| 2014 |
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). |
Immunoblot, immunostaining, alkali extraction, digitonin fractionation of isolated mitochondria, deletion-mutant analysis |
PeerJ |
High |
24765583
|
| 2015 |
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. |
Cationic silica-bead plasma-membrane enrichment + shotgun proteomics, lentiviral shRNA knockdown, co-immunoprecipitation, immunofluorescence, zebrafish morpholino knockdown |
Nature communications |
High |
26403541
|
| 2017 |
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. |
Immunofluorescence, subcellular fractionation/immunoblot, siRNA knockdown, enzymatic respirometry, oxygen consumption rate measurement |
European journal of human genetics |
Medium |
28295037
|
| 2022 |
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. |
rAAV9 shRNA knockdown in mice, immunoprecipitation, super-resolution microscopy, echocardiography, electrocardiography, optical mapping, whole-cell patch clamp electrophysiology |
Nature communications |
High |
36257954
|
| 2022 |
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. |
CHD6 knockdown and knockout (mouse Villin-specific), chromatin immunoprecipitation, co-immunoprecipitation of CHD6–TCF4, reporter assays, xenograft models, patient-derived xenograft drug treatment |
Cell discovery |
Medium |
36473865
|
| 2023 |
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. |
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 |
bioRxivpreprint |
Medium |
37873405
|
| 2024 |
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. |
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 |
Oncogene |
Medium |
38341472
|
| 2025 |
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. |
Proximity biotinylation proteomic screening, Ca2+ efflux assays, pharmacological and genetic NCLX inhibition/deletion, AAV-shRNA mouse knockdown, in vivo cardiac and neuromuscular functional assays |
Nature metabolism |
High |
40200126
|
| 2025 |
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. |
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 |
Nature cell biology |
High |
40691517
|
| 2025 |
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. |
Mitochondrial Ca2+ imaging (overexpression and knockdown), CGP-37157 pharmacological inhibition, C. elegans genetic deletion and MCU-1 epistasis, genetic suppression assay |
Nature communications |
High |
41408045
|
| 2025 |
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. |
siRNA knockdown, ectopic overexpression, OXPHOS/ROS measurements, HIF1α reporter/Western blot, MYC/TET3 inhibitor treatment, in vivo tumor models |
Advanced science |
Medium |
40546127
|
| 2025 |
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. |
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 |
Nature communications |
High |
41980949
|
| 2020 |
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. |
siRNA knockdown in human cells, ROS assay, qRT-PCR, Western blot for UPRmt markers and mitochondrial import receptors |
Biochemistry and biophysics reports |
Medium |
33319071
|