Mitophagy is a quality-control process that selectively recognizes and removes damaged, dysfunctional, or physiologically designated mitochondria. Its core processes include damage sensing, selective labeling, autophagic membrane sequestration, and lysosomal degradation. The PINK1–Parkin ubiquitin-dependent pathway and the BNIP3-, NIX-, and FUNDC1-mediated receptor-dependent pathways constitute the major recognition mechanisms.

Keywords: mitophagy; PINK1; Parkin; BNIP3; NIX; FUNDC1; mitochondrial quality control; autophagic flux

1 Definition and Functional Positioning of Mitophagy

1.1 Definition of Mitophagy

(1) Selective Clearance of Damaged Mitochondria

Mitophagy is a form of selective autophagy in which the substrates are primarily damaged, dysfunctional, or physiologically programmed mitochondria requiring removal. After mitochondrial membrane potential decreases, electron transport becomes impaired, oxidative stress develops, or other homeostatic abnormalities occur, mitochondria can establish connections with LC3/GABARAP-positive autophagic membranes through outer-membrane ubiquitin signals or mitophagy receptors. The isolation membrane extends around the target mitochondrion and closes to form a mitophagosome, which subsequently fuses with lysosomes to complete degradation of mitochondrial components.

(2) Physiological Mitochondrial Remodeling

Mitophagy also participates in erythrocyte maturation, hypoxic adaptation, cell differentiation, and metabolic remodeling. These processes do not necessarily require severe mitochondrial injury but instead programmatically adjust mitochondrial quantity and composition, allowing the mitochondrial pool to adapt to specific cellular states.

1.2 Functional Positioning in Mitochondrial Quality Control

(1) Damage Repair and Selective Clearance

Mild mitochondrial damage can be compensated for by mechanisms such as protein quality control and mitochondrial fusion. Localized dysfunctional regions can be separated from the healthy mitochondrial network through mitochondrial dynamic remodeling, whereas persistently or irreversibly damaged mitochondria enter selective degradation pathways. Mitochondrial fission can facilitate isolation and clearance of damaged regions in some models, but not all mitophagy strictly depends on DRP1-mediated fission. Mitochondrial fragmentation itself also does not prove that mitophagy has occurred.

(2) Balance Between Clearance and Biogenesis

Insufficient mitophagy can lead to accumulation of abnormal mitochondria accompanied by increased ROS, impaired ATP production, and mtDNA-related stress. Conversely, if mitochondrial clearance persistently exceeds mitochondrial replenishment capacity, total mitochondrial mass and cellular energy supply may decline. Mitophagy and mitochondrial biogenesis therefore jointly maintain mitochondrial quantity and quality homeostasis.

1.3 Mitophagy and General Macroautophagy

General macroautophagy can broadly degrade cytoplasmic proteins, organelles, and other cellular components, whereas mitophagy adds a selective recognition step targeting specific mitochondria. PINK1–Parkin establishes selective outer-mitochondrial-membrane ubiquitination, whereas mitochondrial outer-membrane receptors such as BNIP3, NIX, and FUNDC1 can directly connect mitochondria with LC3/GABARAP, thereby preferentially directing selected mitochondria into the autophagic degradation system.

Table 1 Major Differences Between Mitophagy and General Macroautophagy

2 Molecular Mechanisms of Mitophagy

Mitophagy sequentially involves damage sensing, selective labeling, recruitment of autophagic membranes, mitochondrial sequestration, and lysosomal degradation. Different damage conditions can generate PINK1–Parkin-mediated ubiquitin signals or establish direct connections between mitochondria and autophagic membranes through receptors such as BNIP3, NIX, and FUNDC1.

2.1 Mitochondrial Damage Sensing and Dynamic Remodeling

Reduced mitochondrial membrane potential, abnormal electron transport chain function, increased ROS, impaired proteostasis, and mtDNA damage can all alter mitochondrial quality-control status. DRP1 regulates mitochondrial fission with the participation of factors such as MFF and MID49/MID51 and can facilitate separation of locally damaged regions from the mitochondrial network in some stress models. Mitochondrial fission and autophagic clearance are functionally coupled but do not have a fixed sequential dependency. DRP1 activation, mitochondrial shortening, or fragmentation should therefore be evaluated separately from subsequent autophagic delivery and degradation.

2.2 PINK1–Parkin Ubiquitin-Dependent Mitophagy

(1) PINK1 Sensing of Abnormal Membrane Potential

When mitochondrial membrane potential is normal, PINK1 enters mitochondria through the mitochondrial protein-import machinery and is processed by mitochondrial processing peptidases, PARL, and related proteases before undergoing rapid turnover. Following mitochondrial depolarization, normal PINK1 import and processing are disrupted, allowing unprocessed PINK1 to stably accumulate on the outer mitochondrial membrane and maintain kinase activity, thereby converting membrane-potential abnormalities into damage signals recognizable by downstream quality-control systems.

(2) Phosphorylated Ubiquitin and Parkin Activation

Outer-membrane PINK1 phosphorylates ubiquitin at Ser65 and promotes conversion of Parkin from an autoinhibited state to an activated state. Activated Parkin is recruited to damaged mitochondria and ubiquitinates multiple outer-membrane proteins, including MFN1, MFN2, and VDAC. Newly generated ubiquitin chains then serve as additional PINK1 substrates, forming a PINK1–phospho-ubiquitin–Parkin signal-amplification loop that converts initial mitochondrial damage into a high-density outer-membrane ubiquitin signal.

(3) OPTN, NDP52, and TBK1

Ubiquitinated mitochondria can recruit selective autophagy receptors such as OPTN and NDP52. These receptors recognize target mitochondria through ubiquitin-binding regions and connect them to LC3/GABARAP through LIR-related structures. TBK1 participates in phosphorylation-dependent regulation of receptors such as OPTN, strengthening the connection between damaged mitochondria and the autophagic system. PINK1–Parkin mainly performs selective labeling and recruitment of autophagy machinery, whereas final mitochondrial degradation still depends on the autophagosome–lysosome system.

2.3 Receptor-Dependent Mitophagy

(1) BNIP3

BNIP3 is localized to the outer mitochondrial membrane, and its cytoplasmic LIR domain can bind LC3/GABARAP, thereby establishing a connection between mitochondria and autophagic membranes without requiring Parkin-mediated extensive outer-membrane ubiquitination. BNIP3 mainly participates in selective mitochondrial clearance under hypoxia, metabolic stress, and certain tissue-injury conditions.

(2) NIX/BNIP3L

NIX is structurally related to BNIP3 and similarly connects to LC3/GABARAP through an LIR domain. NIX has an important role in mitochondrial removal during erythrocyte maturation and also participates in mitophagy associated with hypoxia, cell differentiation, and metabolic remodeling. Its LIR activity can be regulated by post-translational modifications.

(3) FUNDC1

FUNDC1 is an outer-mitochondrial-membrane LIR-containing receptor whose interaction with LC3 is regulated by phosphorylation status. Under hypoxic conditions, dephosphorylation of FUNDC1-related sites can enhance its interaction with LC3, allowing target mitochondria to recruit autophagic membranes more effectively and enter the clearance process.

2.4 Functional Differences Between Ubiquitin-Dependent and Receptor-Dependent Pathways

The PINK1–Parkin pathway has been extensively studied under conditions of pronounced depolarization and severe mitochondrial injury, whereas BNIP3, NIX, and FUNDC1 are more prominent during hypoxia, differentiation, and specific metabolic adaptations. Basal mitochondrial turnover is not entirely dependent on PINK1/Parkin, and multiple parallel mechanisms of selective mitochondrial clearance can operate under physiological conditions.

Table 2 Two Major Mitophagy Recognition Mechanisms

2.5 Autophagic Membrane Formation and Mitochondrial Sequestration

The ULK1 complex participates in autophagy initiation, the BECN1–VPS34-related complex promotes PI3P generation and isolation-membrane formation, and ATG7, ATG3, and the ATG12–ATG5–ATG16L1 system participate in LC3/GABARAP lipidation. OPTN, NDP52, BNIP3, NIX, or FUNDC1 connects target mitochondria to LC3/GABARAP-positive membrane structures, after which the isolation membrane extends along the mitochondrial surface and closes to form a double-membrane mitophagosome.

2.6 Lysosomal Degradation and Mitochondrial Renewal

(1) Mitophagosome Degradation

After mature mitophagosomes fuse with lysosomes, mitochondrial outer membranes, inner membranes, respiratory-chain components, and matrix proteins are degraded in the acidic environment by lysosomal hydrolytic systems. When lysosomal acidification or autophagosome–lysosome fusion is impaired, LC3-II accumulation and increased mitochondria–autophagosome colocalization may occur even though actual mitochondrial degradation flux is reduced.

(2) Mitochondrial Biogenesis and Replenishment

Regulatory networks including PGC-1α, NRF1/NRF2, and TFAM participate in mitochondrial biogenesis and replenishment of mitochondrial components. Complete mitochondrial quality control therefore includes damage sensing, dynamic remodeling, selective labeling, autophagic membrane sequestration, lysosomal degradation, and mitochondrial biogenesis. The balance between clearance rate and replenishment capacity determines mitochondrial homeostasis.

3 Establishment of Mitophagy Models

3.1 Depolarization-Induced Models

(1) CCCP and FCCP

CCCP and FCCP dissipate the proton gradient across the inner mitochondrial membrane and reduce membrane potential and are commonly used in PINK1–Parkin-related mitophagy research. PINK1 stabilization, Parkin translocation, and outer-membrane ubiquitination can be detected after treatment. High concentrations or prolonged treatment may cause substantial ATP depletion and cell death, so model conditions should be determined by jointly evaluating membrane potential, ATP, and cell viability.

(2) Valinomycin

Valinomycin alters transmembrane ion gradients and mitochondrial membrane potential through selective K⁺ transport. Its mechanism of membrane-potential disruption differs from protonophores and can therefore be used to compare how different modes of depolarization affect damage recognition and mitochondrial clearance.

3.2 Electron Transport Chain Injury Models

(1) Rotenone

Rotenone inhibits mitochondrial electron transport chain complex I, causing respiratory dysfunction, increased ROS, and mitochondrial damage. This model is suitable for mechanistic studies linking complex I dysfunction, oxidative stress, and mitochondrial quality control and has a different injury context from rapid depolarization systems such as CCCP.

(2) Antimycin A and Oligomycin

Antimycin A inhibits electron transport through complex III and promotes ROS generation, whereas oligomycin inhibits F₀F₁-ATP synthase and restricts ATP production. These treatments can respectively establish electron-transport dysfunction and energy-production restriction and can be used to compare the effects of different metabolic injuries on mitophagy initiation and clearance efficiency.

3.3 Autophagic Flux Intervention

(1) Bafilomycin A1 and Chloroquine

Bafilomycin A1 interferes with late-stage autophagy by affecting V-ATPase and lysosomal acidification, whereas chloroquine alters lysosomal function and inhibits degradation of autophagic substrates. Comparing LC3, mitochondrial proteins, and mitochondria–lysosome delivery before and after late-stage blockade can help determine mitochondrial degradation flux.

(2) 3-MA and Rapamycin

3-MA is commonly used to interfere with PI3K-related autophagic processes, whereas Rapamycin promotes autophagy through mTOR-related mechanisms. Both can be used to analyze the relationship between mitochondrial clearance and the core autophagy machinery, but neither is a mitophagy-specific tool.

3.4 Intervention in Mitochondrial Dynamics

Mdivi-1 is commonly used in studies of mitochondrial dynamics, but its experimental effects should not be simply equated with specific DRP1 inhibition. Mdivi-1 can also affect mitochondrial complex I, respiration, and ROS generation. Related experiments should therefore combine DRP1 status, mitochondrial morphology, respiratory function, and genetic intervention for mechanistic confirmation.

Table 3 Common Mitophagy Models and Intervention Methods

4 Experimental Evaluation and Flux Analysis of Mitophagy

4.1 Activation of the PINK1–Parkin Pathway

(1) PINK1 and Parkin

Accumulation of PINK1 on the outer mitochondrial membrane reflects damage recognition following depolarization, while translocation of Parkin from the cytoplasm to mitochondria reflects initiation of the downstream ubiquitination program. Western blotting, immunofluorescence, and mitochondrial/cytoplasmic fractionation can all be used for evaluation. Both indicators represent early events in mitophagy and cannot directly demonstrate that mitochondria have completed degradation.

(2) Phosphorylated Ubiquitin and Receptor Recruitment

Ser65-phosphorylated ubiquitin, ubiquitination of outer-mitochondrial-membrane proteins, and OPTN/NDP52–TBK1-related alterations can further evaluate selective labeling and receptor recruitment. Complete mechanistic validation should cover PINK1/Parkin, ubiquitin signaling, autophagy receptors, autophagic membranes, and lysosomal degradation.

4.2 LC3, p62, and Mitochondrial Sequestration

Colocalization of LC3 with mitochondrial markers such as TOMM20 and VDAC can reflect spatial association between mitochondria and autophagic membranes, while p62/SQSTM1 can assist in evaluating turnover of ubiquitinated substrates. Increased LC3-II may result from either enhanced autophagosome formation or impaired lysosomal degradation. Mitochondria–LC3 colocalization mainly reflects recruitment or sequestration and cannot independently demonstrate increased complete mitophagic flux.

4.3 Mitochondria–Lysosome Delivery

(1) Colocalization of Mitochondria and Lysosomes

Combined detection using mitochondrial fluorescent probes with lysosomal acidic-compartment probes, LAMP1, or LAMP2 can visualize the spatial relationship between mitochondria and lysosomes. Increased colocalization supports mitochondrial delivery to lysosomes, but static colocalization cannot directly distinguish increased delivery from substrate accumulation caused by impaired downstream degradation.

(2) mt-Keima and mito-QC

mt-Keima evaluates mitochondria–lysosome delivery through pH-dependent spectral changes that occur when mitochondria enter acidic compartments. mito-QC uses differences in the stability of GFP and mCherry in acidic environments to identify mitochondria that have entered lysosomes. These reporter systems more directly reflect the key process of mitochondrial entry into acidic degradation compartments.

4.4 Mitochondrial Mass and Function

Decreases in mitochondrial proteins such as TOMM20, VDAC, and COX IV can suggest reduced mitochondrial mass but may also result from reduced mitochondrial biogenesis, altered protein expression, or cell death. JC-1, ATP, and ROS are used to evaluate membrane potential, energy production, and oxidative stress, respectively, and can determine whether changes in mitochondrial quantity are accompanied by altered functional status.

4.5 Determination of Mitophagic Flux

Further accumulation of mitophagy-related substrates after Bafilomycin A1 or chloroquine treatment supports ongoing lysosomal degradation under the original condition. If substrate accumulation after downstream blockade is limited, pre-existing restriction of the late autophagic stage should be considered. PINK1 accumulation, Parkin translocation, LC3-II elevation, mitochondria–LC3 colocalization, and TOMM20 reduction each reflect different stages and cannot individually replace complete flux evaluation.

5 Mitophagy in Disease and Aging

5.1 Neurodegenerative Diseases

Functional abnormalities of PINK1 and PRKN/Parkin are closely associated with hereditary Parkinson’s disease. Neurons are highly dependent on mitochondrial oxidative phosphorylation and have complex requirements for axonal transport and local energy supply. Abnormal mitochondrial quality control can affect ROS homeostasis, energy supply, and neuronal survival. The PINK1–Parkin pathway therefore represents an important mechanistic axis in studies of mitochondrial quality control in neurodegenerative diseases.

5.2 Cardiovascular Injury

Cardiomyocytes have high mitochondrial density and continuous energy requirements. Ischemia, reperfusion, and hypoxia can cause abnormalities in membrane potential, redox status, and calcium homeostasis. Moderate mitophagy helps limit accumulation of damaged mitochondria, whereas imbalance between clearance and mitochondrial biogenesis can affect total mitochondrial mass and ATP supply. Related studies should therefore simultaneously evaluate mitochondrial clearance, biogenesis, and energy status.

5.3 Aging

During aging, mtDNA damage, oxidative stress, proteostasis abnormalities, and changes in lysosomal function can all reduce mitochondrial renewal efficiency. Insufficient mitophagy can increase the burden of abnormal mitochondria, while enhanced clearance without sufficient biogenesis can likewise reduce mitochondrial reserves. The balance of mitochondrial turnover therefore reflects overall quality-control status more effectively than any single autophagy indicator.

6 Products Related to Mitophagy

6.1 Products Related to Model Construction and Pathway Regulation

6.2 Products Related to Autophagy, Mitochondrial Dynamics, and Lysosomal Detection

6.3 Products for Evaluation of Mitochondrial Function, Oxidative Stress, and Cellular Status

Mitophagy removes abnormal mitochondria through damage sensing, selective recognition, autophagic membrane sequestration, and lysosomal degradation. PINK1–Parkin and the BNIP3-, NIX-, and FUNDC1-mediated pathways participate in selective recognition under different stress and physiological conditions. Experimental evaluation requires integrated analysis of upstream signaling, mitochondrial sequestration, lysosomal delivery, degradation flux, and mitochondrial function, together with mitochondrial biogenesis to determine the overall state of mitochondrial quality control.

References

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For more related articles, please see below:

[1] Experiments on oxidation and phosphorylation in isolated mitochondria

[2] Experiments on live staining of mitochondria in cells

[3] Experiments on the hierarchical separation of mitochondria

[4] Oxidative Stress and Experimental Interpretation Framework for ROS, JC-1, MPTP, and Calcium Indicators in Mitochondrial Function Assessment

[5] Mitochondrial Stress Signaling and Disease