Adenine Nucleotide Translocase: From Nucleotide Carrier to a Modulator of Mitochondrial Bioenergetics, Quality Control, and Cellular Communication

Through control of ADP/ATP exchange, proton leak, and membrane potential (ΔΨm), ANT sets key upstream parameters for mitochondrial dynamics. Inner membrane fusion depends on OPA1 activity and an intact bioenergetic state [68]. ANT proteins operate in cardiolipin-rich inner membrane microdomains together with OPA1 and nucleoside diphosphate kinase D (NDPK-D/NME4), where ANT-supplied ATP supports local GTP generation for OPA1-dependent membrane remodeling and cristae organization (Figure 2) [69]. Loss of membrane potential disrupts this module, reduces fusion competence, and promotes fragmentation and mitophagy.

Conversely, increased ANT-dependent proton conductance and permeability transition sensitivity promote fission-prone states. Elevated proton leak, ROS production, and mPTP susceptibility shift network behavior toward fragmentation without requiring primary changes in core fission protein abundance [70]. Disruption of inner membrane regulatory assemblies that constrain ANT activity similarly enhances uncoupling and cell death sensitivity and secondarily promotes network fragmentation. Energetic stress states associated with MFN2 dysfunction show comparable features, including increased inner membrane leak and carrier dysregulation [69].

ANT abundance and isoform composition provide an additional scaling layer [70]. Changes in ANT protein levels track with respiratory capacity and mitochondrial mass and indirectly influence network structure through effects on energetic flux, redox balance, and cristae stability. Moderate ANT1 upregulation, for example, is sufficient to induce fragmentation and cristae distortion in muscle models, whereas ANT2 can help preserve ΔΨm under metabolic stress by importing glycolytic ATP, thereby stabilizing stressed mitochondrial populations.

Overall, ANT-controlled energetic and membrane parameters act as upstream constraints that bias mitochondrial networks toward fusion competence, adaptive fragmentation, or quality-control routing. By shaping membrane potential, inner membrane integrity, and fragmentation state, ANT therefore links mitochondrial network remodeling to the selective routing of dysfunctional organelles into mitophagy.

Mitophagy eliminates dysfunctional mitochondria through autophagy-dependent turnover, thereby limiting the accumulation of bioenergetically compromised organelles associated with cancer, neurodegeneration, cardiovascular disease, and aging [71,72,73,74]. Genetic and mechanistic studies suggest that ANT proteins contribute critically to this process. Deletion of ANT1 or ANT2 markedly suppresses mitophagy despite mitochondrial depolarization, whereas pharmacological ANT inhibition promotes mitophagy, demonstrating a transport-independent regulatory role [75].

Mechanistically, ANT1/2 regulates mitophagy initiation through stress-responsive control of mitochondrial protein import. Upon mitochondrial depolarization, ANT1 and ANT2 are required for inhibition of the TIMM23 pre-sequence translocase, preventing continued matrix import and proteolytic processing of PINK1 and thereby stabilizing PINK1 at the outer mitochondrial membrane to enable Parkin recruitment (Figure 3) [75,76]. ANT1/2 physically associates with TIMM23 and its matrix-facing regulatory component TIMM44 in co-immunoprecipitation assays. Disease-associated ANT1 variants that fail to support mitophagy show reduced TIMM23/TIMM44 association. Loss of ANT1/2 markedly impairs PINK1 accumulation under depolarizing conditions, indicating that ANT acts upstream of canonical PINK1–Parkin signaling via TIMM44-associated translocase complexes [75]. Structural and regulatory functions rather than carrier activity are critical, as transport-deficient ANT1 variants restore mitophagy, whereas disease-associated mutants do not.

In addition, ANT1 abundance is controlled by ubiquitin-dependent quality control pathways that directly tune mitophagy efficiency. The deubiquitinase USP34 interacts with ANT1, limits its polyubiquitination, and stabilizes ANT1 protein levels. USP34 loss accelerates ANT1 degradation, reduces PINK1–Parkin–LC3 (receptor of the phagophore membrane) signaling, and impairs mitophagosome formation, whereas ANT1 overexpression restores mitophagy activity [77]. This regulatory axis places ANT1 functionally upstream of canonical mitophagy signaling and identifies ANT1 stabilization as a rate-limiting step in stress-induced mitochondrial quality control.

This pathway is further tuned by post-translational modification, as GSNOR-dependent denitrosylation of ANT1 at C160 is required to sustain cardiac mitophagy [78]. Environmental suppression of ANT1, such as after benzo[a]pyrene exposure, correspondingly reduces PINK1–Parkin signaling and mitophagic flux [79].

Isoform-linked pathways extend this framework. ANT2 is transcriptionally induced after nerve injury and promotes PINK1 stabilization and mitophagy, supporting mitochondrial clearance and axonal recovery [80]. In cancer, ANT3 associates with TOMM20/TIMM22-related assemblies, restricts PINK1 import, and enhances Parkin-dependent mitophagy independently of nucleotide transport, contributing to proteotoxic stress tolerance and proteasome inhibitor resistance [81]. Thus, distinct ANT isoforms can either lower or raise the threshold for mitophagy in a context-dependent manner, aligning organelle turnover with tissue-specific stress demands. All isoforms share conserved carrier architecture and cardiolipin binding. However, available data suggest that they may engage TIMM23/TIMM44 versus TOMM20/TIMM22 assemblies to different extents (Figure 3). ANT1/2 has been reported to associate with TIMM23/TIMM44-dependent import control in post-mitotic tissues, whereas ANT3 has been observed in proximity to TOMM20/TIMM22-containing complexes in proliferative contexts. However, strict isoform specificity has not yet been established. Current evidence is therefore most consistent with a shared ANT-dependent import-gating mechanism whose quantitative contribution may vary with isoform expression and cellular context.

ANT proteins are synthesized in the cytosol and inserted into the inner membrane via TOMM and TIMM22 complexes, placing them in close proximity to mitochondrial protein import machinery [82].

Recent work further shows that pathogenic ANT1 variants induce mitochondrial protein import stress and compensatory remodeling of TIMM22 pathway components, linking carrier import load, inner membrane proteostasis, and autophagy-related transcriptional programs. These findings support the concept that ANT is functionally embedded in mitochondrial import quality control, which can influence mitophagy thresholds under stress [83].

ANT-dependent mitophagy is closely linked to inner membrane integrity. MTFP1 stabilizes ANT-containing inner membrane assemblies and preserves cristae structure. Its loss destabilizes ANT complexes, increases membrane leakiness, sensitizes mitochondria to permeability transition, and reduces mitophagy competence independently of outer membrane fission pathways [70]. These findings link ANT-mediated import gating mechanisms with the structural stability of the inner membrane as a prerequisite for efficient mitophagy.

Together, these findings position ANT as an inner membrane damage sensor that gates PINK1–Parkin–dependent mitophagy through protein import control rather than nucleotide exchange, thereby coupling mitochondrial energetic state to selective organelle turnover and network remodeling.

Through structural, metabolic, and regulatory interactions at the inner mitochondrial membrane, ANT links bioenergetic state to the stability and organization of mitochondrial DNA (mtDNA) and associated transcriptional machinery.

Several heterozygous missense mutations in the ANT1 gene have been identified in patients with autosomal dominant progressive external ophthalmoplegia, mitochondrial myopathy, and cardiomyopathy [84,85,86,87]. Among these, substitution of a conserved alanine by proline at position 114 (A114P) is associated with secondary accumulation of mtDNA deletions in postmitotic tissues [85]. Although the precise mechanism underlying this phenotype remains unresolved, impaired ANT1 function has been proposed to disturb mitochondrial adenine nucleotide homeostasis. This may indirectly impair mtDNA replication fidelity. Beyond nucleotide imbalance, multiple secondary mechanisms may exacerbate ANT1-associated mtDNA instability. These include increased susceptibility to oxidative damage due to protein misfolding and elevated oxidative stress, all of which negatively affect mtDNA maintenance. Consistent with this model, analogous mutations in the yeast ANT homolog AAC2, including A128P, A106D, and M114P, destabilize mtDNA by uncoupling the inner mitochondrial membrane, impairing electron transport, reducing membrane potential (ΔΨm), and increasing sensitivity to uncoupling agents [87].

Animal models further support a protective role for ANT1 in mtDNA maintenance. ANT1-deficient mice display elevated levels of mtDNA deletions, particularly in tissues with limited antioxidant capacity [88]. In contrast, skeletal muscle with robust antioxidant defenses exhibits fewer mtDNA rearrangements, underscoring the contribution of oxidative stress to mtDNA instability and highlighting ANT1-dependent preservation of mitochondrial genome integrity under metabolic stress.

Collectively, ANT1 mutations promote mtDNA instability by perturbing inner membrane integrity and redox balance, thereby predisposing postmitotic tissues to mtDNA damage and deletions. Beyond preserving mtDNA stability, ANT has also been implicated in the organization and maintenance of mitochondrial nucleoids.

Evidence increasingly links ANT to mitochondrial genome organization through its presence in nucleoid-associated inner membrane domains and its interaction with structural scaffold proteins. Mitochondrial nucleoids are membrane-anchored nucleoprotein complexes that coordinate mtDNA replication and transcription and are functionally coupled to inner membrane activity. Core nucleoid components include TFAM, POLRMT, POLG, Twinkle, and mtSSB [89]. Proteomic and fractionation studies place ANT isoforms, including ANT1, together with prohibitin (PHB) complexes in nucleoid-associated membrane regions, where they are proposed to contribute to membrane anchoring and local nucleotide supply [90]. Beyond structural proximity, ANT participates in stress-responsive mitochondrial–nuclear signaling through dynamic inner membrane scaffold complexes. ANT associates with PHB2, PHB1, VDAC, hematopoietic cell-specific Lyn substrate 1-associated protein X1 (Hax-1), and OPA1 in multiprotein assemblies that stabilize cristae structure, maintain ΔΨm, and modulate apoptotic sensitivity (Figure 4) [91,92]. Disruption of these complexes during mitochondrial stress promotes PHB2 release from the inner membrane. Once relocalized, PHB2 translocates to the nucleus, where it acts as a transcriptional coregulator through interactions with p53, estrogen receptor α, and RB/E2F complexes to modulate gene programs involved in cell cycle control, apoptosis, and metabolic adaptation [93]. In parallel, prohibitin assemblies also interface with mitophagy and mitochondrial protease systems, linking ANT-associated membrane domains to quality control pathways [94,95]. These observations connect ANT-containing inner membrane assemblies with nucleoid organization, stress-induced nuclear transcriptional responses, and broader signaling outputs.

ANT is therefore positioned to link mitochondrial structural state and genome-associated functions with adaptive gene regulation.

Mitochondrial genome organization and RNA handling encode mitochondrial state at the molecular level, but their functional relevance depends on how these signals are converted into cellular signaling networks. By controlling nucleotide flux, membrane potential, and permeability, ANT links mitochondrial bioenergetics to stress signaling, immune function, and immunometabolic adaptation. The following section considers these ANT-dependent pathways across three interconnected layers: kinase-responsive regulation, nucleic acid- and inflammasome-associated danger signaling, and downstream immunometabolic adaptation.

Multiple signaling pathways converge on ANT, positioning it as a mitochondrial integration node where kinase, metabolic, and calcium-dependent signals are translated into changes in nucleotide exchange, membrane potential (ΔΨm), and permeability. ANT-dependent mitochondrial state changes also feed back into cytosolic signaling networks. ANT thus operates within a bidirectional signaling axis as both a downstream target and an upstream modulator of stress and survival pathways. The signaling mechanisms discussed below are organized into three interconnected axes: kinase-dependent regulation, post-translational control of ANT activity, and ANT-dependent feedback to calcium- and survival-associated signaling pathways.

Among these pathways, the PI3K/AKT axis is the most mechanistically defined ANT-linked signaling route. Under stress conditions such as ischemia, AKT translocates to mitochondria and associates with ANT [96]. Downstream, AKT regulates GSK-3β, which phosphorylates ANT—most prominently at serine 62—thereby modulating nucleotide transport, ΔΨm stability, oxidative stress, and mPTP sensitivity [97,98]. In this direction, kinase signaling tunes ANT-dependent mitochondrial function. In the opposite direction, ANT-controlled ΔΨm and ROS output influence AKT/GSK-3β pathway activity and permeability transition thresholds, establishing functional feedback between mitochondrial carrier state and survival kinase signaling [55]. In addition, ANT2 knockdown disrupts the HER2–HSP90 complex, accelerates receptor degradation, and suppresses PI3K/AKT signaling [99]. This bidirectional coupling is particularly evident in cardiomyocytes with elevated ANT1 expression. ANT1 overexpression enhances AKT phosphorylation and establishes a cytoprotective feedback circuit under hypoxic stress [100]. A key amplifier is HSP27. Increased HSP27 release activates TLR4 signaling and further augments AKT activity, reinforcing ΔΨm preservation, reducing ROS and caspase activation, and promoting HIF-1α–dependent VEGF and ANT1 expression [55,100]. Thus, ANT1-dependent mitochondrial adaptation actively reinforces pro-survival signaling rather than merely responding to upstream kinase input. Additional kinase pathways—including SRC family kinases and receptor tyrosine kinase-dependent networks—also regulate ANT through phosphorylation or complex stabilization, while ANT-dependent bioenergetic and redox outputs feed back into proliferation and migration signaling modules [55,101,102,103].

Because isoform expression and signaling context vary across tissues, these interactions should be interpreted as context-dependent rather than strictly isoform-fixed.

Post-translational modification provides an additional regulatory layer within this two-way signaling architecture. In prostate cancer cells, the PAK6–SIRT4–ANT2 axis fine-tunes ANT2 activity and mitochondrial apoptosis [104]. PAK6 promotes ubiquitin-dependent SIRT4 degradation and directly modifies ANT2 by phosphorylation (Thr107) and increased acetylation (Lys105), resulting in apoptosis suppression and tumor-supportive signaling. A broader overview of ANT post-translational modifications is provided in a recent review [5].

ANT-linked signaling is further integrated with calcium handling and excitation–contraction coupling. Altered sarcoplasmic reticulum Ca2+ buffering modifies mitochondrial gene expression, including ANT transcripts [105]. Lipid stress increases ANT-dependent ROS production and promotes oxidative RyR2 modification and sarcoplasmic reticulum Ca2+ leak [106], whereas pharmacological ANT inhibition suppresses sarcoplasmic reticulum Ca2+ release in skeletal muscle [107]. Consistent with this coupling, cardiac ANT1 overexpression is associated with enhanced contractile performance and improved calcium cycling, including increased SERCA2a expression and sarcoplasmic reticulum Ca2+ uptake capacity [108,109,110].

Collectively, this evidence supports a two-directional signaling architecture in which ANT is both shaped by kinase and stress pathways and, in turn, reshapes downstream survival and calcium-dependent signaling outputs.

ANT is also embedded in post-transcriptional regulatory networks formed by microRNAs that fine-tune isoform expression and function (Table 1). miR-2861 directly binds the coding sequence of ANT1, suppressing its expression and sensitizing cardiomyocytes to necrotic cell death under oxidative stress [111]. Conversely, inhibition of miR-2861 protects against H2O2-induced necrosis and ischemia–reperfusion injury in vivo.

ANT2 participates in distinct miRNA-dependent circuits. Proteomic analyses identified ANT2 as a binding partner of miR-29b, which co-localizes with ANT2 in perinuclear clusters and influences nuclear architecture during mitosis [112]. Loss of ANT2 phenocopies miR-29b inhibition, leading to abnormal nuclear morphology and impaired nuclear uptake of endogenous miR-29b, revealing a role for ANT2 in coordinating mitochondrial function with cell-cycle–dependent nuclear organization. These findings suggest that ANT2 may contribute not only to intramitochondrial nucleoid-associated processes (see Section 5) but also to miRNA-dependent coordination between mitochondrial state and nuclear architecture.

In cancer, ANT2–miRNA interactions critically shape oncogenic signaling. ANT2 suppression induces miR-636, a tumor-suppressive miRNA targeting Ras, thereby inhibiting proliferation and tumor growth in hepatocellular carcinoma models [113]. In parallel, ANT2 knockdown reduces oncogenic miRNAs including miR-19a, miR-96, miR-21, and miR-221/222, restoring SOCS1 expression, suppressing STAT3 activity, and attenuating PI3K/AKT signaling [114,115,116]. ANT2 upregulation in sorafenib-resistant HCC cells promotes cancer-initiating cell traits and metastasis, whereas restoration of miR-137 reverses these phenotypes [117]. These data position ANT2 as a node at which miRNA networks intersect with metabolic reprogramming, survival, and therapy resistance.

Pathogens also exploit this axis: during human cytomegalovirus infection, the viral miRNA hcmv-miR-UL36-5p suppresses ANT3 expression, thereby inhibiting apoptosis and promoting viral persistence [118]. Together, these data support a reciprocal relationship between ANT isoforms and miRNA networks, in which miRNAs modulate ANT expression and activity, while ANT-dependent mitochondrial states reshape miRNA-controlled programs linking metabolism, nuclear signaling, oncogenesis, and host–pathogen responses. These post-transcriptional circuits converge functionally with inflammatory cytokine pathways, which regulate ANT expression and mitochondrial stress responses at the transcriptional and signaling level.