Autophagy is an essential lysosome-dependent quality-control process through which cells degrade and recycle intracellular material. Based on how substrates enter degradative compartments, autophagy is primarily classified into macroautophagy, microautophagy, and chaperone-mediated autophagy. These pathways remove substrates through autophagosome sequestration, direct uptake by endosomal or lysosomal membranes, and chaperone-mediated translocation, respectively, collectively regulating proteostasis, organelle turnover, nutrient adaptation, and disease-associated cellular states.

Keywords: autophagy; macroautophagy; microautophagy; chaperone-mediated autophagy; selective autophagy; LC3; LAMP2A; mitophagy; lysosome

1 Basic Concepts of Autophagy

1.1 Biological Basis of Autophagy

Autophagy comprises catabolic processes that deliver intracellular proteins, protein aggregates, cytoplasmic components, or organelles to lysosomes for degradation. After entering lysosomes, substrates are broken down by acidic hydrolases such as cathepsins into amino acids, fatty acids, and other small molecules that re-enter cellular metabolic networks. Autophagy therefore functions in both material recycling and quality control and is regulated by nutrient deprivation, energetic stress, oxidative stress, protein-folding abnormalities, organelle damage, infection, and other stimuli.

1.2 Autophagy and the Ubiquitin–Proteasome System

The ubiquitin–proteasome system primarily processes short-lived regulatory proteins and abnormal proteins that can be unfolded and transported into the proteasome chamber. By contrast, the autophagy–lysosome system can process protein aggregates and larger intracellular structures such as mitochondria and the endoplasmic reticulum. Although the two degradation systems differ in substrate range and delivery mechanisms, they intersect through ubiquitination, molecular chaperones, and selective autophagy receptors to jointly maintain protein and organelle homeostasis.

2 Three Major Forms of Autophagy

According to the route by which substrates enter lysosomes or related degradative compartments, mammalian autophagy is mainly divided into macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). All three ultimately use lysosomes to degrade substrates, but they differ substantially in membrane architecture, substrate-selection mechanisms, and core molecular machinery.

2.1 Macroautophagy

(1) Autophagosome Formation and Substrate Delivery

Macroautophagy is the most extensively studied form of autophagy and is characterized by the formation of double-membrane autophagosomes. Following autophagy initiation, a cup-shaped isolation membrane forms, expands around cytoplasmic material, and eventually closes to produce a complete autophagosome. The mature autophagosome then fuses with a lysosome to form an acidic autolysosome, in which proteins, lipids, and organelle components are hydrolyzed and recycled into cellular metabolism.

(2) Nonselective Macroautophagy

Nutrient deprivation or energetic stress can induce the relatively broad sequestration of cytoplasmic material through macroautophagy. Degradation of cytoplasmic proteins and other cellular components releases amino acids, fatty acids, and additional metabolic substrates, helping cells maintain essential metabolic functions under nutrient-limited conditions. Macroautophagy therefore provides an important mechanistic connection among nutrient sensing, material recycling, and metabolic adaptation.

(3) Selective Macroautophagy

Macroautophagy can also selectively eliminate specific substrates. Damaged mitochondria, abnormal protein aggregates, excess endoplasmic reticulum, damaged lysosomes, and intracellular pathogens can be connected to autophagic membranes through specific autophagy receptors and preferentially sequestered and degraded. Mitophagy, aggrephagy, ER-phagy, lysophagy, and xenophagy are all forms of selective macroautophagy.

(4) Major Molecular Characteristics of Macroautophagy

Macroautophagy depends on the ULK1 complex, the Beclin 1–VPS34 complex, ATG conjugation systems, and LC3/GABARAP family proteins to mediate initiation, membrane nucleation, membrane elongation, and autophagosome maturation. LC3 lipidation and double-membrane autophagosome formation are important molecular and structural features of canonical macroautophagy. However, an increase in LC3-II may reflect either enhanced autophagosome formation or impaired downstream degradation and must therefore be interpreted together with dynamic autophagic-flux measurements.

2.2 Microautophagy

(1) Direct Uptake of Cytoplasmic Components by Lysosomal Membranes

Microautophagy does not require the formation of an independent double-membrane autophagosome. Instead, the limiting membrane of late endosomes or lysosomes undergoes invagination, protrusion, or other forms of membrane remodeling to directly engulf adjacent cytoplasmic components. Compared with macroautophagy, this process bypasses the formation of a separate autophagosome and its subsequent fusion with lysosomes.

(2) Endosomal Microautophagy

Endosomal microautophagy (eMI) primarily occurs in late endosomes or multivesicular bodies. The endosomal limiting membrane buds inward to form intraluminal vesicles, allowing cytoplasmic substrates to enter the endosomal lumen and subsequently undergo lysosomal degradation. ESCRT-associated proteins, including TSG101, VPS4, and members of the CHMP family, participate in membrane deformation, budding, and scission during certain forms of endosomal microautophagy.

(3) Selective Substrate Recognition in Microautophagy

Some forms of endosomal microautophagy do not take up cytoplasmic material entirely at random. HSC70/HSPA8 can recognize certain cytoplasmic proteins containing KFERQ-like motifs and facilitate their entry into late endosomes, creating some overlap with CMA at the level of substrate recognition. Nevertheless, endosomal microautophagy depends on invagination of the endosomal limiting membrane and vesicle formation, whereas CMA uses LAMP2A to translocate substrates directly across the lysosomal membrane. The two pathways therefore represent distinct membrane-transport processes.

(4) Research Characteristics of Microautophagy

Mammalian microautophagy comprises multiple molecular forms, and no single core marker currently covers all of them. Related studies generally need to evaluate endosomal or lysosomal membrane remodeling, ESCRT dependency, the route by which substrates enter degradative compartments, and the resulting degradation outcome.

2.3 Chaperone-Mediated Autophagy

(1) KFERQ-Like Motifs Determine Substrate Selectivity

Chaperone-mediated autophagy is a highly selective lysosomal protein-degradation pathway. Classical CMA substrates possess KFERQ-like recognition motifs, although an exact KFERQ pentapeptide is not required in the protein sequence. Instead, the motif consists of amino acids with a defined combination of charge, polarity, and hydrophobicity. Conformational changes and post-translational modifications such as phosphorylation and acetylation may generate or expose potential recognition motifs, thereby altering a protein’s capacity to enter CMA.

(2) HSC70 Recognizes and Delivers Substrates

Cytosolic HSC70/HSPA8 recognizes exposed KFERQ-like motifs and cooperates with cochaperones to maintain substrates in a state suitable for delivery and unfolding. This mechanism enables CMA to preferentially recognize specific soluble cytoplasmic proteins rather than randomly engulfing cytoplasmic material.

(3) LAMP2A Mediates Translocation Across the Lysosomal Membrane

After the substrate–HSC70 complex reaches the lysosomal surface, it binds to the cytoplasmic region of LAMP2A. LAMP2A is a specific splice isoform encoded by the LAMP2 gene and is the key receptor for translocation of classical CMA substrates. Following substrate binding, LAMP2A dynamically assembles into a translocation complex that allows the unfolded substrate to cross the lysosomal membrane.

(4) Substrate Unfolding and Lysosomal Degradation

Fully folded proteins cannot pass directly through the CMA translocation machinery and must therefore be unfolded with the assistance of molecular chaperones. Lysosomal HSC70 further supports substrate internalization, after which the translocated protein is degraded by acidic hydrolases. CMA primarily processes soluble cytoplasmic proteins capable of unfolding and cannot directly eliminate intact mitochondria, endoplasmic reticulum, or large insoluble protein aggregates.

2.4 Major Differences Among the Three Forms of Autophagy

3 Key Regulatory Mechanisms of Macroautophagy

3.1 Regulation of Autophagy Initiation by the mTORC1–AMPK–ULK1 Axis

mTORC1 and AMPK form a major regulatory axis that connects nutrient and energy status to macroautophagy initiation. When nutrients, growth factors, and energy are abundant, activated mTORC1 inhibits the ULK1 complex and favors anabolic metabolism. Under energetic stress, AMPK is activated and can inhibit mTORC1 while directly regulating ULK1 to promote autophagy initiation.

The basic regulatory relationship can be summarized as follows: nutrient abundance → mTORC1 activation → ULK1 inhibition → reduced autophagy; energetic stress → AMPK activation/mTORC1 suppression → ULK1 activation → autophagy initiation.

3.2 The Beclin 1–VPS34 Complex Controls Autophagic Membrane Nucleation

Activation of the ULK1 complex promotes recruitment and activation of the class III PI3K complex containing VPS34, Beclin 1, ATG14, and other components at autophagosome-formation sites. VPS34 generates phosphatidylinositol 3-phosphate (PI3P), which recruits downstream effectors such as WIPI proteins and establishes a membrane platform for isolation-membrane formation and expansion. The Beclin 1–VPS34 axis therefore converts upstream autophagy-initiation signals into the membrane-biogenesis process.

3.3 ATG Conjugation Systems and LC3/GABARAP Lipidation

ATG12 conjugates to ATG5 to form the ATG12–ATG5–ATG16L1 complex, which facilitates lipidation of LC3/GABARAP family proteins. LC3 is processed by ATG4 to generate LC3-I and is subsequently conjugated to phosphatidylethanolamine through reactions involving ATG7 and ATG3, producing membrane-associated LC3-II.

LC3/GABARAP family proteins regulate autophagic membrane dynamics and provide docking platforms for selective autophagy receptors, making them important nodes connecting autophagic membrane formation with substrate selection.

3.4 Autophagosome–Lysosome Fusion and Substrate Degradation

Mature autophagosomes must fuse successfully with lysosomes to complete substrate clearance. Rab7, the HOPS complex, and membrane-transport and SNARE machinery such as STX17–SNAP29–VAMP8 participate in autophagosome–lysosome contact and fusion. The acidic lysosomal environment and hydrolases such as cathepsins then degrade the inner autophagosomal membrane and its enclosed substrates. Consequently, increased autophagosome formation does not necessarily indicate enhanced overall autophagic degradation; autophagosome generation and lysosomal degradation must be evaluated as a continuous process.

4 Selective Macroautophagy

4.1 Selective Autophagy Receptors Connect Substrates to Autophagic Membranes

Selective autophagy receptors such as p62/SQSTM1, NBR1, OPTN, and NDP52 recognize specific substrates and connect them to LC3/GABARAP-positive autophagic membranes through LC3-interacting regions. Many receptors can bind both ubiquitinated substrates and autophagic membranes, allowing ubiquitin-tagged abnormal structures to preferentially enter autophagosomes. Selective macroautophagy therefore extends broad material recycling into a substrate-specific cellular quality-control system.

4.2 PINK1–Parkin-Mediated Mitophagy

(1) PINK1 Establishes the Mitochondrial Damage Signal

When mitochondrial membrane potential and protein import remain intact, PINK1 is continuously imported into mitochondria and rapidly turned over. Severe depolarization or impaired protein import causes PINK1 to accumulate stably on the outer mitochondrial membrane, converting mitochondrial damage into a molecular signal that can be further amplified.

(2) Parkin Amplifies the Ubiquitination Signal

Outer-membrane PINK1 phosphorylates ubiquitin and promotes activation of the E3 ubiquitin ligase Parkin. Activated Parkin subsequently ubiquitinates multiple outer mitochondrial membrane proteins, generating an amplified ubiquitin signal that enables damaged mitochondria to be recognized by the selective autophagy machinery.

(3) Selective Autophagy Receptors Complete Mitochondrial Delivery

Receptors such as OPTN and NDP52 recognize ubiquitin signals on mitochondria and connect them to LC3/GABARAP-associated autophagic machinery, allowing damaged mitochondria to be sequestered by isolation membranes and delivered to lysosomes. This process can be summarized as follows: mitochondrial damage → PINK1 accumulation → Parkin activation → ubiquitination of outer-membrane proteins → OPTN/NDP52 recruitment → autophagosomal sequestration → lysosomal clearance.

4.3 Aggrephagy

Aggrephagy primarily processes large abnormal protein aggregates that cannot be directly degraded by proteasomes. Receptors such as p62/SQSTM1 and NBR1 recognize ubiquitinated protein aggregates and connect them to LC3/GABARAP-positive autophagic membranes for incorporation into autophagosomes. This process is particularly important for maintaining proteostasis in long-lived cells such as neurons.

4.4 ER-Phagy

ER-phagy uses receptors such as FAM134B, TEX264, and CCPG1 to connect specific regions of the endoplasmic reticulum to autophagic membranes. It contributes to the removal of excess or damaged endoplasmic reticulum and to the remodeling of ER architecture. ER-phagy is closely associated with recovery from ER stress, secretory-protein quality control, and regulation of organelle abundance.

4.5 Other Forms of Selective Macroautophagy

5 Homeostatic Functions of Autophagy

5.1 Nutrient Sensing and Metabolic Adaptation

When nutrient and energy availability declines, pathways such as mTORC1–AMPK–ULK1 adjust macroautophagic activity, allowing cytoplasmic proteins, lipids, and other cellular components to undergo degradation and recycling. This process supplies amino acids, fatty acids, and other metabolic substrates. CMA can also reconfigure metabolic networks by selectively regulating the stability of metabolic enzymes and regulatory proteins. Autophagy therefore participates not only in material recycling but also in remodeling cellular metabolic states.

5.2 Protein and Organelle Quality Control

Aggrephagy, CMA, and the ubiquitin–proteasome system process protein substrates with different physical states and molecular characteristics. Mitophagy, ER-phagy, pexophagy, and lysophagy maintain organelle homeostasis by selectively removing dysfunctional organelles. Timely elimination of damaged structures limits the persistent accumulation of ROS, misfolded proteins, and organelle-derived stress signals and is fundamental to the long-term homeostatic functions of autophagy.

5.3 Immune and Inflammatory Regulation

Selective autophagy can eliminate certain intracellular pathogens through xenophagy and regulate inflammation through mitochondrial quality control, inflammasome modulation, and turnover of innate immune-signaling proteins. Accumulation of damaged mitochondria can increase ROS production and mitochondrial nucleic acid release, whereas effective mitophagy limits these immunostimulatory signals. Autophagy therefore provides an important connection between organelle quality control and innate immune homeostasis.

6 Autophagy and Disease

6.1 Autophagy and Aging

Aging is associated with impaired lysosomal function, reduced protein quality-control capacity, and altered efficiency of multiple autophagic pathways. These changes promote the gradual accumulation of abnormal proteins and damaged organelles and further disrupt redox homeostasis, inflammatory regulation, and metabolism. Macroautophagy, mitophagy, and CMA operate at different levels of quality control, and age-related reductions in LAMP2A stability and CMA activity represent important areas of aging research.

6.2 Autophagy and Neurodegenerative Diseases

Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are frequently associated with abnormal protein aggregation, mitochondrial dysfunction, and impaired lysosomal degradation. Aggrephagy contributes to the clearance of large abnormal protein structures, CMA regulates the turnover of certain soluble proteins including α-synuclein, and defects in PINK1–Parkin-associated mitochondrial quality control are closely linked to hereditary Parkinson’s disease. Imbalances in different autophagic pathways can increase the long-term stress burden on neurons by disrupting both proteostasis and organelle homeostasis.

6.3 Autophagy and Cancer

The role of autophagy in cancer is strongly dependent on disease stage and genetic context. In normal cells and during early tumorigenesis, autophagy maintains cellular homeostasis by removing damaged mitochondria and abnormal proteins and limiting ROS and chronic inflammation. In some established tumors, however, autophagy supports metabolic adaptation under hypoxic and nutrient-limited conditions and alleviates proteotoxic and organelle stress induced by treatment. Autophagy therefore cannot be classified simply as a tumor-promoting or tumor-suppressive mechanism; its function depends on tumor type, driver mutations, metabolic state, and treatment context.

6.4 Autophagy and Metabolic Diseases

The liver, adipose tissue, skeletal muscle, and pancreatic β cells all depend on autophagy to maintain metabolic homeostasis. Lipophagy contributes to lipid mobilization, mitophagy maintains mitochondrial quality and oxidative metabolic capacity, and CMA alters metabolic networks by selectively regulating the turnover of metabolic enzymes and transcriptional regulators. Autophagic dysfunction can form a mutually reinforcing pathological network with lipid accumulation, mitochondrial dysfunction, ER stress, and altered insulin responses.

7 Advances in Autophagy Research

7.1 PINK1–Parkin-Mediated Recognition of Mitochondrial Damage

Research on PINK1–Parkin has established a continuous molecular connection among mitochondrial protein-import status, changes in membrane potential, ubiquitin phosphorylation, and recruitment of selective autophagy receptors. After becoming stabilized on the outer mitochondrial membrane, PINK1 phosphorylates ubiquitin and activates Parkin to generate an amplified ubiquitin signal. This enables dysfunctional mitochondria to be recognized by autophagy receptors such as OPTN and NDP52. The mechanism directly connects mitochondrial damage sensing with autophagic clearance and continues to advance research on Parkinson’s disease and mitochondrial quality control.

7.2 HSC70- and ESCRT-Dependent Endosomal Microautophagy

Mammalian microautophagy research is progressing from morphological observation toward mechanistic analysis of substrate selection and membrane transport. Certain forms of endosomal microautophagy use HSC70 to recognize proteins with KFERQ-like characteristics and depend on ESCRT-associated membrane remodeling to deliver substrates into the lumen of late endosomes. These findings demonstrate that microautophagy is not exclusively a random cytoplasmic-uptake process and that some forms can mediate selective protein degradation.

7.3 LAMP2A-Mediated CMA and Age-Related Proteostasis

Maintaining LAMP2A levels and CMA activity in aged tissues can reduce abnormal protein accumulation and improve tissue homeostasis, suggesting that CMA decline contributes to age-related cellular dysfunction. Further studies have shown that CMA can regulate metabolism and cellular state by altering the stability of metabolic enzymes, transcriptional regulators, and disease-associated substrates, making it an important mechanism connecting proteostasis, aging, and metabolic regulation.

7.4 Tumor Autophagy Dependency and Autophagy–Lysosome-Targeting Strategies

Cancer autophagy research is shifting from broadly inducing or inhibiting autophagy toward identifying autophagic dependencies in defined genetic and metabolic contexts. Certain tumors exhibit increased dependence on the autophagy–lysosome system under conditions involving RAS/MAPK abnormalities, nutrient limitation, mitochondrial stress, or drug treatment. Current studies increasingly focus on how tumor genotype, tissue of origin, metabolic state, and therapeutic pressure influence autophagic dependency and explore combinations of autophagy modulation with other anticancer treatments.

8 Products

8.1 Products for Macroautophagy Research

8.3 Products for Microautophagy-Related Research

8.4 Products for Chaperone-Mediated Autophagy Research

8.5 Products for Lysosomal Function and Terminal Autophagic Degradation Research

Macroautophagy, microautophagy, and CMA connect to the lysosomal degradation system through distinct substrate-recognition and delivery mechanisms. Accurately distinguishing their molecular mechanisms and experimental boundaries is fundamental to understanding their physiological functions and roles in disease.

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