{"gene":"TRAK1","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2005,"finding":"OIP106 (TRAK1) associates with kinesin heavy chain (KIF5C, KIF5A, KIF5B) and mitochondria; the interaction domain was localized to residues 124–283 of GRIF-1 (the TRAK1 paralog), and both OIP106 and GRIF-1 co-immunoprecipitate endogenous kinesin in HEK293 cells and native tissue.","method":"Co-immunoprecipitation from HEK293 cells and native brain/heart tissue; yeast two-hybrid; exogenous KIF5C co-expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP in multiple cell/tissue contexts plus yeast two-hybrid domain mapping, replicated across tissues and cell lines","pmids":["15644324"],"is_preprint":false},{"year":2005,"finding":"Trak1 protein interacts with GABA-A receptors; loss-of-function mutation in Trak1 causes dramatically reduced GABA-A receptor levels in CNS motor neurons, resulting in hypertonia.","method":"Positional cloning of hyrt mutant mice; co-immunoprecipitation showing Trak1–GABA-A receptor interaction; immunohistochemistry quantifying receptor levels","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function mouse model with defined cellular phenotype plus Co-IP interaction, single lab","pmids":["16380713"],"is_preprint":false},{"year":2008,"finding":"Trak1 interacts with Hrs (hepatocyte-growth-factor-regulated tyrosine kinase substrate) on early endosomes; both overexpression and siRNA knockdown of Trak1 block endosome-to-lysosome trafficking, inhibiting EGFR degradation.","method":"Co-immunoprecipitation; double-label immunofluorescence confocal microscopy; siRNA knockdown; EGFR degradation assay","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction plus bidirectional functional perturbation (OE and KD) with defined trafficking phenotype, single lab","pmids":["18675823"],"is_preprint":false},{"year":2013,"finding":"DISC1 associates with TRAK1, and TRAK1 in turn links to Miro1/2 on the outer mitochondrial membrane, forming a mitochondrial transport complex; DISC1 promotes anterograde axonal mitochondrial transport through this complex.","method":"Co-immunoprecipitation; live-cell axonal mitochondrial trafficking assay in neurons; expression of disease-associated DISC1-37W variant","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP defining complex components plus functional transport assay in neurons, single lab","pmids":["24092329"],"is_preprint":false},{"year":2017,"finding":"Trak1 interacts and colocalizes with mitofusins (Mfn1/Mfn2) on the outer mitochondrial membrane and promotes mitochondrial tethering and fusion; depletion of Trak1 causes mitochondrial fragmentation, and the hypertonia-associated truncation mutation impairs Trak1 mitochondrial localization and its ability to support fusion.","method":"Co-immunoprecipitation; confocal colocalization; siRNA knockdown; overexpression; mitochondrial morphology assay; stress-induced hyperfusion assay","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus bidirectional perturbation with morphological readouts and disease mutant analysis, single lab","pmids":["28924745"],"is_preprint":false},{"year":2017,"finding":"Homozygous truncating variants in TRAK1 cause aberrant splicing and loss of TRAK1 protein; TRAK1-deficient patient fibroblasts show irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration.","method":"Whole-exome sequencing; RT-PCR splice analysis; mitochondrial motility assay; membrane potential measurement (JC-1); oxygen consumption assay in patient fibroblasts","journal":"Brain : a journal of neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — human loss-of-function with multiple orthogonal cellular phenotype readouts, single study","pmids":["28364549"],"is_preprint":false},{"year":2018,"finding":"TRAK1 (not TRAK2) mediates anterograde mitochondrial trafficking specifically required for cell invasion; the Arf6-AMAP1 pathway promotes mitochondrial anterograde transport by localizing ILK to focal adhesions to block RhoT1-TRAK2 retrograde association, while the RhoT1-TRAK1 machinery drives forward mitochondrial movement needed to avoid ROS accumulation during invasion.","method":"siRNA knockdown of TRAK1 vs TRAK2; live-cell mitochondrial trafficking assays; invasion assays; ROS measurement; co-immunoprecipitation of RhoT1-TRAK complexes","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — selective KD distinguishing TRAK1 vs TRAK2 with multiple functional readouts, single lab","pmids":["29992963"],"is_preprint":false},{"year":2020,"finding":"TRAK1 activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces; TRAK1 directly interacts with microtubules, providing an additional anchor for the kinesin-1–TRAK1 complex that facilitates navigation around obstacles, passage through tau islands, and increased run lengths in cell lysate; TRAK1 also enables mitochondrial transport in vitro.","method":"Single-molecule TIRF microscopy reconstitution; in vitro motility assay with purified proteins; microtubule-binding assay; cell lysate transport assay; mitochondrial transport reconstitution in vitro","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins, single-molecule assays, multiple orthogonal methods in one rigorous study","pmids":["32561740"],"is_preprint":false},{"year":2020,"finding":"TRAK1 knockdown increases mitochondrial fission factor (MFF) expression and increases susceptibility to seizures in vivo; overexpression of TRAK1 rescues the dysfunction caused by TRAK1 knockdown, linking TRAK1 to regulation of the mitochondrial fusion-fission balance in epilepsy.","method":"siRNA/shRNA knockdown; overexpression rescue; MFF protein level measurement; in vivo seizure susceptibility assay","journal":"Molecular neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional perturbation with molecular and in vivo phenotypic readouts, single lab","pmids":["33119838"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure of TRAK1(569–623) bound to MIRO1 reveals the complex forms a dimer; TRAK1(569–623) binds in a cleft between MIRO1's nGTPase and first EF-hand pair; a second binding site involves TRAK1(425–428) inserting into a pocket between the second EF-hand pair and cGTPase; both sites are required for TRAK1 mitochondrial localization in cells and binding is independent of calcium or nucleotide state.","method":"Cryo-electron microscopy structure determination; site-directed mutagenesis; binding assays; cell-based mitochondrial localization assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with mutagenesis validation and cellular functional confirmation in a single rigorous study","pmids":["40615373"],"is_preprint":false},{"year":2026,"finding":"AMPK phosphorylates TRAK1 in response to decreased ATP-to-AMP ratio (energy stress), arresting mitochondrial movement; this arrest is accompanied by accumulation of actin fibers adjacent to mitochondria that anchor them against motor activity.","method":"Pharmacological AMPK activation (antimycin A); phosphorylation assays identifying TRAK1 as AMPK substrate; live-cell mitochondrial motility imaging in neurons and cell lines; actin fiber visualization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct phosphorylation of TRAK1 by AMPK demonstrated with multiple cellular readouts across neurons and cell lines, single rigorous study","pmids":["41615403"],"is_preprint":false}],"current_model":"TRAK1 is a mitochondrial adaptor protein that bridges the outer mitochondrial membrane anchor MIRO1 (via two structurally defined binding sites) to kinesin-1 and dynein-dynactin motors, directly activating kinesin-1 and enabling robust anterograde mitochondrial transport along microtubules even under crowded conditions; its activity is regulated by AMPK-mediated phosphorylation during energy stress to arrest mitochondria, and it additionally promotes mitochondrial fusion via interaction with mitofusins, regulates endosome-to-lysosome trafficking through Hrs, and maintains GABA-A receptor homeostasis."},"narrative":{"mechanistic_narrative":"TRAK1 is a mitochondrial adaptor protein that couples the outer mitochondrial membrane GTPase MIRO1 (RhoT1) to microtubule motors, organizing the machinery for directed mitochondrial transport along the cytoskeleton [PMID:24092329, PMID:32561740]. It binds kinesin heavy chains (KIF5A/B/C) through an N-proximal region and links to MIRO1/2 on the mitochondrial surface, assembling a transport complex that drives anterograde axonal mitochondrial movement [PMID:15644324, PMID:24092329]. Structurally, TRAK1 engages MIRO1 at two distinct sites — a major interface where TRAK1(569–623) occupies a cleft between the nGTPase and first EF-hand pair, and a second contact via TRAK1(425–428) — both required for mitochondrial localization and independent of calcium or nucleotide state [PMID:40615373]. Beyond passive bridging, TRAK1 directly activates kinesin-1 and binds microtubules itself, providing an additional anchor that confers robust stepping past obstacles such as tau islands and supports reconstituted mitochondrial transport in vitro [PMID:32561740]. This transport activity is switched off during energy stress, when AMPK phosphorylates TRAK1 to arrest mitochondrial movement alongside accumulation of anchoring actin fibers [PMID:41615403]. TRAK1 additionally shapes mitochondrial morphology by interacting with mitofusins (Mfn1/Mfn2) to promote tethering and fusion and by restraining MFF-dependent fission [PMID:28924745, PMID:33119838], and it functions outside mitochondria in endosome-to-lysosome trafficking through Hrs and in maintaining GABA-A receptor levels in CNS motor neurons [PMID:16380713, PMID:18675823]. Homozygous truncating TRAK1 variants cause loss of protein with aberrant mitochondrial distribution, motility, membrane potential, and respiration, establishing TRAK1 as a disease gene [PMID:28364549].","teleology":[{"year":2005,"claim":"Established TRAK1 as a kinesin-associated, mitochondria-linked protein, answering whether it physically engages the anterograde motor machinery.","evidence":"Reciprocal Co-IP from HEK293 cells and native brain/heart tissue plus yeast two-hybrid domain mapping of the KIF5 interaction","pmids":["15644324"],"confidence":"High","gaps":["Did not establish the mitochondrial membrane anchor","No functional transport assay","Direct vs indirect kinesin binding not resolved at the time"]},{"year":2005,"claim":"Linked TRAK1 to neuronal receptor homeostasis, showing a function beyond mitochondrial transport.","evidence":"Positional cloning of hyrt mutant mice, Co-IP of Trak1–GABA-A receptor, and immunohistochemical quantification of receptor loss","pmids":["16380713"],"confidence":"Medium","gaps":["Mechanism connecting TRAK1 to receptor trafficking not defined","Single lab","Relationship to mitochondrial role unclear"]},{"year":2008,"claim":"Identified a role in endosomal cargo sorting via Hrs, distinguishing a vesicle-trafficking function from mitochondrial transport.","evidence":"Co-IP, confocal colocalization, siRNA knockdown, and EGFR degradation assay","pmids":["18675823"],"confidence":"Medium","gaps":["Whether endosomal and mitochondrial roles share machinery unknown","Single lab","No structural basis for Hrs interaction"]},{"year":2013,"claim":"Defined the mitochondrial transport complex by placing TRAK1 between DISC1 and Miro1/2, answering how mitochondria are coupled to motors at the membrane.","evidence":"Reciprocal Co-IP defining complex components plus live-cell axonal mitochondrial trafficking in neurons","pmids":["24092329"],"confidence":"Medium","gaps":["Direct vs scaffolded Miro1 binding not resolved","Single lab","Motor activation mechanism not addressed"]},{"year":2017,"claim":"Expanded TRAK1 function to mitochondrial morphology, showing it promotes fusion rather than only transport.","evidence":"Co-IP with Mfn1/Mfn2, colocalization, bidirectional perturbation, and morphology/hyperfusion assays including disease-mutant analysis","pmids":["28924745"],"confidence":"Medium","gaps":["How fusion and transport roles are coordinated unknown","Single lab","Mechanism of mitofusin activation not defined"]},{"year":2017,"claim":"Established TRAK1 as a human disease gene by linking loss-of-function variants to mitochondrial dysfunction.","evidence":"Whole-exome sequencing, splice analysis, and orthogonal mitochondrial assays (motility, membrane potential, respiration) in patient fibroblasts","pmids":["28364549"],"confidence":"Medium","gaps":["Causal chain from transport defect to respiration deficit not dissected","Single study","Tissue-specific effects unresolved"]},{"year":2018,"claim":"Showed TRAK1 and TRAK2 are functionally non-redundant, with TRAK1 driving anterograde transport required for cell invasion.","evidence":"Selective siRNA knockdown of TRAK1 vs TRAK2, live-cell trafficking, invasion and ROS assays, and RhoT1-TRAK Co-IP","pmids":["29992963"],"confidence":"Medium","gaps":["Structural basis for TRAK1/TRAK2 directional bias unknown","Single lab","Generality beyond invasive cells unclear"]},{"year":2020,"claim":"Resolved the mechanism of motor regulation, demonstrating TRAK1 directly activates kinesin-1 and itself binds microtubules to confer transport robustness.","evidence":"Single-molecule TIRF reconstitution with purified proteins, microtubule-binding and motility assays, and in vitro mitochondrial transport","pmids":["32561740"],"confidence":"High","gaps":["Dynein/dynactin arm of the complex not reconstituted here","Regulation of the activation switch in cells not addressed","MIRO1 contribution to motility not tested in this system"]},{"year":2020,"claim":"Connected TRAK1 to fusion–fission balance and seizure susceptibility, implicating MFF regulation in vivo.","evidence":"siRNA/shRNA knockdown with overexpression rescue, MFF protein measurement, and in vivo seizure susceptibility assay","pmids":["33119838"],"confidence":"Medium","gaps":["Direct vs indirect MFF regulation unknown","Single lab","Mechanistic link to seizure phenotype incomplete"]},{"year":2025,"claim":"Provided the structural basis for TRAK1–MIRO1 anchoring, defining two binding sites required for mitochondrial localization.","evidence":"Cryo-EM structure of TRAK1(569–623)–MIRO1 with mutagenesis and cell-based localization assays","pmids":["40615373"],"confidence":"High","gaps":["Full-length complex with motors not visualized","Calcium/nucleotide independence leaves regulatory input on anchoring unclear","Stoichiometry in cells not resolved"]},{"year":2026,"claim":"Identified the regulatory switch arresting mitochondrial movement, showing AMPK phosphorylates TRAK1 during energy stress.","evidence":"Pharmacological AMPK activation, phosphorylation assays identifying TRAK1 as substrate, live-cell motility imaging, and actin fiber visualization","pmids":["41615403"],"confidence":"High","gaps":["Phosphosite mapping and its effect on motor binding not fully detailed","Link between TRAK1 phosphorylation and actin anchoring mechanistically open","Reversal/dephosphorylation pathway unknown"]},{"year":null,"claim":"How TRAK1's multiple roles — kinesin/dynein transport, mitofusin-driven fusion, endosomal sorting, and GABA-A receptor maintenance — are integrated and differentially regulated within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coordinating transport vs fusion vs endosomal functions","Dynein-dynactin engagement not structurally defined","Switch between anterograde and retrograde states incompletely understood"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3,9]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[3,4,5]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[7]}],"pathway":[],"complexes":["MIRO1-TRAK1-kinesin-1 mitochondrial transport complex"],"partners":["MIRO1","KIF5C","KIF5A","KIF5B","MFN1","MFN2","HGS","DISC1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UPV9","full_name":"Trafficking kinesin-binding protein 1","aliases":["106 kDa O-GlcNAc transferase-interacting protein","Protein Milton"],"length_aa":953,"mass_kda":106.0,"function":"Involved in the regulation of endosome-to-lysosome trafficking, including endocytic trafficking of EGF-EGFR complexes and GABA-A receptors (PubMed:18675823). Involved in mitochondrial motility. When O-glycosylated, abolishes mitochondrial motility. Crucial for recruiting OGT to the mitochondrial surface of neuronal processes (PubMed:24995978). TRAK1 and RHOT form an essential protein complex that links KIF5 to mitochondria for light chain-independent, anterograde transport of mitochondria (By similarity)","subcellular_location":"Cytoplasm; Nucleus; Mitochondrion; Early endosome; Endosome; Mitochondrion membrane; Cytoplasm, cell cortex","url":"https://www.uniprot.org/uniprotkb/Q9UPV9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRAK1","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DYNLL1","stoichiometry":0.2},{"gene":"DYNLL2","stoichiometry":0.2},{"gene":"GSK3B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TRAK1","total_profiled":1310},"omim":[{"mim_id":"618201","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 68; DEE68","url":"https://www.omim.org/entry/618201"},{"mim_id":"608112","title":"TRAFFICKING PROTEIN, KINESIN-BINDING 1; TRAK1","url":"https://www.omim.org/entry/608112"},{"mim_id":"605704","title":"VAMP-ASSOCIATED PROTEIN B AND C; VAPB","url":"https://www.omim.org/entry/605704"},{"mim_id":"308350","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 1; DEE1","url":"https://www.omim.org/entry/308350"},{"mim_id":"184850","title":"STIFF-PERSON SYNDROME; SPS","url":"https://www.omim.org/entry/184850"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Endoplasmic reticulum","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"heart muscle","ntpm":158.2},{"tissue":"skeletal muscle","ntpm":245.8},{"tissue":"tongue","ntpm":189.2}],"url":"https://www.proteinatlas.org/search/TRAK1"},"hgnc":{"alias_symbol":["OIP106","KIAA1042","MILT1"],"prev_symbol":[]},"alphafold":{"accession":"Q9UPV9","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UPV9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UPV9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UPV9-F1-predicted_aligned_error_v6.png","plddt_mean":58.09},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRAK1","jax_strain_url":"https://www.jax.org/strain/search?query=TRAK1"},"sequence":{"accession":"Q9UPV9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UPV9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UPV9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UPV9"}},"corpus_meta":[{"pmid":"15644324","id":"PMC_15644324","title":"GRIF-1 and OIP106, members of a novel gene family of coiled-coil domain proteins: association in vivo and in vitro with kinesin.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15644324","citation_count":137,"is_preprint":false},{"pmid":"24092329","id":"PMC_24092329","title":"DISC1 complexes with TRAK1 and Miro1 to modulate anterograde axonal mitochondrial trafficking.","date":"2013","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24092329","citation_count":85,"is_preprint":false},{"pmid":"29992963","id":"PMC_29992963","title":"Arf6-driven cell invasion is intrinsically linked to TRAK1-mediated mitochondrial anterograde trafficking to avoid oxidative catastrophe.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29992963","citation_count":69,"is_preprint":false},{"pmid":"32561740","id":"PMC_32561740","title":"Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32561740","citation_count":65,"is_preprint":false},{"pmid":"16380713","id":"PMC_16380713","title":"Trak1 mutation disrupts GABA(A) receptor homeostasis in hypertonic mice.","date":"2005","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/16380713","citation_count":53,"is_preprint":false},{"pmid":"28364549","id":"PMC_28364549","title":"Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy.","date":"2017","source":"Brain : a journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/28364549","citation_count":53,"is_preprint":false},{"pmid":"18675823","id":"PMC_18675823","title":"Hypertonia-associated protein Trak1 is a novel regulator of endosome-to-lysosome trafficking.","date":"2008","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18675823","citation_count":38,"is_preprint":false},{"pmid":"28924745","id":"PMC_28924745","title":"Hypertonia-linked protein Trak1 functions with mitofusins to promote mitochondrial tethering and fusion.","date":"2017","source":"Protein & cell","url":"https://pubmed.ncbi.nlm.nih.gov/28924745","citation_count":28,"is_preprint":false},{"pmid":"18986759","id":"PMC_18986759","title":"Identification of TRAK1 (Trafficking protein, kinesin-binding 1) as MGb2-Ag: a novel cancer biomarker.","date":"2008","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/18986759","citation_count":22,"is_preprint":false},{"pmid":"33119838","id":"PMC_33119838","title":"TRAK1-Mediated Abnormality of Mitochondrial Fission Increases Seizure Susceptibility in Temporal Lobe Epilepsy.","date":"2020","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/33119838","citation_count":13,"is_preprint":false},{"pmid":"40615373","id":"PMC_40615373","title":"Structural-functional characterization of the MIRO1-TRAK1 complex.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40615373","citation_count":8,"is_preprint":false},{"pmid":"37399073","id":"PMC_37399073","title":"Recurrent TRAK1::RAF1 Fusions in pediatric low-grade gliomas.","date":"2023","source":"Brain pathology (Zurich, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/37399073","citation_count":7,"is_preprint":false},{"pmid":"38410694","id":"PMC_38410694","title":"Role of TRAK1 variants in epilepsy: genotype-phenotype analysis in a pediatric case of epilepsy with developmental disorder.","date":"2024","source":"Frontiers in molecular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/38410694","citation_count":1,"is_preprint":false},{"pmid":"41615403","id":"PMC_41615403","title":"Energy stress activates AMPK to arrest mitochondria via phosphorylation of TRAK1.","date":"2026","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/41615403","citation_count":1,"is_preprint":false},{"pmid":"27319755","id":"PMC_27319755","title":"Behavioral analysis of the huntingtin-associated protein 1 ortholog trak-1 in Caenorhabditis elegans.","date":"2016","source":"Journal of neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/27319755","citation_count":1,"is_preprint":false},{"pmid":"41028571","id":"PMC_41028571","title":"DNA hypomethylation at specific CG-sites within TRAK1 is linked to the neurocognitive profile in Klinefelter syndrome.","date":"2025","source":"Molecular psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/41028571","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9275,"output_tokens":2741,"usd":0.03447,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10113,"output_tokens":3650,"usd":0.070907,"stage2_stop_reason":"end_turn"},"total_usd":0.105377,"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\": \"OIP106 (TRAK1) associates with kinesin heavy chain (KIF5C, KIF5A, KIF5B) and mitochondria; the interaction domain was localized to residues 124–283 of GRIF-1 (the TRAK1 paralog), and both OIP106 and GRIF-1 co-immunoprecipitate endogenous kinesin in HEK293 cells and native tissue.\",\n      \"method\": \"Co-immunoprecipitation from HEK293 cells and native brain/heart tissue; yeast two-hybrid; exogenous KIF5C co-expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP in multiple cell/tissue contexts plus yeast two-hybrid domain mapping, replicated across tissues and cell lines\",\n      \"pmids\": [\"15644324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Trak1 protein interacts with GABA-A receptors; loss-of-function mutation in Trak1 causes dramatically reduced GABA-A receptor levels in CNS motor neurons, resulting in hypertonia.\",\n      \"method\": \"Positional cloning of hyrt mutant mice; co-immunoprecipitation showing Trak1–GABA-A receptor interaction; immunohistochemistry quantifying receptor levels\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function mouse model with defined cellular phenotype plus Co-IP interaction, single lab\",\n      \"pmids\": [\"16380713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Trak1 interacts with Hrs (hepatocyte-growth-factor-regulated tyrosine kinase substrate) on early endosomes; both overexpression and siRNA knockdown of Trak1 block endosome-to-lysosome trafficking, inhibiting EGFR degradation.\",\n      \"method\": \"Co-immunoprecipitation; double-label immunofluorescence confocal microscopy; siRNA knockdown; EGFR degradation assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction plus bidirectional functional perturbation (OE and KD) with defined trafficking phenotype, single lab\",\n      \"pmids\": [\"18675823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DISC1 associates with TRAK1, and TRAK1 in turn links to Miro1/2 on the outer mitochondrial membrane, forming a mitochondrial transport complex; DISC1 promotes anterograde axonal mitochondrial transport through this complex.\",\n      \"method\": \"Co-immunoprecipitation; live-cell axonal mitochondrial trafficking assay in neurons; expression of disease-associated DISC1-37W variant\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP defining complex components plus functional transport assay in neurons, single lab\",\n      \"pmids\": [\"24092329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Trak1 interacts and colocalizes with mitofusins (Mfn1/Mfn2) on the outer mitochondrial membrane and promotes mitochondrial tethering and fusion; depletion of Trak1 causes mitochondrial fragmentation, and the hypertonia-associated truncation mutation impairs Trak1 mitochondrial localization and its ability to support fusion.\",\n      \"method\": \"Co-immunoprecipitation; confocal colocalization; siRNA knockdown; overexpression; mitochondrial morphology assay; stress-induced hyperfusion assay\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus bidirectional perturbation with morphological readouts and disease mutant analysis, single lab\",\n      \"pmids\": [\"28924745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Homozygous truncating variants in TRAK1 cause aberrant splicing and loss of TRAK1 protein; TRAK1-deficient patient fibroblasts show irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration.\",\n      \"method\": \"Whole-exome sequencing; RT-PCR splice analysis; mitochondrial motility assay; membrane potential measurement (JC-1); oxygen consumption assay in patient fibroblasts\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human loss-of-function with multiple orthogonal cellular phenotype readouts, single study\",\n      \"pmids\": [\"28364549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRAK1 (not TRAK2) mediates anterograde mitochondrial trafficking specifically required for cell invasion; the Arf6-AMAP1 pathway promotes mitochondrial anterograde transport by localizing ILK to focal adhesions to block RhoT1-TRAK2 retrograde association, while the RhoT1-TRAK1 machinery drives forward mitochondrial movement needed to avoid ROS accumulation during invasion.\",\n      \"method\": \"siRNA knockdown of TRAK1 vs TRAK2; live-cell mitochondrial trafficking assays; invasion assays; ROS measurement; co-immunoprecipitation of RhoT1-TRAK complexes\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — selective KD distinguishing TRAK1 vs TRAK2 with multiple functional readouts, single lab\",\n      \"pmids\": [\"29992963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRAK1 activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces; TRAK1 directly interacts with microtubules, providing an additional anchor for the kinesin-1–TRAK1 complex that facilitates navigation around obstacles, passage through tau islands, and increased run lengths in cell lysate; TRAK1 also enables mitochondrial transport in vitro.\",\n      \"method\": \"Single-molecule TIRF microscopy reconstitution; in vitro motility assay with purified proteins; microtubule-binding assay; cell lysate transport assay; mitochondrial transport reconstitution in vitro\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins, single-molecule assays, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"32561740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRAK1 knockdown increases mitochondrial fission factor (MFF) expression and increases susceptibility to seizures in vivo; overexpression of TRAK1 rescues the dysfunction caused by TRAK1 knockdown, linking TRAK1 to regulation of the mitochondrial fusion-fission balance in epilepsy.\",\n      \"method\": \"siRNA/shRNA knockdown; overexpression rescue; MFF protein level measurement; in vivo seizure susceptibility assay\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional perturbation with molecular and in vivo phenotypic readouts, single lab\",\n      \"pmids\": [\"33119838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structure of TRAK1(569–623) bound to MIRO1 reveals the complex forms a dimer; TRAK1(569–623) binds in a cleft between MIRO1's nGTPase and first EF-hand pair; a second binding site involves TRAK1(425–428) inserting into a pocket between the second EF-hand pair and cGTPase; both sites are required for TRAK1 mitochondrial localization in cells and binding is independent of calcium or nucleotide state.\",\n      \"method\": \"Cryo-electron microscopy structure determination; site-directed mutagenesis; binding assays; cell-based mitochondrial localization assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with mutagenesis validation and cellular functional confirmation in a single rigorous study\",\n      \"pmids\": [\"40615373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"AMPK phosphorylates TRAK1 in response to decreased ATP-to-AMP ratio (energy stress), arresting mitochondrial movement; this arrest is accompanied by accumulation of actin fibers adjacent to mitochondria that anchor them against motor activity.\",\n      \"method\": \"Pharmacological AMPK activation (antimycin A); phosphorylation assays identifying TRAK1 as AMPK substrate; live-cell mitochondrial motility imaging in neurons and cell lines; actin fiber visualization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct phosphorylation of TRAK1 by AMPK demonstrated with multiple cellular readouts across neurons and cell lines, single rigorous study\",\n      \"pmids\": [\"41615403\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRAK1 is a mitochondrial adaptor protein that bridges the outer mitochondrial membrane anchor MIRO1 (via two structurally defined binding sites) to kinesin-1 and dynein-dynactin motors, directly activating kinesin-1 and enabling robust anterograde mitochondrial transport along microtubules even under crowded conditions; its activity is regulated by AMPK-mediated phosphorylation during energy stress to arrest mitochondria, and it additionally promotes mitochondrial fusion via interaction with mitofusins, regulates endosome-to-lysosome trafficking through Hrs, and maintains GABA-A receptor homeostasis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRAK1 is a mitochondrial adaptor protein that couples the outer mitochondrial membrane GTPase MIRO1 (RhoT1) to microtubule motors, organizing the machinery for directed mitochondrial transport along the cytoskeleton [#3, #7]. It binds kinesin heavy chains (KIF5A/B/C) through an N-proximal region and links to MIRO1/2 on the mitochondrial surface, assembling a transport complex that drives anterograde axonal mitochondrial movement [#0, #3]. Structurally, TRAK1 engages MIRO1 at two distinct sites — a major interface where TRAK1(569–623) occupies a cleft between the nGTPase and first EF-hand pair, and a second contact via TRAK1(425–428) — both required for mitochondrial localization and independent of calcium or nucleotide state [#9]. Beyond passive bridging, TRAK1 directly activates kinesin-1 and binds microtubules itself, providing an additional anchor that confers robust stepping past obstacles such as tau islands and supports reconstituted mitochondrial transport in vitro [#7]. This transport activity is switched off during energy stress, when AMPK phosphorylates TRAK1 to arrest mitochondrial movement alongside accumulation of anchoring actin fibers [#10]. TRAK1 additionally shapes mitochondrial morphology by interacting with mitofusins (Mfn1/Mfn2) to promote tethering and fusion and by restraining MFF-dependent fission [#4, #8], and it functions outside mitochondria in endosome-to-lysosome trafficking through Hrs and in maintaining GABA-A receptor levels in CNS motor neurons [#1, #2]. Homozygous truncating TRAK1 variants cause loss of protein with aberrant mitochondrial distribution, motility, membrane potential, and respiration, establishing TRAK1 as a disease gene [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established TRAK1 as a kinesin-associated, mitochondria-linked protein, answering whether it physically engages the anterograde motor machinery.\",\n      \"evidence\": \"Reciprocal Co-IP from HEK293 cells and native brain/heart tissue plus yeast two-hybrid domain mapping of the KIF5 interaction\",\n      \"pmids\": [\"15644324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the mitochondrial membrane anchor\", \"No functional transport assay\", \"Direct vs indirect kinesin binding not resolved at the time\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked TRAK1 to neuronal receptor homeostasis, showing a function beyond mitochondrial transport.\",\n      \"evidence\": \"Positional cloning of hyrt mutant mice, Co-IP of Trak1–GABA-A receptor, and immunohistochemical quantification of receptor loss\",\n      \"pmids\": [\"16380713\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting TRAK1 to receptor trafficking not defined\", \"Single lab\", \"Relationship to mitochondrial role unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified a role in endosomal cargo sorting via Hrs, distinguishing a vesicle-trafficking function from mitochondrial transport.\",\n      \"evidence\": \"Co-IP, confocal colocalization, siRNA knockdown, and EGFR degradation assay\",\n      \"pmids\": [\"18675823\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether endosomal and mitochondrial roles share machinery unknown\", \"Single lab\", \"No structural basis for Hrs interaction\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the mitochondrial transport complex by placing TRAK1 between DISC1 and Miro1/2, answering how mitochondria are coupled to motors at the membrane.\",\n      \"evidence\": \"Reciprocal Co-IP defining complex components plus live-cell axonal mitochondrial trafficking in neurons\",\n      \"pmids\": [\"24092329\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs scaffolded Miro1 binding not resolved\", \"Single lab\", \"Motor activation mechanism not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Expanded TRAK1 function to mitochondrial morphology, showing it promotes fusion rather than only transport.\",\n      \"evidence\": \"Co-IP with Mfn1/Mfn2, colocalization, bidirectional perturbation, and morphology/hyperfusion assays including disease-mutant analysis\",\n      \"pmids\": [\"28924745\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How fusion and transport roles are coordinated unknown\", \"Single lab\", \"Mechanism of mitofusin activation not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established TRAK1 as a human disease gene by linking loss-of-function variants to mitochondrial dysfunction.\",\n      \"evidence\": \"Whole-exome sequencing, splice analysis, and orthogonal mitochondrial assays (motility, membrane potential, respiration) in patient fibroblasts\",\n      \"pmids\": [\"28364549\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from transport defect to respiration deficit not dissected\", \"Single study\", \"Tissue-specific effects unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed TRAK1 and TRAK2 are functionally non-redundant, with TRAK1 driving anterograde transport required for cell invasion.\",\n      \"evidence\": \"Selective siRNA knockdown of TRAK1 vs TRAK2, live-cell trafficking, invasion and ROS assays, and RhoT1-TRAK Co-IP\",\n      \"pmids\": [\"29992963\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for TRAK1/TRAK2 directional bias unknown\", \"Single lab\", \"Generality beyond invasive cells unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the mechanism of motor regulation, demonstrating TRAK1 directly activates kinesin-1 and itself binds microtubules to confer transport robustness.\",\n      \"evidence\": \"Single-molecule TIRF reconstitution with purified proteins, microtubule-binding and motility assays, and in vitro mitochondrial transport\",\n      \"pmids\": [\"32561740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynein/dynactin arm of the complex not reconstituted here\", \"Regulation of the activation switch in cells not addressed\", \"MIRO1 contribution to motility not tested in this system\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected TRAK1 to fusion–fission balance and seizure susceptibility, implicating MFF regulation in vivo.\",\n      \"evidence\": \"siRNA/shRNA knockdown with overexpression rescue, MFF protein measurement, and in vivo seizure susceptibility assay\",\n      \"pmids\": [\"33119838\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect MFF regulation unknown\", \"Single lab\", \"Mechanistic link to seizure phenotype incomplete\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided the structural basis for TRAK1–MIRO1 anchoring, defining two binding sites required for mitochondrial localization.\",\n      \"evidence\": \"Cryo-EM structure of TRAK1(569–623)–MIRO1 with mutagenesis and cell-based localization assays\",\n      \"pmids\": [\"40615373\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length complex with motors not visualized\", \"Calcium/nucleotide independence leaves regulatory input on anchoring unclear\", \"Stoichiometry in cells not resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified the regulatory switch arresting mitochondrial movement, showing AMPK phosphorylates TRAK1 during energy stress.\",\n      \"evidence\": \"Pharmacological AMPK activation, phosphorylation assays identifying TRAK1 as substrate, live-cell motility imaging, and actin fiber visualization\",\n      \"pmids\": [\"41615403\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosite mapping and its effect on motor binding not fully detailed\", \"Link between TRAK1 phosphorylation and actin anchoring mechanistically open\", \"Reversal/dephosphorylation pathway unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TRAK1's multiple roles — kinesin/dynein transport, mitofusin-driven fusion, endosomal sorting, and GABA-A receptor maintenance — are integrated and differentially regulated within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model coordinating transport vs fusion vs endosomal functions\", \"Dynein-dynactin engagement not structurally defined\", \"Switch between anterograde and retrograde states incompletely understood\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 9]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0009609507\", \"supporting_discovery_ids\": []}\n    ],\n    \"complexes\": [\"MIRO1-TRAK1-kinesin-1 mitochondrial transport complex\"],\n    \"partners\": [\"MIRO1\", \"KIF5C\", \"KIF5A\", \"KIF5B\", \"MFN1\", \"MFN2\", \"HGS\", \"DISC1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}