GO:0141046 Atg8-family conjugating enzyme activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0141046 describes the E2-like enzymatic step that transfers an Atg8-family modifier (such as LC3B or GABARAP) from an E1-like Atg7 intermediate onto phosphatidylethanolamine (PE) or phosphatidylserine (PS) on a membrane.
• The reaction is isoenergetic: X-Atg8 + Y = Y-Atg8 + X, where Y is PE or PS and X is the E1-like enzyme.
• Atg3 is the canonical conjugating enzyme for this activity, and its high-plasticity regions undergo multiple structural rearrangements for efficient Atg8-PE conjugation.
• The Atg8-family includes LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2, which are central coordinators in autophagy.
• Neddylation stabilizes LC3B by antagonizing its ubiquitin-mediated degradation, linking this conjugating activity to broader ubiquitin-like protein regulation.
• Dysregulation of Atg8-family conjugation is implicated in autophagy-related pathologies including skin disorders and cancer, making it a target for CRISPR-based functional studies.
Description
GO:0141046, Atg8-family conjugating enzyme activity, is a molecular_function term in the Gene Ontology that captures the E2-like step of ubiquitin-like conjugation in autophagy. The reaction is defined as the isoenergetic transfer of an Atg8 family modifier from one protein to a phosphatidylethanolamine (PE) or phosphatidylserine (PS) on a membrane, via the reaction X-Atg8 + Y = Y-Atg8 + X. This activity is essential for lipidation of Atg8-family proteins, a prerequisite for autophagosome formation and selective autophagy. Researchers study this term because it sits at the intersection of ubiquitin-like protein chemistry, membrane biology, and human disease. The conjugating enzyme Atg3 catalyzes the transfer, and its structural plasticity is key for efficient Atg8-PE conjugation during autophagy. Understanding GO:0141046 helps interpret genetic screens, proteomic datasets, and disease models where autophagy flux is perturbed.
Atg8-family conjugating enzyme activity At A Glance
| GO ID | GO:0141046 |
|---|---|
| GO term | Atg8-family conjugating enzyme activity |
| Ontology | molecular_function |
| Synonym | APG8 conjugating enzyme activity; Atg8 conjugating enzyme activity; Atg8-like conjugating activity; E2 |
| Major function | Isoenergetic transfer of an Atg8 family modifier from one protein to a phosphatidylethanolamine or phosphatidylserine on a membrane |
| Reaction | X-Atg8 + Y = Y-Atg8 + X |
| Canonical enzyme | Atg3 |
| Substrates | Atg8-family proteins (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, GABARAPL2) and PE/PS lipids |
| Related process | Autophagy and selective autophagy |
What Is GO:0141046?
In our own words, GO:0141046 describes the catalytic activity of an E2-like conjugating enzyme that moves an Atg8-family modifier from a thioester-linked E1 intermediate onto a membrane lipid, either phosphatidylethanolamine or phosphatidylserine. The reaction is isoenergetic, meaning no additional ATP is consumed at this step; the energy comes from the preceding E1 activation. The modifier becomes covalently attached to the lipid, converting a soluble Atg8-family protein into a membrane-anchored form. This activity is synonymous with APG8 conjugating enzyme activity, Atg8 conjugating enzyme activity, Atg8-like conjugating activity, and E2 in the context of autophagy conjugation systems.
Why Is Atg8-family conjugating enzyme activity Important in Cell Biology?
GO:0141046 is important because it represents the committed lipidation step that anchors Atg8-family proteins to autophagic membranes, enabling autophagosome expansion and cargo recruitment. Without this conjugating activity, Atg8-family proteins remain soluble and cannot support selective autophagy, which is critical for cellular homeostasis. The activity is also a node for crosstalk with other ubiquitin-like modifications; for example, neddylation stabilizes LC3B by antagonizing its ubiquitin-mediated degradation and promoting autophagy in skin. Because Atg3 structural rearrangements are required for efficient conjugation, mutations or regulatory changes in this step can alter autophagy flux and contribute to disease. Studying GO:0141046 therefore informs cancer biology, neurodegeneration, and skin disorders where autophagy is dysregulated.
• Defines the E2-like step that covalently attaches Atg8-family proteins to PE/PS on membranes.
• Essential for autophagosome formation and selective autophagy.
• Links ubiquitin-like conjugation chemistry to membrane remodeling.
• Atg3 structural plasticity is required for efficient Atg8-PE conjugation.
• Neddylation crosstalk stabilizes LC3B and promotes autophagy in skin.
• Provides a mechanistic explanation for autophagy flux changes in disease.
• Enables interpretation of CRISPR screens targeting autophagy genes.
• Supports development of chemical probes for ubiquitin-like proteins.
• Relevant to cancer, neurodegeneration, and skin disorders.
• Guides experimental design for lipidation assays and imaging.
What Happens During Atg8-family conjugating enzyme activity?
Activation and transfer to the conjugating enzyme
In simple terms: First, the Atg8 protein is activated and handed to the conjugating enzyme.
The Atg8-family modifier is first activated by an E1-like enzyme (Atg7) in an ATP-dependent manner, forming a thioester intermediate. The conjugating enzyme (Atg3) then receives the Atg8 protein through a trans-thioesterification reaction, positioning it for transfer to the membrane lipid.
Membrane lipid targeting
In simple terms: The conjugating enzyme brings the Atg8 protein to a lipid in the membrane.
The conjugating enzyme must interact with a membrane containing phosphatidylethanolamine (PE) or phosphatidylserine (PS). The reaction is isoenergetic: X-Atg8 + Y = Y-Atg8 + X, where Y is PE or PS. This step anchors the Atg8-family protein to the membrane, a prerequisite for autophagosome formation.
Conjugation and membrane anchoring
In simple terms: The Atg8 protein becomes covalently attached to the lipid, anchoring it to the membrane.
The conjugating enzyme catalyzes the formation of an amide bond between the C-terminal glycine of the Atg8-family protein and the amine group of PE or PS. This covalent lipidation converts the soluble Atg8-family protein into a membrane-bound form that can recruit cargo receptors and promote autophagosome expansion.
Structural rearrangements in the conjugating enzyme
In simple terms: The conjugating enzyme changes shape to do its job efficiently.
Multiple structural rearrangements mediated by high-plasticity regions in Atg3 are key for efficient conjugation of Atg8 to PE during autophagy. These conformational changes allow the enzyme to accommodate the Atg8 protein and the membrane lipid, facilitating the transfer reaction.
Crosstalk with ubiquitin-like modifications
In simple terms: Other ubiquitin-like modifications can affect this step.
Neddylation modification stabilizes LC3B by antagonizing its ubiquitin-mediated degradation and promoting autophagy in skin. This indicates that the Atg8-family conjugating enzyme activity is integrated with other ubiquitin-like protein pathways, influencing the availability of Atg8-family substrates.
Key Genes Involved in GO:0141046 Atg8-family conjugating enzyme activity
The following genes and proteins are central to Atg8-family conjugating enzyme activity (GO:0141046) and its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ATG3 | Canonical E2-like conjugating enzyme for Atg8-family proteins | Structural and kinetic studies of lipidation |
| ATG7 | E1-like activating enzyme for Atg8-family proteins | Upstream activation step |
| MAP1LC3B | Atg8-family modifier (LC3B) conjugated to PE | Autophagy marker and neddylation crosstalk |
| MAP1LC3A | Atg8-family modifier (LC3A) | Autophagy flux studies |
| MAP1LC3C | Atg8-family modifier (LC3C) | Selective autophagy |
| GABARAP | Atg8-family modifier | Autophagosome maturation |
| GABARAPL1 | Atg8-family modifier | Autophagy and cancer |
| GABARAPL2 | Atg8-family modifier | Autophagy and membrane trafficking |
| ATG4A | Cysteine protease that primes Atg8-family proteins | Regulates substrate availability |
| ATG4B | Cysteine protease that primes Atg8-family proteins | Regulates substrate availability |
| ATG4C | Cysteine protease that primes Atg8-family proteins | Regulates substrate availability |
| ATG4D | Cysteine protease that primes Atg8-family proteins | Regulates substrate availability |
| ATG5 | Component of the Atg12-Atg5-Atg16L1 complex | Facilitates Atg8 lipidation |
| ATG12 | Ubiquitin-like protein conjugated to Atg5 | Required for Atg8 lipidation |
| ATG16L1 | Scaffold for the Atg12-Atg5 complex | Determines site of lipidation |
| NEDD8 | Ubiquitin-like modifier | Stabilizes LC3B and promotes autophagy |
| UBB | Ubiquitin | Antagonizes LC3B stability |
How Is Atg8-family conjugating enzyme activity Regulated?
The activity of Atg8-family conjugating enzymes is regulated at multiple levels. The availability of Atg8-family substrates is controlled by ATG4 proteases, which cleave the C-terminal extension to expose the glycine required for conjugation. The E1-like enzyme Atg7 and the Atg12-Atg5-Atg16L1 complex are required for efficient transfer to the conjugating enzyme. Neddylation modification stabilizes LC3B by antagonizing its ubiquitin-mediated degradation, thereby promoting autophagy in skin. Additionally, structural rearrangements in Atg3 mediated by high-plasticity regions are key for efficient conjugation of Atg8 to PE during autophagy. These regulatory layers ensure that lipidation occurs at the right time and place.
Atg8-family conjugating enzyme activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MAP1LC3B | Skin autophagy and neddylation crosstalk | Keratinocyte knockout and point-mutation models |
| ATG3 | Autophagy deficiency and cancer | Cancer cell lines with ATG3 knockout |
| ATG7 | Neurodegeneration and autophagy | Neuronal knockout models |
| GABARAPL1 | Cancer and autophagy | Tumor xenografts with overexpression |
| ATG16L1 | Inflammatory bowel disease | Intestinal organoids with knock-in |
Autophagy dysregulation in skin disorders
Neddylation modification stabilizes LC3B by antagonizing its ubiquitin-mediated degradation and promoting autophagy in skin. This links Atg8-family conjugating enzyme activity to skin homeostasis and disease, where impaired autophagy can contribute to pathology.
Cancer and selective autophagy
Selective autophagy goes exclusive, and Atg8-family proteins are central coordinators in autophagy. Dysregulation of this conjugating activity can alter cargo recognition and degradation, influencing cancer cell survival and drug resistance.
Neurodegeneration
Impaired autophagy is a hallmark of neurodegenerative diseases. Because Atg8-family conjugating enzyme activity is required for autophagosome formation, its dysfunction may contribute to the accumulation of toxic protein aggregates.
From Atg8-family conjugating enzyme activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of ATG3 abolish Atg8 lipidation? | ATG3 knockout cell line |
| Does a point mutation in ATG3 affect conjugation efficiency? | ATG3 point-mutation knock-in |
| Where does LC3B localize during autophagy? | LC3B tagged knock-in |
| Does neddylation regulate LC3B stability? | NEDD8 overexpression and knockout |
| Can GABARAPL1 overexpression enhance autophagy? | GABARAPL1 overexpression cell model |
| Which genes are required for selective autophagy? | CRISPR library screening |
How to Study the Atg8-family conjugating enzyme activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro lipidation assay | Transfer of Atg8 to PE/PS | Enzyme kinetics and mutant analysis |
| GFP-LC3B imaging | Autophagosome formation | Autophagy flux in cells |
| Western blot for LC3B-II | Lipidated LC3B levels | Autophagy induction |
| Mass spectrometry | Atg8-family conjugates | Proteomic profiling |
| CRISPR knockout screen | Genes required for conjugation | Functional genomics |
| Structural biology (cryo-EM) | Atg3 conformational changes | Mechanistic studies |
| Chemical probes | Ubiquitin-like protein activity | Probe development |
Lipidation assays
In vitro and in vivo lipidation assays using recombinant Atg3 and Atg8-family proteins can measure the transfer of Atg8 to PE or PS. These assays are essential to confirm the conjugating enzyme activity and to test structural mutants.
Fluorescence imaging
Tagged Atg8-family proteins (e.g., GFP-LC3B) allow visualization of autophagosome formation and colocalization with membranes. This method is widely used to assess autophagy flux and the impact of mutations in conjugating enzymes.
Proteomics and ubiquitin-like protein profiling
Mass spectrometry-based proteomics can identify Atg8-family conjugates and crosstalk with ubiquitin and neddylation. Chemical probes for ubiquitin-like proteins are expanding the toolkit for studying these modifications.
CRISPR screening
Genome-wide CRISPR knockout screens can identify genes required for Atg8-family conjugating enzyme activity and autophagy. Hits can be validated with targeted knockouts or point mutations.
How CRISPR Can Be Used to Study GO:0141046 Atg8-family conjugating enzyme activity
Knockout
CRISPR knockout of ATG3 or ATG7 abolishes Atg8-family conjugating enzyme activity, providing a clean background to study downstream effects on autophagy and disease models.
Point Mutation
Point mutations in ATG3 can be introduced to dissect the catalytic mechanism and structural rearrangements required for efficient Atg8-PE conjugation.
Knock-in
Knock-in of tagged Atg8-family proteins (e.g., GFP-LC3B) allows real-time imaging of lipidation and autophagosome dynamics in live cells.
Overexpression
Overexpression of GABARAPL1 or LC3B can enhance autophagy flux and protect against degradation, as shown for neddylation-mediated stabilization of LC3B.
How EDITGENE Supports Atg8-family conjugating enzyme activity Research
Researchers studying Atg8-family conjugating enzyme activity-related genes often need to determine whether a candidate gene is causally involved in autophagy, membrane lipidation, or disease. EDITGENE provides a comprehensive suite of CRISPR services to generate precisely engineered cell models for such functional studies.
Contact EDITGENE today to design your custom CRISPR model for Atg8-family conjugating enzyme activity research.
Frequently Asked Questions About Atg8-family conjugating enzyme activity
What is GO:0141046?
GO:0141046 is the Gene Ontology molecular_function term for Atg8-family conjugating enzyme activity, the E2-like step that transfers an Atg8-family modifier to phosphatidylethanolamine or phosphatidylserine on a membrane.
What genes are involved in Atg8-family conjugating enzyme activity?
Key genes include ATG3 (the conjugating enzyme), ATG7 (E1-like), and Atg8-family members such as MAP1LC3B, GABARAP, and GABARAPL1.
What is the reaction catalyzed by Atg8-family conjugating enzyme activity?
The reaction is X-Atg8 + Y = Y-Atg8 + X, where Y is phosphatidylethanolamine or phosphatidylserine.
Which enzyme carries out Atg8-family conjugating enzyme activity?
Atg3 is the canonical conjugating enzyme that catalyzes the transfer of Atg8-family proteins to PE or PS.
How is Atg8-family conjugating enzyme activity regulated?
It is regulated by ATG4 proteases, the Atg12-Atg5-Atg16L1 complex, and crosstalk with neddylation and ubiquitination.
What diseases are linked to Atg8-family conjugating enzyme activity?
Dysregulation is linked to skin disorders, cancer, and neurodegeneration through impaired autophagy.
What methods are used to study Atg8-family conjugating enzyme activity?
Common methods include in vitro lipidation assays, GFP-LC3B imaging, western blot for LC3B-II, proteomics, and CRISPR screens.
Can CRISPR knockout be used to study Atg8-family conjugating enzyme activity?
Yes, knockout of ATG3 or ATG7 abolishes the activity and is widely used to study autophagy.
What is the role of neddylation in Atg8-family conjugating enzyme activity?
Neddylation stabilizes LC3B by antagonizing its ubiquitin-mediated degradation, promoting autophagy in skin.
Why is Atg8-family conjugating enzyme activity important for selective autophagy?
It anchors Atg8-family proteins to membranes, enabling cargo recruitment and autophagosome formation during selective autophagy.
Conclusion
GO:0141046, Atg8-family conjugating enzyme activity, is a central molecular function in autophagy that mediates the lipidation of Atg8-family proteins. Its mechanism, regulation, and crosstalk with ubiquitin-like modifications are critical for understanding autophagy-related diseases. CRISPR-based models and EDITGENE services provide powerful tools to dissect this activity and its role in health and disease.
References
- 1. Xu L et al.. 2025. Neddylation modification stabilizes LC3B by antagonizing its ubiquitin-mediated degradation and promoting autophagy in skin.. Proc Natl Acad Sci U S A 122(15):e2411429122 PMID: 40208944
- 2. Johansen T et al.. 2014. Selective autophagy goes exclusive.. Nat Cell Biol 16(5):395-7 PMID: 24914435
- 3. Popelka H et al.. 2021. Multiple structural rearrangements mediated by high-plasticity regions in Atg3 are key for efficient conjugation of Atg8 to PE during autophagy.. Autophagy 17(8):1805-1808 PMID: 34338142
- 4. Chanda S et al.. 2026. From Covalent Traps to Fluorescent Beacons: The Expanding Arsenal of Chemical Probes for Studying Ubiquitin and Ubiquitin-Like Proteins.. Angew Chem Int Ed Engl 65(13):e20118 PMID: 41673774
- 5. Mohan J et al.. 2018. Human ubiquitin-like proteins as central coordinators in autophagy.. Interface Focus 8(5):20180025 PMID: 30443326
