{"gene":"TIMM21","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2005,"finding":"Tim21 (yeast ortholog of TIMM21) is a subunit of the TIM23 presequence translocase that interacts with the TOM complex and is specific to the TOM-tethered, sorting-active form of TIM23. The presequence translocase switches between a Tim21-containing form (for inner membrane insertion/TOM coupling) and a Tim21-free, PAM-bound form (for matrix translocation), with Tim17 playing a crucial role in mediating this switch by promoting inner membrane insertion and binding Pam18.","method":"Co-immunoprecipitation, genetic interaction analysis, import assays in yeast mitochondria","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, functional import assays, replicated across multiple experiments in a high-impact study; foundational mechanism paper","pmids":["15797382"],"is_preprint":false},{"year":2006,"finding":"The IMS-exposed C-terminal domain of Tim21 (yeast ortholog) directly binds the IMS domain of the TOM complex receptor Tom22, physically connecting the TOM and TIM23 translocases. The crystal structure of the Tim21 binding domain was solved at 1.6 Å resolution, revealing a novel α/β-mixed fold with two α-helices flanked by an eight-stranded β-sheet. Negatively charged residues of Tom22 are important for this interaction.","method":"X-ray crystallography (1.6 Å resolution), binding assays, mutagenesis of Tom22","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and binding assays in a single rigorous study","pmids":["17099692"],"is_preprint":false},{"year":2006,"finding":"Tim21 (yeast ortholog) is a subunit of the sorting-active form of the presequence translocase and directly recruits proton-pumping respiratory-chain complexes (e.g., complex III/IV), stimulating preprotein insertion into the inner membrane in a membrane-potential-dependent manner. This represents a physical coupling between the presequence translocase and the respiratory chain.","method":"Co-immunoprecipitation, import assays, native gel electrophoresis, Tim21-deletion yeast strains","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, functional import assays with deletion mutants, replicated findings","pmids":["17113393"],"is_preprint":false},{"year":2007,"finding":"The respiratory chain-bound (Tim21-containing, sorting-form) presequence translocase is actively engaged in preprotein translocation, containing preproteins in transit destined for both the inner membrane and matrix. The motor subunits Pam16/18 interact with the respiratory chain independently of Tim21, indicating a stepwise assembly of the PAM motor rather than en bloc recruitment.","method":"Co-immunoprecipitation, cross-linking, import intermediate trapping assays in yeast","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal biochemical methods (co-IP, cross-linking, import trapping), mechanistically resolves translocase assembly order","pmids":["18070913"],"is_preprint":false},{"year":2012,"finding":"Human TIM21 (TIMM21) is present not only in the TIM23 inner-membrane presequence translocase but also in major respiratory-chain assembly intermediates (termed MITRAC complexes) containing both mitochondria-synthesized and imported subunits. TIM21 is dispensable for general presequence protein import but is specifically required for integration of early-assembling, presequence-containing subunits into respiratory-chain complexes (cytochrome c oxidase assembly intermediates).","method":"Mass spectrometry, co-immunoprecipitation, siRNA knockdown of TIM21, pulse-chase import assays in human cells","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, MS identification, siRNA KD with specific assembly phenotype; human cell system with multiple orthogonal methods","pmids":["23260140"],"is_preprint":false},{"year":2012,"finding":"Mgr2 (yeast) is a new TIM23 subunit required for binding of Tim21 to TIM23-CORE, establishing a binding chain of TIM23(CORE)–Mgr2/Tim21–respiratory chain. Loss of Mgr2 specifically destabilizes the Tim21-containing sorting form and impairs TOM-TIM23 coupling and presequence preprotein import.","method":"Co-immunoprecipitation, deletion mutant analysis, native gel electrophoresis, import assays in yeast","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, genetic deletion with defined import phenotype, multiple orthogonal methods","pmids":["22613836"],"is_preprint":false},{"year":2013,"finding":"The presequence receptor Tim50 interacts with Tim21 (yeast ortholog) in a signal-sensitive manner, involving the IMS domain of the Tim23 channel. Upon recognition of an incoming presequence signal, Tim21 is released from Tim50, which promotes recruitment of Pam17 and triggers formation of the motor-associated (matrix-import) form of the TIM23 complex.","method":"Co-immunoprecipitation, cross-linking, import assays with presequence peptides, yeast genetics","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — signal-dependent co-IP, cross-linking, and import assays with multiple controls establishing dynamic mechanism","pmids":["23403928"],"is_preprint":false},{"year":2013,"finding":"The TM2 transmembrane helix of Tim23 is involved in the recruitment of Tim21 (yeast ortholog) to the Tim23 channel, while the C-terminal sequence of Tim23 functions as an inhibitor of Tim21 binding. The matrix-exposed loop L1 of Tim23 generates specificity in association of Tim21 with the core complex, and Tim21 antagonistically determines the bound and free forms of the PAM subcomplex during import.","method":"Site-directed mutagenesis, co-immunoprecipitation, yeast growth assays, in vitro binding assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with co-IP, single lab, defines Tim21 binding domain on Tim23","pmids":["24061477"],"is_preprint":false},{"year":2014,"finding":"NMR spectroscopy at single-residue resolution revealed that the IMS domain of Tim23 (yeast ortholog) contains multiple binding sites that can interact with Tim21(IMS), Tim50(IMS), and Tom22(IMS), supporting a central role of Tim23(IMS) in formation and regulation of the presequence translocase. The atomic details of the dynamic Tim23(IMS)–Tim21(IMS) complex were characterized.","method":"Nuclear magnetic resonance (NMR) spectroscopy, chemical shift perturbation mapping","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with single-residue interaction mapping, single lab but rigorous structural method","pmids":["25263020"],"is_preprint":false},{"year":2017,"finding":"Tim17 (yeast ortholog) uses conserved G/AXXXG/A motifs in its transmembrane regions to interact with Tim23 and regulate assembly of TIM23 into either the PAM-containing (matrix import) or Tim21-containing (sorting) form. Disruption of these motifs destabilizes the TIM23 channel and abrogates recruitment of Tim21 to the sorting complex.","method":"Site-directed mutagenesis, co-immunoprecipitation, yeast viability assays, import assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with co-IP and functional import assays, single lab","pmids":["27994013"],"is_preprint":false},{"year":2018,"finding":"Human TIM21 (TIMM21) undergoes dynamic changes during respiratory chain biogenesis and its behavior is influenced by ROMO1, a new TIM23 constituent. In the absence of ROMO1, TIM21 dynamics during respiratory chain biogenesis are altered, although ROMO1 is dispensable for general presequence import. This establishes a functional link between ROMO1 and TIM21 within the human TIM23 translocase in the context of respiratory chain assembly.","method":"ROMO1 knockout cell line, mass spectrometry identification of TIM23 complex, co-immunoprecipitation, import assays in human cells","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cell line with MS and co-IP, single lab, mechanistic link to ROMO1 established but details of TIM21 dynamics are partially characterized","pmids":["30598479"],"is_preprint":false}],"current_model":"TIMM21/Tim21 is a peripheral subunit of the mitochondrial inner-membrane presequence translocase (TIM23 complex) that, via its IMS-exposed domain, bridges TOM and TIM23 by directly binding Tom22, recruits respiratory-chain complexes to drive membrane-potential-dependent inner-membrane protein sorting, and is dynamically released from Tim50 upon presequence recognition to allow PAM motor recruitment for matrix translocation; in humans, TIM21 additionally localizes to MITRAC respiratory-chain assembly intermediates where it is specifically required for integration of presequence-containing subunits into respiratory-chain complexes, establishing a molecular link between protein import and respiratory-chain biogenesis."},"narrative":{"mechanistic_narrative":"TIMM21 (Tim21) is a peripheral subunit of the mitochondrial inner-membrane presequence translocase (TIM23 complex) that physically couples protein import to respiratory-chain biogenesis [PMID:15797382, PMID:23260140]. It is specific to the sorting-active, TOM-tethered form of TIM23, where its IMS-exposed C-terminal domain—a novel α/β-mixed fold—directly binds negatively charged residues of the TOM receptor Tom22 to bridge the TOM and TIM23 translocases [PMID:15797382, PMID:17099692]. The same domain engages multiple binding sites on the Tim23 IMS region, which it shares with Tim50, defining a dynamic, signal-sensitive interaction network: upon recognition of an incoming presequence, Tim21 is released from Tim50, promoting Pam17 recruitment and conversion to the PAM motor-associated form competent for matrix translocation [PMID:23403928, PMID:25263020]. Recruitment of Tim21 to the TIM23 core depends on the Tim23 TM2 helix and on Mgr2, which links Tim21 to the core and to the respiratory chain, while Tim17 G/AXXXG/A motifs govern the switch between sorting and matrix-import forms [PMID:22613836, PMID:24061477, PMID:27994013]. Beyond bridging translocases, the sorting-form translocase recruits proton-pumping respiratory-chain complexes to stimulate membrane-potential-dependent inner-membrane insertion of preproteins [PMID:17113393, PMID:18070913]. In human cells, TIMM21 is additionally a component of MITRAC respiratory-chain assembly intermediates and, although dispensable for general presequence import, is specifically required for integration of early presequence-containing subunits into respiratory-chain complexes such as cytochrome c oxidase, with its dynamics modulated by ROMO1 [PMID:23260140, PMID:30598479].","teleology":[{"year":2005,"claim":"Established that the presequence translocase exists in two functionally distinct forms and identified Tim21 as the marker of the TOM-coupled, sorting-active form, framing TIM23 as a modular machine that switches between membrane insertion and matrix translocation.","evidence":"Co-IP, genetic interaction analysis, and import assays in yeast mitochondria","pmids":["15797382"],"confidence":"High","gaps":["Molecular contacts mediating TOM-TIM23 coupling not defined at the residue level","Trigger for the form switch not yet identified"]},{"year":2006,"claim":"Defined the structural and physical basis of TOM-TIM23 coupling by showing the Tim21 IMS domain directly binds Tom22, and solved the domain fold, converting a functional association into an atomic interaction.","evidence":"1.6 Å X-ray crystallography, binding assays, and Tom22 mutagenesis","pmids":["17099692"],"confidence":"High","gaps":["Stoichiometry and affinity in the intact translocase not measured","Does not address how this interaction is regulated during import"]},{"year":2006,"claim":"Revealed that Tim21 couples the sorting translocase to the respiratory chain, mechanistically linking the membrane potential generated by proton-pumping complexes to preprotein insertion.","evidence":"Co-IP, import assays, native gels, and Tim21-deletion yeast strains","pmids":["17113393"],"confidence":"High","gaps":["Direct binding interface between Tim21 and respiratory complexes not resolved","Whether the same coupling operates in mammals not addressed here"]},{"year":2007,"claim":"Showed the respiratory chain-bound translocase is actively translocating preproteins and that PAM motor subunits assemble stepwise rather than en bloc, refining the order of translocase remodeling during import.","evidence":"Co-IP, cross-linking, and import intermediate trapping in yeast","pmids":["18070913"],"confidence":"High","gaps":["Precise timing of Tim21 departure relative to PAM assembly not pinned down","How preproteins are partitioned between inner-membrane and matrix fates not resolved"]},{"year":2012,"claim":"Extended the function to human cells, placing TIMM21 in MITRAC respiratory-chain assembly intermediates and assigning it a specific role in integrating presequence-containing subunits, establishing a conserved import–respiratory biogenesis link.","evidence":"Mass spectrometry, co-IP, siRNA knockdown, and pulse-chase import assays in human cells","pmids":["23260140"],"confidence":"High","gaps":["Direct human TIM21 binding partners within MITRAC not fully enumerated","Whether human TIM21 binds Tom22 as in yeast not tested here"]},{"year":2012,"claim":"Identified Mgr2 as the bridge linking Tim21 to the TIM23 core and the respiratory chain, defining a TIM23(CORE)–Mgr2/Tim21–respiratory chain binding chain and a structural requirement for the sorting form.","evidence":"Co-IP, deletion mutant analysis, native gels, and import assays in yeast","pmids":["22613836"],"confidence":"High","gaps":["Human ortholog of Mgr2 in this chain not addressed","Structural detail of the Mgr2-Tim21 contact unknown"]},{"year":2013,"claim":"Demonstrated that Tim50 release of Tim21 is signal-sensitive, providing the mechanistic trigger by which presequence recognition converts the sorting form into the matrix-import (PAM-bound) form.","evidence":"Signal-dependent co-IP, cross-linking, and import assays with presequence peptides in yeast","pmids":["23403928"],"confidence":"High","gaps":["Conformational change in Tim23 IMS that drives the switch not visualized","Whether the same signal-sensitivity operates in human TIM21 not tested"]},{"year":2013,"claim":"Mapped the determinants on Tim23 governing Tim21 recruitment—TM2 promotes binding while the C-terminus inhibits it—showing how the channel itself controls assembly of the sorting versus motor forms.","evidence":"Site-directed mutagenesis, co-IP, and in vitro binding assays in yeast","pmids":["24061477"],"confidence":"Medium","gaps":["Single-lab mutagenesis study without structural confirmation of the regulatory contacts","Functional consequence in mammalian cells not examined"]},{"year":2014,"claim":"Resolved at single-residue resolution that the Tim23 IMS domain carries overlapping binding sites for Tim21, Tim50, and Tom22, explaining how the channel acts as a hub coordinating competing interactions during translocase remodeling.","evidence":"NMR spectroscopy with chemical shift perturbation mapping","pmids":["25263020"],"confidence":"High","gaps":["Dynamics measured on isolated domains, not the intact membrane-embedded translocase","Order and kinetics of site occupancy during active import not determined"]},{"year":2017,"claim":"Showed that Tim17 G/AXXXG/A transmembrane motifs control TIM23 assembly into either the PAM or Tim21 form, identifying a membrane-helix code that determines translocase fate and Tim21 recruitment.","evidence":"Site-directed mutagenesis, co-IP, viability and import assays in yeast","pmids":["27994013"],"confidence":"Medium","gaps":["Single-lab study; structural basis of motif-driven assembly not solved","Mammalian relevance not tested"]},{"year":2018,"claim":"Linked human TIM21 dynamics during respiratory-chain biogenesis to the TIM23 constituent ROMO1, identifying a new modulator of TIM21 behavior specific to assembly rather than general import.","evidence":"ROMO1 knockout cells, mass spectrometry, co-IP, and import assays in human cells","pmids":["30598479"],"confidence":"Medium","gaps":["Mechanism by which ROMO1 alters TIM21 dynamics only partially characterized","Direct ROMO1-TIM21 contact not structurally defined"]},{"year":null,"claim":"How the regulatory logic and structural transitions defined in yeast map onto the human TIM21 MITRAC function, and the atomic mechanism by which TIM21 integrates presequence-containing subunits into respiratory complexes, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of intact human TIM21-containing TIM23 or MITRAC complex","Direct human binding partners and substrate-handoff mechanism in respiratory assembly not fully resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2,4]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[2,4,10]}],"complexes":["TIM23 presequence translocase (sorting form)","MITRAC respiratory-chain assembly intermediate"],"partners":["TOMM22","TIMM50","TIMM23","MGR2","TIMM17","ROMO1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BVV7","full_name":"Mitochondrial import inner membrane translocase subunit Tim21","aliases":["TIM21-like protein, mitochondrial"],"length_aa":248,"mass_kda":28.2,"function":"Participates in the translocation of transit peptide-containing proteins across the mitochondrial inner membrane. Also required for assembly of mitochondrial respiratory chain complex I and complex IV as component of the MITRAC (mitochondrial translation regulation assembly intermediate of cytochrome c oxidase complex) complex. Probably shuttles between the presequence translocase and respiratory-chain assembly intermediates in a process that promotes incorporation of early nuclear-encoded subunits into these complexes","subcellular_location":"Mitochondrion membrane","url":"https://www.uniprot.org/uniprotkb/Q9BVV7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TIMM21","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TIMM21","total_profiled":1310},"omim":[{"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":"615180","title":"TRANSLOCASE OF INNER MITOCHONDRIAL MEMBRANE 21; TIMM21","url":"https://www.omim.org/entry/615180"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TIMM21"},"hgnc":{"alias_symbol":["HSPC154","TIM21"],"prev_symbol":["C18orf55"]},"alphafold":{"accession":"Q9BVV7","domains":[{"cath_id":"3.10.450.320","chopping":"137-245","consensus_level":"high","plddt":90.6822,"start":137,"end":245},{"cath_id":"1.20.5","chopping":"106-134","consensus_level":"medium","plddt":88.4879,"start":106,"end":134}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BVV7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BVV7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BVV7-F1-predicted_aligned_error_v6.png","plddt_mean":73.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TIMM21","jax_strain_url":"https://www.jax.org/strain/search?query=TIMM21"},"sequence":{"accession":"Q9BVV7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BVV7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BVV7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BVV7"}},"corpus_meta":[{"pmid":"15797382","id":"PMC_15797382","title":"Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17.","date":"2005","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/15797382","citation_count":281,"is_preprint":false},{"pmid":"23260140","id":"PMC_23260140","title":"MITRAC links mitochondrial protein translocation to respiratory-chain assembly and translational regulation.","date":"2012","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/23260140","citation_count":201,"is_preprint":false},{"pmid":"17113393","id":"PMC_17113393","title":"A role for Tim21 in membrane-potential-dependent preprotein sorting in mitochondria.","date":"2006","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/17113393","citation_count":147,"is_preprint":false},{"pmid":"18070913","id":"PMC_18070913","title":"Sorting switch of mitochondrial presequence translocase involves coupling of motor module to respiratory chain.","date":"2007","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/18070913","citation_count":90,"is_preprint":false},{"pmid":"22613836","id":"PMC_22613836","title":"Mgr2 promotes coupling of the mitochondrial presequence translocase to partner complexes.","date":"2012","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/22613836","citation_count":81,"is_preprint":false},{"pmid":"17099692","id":"PMC_17099692","title":"The Tim21 binding domain connects the preprotein translocases of both mitochondrial membranes.","date":"2006","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/17099692","citation_count":55,"is_preprint":false},{"pmid":"23403928","id":"PMC_23403928","title":"Signal recognition initiates reorganization of the presequence translocase during protein import.","date":"2013","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/23403928","citation_count":55,"is_preprint":false},{"pmid":"30598479","id":"PMC_30598479","title":"ROMO1 is a constituent of the human presequence translocase required for YME1L protease import.","date":"2018","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30598479","citation_count":52,"is_preprint":false},{"pmid":"27650541","id":"PMC_27650541","title":"Unique long non-coding RNA expression signature in ETV6/RUNX1-driven B-cell precursor acute lymphoblastic leukemia.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27650541","citation_count":30,"is_preprint":false},{"pmid":"17199091","id":"PMC_17199091","title":"Proteomic analysis of proteins differentially expressed in preeclamptic trophoblasts.","date":"2007","source":"Gynecologic and obstetric investigation","url":"https://pubmed.ncbi.nlm.nih.gov/17199091","citation_count":30,"is_preprint":false},{"pmid":"23051955","id":"PMC_23051955","title":"Establishment of paclitaxel-resistant cell line and the underlying mechanism on drug resistance.","date":"2012","source":"International journal of gynecological cancer : official journal of the International Gynecological Cancer Society","url":"https://pubmed.ncbi.nlm.nih.gov/23051955","citation_count":26,"is_preprint":false},{"pmid":"25263020","id":"PMC_25263020","title":"Molecular basis of the dynamic structure of the TIM23 complex in the mitochondrial intermembrane space.","date":"2014","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/25263020","citation_count":23,"is_preprint":false},{"pmid":"24061477","id":"PMC_24061477","title":"Molecular insights revealing interaction of Tim23 and channel subunits of presequence translocase.","date":"2013","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24061477","citation_count":21,"is_preprint":false},{"pmid":"31134102","id":"PMC_31134102","title":"SnRK1 Kinase and the NAC Transcription Factor SOG1 Are Components of a Novel Signaling Pathway Mediating the Low Energy Response Triggered by ATP Depletion.","date":"2019","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/31134102","citation_count":20,"is_preprint":false},{"pmid":"27994013","id":"PMC_27994013","title":"Role of Tim17 Transmembrane Regions in Regulating the Architecture of Presequence Translocase and Mitochondrial DNA Stability.","date":"2017","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/27994013","citation_count":18,"is_preprint":false},{"pmid":"21611166","id":"PMC_21611166","title":"In silico survey of the mitochondrial protein uptake and maturation systems in the brown alga Ectocarpus siliculosus.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21611166","citation_count":7,"is_preprint":false},{"pmid":"31449839","id":"PMC_31449839","title":"Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study.","date":"2019","source":"Biochimica et biophysica acta. General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/31449839","citation_count":5,"is_preprint":false},{"pmid":"31445064","id":"PMC_31445064","title":"Conformational ensemble of an intrinsically flexible loop in mitochondrial import protein Tim21 studied by modeling and molecular dynamics simulations.","date":"2019","source":"Biochimica et biophysica acta. General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/31445064","citation_count":4,"is_preprint":false},{"pmid":"40457403","id":"PMC_40457403","title":"Genome-to-genome analysis reveals associations between human and mycobacterial genetic variation in tuberculosis patients from Tanzania.","date":"2025","source":"BMC medical genomics","url":"https://pubmed.ncbi.nlm.nih.gov/40457403","citation_count":3,"is_preprint":false},{"pmid":"41639907","id":"PMC_41639907","title":"Proteomic profiles in inclusion body myositis and polymyositis with mitochondrial pathology.","date":"2026","source":"Acta neuropathologica communications","url":"https://pubmed.ncbi.nlm.nih.gov/41639907","citation_count":1,"is_preprint":false},{"pmid":"37689677","id":"PMC_37689677","title":"NLG1, encoding a mitochondrial membrane protein, controls leaf and grain development in rice.","date":"2023","source":"BMC plant biology","url":"https://pubmed.ncbi.nlm.nih.gov/37689677","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11313,"output_tokens":2799,"usd":0.037962,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10216,"output_tokens":3633,"usd":0.070953,"stage2_stop_reason":"end_turn"},"total_usd":0.108915,"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\": 2005,\n      \"finding\": \"Tim21 (yeast ortholog of TIMM21) is a subunit of the TIM23 presequence translocase that interacts with the TOM complex and is specific to the TOM-tethered, sorting-active form of TIM23. The presequence translocase switches between a Tim21-containing form (for inner membrane insertion/TOM coupling) and a Tim21-free, PAM-bound form (for matrix translocation), with Tim17 playing a crucial role in mediating this switch by promoting inner membrane insertion and binding Pam18.\",\n      \"method\": \"Co-immunoprecipitation, genetic interaction analysis, import assays in yeast mitochondria\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, functional import assays, replicated across multiple experiments in a high-impact study; foundational mechanism paper\",\n      \"pmids\": [\"15797382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The IMS-exposed C-terminal domain of Tim21 (yeast ortholog) directly binds the IMS domain of the TOM complex receptor Tom22, physically connecting the TOM and TIM23 translocases. The crystal structure of the Tim21 binding domain was solved at 1.6 Å resolution, revealing a novel α/β-mixed fold with two α-helices flanked by an eight-stranded β-sheet. Negatively charged residues of Tom22 are important for this interaction.\",\n      \"method\": \"X-ray crystallography (1.6 Å resolution), binding assays, mutagenesis of Tom22\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and binding assays in a single rigorous study\",\n      \"pmids\": [\"17099692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Tim21 (yeast ortholog) is a subunit of the sorting-active form of the presequence translocase and directly recruits proton-pumping respiratory-chain complexes (e.g., complex III/IV), stimulating preprotein insertion into the inner membrane in a membrane-potential-dependent manner. This represents a physical coupling between the presequence translocase and the respiratory chain.\",\n      \"method\": \"Co-immunoprecipitation, import assays, native gel electrophoresis, Tim21-deletion yeast strains\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, functional import assays with deletion mutants, replicated findings\",\n      \"pmids\": [\"17113393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The respiratory chain-bound (Tim21-containing, sorting-form) presequence translocase is actively engaged in preprotein translocation, containing preproteins in transit destined for both the inner membrane and matrix. The motor subunits Pam16/18 interact with the respiratory chain independently of Tim21, indicating a stepwise assembly of the PAM motor rather than en bloc recruitment.\",\n      \"method\": \"Co-immunoprecipitation, cross-linking, import intermediate trapping assays in yeast\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal biochemical methods (co-IP, cross-linking, import trapping), mechanistically resolves translocase assembly order\",\n      \"pmids\": [\"18070913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human TIM21 (TIMM21) is present not only in the TIM23 inner-membrane presequence translocase but also in major respiratory-chain assembly intermediates (termed MITRAC complexes) containing both mitochondria-synthesized and imported subunits. TIM21 is dispensable for general presequence protein import but is specifically required for integration of early-assembling, presequence-containing subunits into respiratory-chain complexes (cytochrome c oxidase assembly intermediates).\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, siRNA knockdown of TIM21, pulse-chase import assays in human cells\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, MS identification, siRNA KD with specific assembly phenotype; human cell system with multiple orthogonal methods\",\n      \"pmids\": [\"23260140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Mgr2 (yeast) is a new TIM23 subunit required for binding of Tim21 to TIM23-CORE, establishing a binding chain of TIM23(CORE)–Mgr2/Tim21–respiratory chain. Loss of Mgr2 specifically destabilizes the Tim21-containing sorting form and impairs TOM-TIM23 coupling and presequence preprotein import.\",\n      \"method\": \"Co-immunoprecipitation, deletion mutant analysis, native gel electrophoresis, import assays in yeast\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, genetic deletion with defined import phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"22613836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The presequence receptor Tim50 interacts with Tim21 (yeast ortholog) in a signal-sensitive manner, involving the IMS domain of the Tim23 channel. Upon recognition of an incoming presequence signal, Tim21 is released from Tim50, which promotes recruitment of Pam17 and triggers formation of the motor-associated (matrix-import) form of the TIM23 complex.\",\n      \"method\": \"Co-immunoprecipitation, cross-linking, import assays with presequence peptides, yeast genetics\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — signal-dependent co-IP, cross-linking, and import assays with multiple controls establishing dynamic mechanism\",\n      \"pmids\": [\"23403928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The TM2 transmembrane helix of Tim23 is involved in the recruitment of Tim21 (yeast ortholog) to the Tim23 channel, while the C-terminal sequence of Tim23 functions as an inhibitor of Tim21 binding. The matrix-exposed loop L1 of Tim23 generates specificity in association of Tim21 with the core complex, and Tim21 antagonistically determines the bound and free forms of the PAM subcomplex during import.\",\n      \"method\": \"Site-directed mutagenesis, co-immunoprecipitation, yeast growth assays, in vitro binding assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with co-IP, single lab, defines Tim21 binding domain on Tim23\",\n      \"pmids\": [\"24061477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NMR spectroscopy at single-residue resolution revealed that the IMS domain of Tim23 (yeast ortholog) contains multiple binding sites that can interact with Tim21(IMS), Tim50(IMS), and Tom22(IMS), supporting a central role of Tim23(IMS) in formation and regulation of the presequence translocase. The atomic details of the dynamic Tim23(IMS)–Tim21(IMS) complex were characterized.\",\n      \"method\": \"Nuclear magnetic resonance (NMR) spectroscopy, chemical shift perturbation mapping\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with single-residue interaction mapping, single lab but rigorous structural method\",\n      \"pmids\": [\"25263020\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Tim17 (yeast ortholog) uses conserved G/AXXXG/A motifs in its transmembrane regions to interact with Tim23 and regulate assembly of TIM23 into either the PAM-containing (matrix import) or Tim21-containing (sorting) form. Disruption of these motifs destabilizes the TIM23 channel and abrogates recruitment of Tim21 to the sorting complex.\",\n      \"method\": \"Site-directed mutagenesis, co-immunoprecipitation, yeast viability assays, import assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with co-IP and functional import assays, single lab\",\n      \"pmids\": [\"27994013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Human TIM21 (TIMM21) undergoes dynamic changes during respiratory chain biogenesis and its behavior is influenced by ROMO1, a new TIM23 constituent. In the absence of ROMO1, TIM21 dynamics during respiratory chain biogenesis are altered, although ROMO1 is dispensable for general presequence import. This establishes a functional link between ROMO1 and TIM21 within the human TIM23 translocase in the context of respiratory chain assembly.\",\n      \"method\": \"ROMO1 knockout cell line, mass spectrometry identification of TIM23 complex, co-immunoprecipitation, import assays in human cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cell line with MS and co-IP, single lab, mechanistic link to ROMO1 established but details of TIM21 dynamics are partially characterized\",\n      \"pmids\": [\"30598479\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TIMM21/Tim21 is a peripheral subunit of the mitochondrial inner-membrane presequence translocase (TIM23 complex) that, via its IMS-exposed domain, bridges TOM and TIM23 by directly binding Tom22, recruits respiratory-chain complexes to drive membrane-potential-dependent inner-membrane protein sorting, and is dynamically released from Tim50 upon presequence recognition to allow PAM motor recruitment for matrix translocation; in humans, TIM21 additionally localizes to MITRAC respiratory-chain assembly intermediates where it is specifically required for integration of presequence-containing subunits into respiratory-chain complexes, establishing a molecular link between protein import and respiratory-chain biogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TIMM21 (Tim21) is a peripheral subunit of the mitochondrial inner-membrane presequence translocase (TIM23 complex) that physically couples protein import to respiratory-chain biogenesis [#0, #4]. It is specific to the sorting-active, TOM-tethered form of TIM23, where its IMS-exposed C-terminal domain—a novel α/β-mixed fold—directly binds negatively charged residues of the TOM receptor Tom22 to bridge the TOM and TIM23 translocases [#0, #1]. The same domain engages multiple binding sites on the Tim23 IMS region, which it shares with Tim50, defining a dynamic, signal-sensitive interaction network: upon recognition of an incoming presequence, Tim21 is released from Tim50, promoting Pam17 recruitment and conversion to the PAM motor-associated form competent for matrix translocation [#6, #8]. Recruitment of Tim21 to the TIM23 core depends on the Tim23 TM2 helix and on Mgr2, which links Tim21 to the core and to the respiratory chain, while Tim17 G/AXXXG/A motifs govern the switch between sorting and matrix-import forms [#5, #7, #9]. Beyond bridging translocases, the sorting-form translocase recruits proton-pumping respiratory-chain complexes to stimulate membrane-potential-dependent inner-membrane insertion of preproteins [#2, #3]. In human cells, TIMM21 is additionally a component of MITRAC respiratory-chain assembly intermediates and, although dispensable for general presequence import, is specifically required for integration of early presequence-containing subunits into respiratory-chain complexes such as cytochrome c oxidase, with its dynamics modulated by ROMO1 [#4, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established that the presequence translocase exists in two functionally distinct forms and identified Tim21 as the marker of the TOM-coupled, sorting-active form, framing TIM23 as a modular machine that switches between membrane insertion and matrix translocation.\",\n      \"evidence\": \"Co-IP, genetic interaction analysis, and import assays in yeast mitochondria\",\n      \"pmids\": [\"15797382\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular contacts mediating TOM-TIM23 coupling not defined at the residue level\", \"Trigger for the form switch not yet identified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the structural and physical basis of TOM-TIM23 coupling by showing the Tim21 IMS domain directly binds Tom22, and solved the domain fold, converting a functional association into an atomic interaction.\",\n      \"evidence\": \"1.6 Å X-ray crystallography, binding assays, and Tom22 mutagenesis\",\n      \"pmids\": [\"17099692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and affinity in the intact translocase not measured\", \"Does not address how this interaction is regulated during import\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealed that Tim21 couples the sorting translocase to the respiratory chain, mechanistically linking the membrane potential generated by proton-pumping complexes to preprotein insertion.\",\n      \"evidence\": \"Co-IP, import assays, native gels, and Tim21-deletion yeast strains\",\n      \"pmids\": [\"17113393\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding interface between Tim21 and respiratory complexes not resolved\", \"Whether the same coupling operates in mammals not addressed here\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed the respiratory chain-bound translocase is actively translocating preproteins and that PAM motor subunits assemble stepwise rather than en bloc, refining the order of translocase remodeling during import.\",\n      \"evidence\": \"Co-IP, cross-linking, and import intermediate trapping in yeast\",\n      \"pmids\": [\"18070913\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise timing of Tim21 departure relative to PAM assembly not pinned down\", \"How preproteins are partitioned between inner-membrane and matrix fates not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended the function to human cells, placing TIMM21 in MITRAC respiratory-chain assembly intermediates and assigning it a specific role in integrating presequence-containing subunits, establishing a conserved import–respiratory biogenesis link.\",\n      \"evidence\": \"Mass spectrometry, co-IP, siRNA knockdown, and pulse-chase import assays in human cells\",\n      \"pmids\": [\"23260140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct human TIM21 binding partners within MITRAC not fully enumerated\", \"Whether human TIM21 binds Tom22 as in yeast not tested here\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified Mgr2 as the bridge linking Tim21 to the TIM23 core and the respiratory chain, defining a TIM23(CORE)–Mgr2/Tim21–respiratory chain binding chain and a structural requirement for the sorting form.\",\n      \"evidence\": \"Co-IP, deletion mutant analysis, native gels, and import assays in yeast\",\n      \"pmids\": [\"22613836\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human ortholog of Mgr2 in this chain not addressed\", \"Structural detail of the Mgr2-Tim21 contact unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated that Tim50 release of Tim21 is signal-sensitive, providing the mechanistic trigger by which presequence recognition converts the sorting form into the matrix-import (PAM-bound) form.\",\n      \"evidence\": \"Signal-dependent co-IP, cross-linking, and import assays with presequence peptides in yeast\",\n      \"pmids\": [\"23403928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational change in Tim23 IMS that drives the switch not visualized\", \"Whether the same signal-sensitivity operates in human TIM21 not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapped the determinants on Tim23 governing Tim21 recruitment—TM2 promotes binding while the C-terminus inhibits it—showing how the channel itself controls assembly of the sorting versus motor forms.\",\n      \"evidence\": \"Site-directed mutagenesis, co-IP, and in vitro binding assays in yeast\",\n      \"pmids\": [\"24061477\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab mutagenesis study without structural confirmation of the regulatory contacts\", \"Functional consequence in mammalian cells not examined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved at single-residue resolution that the Tim23 IMS domain carries overlapping binding sites for Tim21, Tim50, and Tom22, explaining how the channel acts as a hub coordinating competing interactions during translocase remodeling.\",\n      \"evidence\": \"NMR spectroscopy with chemical shift perturbation mapping\",\n      \"pmids\": [\"25263020\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics measured on isolated domains, not the intact membrane-embedded translocase\", \"Order and kinetics of site occupancy during active import not determined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed that Tim17 G/AXXXG/A transmembrane motifs control TIM23 assembly into either the PAM or Tim21 form, identifying a membrane-helix code that determines translocase fate and Tim21 recruitment.\",\n      \"evidence\": \"Site-directed mutagenesis, co-IP, viability and import assays in yeast\",\n      \"pmids\": [\"27994013\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study; structural basis of motif-driven assembly not solved\", \"Mammalian relevance not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked human TIM21 dynamics during respiratory-chain biogenesis to the TIM23 constituent ROMO1, identifying a new modulator of TIM21 behavior specific to assembly rather than general import.\",\n      \"evidence\": \"ROMO1 knockout cells, mass spectrometry, co-IP, and import assays in human cells\",\n      \"pmids\": [\"30598479\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which ROMO1 alters TIM21 dynamics only partially characterized\", \"Direct ROMO1-TIM21 contact not structurally defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the regulatory logic and structural transitions defined in yeast map onto the human TIM21 MITRAC function, and the atomic mechanism by which TIM21 integrates presequence-containing subunits into respiratory complexes, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of intact human TIM21-containing TIM23 or MITRAC complex\", \"Direct human binding partners and substrate-handoff mechanism in respiratory assembly not fully resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [2, 4, 10]}\n    ],\n    \"complexes\": [\n      \"TIM23 presequence translocase (sorting form)\",\n      \"MITRAC respiratory-chain assembly intermediate\"\n    ],\n    \"partners\": [\n      \"TOMM22\",\n      \"TIMM50\",\n      \"TIMM23\",\n      \"MGR2\",\n      \"TIMM17\",\n      \"ROMO1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}