ATG4C (autophagy related 4C cysteine peptidase, autophagin-3) is a cytoplasmic papain-like cysteine protease of peptidase family C54 that processes ATG8-family ubiquitin-like proteins (LC3s and GABARAPs). It can cleave the C-terminal residue of pro-ATG8 to expose the glycine used for phosphatidylethanolamine conjugation, although this priming activity is weak compared with ATG4B, and it removes phosphatidylethanolamine from membrane-anchored ATG8 proteins (delipidation), an activity in which it is comparable to the other ATG4 paralogs. Through these reactions it supports autophagosome formation and the recycling of ATG8 proteins during macroautophagy. The donkey protein is a full-length (458-residue) member of the ATG4C subfamily (PANTHER PTHR22624:SF38), 95% identical to human ATG4C, with the Cys-Asp-His catalytic triad and the C-terminal LIR motif conserved.
Summary: ATG4 processing and recycling of ATG8 proteins is required for phagophore expansion and autophagosome formation.
Reason: Human ATG4C has IDA support for autophagosome assembly, and ATG8 priming/delipidation by ATG4 proteases is a step of autophagosome biogenesis. The donkey protein conserves the catalytic triad and aligns at 94.98% identity over its full length.
The Atg4 cysteine proteases are required for processing Atg8 for the latter to be conjugated to phosphatidylethanolamine on autophagosomal membranes, a key step in autophagosome biogenesis.
file:EQUAS/ATG4C/ATG4C-bioinformatics/RESULTS.md
The donkey sequence A0A9L0IIU3 (458 residues) aligns to human Q96DT6 (458 residues) with 94.98% identity across 458 paired residues.
Summary: ATG4 family members drive phagophore growth in PINK1/Parkin mitophagy, but ATG4C is the paralog reported as weakly or not involved.
Reason: The family-level IBA rests on ATG4A/B/D evidence for a protease-independent role in mitophagic phagophore growth (PMID:33773106). The same work, as summarized in the human ATG4C record, found ATG4C weakly or not involved, so a dedicated mitophagy role is not supported for ATG4C. Its general ATG8 processing may still contribute to mitophagy, so the term is marked over-annotated rather than removed.
Summary: ATG4C cleaves ATG8 proteins after the conserved glycine using a Cys-Asp-His catalytic triad.
Reason: Human ATG4C has IDA support for this activity (weak priming of MAP1LC3 and GABARAPL2; delipidation is also a hydrolysis at the glycine). The nucleophile Cys-111 and Asp-345/His-347 are conserved in the donkey protein.
Reason: The human ATG4C record gives cytoplasm by similarity to mouse ATG4B (a paralog), and the location is consistent with the cytoplasmic site of ATG8 priming and delipidation on phagophore and autophagosome membranes. ATG4C has no transmembrane segment or targeting signal, so cytoplasm is as precise as the evidence allows.
Summary: Correct but general parent of the endopeptidase activity, from the Peptidase C54 InterPro entries.
Reason: The catalytic triad is conserved and human ATG4C has experimental support for cysteine peptidase activity, so the term is true. It is less specific than the evidence supports; the specific descendant, cysteine-type endopeptidase activity (GO:0004197), is already carried by this gene from the ATG4 PANTHER node, so the row is refined to that term.
Summary: ATG4 proteases perform the C-terminal processing of pro-ATG8 proteins.
Reason: ATG4C itself does the cleaving, so it participates in this processing step; its priming activity is weaker than that of ATG4B but demonstrable.
Reason: Reconstituted delipidation assays show that all four ATG4 paralogs act efficiently on membrane-anchored ATG8 proteins, and human ATG4C displays stronger delipidation than priming activity. This is the best-supported molecular function of ATG4C.
GO:0034727 piecemeal microautophagy of the nucleus
IEA GO_REF:0000118
MARK AS OVER ANNOTATED
Summary: Piecemeal microautophagy of the nucleus is a yeast process at the nucleus-vacuole junction; it is not described for mammalian ATG4C.
Reason: The PAINT node behind this transfer (also carried by human ATG4C as IBA) spans the whole ATG4 family, and the experimental grounding for the term is yeast Atg4. Piecemeal microautophagy of the nucleus is defined at the yeast nucleus-vacuole junction, a structure not described in mammals, and there is no ATG4C-specific evidence for it. ATG4C's documented role is general ATG8 processing in macroautophagy, so the term over-extends the family node to a mammalian paralog. It is marked over-annotated rather than removed because ATG8 processing is the step Atg4 contributes in yeast, and the call rests on the absence of a mammalian counterpart rather than on contrary evidence.
Summary: Aggrephagy uses the general ATG8 conjugation machinery to which ATG4C contributes.
Reason: There is no ATG4C-specific aggrephagy evidence; the term follows from its general role in ATG8 processing during macroautophagy, as judged for the ATG4D paralog in this repository.
ISS file:EQUAS/ATG4C/ATG4C-bioinformatics/human_Q96DT6-uniprot.txt
NEW
Summary: ATG4C itself catalyses the release of phosphatidylethanolamine from lipidated ATG8 proteins, so it performs protein delipidation.
Reason: The donkey GOA set carries the molecular function (protein-phosphatidylethanolamide deconjugating activity) but not the process it executes. ATG4C is the enzyme that performs this step, not a substrate of it, so the participation test is met. Human ATG4C has experimental support for delipidation of PE-conjugated ATG8 proteins in a reconstituted assay, and the donkey protein sits in the same PANTHER subfamily (PTHR22624:SF38, ATG4C orthologs), aligns to human over its full length at 95% identity, and conserves the catalytic triad and the C-terminal LIR motif needed for recognition of lipidated substrates. Comparator ATG4 paralogs (human ATG4D) carry the same process term by direct assay.
Catalyzes delipidation of PE-conjugated forms of ATG8 proteins during macroautophagy
file:EQUAS/ATG4C/ATG4C-bioinformatics/RESULTS.md
with 94.98% identity across 458 paired residues
Core Functions
Delipidates PE-conjugated ATG8-family proteins (and weakly primes pro-ATG8 by C-terminal cleavage) in the cytoplasm, supporting autophagosome assembly and ATG8 recycling.
Q: Given its weak priming activity, is ATG4C's physiological role in mammals predominantly delipidation and ATG8 recycling, and which ATG8 paralogs does it act on in vivo?
Suggested Experiments
Experiment: Compare ATG8 lipidation and delipidation kinetics in ATG4B-null versus ATG4B/ATG4C double-null cells reconstituted with wild-type or C111S ATG4C, to separate priming from delipidation contributions.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Assessment of 3 ProtNLM2 GO predictions for donkey ATG4C (A0A9L0IIU3), using the human Swiss-Prot record Q96DT6, cached ATG4 biochemistry papers, and a donkey-human comparison (94.98% identity, full length, catalytic Cys111/Asp345/His347 conserved). Protein delipidation is correct and absent from the donkey GOA record as a process (COR). Proteolysis and autophagy are correct but less precise than the protein processing, cysteine-type endopeptidase and autophagosome assembly annotations already present (LSP). ProtNLM2 emitted no non-GO function-text or subcellular-location output for this accession in the frozen snapshot.
LSP — Less precise than existing annotation Review score: 2/2
Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-10-14
Review rationale: ATG4C is a C54-family cysteine protease that cleaves ATG8 proteins, and the donkey protein conserves the catalytic nucleophile. Proteolysis is therefore correct, but the donkey GOA record already carries protein processing (a child of proteolysis) and cysteine-type endopeptidase activity, so the prediction is less precise than existing annotations.
Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-10-14
Review rationale: All four mammalian ATG4 paralogs delipidate membrane-anchored ATG8 proteins in reconstituted assays, and human ATG4C displays stronger delipidation than priming activity in the curated human UniProt function statement. The donkey protein is a full-length ortholog with the catalytic triad conserved. The donkey GOA record has the corresponding molecular function (protein-phosphatidylethanolamine deconjugating activity) but not this process term, so the prediction adds a correct, target-novel process annotation.
Supporting Evidence:
PMID:29458288: "all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates"
LSP — Less precise than existing annotation Review score: 2/2
Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-10-14
Review rationale: ATG4C acts in autophagy by priming and delipidating ATG8 proteins, so the term is correct. The donkey GOA record already has more specific autophagy annotations (autophagosome assembly, aggrephagy), so the prediction is less precise than existing annotations.