{"gene":"TMEM65","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2014,"finding":"TMEM65 is an integral protein of the mitochondrial inner membrane. Immunoblot, immunostaining, alkali extraction, and digitonin extraction of isolated mitochondria established its mitochondrial inner-membrane localization. The N-terminal region (residues 1–20) is sufficient for mitochondrial targeting, and the mitochondrial targeting signal is cleaved between residues 35 and 64 by matrix processing protease (MPP).","method":"Immunoblot, immunostaining, alkali extraction, digitonin fractionation of isolated mitochondria, deletion-mutant analysis","journal":"PeerJ","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal biochemical fractionation methods in a dedicated localization study; replicated by subsequent independent labs","pmids":["24765583"],"is_preprint":false},{"year":2015,"finding":"TMEM65 localizes to the intercalated disc (ICD) of cardiomyocytes and physically interacts with connexin 43 (Cx43). shRNA-mediated Tmem65 knockdown in mouse neonatal cardiomyocytes causes internalization of Cx43 away from the ICD, shortens Cx43 half-life through increased degradation, and abolishes Cx43 gap-junction function. Morpholino knockdown in zebrafish recapitulates gap-junction dysfunction and cardiac morphology defects.","method":"Cationic silica-bead plasma-membrane enrichment + shotgun proteomics, lentiviral shRNA knockdown, co-immunoprecipitation, immunofluorescence, zebrafish morpholino knockdown","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro KD with defined molecular phenotype, and in vivo (zebrafish) validation; independently extended by subsequent mouse in vivo study","pmids":["26403541"],"is_preprint":false},{"year":2017,"finding":"A homozygous splice variant (c.472+1G>A) in TMEM65 causes mitochondrial encephalomyopathy in a patient. Subcellular fractionation confirmed TMEM65 protein in the inner mitochondrial membrane of patient fibroblasts. siRNA knockdown of TMEM65 in fibroblasts severely reduced mitochondrial content and oxygen consumption rate, establishing a direct role for TMEM65 in mitochondrial respiratory chain function.","method":"Immunofluorescence, subcellular fractionation/immunoblot, siRNA knockdown, enzymatic respirometry, oxygen consumption rate measurement","journal":"European journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined cellular phenotype plus patient-derived validation, single lab, two orthogonal functional readouts","pmids":["28295037"],"is_preprint":false},{"year":2022,"finding":"Tmem65 physically interacts with the voltage-gated sodium channel β1 subunit (SCN1B/β1) at the ICD, and this interaction is required for the establishment of the perinexal nanodomain and for correct localization of NaV1.5 and Cx43 to ICDs. AAV9-shRNA-mediated Tmem65 knockdown (90% reduction) in mouse hearts caused eccentric hypertrophic cardiomyopathy progressing to dilated cardiomyopathy, slowed conduction, prolonged PR intervals and QRS duration, and reduced Ca2+ and K+ currents in cardiomyocytes.","method":"rAAV9 shRNA knockdown in mice, immunoprecipitation, super-resolution microscopy, echocardiography, electrocardiography, optical mapping, whole-cell patch clamp electrophysiology","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KD mouse model with multiple orthogonal functional and structural readouts plus Co-IP for binding partner; single lab but comprehensive","pmids":["36257954"],"is_preprint":false},{"year":2022,"finding":"CHD6 chromatin remodeler binds the TCF4 transcription factor at the TMEM65 promoter and drives transcriptional expression of TMEM65; TMEM65 in turn affects mitochondrial dynamics and ATP production. EGF signaling stabilizes CHD6 by preventing GSK3β-mediated phosphodegron formation and FBXW7-mediated ubiquitination, while Wnt/TCF4-β-catenin signaling transcriptionally activates CHD6 itself.","method":"CHD6 knockdown and knockout (mouse Villin-specific), chromatin immunoprecipitation, co-immunoprecipitation of CHD6–TCF4, reporter assays, xenograft models, patient-derived xenograft drug treatment","journal":"Cell discovery","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — ChIP and Co-IP support CHD6–TCF4 binding at TMEM65 promoter; in vivo KO mouse and PDX models; single lab","pmids":["36473865"],"is_preprint":false},{"year":2023,"finding":"TMEM65 is an NCLX-proximal protein at the mitochondrial inner membrane that potently enhances Na+-dependent mitochondrial Ca2+ efflux. Pharmacological NCLX inhibition or genetic NCLX deletion abolishes the TMEM65-dependent increase in Ca2+ efflux. Loss-of-function studies show TMEM65 is required for Na+-dependent mitochondrial Ca2+ efflux. Co-fractionation and in silico structural modeling suggest TMEM65 and NCLX exist in a common macromolecular complex. Tmem65 knockdown in mice causes mitochondrial Ca2+ overload in heart and skeletal muscle and impairs cardiac and neuromuscular function. TMEM65 deletion causes excessive mitochondrial permeability transition; TMEM65 overexpression protects against necrotic cell death during Ca2+ stress.","method":"Proximity biotinylation (BioID) proteomics, Ca2+ flux assays, pharmacological NCLX inhibition (CGP-37157), genetic NCLX knockout, co-fractionation, in silico structural modeling, AAV-shRNA mouse knockdown, mitochondrial permeability transition assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity proteomics plus functional Ca2+ flux assays plus in vivo mouse KD; preprint, not yet peer-reviewed at this stage but superseded by peer-reviewed version","pmids":["37873405"],"is_preprint":true},{"year":2024,"finding":"TMEM65 directly binds YWHAZ (14-3-3ζ) in the cytoplasm, inhibiting ubiquitin-mediated degradation of YWHAZ, which in turn activates the PI3K–Akt–mTOR signaling pathway (evidenced by increased p-Akt, p-GSK-3β, p-mTOR). TMEM65 oncogenic effects in gastric cancer are partly dependent on YWHAZ.","method":"Co-immunoprecipitation (TMEM65–YWHAZ), Western blot for pathway activation markers, siRNA knockdown, ectopic overexpression, in vivo xenograft and lung-metastasis models, VNP-encapsulated siRNA delivery","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct binding by Co-IP, functional epistasis via YWHAZ rescue, multiple cellular phenotypes; single lab","pmids":["38341472"],"is_preprint":false},{"year":2025,"finding":"TMEM65 is identified as a direct NCLX binding partner that enhances Na+-dependent mitochondrial Ca2+ efflux. Genetic deletion of NCLX ablates TMEM65-dependent Ca2+ efflux, and TMEM65 knockdown in mice promotes mitochondrial Ca2+ overload in heart and skeletal muscle. TMEM65 loss impairs cardiac and neuromuscular function, and TMEM65 deletion causes mitochondrial permeability transition and cell death.","method":"Proximity biotinylation proteomic screening, Ca2+ efflux assays, pharmacological and genetic NCLX inhibition/deletion, AAV-shRNA mouse knockdown, in vivo cardiac and neuromuscular functional assays","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — proximity proteomics for interaction identification, multiple orthogonal functional Ca2+ efflux assays, genetic deletion controls, in vivo mouse models; peer-reviewed, multiple complementary methods","pmids":["40200126"],"is_preprint":false},{"year":2025,"finding":"TMEM65 itself functions as the mitochondrial Na+/Ca2+ exchanger (mito-NCX). TMEM65 forms a homodimer containing putative ion-coordinating residues essential for function. Heterologous expression of TMEM65 alone induces Na+/Ca2+ exchange in cells lacking native mito-NCX activity. Purified, liposome-reconstituted TMEM65 exhibits key mito-NCX features (Na+-dependent Ca2+ exchange). The CGP-37157 binding site on TMEM65 was identified. TMEM65 deletion elevates mitochondrial Ca2+ and primes mitochondrial permeability transition.","method":"Biochemical homodimerization assays, site-directed mutagenesis of ion-coordinating residues, heterologous expression in mito-NCX-null cells, protein purification and liposome reconstitution, Ca2+ transport assay, CGP-37157 binding-site mapping, Ca2+ imaging","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified protein in liposomes plus heterologous expression plus mutagenesis; multiple orthogonal methods establishing catalytic identity","pmids":["40691517"],"is_preprint":false},{"year":2025,"finding":"TMEM65 overexpression specifically enhances Na+- and Li+-dependent mitochondrial Ca2+ extrusion in an NCLX-independent manner; this effect is inhibited by CGP-37157. TMEM65 downregulation chronically elevates basal mitochondrial [Ca2+] and impairs efflux upon stimulation. In C. elegans, deletion of TMEM65 homologs causes necrotic lesions under mild thermal stress that are suppressed by genetic inhibition of MCU-1, placing TMEM65 downstream of MCU in a Ca2+ overload pathway.","method":"Mitochondrial Ca2+ imaging (overexpression and knockdown), CGP-37157 pharmacological inhibition, C. elegans genetic deletion and MCU-1 epistasis, genetic suppression assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct Ca2+ flux measurements with gain- and loss-of-function, pharmacological validation, genetic epistasis in C. elegans; multi-method single study","pmids":["41408045"],"is_preprint":false},{"year":2025,"finding":"TMEM65 enhances mitochondrial oxidative phosphorylation (OXPHOS) and ROS production. Elevated ROS induces HIF1α, which transcriptionally activates SERPINB3, enhancing cancer stemness. MYC and TET3 coordinately upregulate TMEM65 transcription in triple-negative breast cancer. Pharmacological or siRNA-mediated inhibition of MYC or TET3 attenuates TMEM65-driven tumor progression.","method":"siRNA knockdown, ectopic overexpression, OXPHOS/ROS measurements, HIF1α reporter/Western blot, MYC/TET3 inhibitor treatment, in vivo tumor models","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional pathway placement via KD/OE with defined molecular readouts; single lab; indirect evidence for HIF1α–SERPINB3 axis","pmids":["40546127"],"is_preprint":false},{"year":2025,"finding":"Conditional whole-body and nervous system-specific Tmem65 knockout mice exhibit severe growth retardation and seizure-associated sudden death at ~3 weeks; skeletal muscle-specific knockout produces adult-onset myopathy with elevated mitochondrial Ca2+. TMEM65 ablation causes loss of Na+-dependent mitochondrial Ca2+ export. Genetic epistasis: MCU knockout rescues early lethality of whole-body Tmem65 knockout, extending lifespan from ~3 weeks to >1 year, placing TMEM65 function downstream of MCU in the mitochondrial Ca2+ overload pathway.","method":"Conditional knockout mouse generation (whole-body, neuronal, skeletal muscle-specific), mitochondrial Ca2+ measurements, Na+-dependent Ca2+ export assay, MCU double-knockout epistasis, survival analysis, electrophysiology, histology","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO mouse models with tissue-specific phenotypes, direct Ca2+ flux measurements, genetic epistasis with MCU rescue; multiple orthogonal in vivo methods","pmids":["41980949"],"is_preprint":false},{"year":2020,"finding":"TMEM65 knockdown in human cultured cells induces mild ROS generation, oxidative stress response (upregulation of NFE2L2, SESN3), mild apoptosis, and mitochondrial unfolded protein response (UPRmt) with upregulation of HSPD1, LONP1. TOMM22 and HSPA9 protein levels are upregulated in an ATF5-independent manner following TMEM65 depletion.","method":"siRNA knockdown in human cells, ROS assay, qRT-PCR, Western blot for UPRmt markers and mitochondrial import receptors","journal":"Biochemistry and biophysics reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — KD with multiple cellular stress readouts; single lab, single method (siRNA KD) with multiple marker readouts","pmids":["33319071"],"is_preprint":false}],"current_model":"TMEM65 is a mitochondrial inner-membrane protein that functions as the mitochondrial Na+/Ca2+ exchanger (mito-NCX): it forms a homodimer with ion-coordinating residues essential for Na+-dependent Ca2+ efflux, interacts with NCLX to enhance Ca2+ export, and its loss causes mitochondrial Ca2+ overload, permeability transition, and cell death—phenotypes rescued in vivo by MCU knockout—while in the heart TMEM65 additionally localizes to intercalated discs where it interacts with Cx43 and the sodium channel β1 subunit to maintain gap-junction function, perinexal nanodomain structure, and normal cardiac conduction."},"narrative":{"mechanistic_narrative":"TMEM65 is an integral protein of the mitochondrial inner membrane, targeted there by an N-terminal signal that is cleaved by the matrix processing protease, and it serves as the principal effector of Na+-dependent mitochondrial Ca2+ extrusion [PMID:24765583, PMID:40691517]. Reconstitution of purified TMEM65 in liposomes demonstrates that the protein itself functions as the mitochondrial Na+/Ca2+ exchanger (mito-NCX): it forms a homodimer with ion-coordinating residues essential for activity, drives Na+/Ca2+ exchange when expressed in cells lacking native mito-NCX, and harbors the binding site for the inhibitor CGP-37157 [PMID:40691517]. Consistent with this catalytic identity, TMEM65 enhances Na+- and Li+-dependent Ca2+ efflux that is blocked by CGP-37157 [PMID:41408045], and proximity proteomics and co-fractionation place it adjacent to and in direct association with NCLX within a common macromolecular complex [PMID:37873405, PMID:40200126]. Loss of TMEM65 ablates Na+-dependent Ca2+ export, drives mitochondrial Ca2+ overload, primes the permeability transition, and causes necrotic cell death, whereas overexpression protects against Ca2+ stress [PMID:40691517, PMID:41980949]. Genetic epistasis establishes TMEM65 downstream of the mitochondrial calcium uniporter: MCU knockout rescues the early lethality of whole-body Tmem65-null mice and suppresses thermal-stress necrosis in C. elegans lacking TMEM65 homologs [PMID:41408045, PMID:41980949]. Tissue-specific ablation produces seizure-associated sudden death and neuronal phenotypes, skeletal myopathy, and impaired cardiac and neuromuscular function [PMID:40200126, PMID:41980949], and a homozygous splice variant causes human mitochondrial encephalomyopathy with reduced respiratory capacity [PMID:28295037]. In cardiomyocytes TMEM65 additionally localizes to the intercalated disc, where it interacts with connexin 43 to maintain gap-junction stability and with the sodium-channel β1 subunit (SCN1B) to organize the perinexal nanodomain and proper localization of NaV1.5 and Cx43, such that its loss slows cardiac conduction and produces cardiomyopathy [PMID:26403541, PMID:36257954].","teleology":[{"year":2014,"claim":"Establishing where TMEM65 resides was the first step in assigning it any function; the work fixed it as an inner-membrane protein with a cleavable targeting signal.","evidence":"Orthogonal biochemical fractionation (alkali/digitonin extraction), immunostaining, and deletion-mutant targeting analysis of isolated mitochondria","pmids":["24765583"],"confidence":"High","gaps":["No molecular function or transport activity assigned","Topology and oligomeric state within the inner membrane not defined"]},{"year":2015,"claim":"Beyond mitochondria, TMEM65 was found at the cardiac intercalated disc as a Cx43 partner, defining an unexpected role in gap-junction maintenance and cardiac conduction.","evidence":"Plasma-membrane proteomics, reciprocal Co-IP, shRNA knockdown in neonatal cardiomyocytes, and zebrafish morpholino knockdown","pmids":["26403541"],"confidence":"High","gaps":["Mechanism linking a mitochondrial inner-membrane protein to an ICD pool unresolved","Whether ICD localization reflects a distinct protein population not addressed"]},{"year":2017,"claim":"A human loss-of-function variant tied TMEM65 to disease and to respiratory-chain capacity, confirming functional importance in patient cells.","evidence":"Patient fibroblast fractionation, siRNA knockdown, and oxygen consumption/respirometry","pmids":["28295037"],"confidence":"Medium","gaps":["Molecular basis of the respiratory defect not mechanistically defined","Single patient/single lab"]},{"year":2022,"claim":"The cardiac role was extended in vivo: TMEM65 binds the sodium-channel β1 subunit and is required for perinexal nanodomain assembly and conduction, linking its loss to progressive cardiomyopathy.","evidence":"AAV9-shRNA mouse knockdown, Co-IP, super-resolution microscopy, ECG/echocardiography, optical mapping, and patch-clamp electrophysiology","pmids":["36257954"],"confidence":"High","gaps":["How TMEM65 organizes the nanodomain at the molecular level unknown","Relationship between ICD and mitochondrial functions not reconciled"]},{"year":2022,"claim":"Transcriptional control of TMEM65 was placed under a CHD6–TCF4 axis coupled to mitochondrial dynamics and ATP output.","evidence":"ChIP and Co-IP at the TMEM65 promoter, CHD6 knockdown/knockout mice, reporter assays, and xenograft/PDX models","pmids":["36473865"],"confidence":"Medium","gaps":["Direct mechanism by which TMEM65 affects mitochondrial dynamics not shown","Generality of this regulation across tissues unclear"]},{"year":2023,"claim":"Proximity proteomics reframed TMEM65 as an NCLX-proximal regulator of mitochondrial Ca2+ efflux, with loss causing Ca2+ overload and permeability transition.","evidence":"BioID proteomics, Ca2+ flux assays with pharmacological/genetic NCLX inhibition, co-fractionation, in silico modeling, and AAV-shRNA mouse knockdown (preprint)","pmids":["37873405"],"confidence":"Medium","gaps":["Whether TMEM65 is a regulator versus the transporter itself unresolved at this stage","Direct binding not yet distinguished from proximity"]},{"year":2025,"claim":"Peer-reviewed work confirmed TMEM65 as a direct NCLX binding partner enhancing Na+-dependent Ca2+ efflux, with in vivo overload phenotypes in heart and muscle.","evidence":"Proximity proteomics, Ca2+ efflux assays, pharmacological/genetic NCLX manipulation, and AAV-shRNA mouse models with cardiac/neuromuscular assays","pmids":["40200126"],"confidence":"High","gaps":["Did not establish whether TMEM65 has intrinsic transport activity","Stoichiometry of the TMEM65–NCLX complex undefined"]},{"year":2025,"claim":"The catalytic identity was resolved: purified, liposome-reconstituted TMEM65 is itself the mitochondrial Na+/Ca2+ exchanger, acting as a homodimer with defined ion-coordinating residues.","evidence":"Homodimerization assays, site-directed mutagenesis, heterologous expression in mito-NCX-null cells, protein purification with liposome reconstitution, and CGP-37157 binding-site mapping","pmids":["40691517"],"confidence":"High","gaps":["Atomic structure of the transporter not reported","Functional relationship between intrinsic TMEM65 activity and NCLX requires reconciliation"]},{"year":2025,"claim":"Pharmacology and cross-species genetics confirmed TMEM65-dependent Na+/Li+-driven Ca2+ extrusion and positioned TMEM65 downstream of MCU in a Ca2+-overload death pathway.","evidence":"Mitochondrial Ca2+ imaging with gain/loss-of-function, CGP-37157 inhibition, and C. elegans MCU-1 epistasis","pmids":["41408045"],"confidence":"High","gaps":["NCLX-independent versus NCLX-dependent activity not fully reconciled with binding data","Conditions selecting one mode over another unclear"]},{"year":2025,"claim":"Conditional knockout mice defined tissue-specific physiological consequences and cemented the MCU-downstream placement via MCU-rescue of lethality.","evidence":"Whole-body, neuronal, and muscle-specific conditional knockouts, Ca2+ flux assays, MCU double-knockout epistasis, survival analysis, and electrophysiology/histology","pmids":["41980949"],"confidence":"High","gaps":["Cell-autonomous basis of seizure/sudden-death phenotype not dissected","Link between Ca2+ handling and ICD/conduction roles not integrated"]},{"year":2025,"claim":"In cancer contexts TMEM65 was connected to OXPHOS/ROS-driven stemness and to oncogenic signaling, defining roles distinct from its core transport function.","evidence":"siRNA/overexpression with OXPHOS/ROS readouts, HIF1α/SERPINB3 assays, MYC/TET3 manipulation (idx 10); Co-IP and YWHAZ-rescue with PI3K–Akt–mTOR markers and xenograft models (idx 6)","pmids":["40546127","38341472"],"confidence":"Medium","gaps":["Whether these effects are downstream of mitochondrial Ca2+ handling unknown","Cytoplasmic YWHAZ interaction not reconciled with inner-membrane localization","Single-lab studies"]},{"year":2020,"claim":"TMEM65 depletion was shown to trigger mitochondrial stress responses, indicating its loss perturbs mitochondrial proteostasis and redox state.","evidence":"siRNA knockdown in human cells with ROS assays, qRT-PCR, and Western blot for UPRmt and import-receptor markers","pmids":["33319071"],"confidence":"Medium","gaps":["Causal link between Ca2+ overload and UPRmt activation not established","Single method (knockdown) with marker readouts"]},{"year":null,"claim":"How TMEM65's intrinsic mito-NCX transport activity, its direct association with NCLX, and its distinct intercalated-disc/Cx43/SCN1B functions are mechanistically integrated remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No atomic structure of the transporter or of the TMEM65–NCLX complex","Mechanism allowing one protein to act at both inner membrane and ICD undefined","Whether cancer-associated signaling roles depend on Ca2+ transport unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[8,9,7,5]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[8,9]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,2]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,3]}],"pathway":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]}],"complexes":[],"partners":["NCLX","GJA1","SCN1B","YWHAZ"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6PI78","full_name":"Transmembrane protein 65","aliases":[],"length_aa":240,"mass_kda":25.5,"function":"Essential for maintaining proper cardiac intercalated disk (ICD) structure and function as well as cardiac conduction velocity in the heart. Its association with SCN1B is required for stabilizing the perinexus in the ICD and for localization of GJA1 and SCN5A to the ICD. May regulate the function of the gap junction protein GJA1 and may contribute to the stability and proper localization of GJA1 to cardiac intercalated disk thereby regulating gap junction communication (By similarity). May also play a role in the regulation of mitochondrial respiration and mitochondrial DNA copy number maintenance (PubMed:28295037)","subcellular_location":"Cell membrane; Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/Q6PI78/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TMEM65","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TMEM65","total_profiled":1310},"omim":[{"mim_id":"616609","title":"TRANSMEMBRANE PROTEIN 65; TMEM65","url":"https://www.omim.org/entry/616609"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TMEM65"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q6PI78","domains":[{"cath_id":"-","chopping":"67-105","consensus_level":"medium","plddt":88.6867,"start":67,"end":105},{"cath_id":"-","chopping":"109-230","consensus_level":"high","plddt":85.6018,"start":109,"end":230}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6PI78","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6PI78-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6PI78-F1-predicted_aligned_error_v6.png","plddt_mean":72.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TMEM65","jax_strain_url":"https://www.jax.org/strain/search?query=TMEM65"},"sequence":{"accession":"Q6PI78","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6PI78.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6PI78/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6PI78"}},"corpus_meta":[{"pmid":"36473865","id":"PMC_36473865","title":"The chromatin remodeler CHD6 promotes colorectal cancer development by regulating TMEM65-mediated mitochondrial dynamics via EGF and Wnt signaling.","date":"2022","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/36473865","citation_count":43,"is_preprint":false},{"pmid":"26403541","id":"PMC_26403541","title":"Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function.","date":"2015","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/26403541","citation_count":39,"is_preprint":false},{"pmid":"24765583","id":"PMC_24765583","title":"TMEM65 is a mitochondrial inner-membrane protein.","date":"2014","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/24765583","citation_count":36,"is_preprint":false},{"pmid":"40200126","id":"PMC_40200126","title":"TMEM65 regulates and is required for NCLX-dependent mitochondrial calcium efflux.","date":"2025","source":"Nature metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/40200126","citation_count":33,"is_preprint":false},{"pmid":"28295037","id":"PMC_28295037","title":"A mutation in the TMEM65 gene results in mitochondrial myopathy with severe neurological manifestations.","date":"2017","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/28295037","citation_count":27,"is_preprint":false},{"pmid":"36257954","id":"PMC_36257954","title":"Tmem65 is critical for the structure and function of the intercalated discs in mouse hearts.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36257954","citation_count":19,"is_preprint":false},{"pmid":"40691517","id":"PMC_40691517","title":"TMEM65 functions as the mitochondrial Na+/Ca2+ exchanger.","date":"2025","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/40691517","citation_count":18,"is_preprint":false},{"pmid":"38341472","id":"PMC_38341472","title":"TMEM65 promotes gastric tumorigenesis by targeting YWHAZ to activate PI3K-Akt-mTOR pathway and is a therapeutic target.","date":"2024","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/38341472","citation_count":16,"is_preprint":false},{"pmid":"37873405","id":"PMC_37873405","title":"TMEM65 regulates NCLX-dependent mitochondrial calcium efflux.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37873405","citation_count":11,"is_preprint":false},{"pmid":"40546127","id":"PMC_40546127","title":"MYC/TET3-Regulated TMEM65 Activates OXPHOS-SERPINB3 Pathway to Promote Progression and Cisplatin Resistance in Triple-Negative Breast Cancer.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/40546127","citation_count":10,"is_preprint":false},{"pmid":"33319071","id":"PMC_33319071","title":"Depletion of TMEM65 leads to oxidative stress, apoptosis, induction of mitochondrial unfolded protein response, and upregulation of mitochondrial protein import receptor TOMM22.","date":"2020","source":"Biochemistry and biophysics reports","url":"https://pubmed.ncbi.nlm.nih.gov/33319071","citation_count":9,"is_preprint":false},{"pmid":"41408045","id":"PMC_41408045","title":"TMEM65-dependent Ca2+ extrusion safeguards mitochondrial homeostasis.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41408045","citation_count":8,"is_preprint":false},{"pmid":"41061666","id":"PMC_41061666","title":"Mitochondrial sodium-calcium exchange-Can TMEM65 do it alone?","date":"2025","source":"Cell metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/41061666","citation_count":6,"is_preprint":false},{"pmid":"40803458","id":"PMC_40803458","title":"Integrative multi-omics analysis N4-acetylcytidine modification landscape and the role of TMEM65 in breast cancer.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40803458","citation_count":2,"is_preprint":false},{"pmid":"41980949","id":"PMC_41980949","title":"Loss of TMEM65 in mice causes mitochondrial disease mediated by mitochondrial Ca2.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41980949","citation_count":1,"is_preprint":false},{"pmid":"41663003","id":"PMC_41663003","title":"Modified Banxia Xiexin Decoction promotes mitochondrial fission in colon cancer cells by inhibiting the CHD6-TMEM65 axis.","date":"2026","source":"Journal of ethnopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41663003","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.31.685856","title":"Deficient Cardiolipin Remodeling Alters Muscle Fiber Composition and Neuromuscular Connectivity in Barth Syndrome","date":"2025-11-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.31.685856","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10734,"output_tokens":4095,"usd":0.046814,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12187,"output_tokens":4242,"usd":0.083492,"stage2_stop_reason":"end_turn"},"total_usd":0.130306,"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\": 2014,\n      \"finding\": \"TMEM65 is an integral protein of the mitochondrial inner membrane. Immunoblot, immunostaining, alkali extraction, and digitonin extraction of isolated mitochondria established its mitochondrial inner-membrane localization. The N-terminal region (residues 1–20) is sufficient for mitochondrial targeting, and the mitochondrial targeting signal is cleaved between residues 35 and 64 by matrix processing protease (MPP).\",\n      \"method\": \"Immunoblot, immunostaining, alkali extraction, digitonin fractionation of isolated mitochondria, deletion-mutant analysis\",\n      \"journal\": \"PeerJ\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal biochemical fractionation methods in a dedicated localization study; replicated by subsequent independent labs\",\n      \"pmids\": [\"24765583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM65 localizes to the intercalated disc (ICD) of cardiomyocytes and physically interacts with connexin 43 (Cx43). shRNA-mediated Tmem65 knockdown in mouse neonatal cardiomyocytes causes internalization of Cx43 away from the ICD, shortens Cx43 half-life through increased degradation, and abolishes Cx43 gap-junction function. Morpholino knockdown in zebrafish recapitulates gap-junction dysfunction and cardiac morphology defects.\",\n      \"method\": \"Cationic silica-bead plasma-membrane enrichment + shotgun proteomics, lentiviral shRNA knockdown, co-immunoprecipitation, immunofluorescence, zebrafish morpholino knockdown\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vitro KD with defined molecular phenotype, and in vivo (zebrafish) validation; independently extended by subsequent mouse in vivo study\",\n      \"pmids\": [\"26403541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A homozygous splice variant (c.472+1G>A) in TMEM65 causes mitochondrial encephalomyopathy in a patient. Subcellular fractionation confirmed TMEM65 protein in the inner mitochondrial membrane of patient fibroblasts. siRNA knockdown of TMEM65 in fibroblasts severely reduced mitochondrial content and oxygen consumption rate, establishing a direct role for TMEM65 in mitochondrial respiratory chain function.\",\n      \"method\": \"Immunofluorescence, subcellular fractionation/immunoblot, siRNA knockdown, enzymatic respirometry, oxygen consumption rate measurement\",\n      \"journal\": \"European journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined cellular phenotype plus patient-derived validation, single lab, two orthogonal functional readouts\",\n      \"pmids\": [\"28295037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Tmem65 physically interacts with the voltage-gated sodium channel β1 subunit (SCN1B/β1) at the ICD, and this interaction is required for the establishment of the perinexal nanodomain and for correct localization of NaV1.5 and Cx43 to ICDs. AAV9-shRNA-mediated Tmem65 knockdown (90% reduction) in mouse hearts caused eccentric hypertrophic cardiomyopathy progressing to dilated cardiomyopathy, slowed conduction, prolonged PR intervals and QRS duration, and reduced Ca2+ and K+ currents in cardiomyocytes.\",\n      \"method\": \"rAAV9 shRNA knockdown in mice, immunoprecipitation, super-resolution microscopy, echocardiography, electrocardiography, optical mapping, whole-cell patch clamp electrophysiology\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KD mouse model with multiple orthogonal functional and structural readouts plus Co-IP for binding partner; single lab but comprehensive\",\n      \"pmids\": [\"36257954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CHD6 chromatin remodeler binds the TCF4 transcription factor at the TMEM65 promoter and drives transcriptional expression of TMEM65; TMEM65 in turn affects mitochondrial dynamics and ATP production. EGF signaling stabilizes CHD6 by preventing GSK3β-mediated phosphodegron formation and FBXW7-mediated ubiquitination, while Wnt/TCF4-β-catenin signaling transcriptionally activates CHD6 itself.\",\n      \"method\": \"CHD6 knockdown and knockout (mouse Villin-specific), chromatin immunoprecipitation, co-immunoprecipitation of CHD6–TCF4, reporter assays, xenograft models, patient-derived xenograft drug treatment\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — ChIP and Co-IP support CHD6–TCF4 binding at TMEM65 promoter; in vivo KO mouse and PDX models; single lab\",\n      \"pmids\": [\"36473865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TMEM65 is an NCLX-proximal protein at the mitochondrial inner membrane that potently enhances Na+-dependent mitochondrial Ca2+ efflux. Pharmacological NCLX inhibition or genetic NCLX deletion abolishes the TMEM65-dependent increase in Ca2+ efflux. Loss-of-function studies show TMEM65 is required for Na+-dependent mitochondrial Ca2+ efflux. Co-fractionation and in silico structural modeling suggest TMEM65 and NCLX exist in a common macromolecular complex. Tmem65 knockdown in mice causes mitochondrial Ca2+ overload in heart and skeletal muscle and impairs cardiac and neuromuscular function. TMEM65 deletion causes excessive mitochondrial permeability transition; TMEM65 overexpression protects against necrotic cell death during Ca2+ stress.\",\n      \"method\": \"Proximity biotinylation (BioID) proteomics, Ca2+ flux assays, pharmacological NCLX inhibition (CGP-37157), genetic NCLX knockout, co-fractionation, in silico structural modeling, AAV-shRNA mouse knockdown, mitochondrial permeability transition assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity proteomics plus functional Ca2+ flux assays plus in vivo mouse KD; preprint, not yet peer-reviewed at this stage but superseded by peer-reviewed version\",\n      \"pmids\": [\"37873405\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM65 directly binds YWHAZ (14-3-3ζ) in the cytoplasm, inhibiting ubiquitin-mediated degradation of YWHAZ, which in turn activates the PI3K–Akt–mTOR signaling pathway (evidenced by increased p-Akt, p-GSK-3β, p-mTOR). TMEM65 oncogenic effects in gastric cancer are partly dependent on YWHAZ.\",\n      \"method\": \"Co-immunoprecipitation (TMEM65–YWHAZ), Western blot for pathway activation markers, siRNA knockdown, ectopic overexpression, in vivo xenograft and lung-metastasis models, VNP-encapsulated siRNA delivery\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct binding by Co-IP, functional epistasis via YWHAZ rescue, multiple cellular phenotypes; single lab\",\n      \"pmids\": [\"38341472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM65 is identified as a direct NCLX binding partner that enhances Na+-dependent mitochondrial Ca2+ efflux. Genetic deletion of NCLX ablates TMEM65-dependent Ca2+ efflux, and TMEM65 knockdown in mice promotes mitochondrial Ca2+ overload in heart and skeletal muscle. TMEM65 loss impairs cardiac and neuromuscular function, and TMEM65 deletion causes mitochondrial permeability transition and cell death.\",\n      \"method\": \"Proximity biotinylation proteomic screening, Ca2+ efflux assays, pharmacological and genetic NCLX inhibition/deletion, AAV-shRNA mouse knockdown, in vivo cardiac and neuromuscular functional assays\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proximity proteomics for interaction identification, multiple orthogonal functional Ca2+ efflux assays, genetic deletion controls, in vivo mouse models; peer-reviewed, multiple complementary methods\",\n      \"pmids\": [\"40200126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM65 itself functions as the mitochondrial Na+/Ca2+ exchanger (mito-NCX). TMEM65 forms a homodimer containing putative ion-coordinating residues essential for function. Heterologous expression of TMEM65 alone induces Na+/Ca2+ exchange in cells lacking native mito-NCX activity. Purified, liposome-reconstituted TMEM65 exhibits key mito-NCX features (Na+-dependent Ca2+ exchange). The CGP-37157 binding site on TMEM65 was identified. TMEM65 deletion elevates mitochondrial Ca2+ and primes mitochondrial permeability transition.\",\n      \"method\": \"Biochemical homodimerization assays, site-directed mutagenesis of ion-coordinating residues, heterologous expression in mito-NCX-null cells, protein purification and liposome reconstitution, Ca2+ transport assay, CGP-37157 binding-site mapping, Ca2+ imaging\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified protein in liposomes plus heterologous expression plus mutagenesis; multiple orthogonal methods establishing catalytic identity\",\n      \"pmids\": [\"40691517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM65 overexpression specifically enhances Na+- and Li+-dependent mitochondrial Ca2+ extrusion in an NCLX-independent manner; this effect is inhibited by CGP-37157. TMEM65 downregulation chronically elevates basal mitochondrial [Ca2+] and impairs efflux upon stimulation. In C. elegans, deletion of TMEM65 homologs causes necrotic lesions under mild thermal stress that are suppressed by genetic inhibition of MCU-1, placing TMEM65 downstream of MCU in a Ca2+ overload pathway.\",\n      \"method\": \"Mitochondrial Ca2+ imaging (overexpression and knockdown), CGP-37157 pharmacological inhibition, C. elegans genetic deletion and MCU-1 epistasis, genetic suppression assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct Ca2+ flux measurements with gain- and loss-of-function, pharmacological validation, genetic epistasis in C. elegans; multi-method single study\",\n      \"pmids\": [\"41408045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM65 enhances mitochondrial oxidative phosphorylation (OXPHOS) and ROS production. Elevated ROS induces HIF1α, which transcriptionally activates SERPINB3, enhancing cancer stemness. MYC and TET3 coordinately upregulate TMEM65 transcription in triple-negative breast cancer. Pharmacological or siRNA-mediated inhibition of MYC or TET3 attenuates TMEM65-driven tumor progression.\",\n      \"method\": \"siRNA knockdown, ectopic overexpression, OXPHOS/ROS measurements, HIF1α reporter/Western blot, MYC/TET3 inhibitor treatment, in vivo tumor models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional pathway placement via KD/OE with defined molecular readouts; single lab; indirect evidence for HIF1α–SERPINB3 axis\",\n      \"pmids\": [\"40546127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Conditional whole-body and nervous system-specific Tmem65 knockout mice exhibit severe growth retardation and seizure-associated sudden death at ~3 weeks; skeletal muscle-specific knockout produces adult-onset myopathy with elevated mitochondrial Ca2+. TMEM65 ablation causes loss of Na+-dependent mitochondrial Ca2+ export. Genetic epistasis: MCU knockout rescues early lethality of whole-body Tmem65 knockout, extending lifespan from ~3 weeks to >1 year, placing TMEM65 function downstream of MCU in the mitochondrial Ca2+ overload pathway.\",\n      \"method\": \"Conditional knockout mouse generation (whole-body, neuronal, skeletal muscle-specific), mitochondrial Ca2+ measurements, Na+-dependent Ca2+ export assay, MCU double-knockout epistasis, survival analysis, electrophysiology, histology\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO mouse models with tissue-specific phenotypes, direct Ca2+ flux measurements, genetic epistasis with MCU rescue; multiple orthogonal in vivo methods\",\n      \"pmids\": [\"41980949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM65 knockdown in human cultured cells induces mild ROS generation, oxidative stress response (upregulation of NFE2L2, SESN3), mild apoptosis, and mitochondrial unfolded protein response (UPRmt) with upregulation of HSPD1, LONP1. TOMM22 and HSPA9 protein levels are upregulated in an ATF5-independent manner following TMEM65 depletion.\",\n      \"method\": \"siRNA knockdown in human cells, ROS assay, qRT-PCR, Western blot for UPRmt markers and mitochondrial import receptors\",\n      \"journal\": \"Biochemistry and biophysics reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — KD with multiple cellular stress readouts; single lab, single method (siRNA KD) with multiple marker readouts\",\n      \"pmids\": [\"33319071\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TMEM65 is a mitochondrial inner-membrane protein that functions as the mitochondrial Na+/Ca2+ exchanger (mito-NCX): it forms a homodimer with ion-coordinating residues essential for Na+-dependent Ca2+ efflux, interacts with NCLX to enhance Ca2+ export, and its loss causes mitochondrial Ca2+ overload, permeability transition, and cell death—phenotypes rescued in vivo by MCU knockout—while in the heart TMEM65 additionally localizes to intercalated discs where it interacts with Cx43 and the sodium channel β1 subunit to maintain gap-junction function, perinexal nanodomain structure, and normal cardiac conduction.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TMEM65 is an integral protein of the mitochondrial inner membrane, targeted there by an N-terminal signal that is cleaved by the matrix processing protease, and it serves as the principal effector of Na+-dependent mitochondrial Ca2+ extrusion [#0, #8]. Reconstitution of purified TMEM65 in liposomes demonstrates that the protein itself functions as the mitochondrial Na+/Ca2+ exchanger (mito-NCX): it forms a homodimer with ion-coordinating residues essential for activity, drives Na+/Ca2+ exchange when expressed in cells lacking native mito-NCX, and harbors the binding site for the inhibitor CGP-37157 [#8]. Consistent with this catalytic identity, TMEM65 enhances Na+- and Li+-dependent Ca2+ efflux that is blocked by CGP-37157 [#9], and proximity proteomics and co-fractionation place it adjacent to and in direct association with NCLX within a common macromolecular complex [#5, #7]. Loss of TMEM65 ablates Na+-dependent Ca2+ export, drives mitochondrial Ca2+ overload, primes the permeability transition, and causes necrotic cell death, whereas overexpression protects against Ca2+ stress [#8, #11]. Genetic epistasis establishes TMEM65 downstream of the mitochondrial calcium uniporter: MCU knockout rescues the early lethality of whole-body Tmem65-null mice and suppresses thermal-stress necrosis in C. elegans lacking TMEM65 homologs [#9, #11]. Tissue-specific ablation produces seizure-associated sudden death and neuronal phenotypes, skeletal myopathy, and impaired cardiac and neuromuscular function [#7, #11], and a homozygous splice variant causes human mitochondrial encephalomyopathy with reduced respiratory capacity [#2]. In cardiomyocytes TMEM65 additionally localizes to the intercalated disc, where it interacts with connexin 43 to maintain gap-junction stability and with the sodium-channel \\u03b21 subunit (SCN1B) to organize the perinexal nanodomain and proper localization of NaV1.5 and Cx43, such that its loss slows cardiac conduction and produces cardiomyopathy [#1, #3].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Establishing where TMEM65 resides was the first step in assigning it any function; the work fixed it as an inner-membrane protein with a cleavable targeting signal.\",\n      \"evidence\": \"Orthogonal biochemical fractionation (alkali/digitonin extraction), immunostaining, and deletion-mutant targeting analysis of isolated mitochondria\",\n      \"pmids\": [\"24765583\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No molecular function or transport activity assigned\", \"Topology and oligomeric state within the inner membrane not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Beyond mitochondria, TMEM65 was found at the cardiac intercalated disc as a Cx43 partner, defining an unexpected role in gap-junction maintenance and cardiac conduction.\",\n      \"evidence\": \"Plasma-membrane proteomics, reciprocal Co-IP, shRNA knockdown in neonatal cardiomyocytes, and zebrafish morpholino knockdown\",\n      \"pmids\": [\"26403541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking a mitochondrial inner-membrane protein to an ICD pool unresolved\", \"Whether ICD localization reflects a distinct protein population not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A human loss-of-function variant tied TMEM65 to disease and to respiratory-chain capacity, confirming functional importance in patient cells.\",\n      \"evidence\": \"Patient fibroblast fractionation, siRNA knockdown, and oxygen consumption/respirometry\",\n      \"pmids\": [\"28295037\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the respiratory defect not mechanistically defined\", \"Single patient/single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The cardiac role was extended in vivo: TMEM65 binds the sodium-channel \\u03b21 subunit and is required for perinexal nanodomain assembly and conduction, linking its loss to progressive cardiomyopathy.\",\n      \"evidence\": \"AAV9-shRNA mouse knockdown, Co-IP, super-resolution microscopy, ECG/echocardiography, optical mapping, and patch-clamp electrophysiology\",\n      \"pmids\": [\"36257954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TMEM65 organizes the nanodomain at the molecular level unknown\", \"Relationship between ICD and mitochondrial functions not reconciled\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Transcriptional control of TMEM65 was placed under a CHD6\\u2013TCF4 axis coupled to mitochondrial dynamics and ATP output.\",\n      \"evidence\": \"ChIP and Co-IP at the TMEM65 promoter, CHD6 knockdown/knockout mice, reporter assays, and xenograft/PDX models\",\n      \"pmids\": [\"36473865\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism by which TMEM65 affects mitochondrial dynamics not shown\", \"Generality of this regulation across tissues unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Proximity proteomics reframed TMEM65 as an NCLX-proximal regulator of mitochondrial Ca2+ efflux, with loss causing Ca2+ overload and permeability transition.\",\n      \"evidence\": \"BioID proteomics, Ca2+ flux assays with pharmacological/genetic NCLX inhibition, co-fractionation, in silico modeling, and AAV-shRNA mouse knockdown (preprint)\",\n      \"pmids\": [\"37873405\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TMEM65 is a regulator versus the transporter itself unresolved at this stage\", \"Direct binding not yet distinguished from proximity\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Peer-reviewed work confirmed TMEM65 as a direct NCLX binding partner enhancing Na+-dependent Ca2+ efflux, with in vivo overload phenotypes in heart and muscle.\",\n      \"evidence\": \"Proximity proteomics, Ca2+ efflux assays, pharmacological/genetic NCLX manipulation, and AAV-shRNA mouse models with cardiac/neuromuscular assays\",\n      \"pmids\": [\"40200126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish whether TMEM65 has intrinsic transport activity\", \"Stoichiometry of the TMEM65\\u2013NCLX complex undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The catalytic identity was resolved: purified, liposome-reconstituted TMEM65 is itself the mitochondrial Na+/Ca2+ exchanger, acting as a homodimer with defined ion-coordinating residues.\",\n      \"evidence\": \"Homodimerization assays, site-directed mutagenesis, heterologous expression in mito-NCX-null cells, protein purification with liposome reconstitution, and CGP-37157 binding-site mapping\",\n      \"pmids\": [\"40691517\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the transporter not reported\", \"Functional relationship between intrinsic TMEM65 activity and NCLX requires reconciliation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Pharmacology and cross-species genetics confirmed TMEM65-dependent Na+/Li+-driven Ca2+ extrusion and positioned TMEM65 downstream of MCU in a Ca2+-overload death pathway.\",\n      \"evidence\": \"Mitochondrial Ca2+ imaging with gain/loss-of-function, CGP-37157 inhibition, and C. elegans MCU-1 epistasis\",\n      \"pmids\": [\"41408045\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NCLX-independent versus NCLX-dependent activity not fully reconciled with binding data\", \"Conditions selecting one mode over another unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Conditional knockout mice defined tissue-specific physiological consequences and cemented the MCU-downstream placement via MCU-rescue of lethality.\",\n      \"evidence\": \"Whole-body, neuronal, and muscle-specific conditional knockouts, Ca2+ flux assays, MCU double-knockout epistasis, survival analysis, and electrophysiology/histology\",\n      \"pmids\": [\"41980949\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-autonomous basis of seizure/sudden-death phenotype not dissected\", \"Link between Ca2+ handling and ICD/conduction roles not integrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In cancer contexts TMEM65 was connected to OXPHOS/ROS-driven stemness and to oncogenic signaling, defining roles distinct from its core transport function.\",\n      \"evidence\": \"siRNA/overexpression with OXPHOS/ROS readouts, HIF1\\u03b1/SERPINB3 assays, MYC/TET3 manipulation (idx 10); Co-IP and YWHAZ-rescue with PI3K\\u2013Akt\\u2013mTOR markers and xenograft models (idx 6)\",\n      \"pmids\": [\"40546127\", \"38341472\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether these effects are downstream of mitochondrial Ca2+ handling unknown\", \"Cytoplasmic YWHAZ interaction not reconciled with inner-membrane localization\", \"Single-lab studies\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"TMEM65 depletion was shown to trigger mitochondrial stress responses, indicating its loss perturbs mitochondrial proteostasis and redox state.\",\n      \"evidence\": \"siRNA knockdown in human cells with ROS assays, qRT-PCR, and Western blot for UPRmt and import-receptor markers\",\n      \"pmids\": [\"33319071\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal link between Ca2+ overload and UPRmt activation not established\", \"Single method (knockdown) with marker readouts\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TMEM65's intrinsic mito-NCX transport activity, its direct association with NCLX, and its distinct intercalated-disc/Cx43/SCN1B functions are mechanistically integrated remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No atomic structure of the transporter or of the TMEM65\\u2013NCLX complex\", \"Mechanism allowing one protein to act at both inner membrane and ICD undefined\", \"Whether cancer-associated signaling roles depend on Ca2+ transport unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [8, 9, 7, 5]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NCLX\", \"GJA1\", \"SCN1B\", \"YWHAZ\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}