondrial MPP+ uptake, rescuing MPP+-induced mitochondrial fragmentation, complex I inhibition, membrane potential reduction, and caspase activation in SH-SY5Y neuronal cells.\",\n      \"method\": \"TIMM22 siRNA knockdown, mitochondrial fractionation, OCT3 localization, MPP+ uptake assays, mitochondrial functional assays\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — knockdown with multiple mitochondrial functional readouts, single lab, single study\",\n      \"pmids\": [\"40660178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Overexpression of TIM22 (and TIM29) reduces intracellular HBV DNA/RNA levels and secreted HBV antigens by reducing HBV core promoter activity through increased expression of SRSF1, which acts as a suppressor of HBV replication.\",\n      \"method\": \"Overexpression in HBV-infected cells, HBV DNA/RNA quantification, core promoter reporter assay, SRSF1 expression analysis\",\n      \"journal\": \"Journal of medical virology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — overexpression with reporter assay, single lab, single method per mechanistic step, indirect pathway connection\",\n      \"pmids\": [\"38294104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Chemical crosslinking mass spectrometry (XL-MS) of the isolated human TIM22 complex defined the molecular arrangement of its subunits, including unexpected crosslinks between the small TIM chaperone complex and core TIM22 subunits, revealing proximity relationships not previously characterized.\",\n      \"method\": \"Chemical crosslinking (BS3) coupled with mass spectrometry of purified human TIM22 complex\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — structural proximity mapping by XL-MS on purified complex, single lab, no mutagenesis validation\",\n      \"pmids\": [\"33125709\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TIMM22 is the pore-forming core subunit of the mitochondrial inner membrane TIM22 carrier translocase, forming a voltage-activated, signal-gated aqueous channel that mediates the membrane insertion of multi-spanning hydrophobic proteins (carrier family proteins, TIM subunits, pyruvate carrier subunits, sideroflexins) bearing internal targeting signals; the complex is assembled with multiple accessory subunits (Tim9, Tim10, Tim12, Tim18/Tim54 in yeast; AGK/Tim29/small TIMs in humans) and associates with MICOS at crista junctions, with Tim22's conserved cysteines forming an intramolecular disulfide bond (assisted by Mia40) that stabilizes the complex, while its primary anabolic function includes facilitating transporter-mediated mitochondrial iron uptake required for Fe-S cluster biogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TIMM22 is the pore-forming core subunit of the mitochondrial inner-membrane TIM22 translocase, which mediates membrane insertion of multi-spanning hydrophobic proteins bearing internal targeting signals along a pathway functionally distinct from the presequence-driven TIM23 route [#0]. As the sole essential membrane-integral subunit, purified Tim22 reconstitutes a voltage-activated, high-conductance aqueous channel that is specifically gated open by internal targeting signals rather than presequences, thereby combining signal recognition, channel formation, and energy transduction in one component [#1, #11]. The active complex is built around accessory subunits that stabilize Tim22 and deliver substrates: the intermembrane-space small TIM chaperones (Tim9-Tim10-Tim12) hand off hydrophobic carriers from the TOM complex to the inner-membrane translocase [#2, #12], while membrane-embedded partners Tim18 and the dual-function respiratory subunit Sdh3 support assembly and architecture of the complex through distinct contacts mapped to Tim22's transmembrane and IMS regions [#3, #7, #17]. In humans the complex incorporates the metazoan-specific subunit TIM29 and acylglycerol kinase (AGK), the latter acting kinase-independently to maintain complex integrity and carrier import [#4, #5, #6], and it docks at crista junctions via association with the MICOS complex to coordinate transport across the intermembrane space [#13]. The translocase imports a broad substrate repertoire including carrier-family proteins, pyruvate carrier (MPC) subunits, and five-transmembrane sideroflexins, linking it to one-carbon metabolism [#14, #15], and its loss reduces mitoferrin levels and mitochondrial iron uptake required for Fe-S cluster biogenesis [#20]. Compound heterozygous TIMM22 mutations cause early-onset mitochondrial myopathy by destabilizing the complex and impairing carrier biogenesis, while AGK mutations cause Sengers syndrome through the same import defect [#16, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that carrier-family proteins use an import route separate from the presequence translocase, defining TIM22 as the founding component of a distinct inner-membrane insertion pathway.\",\n      \"evidence\": \"Genetic depletion of Tim22 in yeast with in organello import assays and native gel fractionation\",\n      \"pmids\": [\"8955274\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the channel mechanism or the accessory subunit composition\", \"Substrate spectrum beyond AAC family unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Resolved how hydrophobic carriers traverse the aqueous intermembrane space by identifying small TIM chaperone complexes that escort substrates to TIM22.\",\n      \"evidence\": \"Protein purification, native gels, reciprocal co-IP, and import assays of Tim9/Tim10/Tim12 complexes in yeast\",\n      \"pmids\": [\"9889188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of substrate handoff not defined\", \"Regulation of chaperone-translocase docking unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identified Tim18 as a membrane-integral subunit functioning in assembly and stabilization of the TIM22 complex.\",\n      \"evidence\": \"Co-IP, native gels, import assays in tim18\\u0394 yeast, and synthetic lethality with small TIM mutants\",\n      \"pmids\": [\"10648604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Tim18 stabilizes the complex not defined\", \"Human ortholog/counterpart not addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstrated that Tim22 alone forms the import channel, unifying signal recognition, pore formation, and energy transduction in the single essential membrane subunit.\",\n      \"evidence\": \"Reconstitution of purified Tim22 in lipid bilayers with electrophysiology plus genetic complementation in yeast\",\n      \"pmids\": [\"11864609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the open/closed channel not resolved\", \"How internal signals physically gate the pore unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed that redox state of the small TIM chaperones regulates substrate translocation, with Hot13p mediating their oxidative assembly.\",\n      \"evidence\": \"In organello import with oxidant/reductant manipulation and native gels in \\u0394Hot13 mitochondria\",\n      \"pmids\": [\"15294910\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without structural validation\", \"Physiological redox triggers in vivo not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Confirmed in intact organelles that TIM22 is a ligand-gated channel, silent without an internal signal peptide and opened by signal presentation in the IMS.\",\n      \"evidence\": \"Electrophysiology of intact mitoplasts with internal signal peptide titration\",\n      \"pmids\": [\"17462993\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab in organello measurement\", \"Quantitative coupling between gating and substrate flux not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined substrate selectivity of the small TIM chaperones and provided a chemical tool (MitoBloCK-1) that blocks early TIM22-pathway translocation without affecting other import routes.\",\n      \"evidence\": \"Chemical-genetic screen, in vitro import assays with inhibitor, validated in mammalian cells\",\n      \"pmids\": [\"20457929\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular target site of MitoBloCK-1 on chaperones not mapped\", \"Full substrate selectivity rules incomplete\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed an unexpected moonlighting role of the respiratory complex II subunit Sdh3 as a TIM22 assembly factor, linking translocase biogenesis to respiratory subunit pools.\",\n      \"evidence\": \"Co-IP, native/blue-native PAGE, and genetic growth analysis in yeast\",\n      \"pmids\": [\"22152483\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Sdh3 partitions between complex II and TIM22 not defined\", \"Human counterpart for this dual function not established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended the MIA disulfide-relay pathway beyond IMS proteins by showing Mia40 assists Tim22 integration into the inner membrane.\",\n      \"evidence\": \"In organello import, disulfide trapping, co-IP, and non-reducing SDS-PAGE\",\n      \"pmids\": [\"23283984\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-study disulfide trapping without reconstitution\", \"Kinetics of the Mia40-Tim22 intermediate not quantified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed that an intramolecular disulfide bond in Tim22, supported by Tim18, stabilizes the protein and is required for complex integrity under substrate or thermal stress.\",\n      \"evidence\": \"Cys\\u2192Ser mutagenesis, non-reducing SDS-PAGE, import assays, and temperature-sensitive growth in yeast\",\n      \"pmids\": [\"24385427\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Conservation and role of the disulfide in the human complex not directly tested here\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified the metazoan-specific subunit Tim29, defining how the human complex is stabilized and physically bridges to the TOM complex for substrate transfer.\",\n      \"evidence\": \"Co-IP, BN-PAGE, siRNA knockdown, in vitro import, and protease-protection topology in human cells\",\n      \"pmids\": [\"27554484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural details of the TIM22-TOM contact not resolved\", \"Whether Tim29 directly gates substrate entry unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established AGK as a kinase-independent TIM22 subunit required for complex integrity and carrier import, mechanistically explaining the carrier-biogenesis defect in Sengers syndrome.\",\n      \"evidence\": \"Mitochondrial interactome MS, co-IP, BN-PAGE, and import/metabolic assays in patient-derived cells (two companion studies)\",\n      \"pmids\": [\"28712724\", \"28712726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a lipid kinase doubles as a structural subunit not fully defined\", \"Separation of AGK's kinase and assembly roles in vivo incomplete\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Uncovered a noncanonical cytotoxic role: granzymes and caspase-3 exploit Tim22 to breach the inner membrane and induce cell death in an mtHsp70-dependent manner.\",\n      \"evidence\": \"Tim22 siRNA knockdown, Sam50 depletion, Tim22 residue mutagenesis, and cell-death/ROS assays\",\n      \"pmids\": [\"28338658\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Whether the death substrates use the canonical signal-gated channel mode unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided direct human genetic proof that TIMM22 is essential for carrier biogenesis, with loss-of-function mutations causing early-onset mitochondrial myopathy.\",\n      \"evidence\": \"Patient fibroblast biochemistry, BN-PAGE, import assays, cybrids, and whole-exome sequencing\",\n      \"pmids\": [\"30452684\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single case\", \"Genotype-phenotype relationship across the mutation spectrum not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Broadened the TIM22 substrate repertoire to include atypical-topology cargos (pyruvate carrier subunits) beyond classical 4/6-TM carriers.\",\n      \"evidence\": \"Temperature-conditional tim22 yeast mutant, quantitative proteomics, import assays, patient cell analysis\",\n      \"pmids\": [\"32142709\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Recognition determinants for atypical cargos not defined\", \"Full substrate census incomplete\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mapped the Tim22 regions governing contacts with membrane-embedded subunits, linking complex architecture to translocase activity and mitochondrial network integrity.\",\n      \"evidence\": \"Tim22 region mutagenesis, co-IP, BN-PAGE, and import assays in yeast\",\n      \"pmids\": [\"32591483\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab domain-mapping without structure\", \"Human subunit contacts not directly tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided proximity-based architecture of the human complex, including unexpected small-TIM-to-core crosslinks.\",\n      \"evidence\": \"Chemical crosslinking (BS3) mass spectrometry of the purified human TIM22 complex\",\n      \"pmids\": [\"33125709\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis validation of crosslinks\", \"No high-resolution structure\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected TIM22 import to one-carbon metabolism by identifying five-TM sideroflexins as human substrates whose loss impairs serine-dependent proliferation.\",\n      \"evidence\": \"Quantitative proteomics of AGK KO cells, import assays, and serine auxotrophy proliferation assays\",\n      \"pmids\": [\"33476211\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demonstration of SFXN insertion mechanism limited\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed reciprocal proteostatic control: Yme1 metalloprotease stabilizes the TIM22 complex and limits accumulation of TIM22-pathway substrates that otherwise impair respiration.\",\n      \"evidence\": \"Yeast genetic epistasis, growth assays, BN-PAGE, and import/degradation assays\",\n      \"pmids\": [\"36601773\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of Yme1-mediated TIM22 stabilization not defined\", \"Single-lab, yeast-only study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a primary anabolic role for TIM22 in mitochondrial iron homeostasis: it sustains mitoferrin levels and iron uptake required for Fe-S cluster biogenesis and growth.\",\n      \"evidence\": \"Mitochondria-focused CRISPR screen, DepMap co-essentiality, iron sensors, mitoferrin localization, and zebrafish timm29 KO rescue\",\n      \"pmids\": [\"41418755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether all iron-dependent phenotypes trace to mitoferrin import alone unresolved\", \"Structural basis of mitoferrin insertion not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed TIM22 controls neuronal toxin susceptibility by setting mitochondrial OCT3 levels and thereby MPP+ uptake.\",\n      \"evidence\": \"TIMM22 siRNA knockdown, mitochondrial fractionation, OCT3 localization, and MPP+ uptake/functional assays in SH-SY5Y cells\",\n      \"pmids\": [\"40660178\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Whether OCT3 is a direct TIM22 substrate not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of the assembled human TIM22 complex and a mechanistic account of how internal signals gate the channel during substrate insertion remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No atomic structure of the gated channel\", \"Physical mechanism coupling signal recognition to membrane insertion undefined\", \"Determinants distinguishing canonical carriers from atypical and cytotoxic cargos unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 1, 14, 15, 20]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 11, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 4, 6, 16]},\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [0, 1, 6, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 1, 6, 14, 15]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 16]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [3, 6, 13, 17]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [14, 15, 20]}\n    ],\n    \"complexes\": [\n      \"TIM22 carrier translocase\",\n      \"MICOS\",\n      \"TIM9-TIM10-TIM12 small TIM complex\"\n    ],\n    \"partners\": [\n      \"TIMM29\",\n      \"AGK\",\n      \"TIM9\",\n      \"TIM10\",\n      \"TIM12\",\n      \"TIM18\",\n      \"SDH3\",\n      \"MIA40\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}