ATG4D

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0000045 autophagosome assembly	IBA GO_REF:0000033	ACCEPT	Summary: ATG4D functions in autophagosome assembly through its role in processing ATG8-family proteins (LC3/GABARAP). The ATG4 family proteins drive phagophore growth, and ATG4D specifically promotes phagophore-ER contacts during the lipid transfer phase of autophagosome formation. IBA annotation is phylogenetically supported. Reason: Core autophagy function well-supported by literature. ATG4D acts in autophagosome biogenesis through ATG8 processing and has additional protease-independent roles in phagophore growth via ATG9A trafficking regulation (PMID:33773106). Supporting Evidence: PMID:33773106 ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system... ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation file:human/ATG4D/ATG4D-deep-research-falcon.md model: Edison Scientific Literature
GO:0004197 cysteine-type endopeptidase activity	IBA GO_REF:0000033	ACCEPT	Summary: ATG4D is a papain-like cysteine endopeptidase belonging to the peptidase C54 family. It cleaves the C-terminal amino acids of ATG8 proteins (LC3/GABARAP) to expose the glycine required for lipidation. Kinetic studies demonstrate endopeptidase activity against MAP1LC3B (Km=13.1 uM) and GABARAPL2 (Km=7.2 uM). Reason: Core molecular function directly demonstrated by biochemical studies. ATG4D contains the catalytic cysteine (Cys144) characteristic of C54 family proteases and has experimentally validated endopeptidase activity (PMID:21177865). Supporting Evidence: PMID:21177865 The data indicated that Atg4B possessed the broadest spectrum against all substrates, followed by Atg4A, whereas Atg4C and Atg4D had minimal activities
GO:0005737 cytoplasm	IBA GO_REF:0000033	ACCEPT	Summary: ATG4D localizes to the cytoplasm where it performs its primary function of processing ATG8-family proteins on autophagosomal membranes. The full-length protein resides in cytoplasm where it functions in delipidation of autophagosomal ATG8. Reason: Cytoplasmic localization is the primary site of ATG4D function. The deep research review confirms ATG4D acts on autophagosomal/autolysosomal membranes in the cytoplasm.
GO:0000423 mitophagy	IBA GO_REF:0000033	ACCEPT	Summary: ATG4D plays a role in mitophagy. ATG4D re-expression restores mitophagy more effectively than ATG4B in ATG4-quadruple knockout cells. The caspase-cleaved form (DeltaN63) targets mitochondria and associates with cardiolipin, supporting function in mitochondrial quality control. Reason: Mitophagy function is well-supported by experimental evidence. ATG4D is involved in phagophore growth during mitophagy independently of its protease activity and of ATG8 proteins by regulating ATG9A trafficking to mitochondria (PMID:33773106). Supporting Evidence: PMID:33773106 ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria
GO:0016485 protein processing	IBA GO_REF:0000033	ACCEPT	Summary: ATG4D processes ATG8-family proteins by cleaving their C-terminus to expose the glycine required for lipidation (priming activity). This protein processing is essential for autophagy. Reason: Core function of ATG4D family. While ATG4D has weak priming activity compared to ATG4B, it contributes to LC3/GABARAP processing in cells, especially GABARAPL1 and GABARAPL2 (PMID:30661429). Supporting Evidence: PMID:30661429 By further depletion of ATG4 isoforms, we discover that ATG4A, ATG4C and ATGD all contribute to the remaining processing activity and thus show overlapping redundancy in cells
GO:0019786 protein-phosphatidylethanolamide deconjugating activity	IBA GO_REF:0000033	ACCEPT	Summary: ATG4D catalyzes delipidation of PE-conjugated ATG8 proteins, removing them from membranes. This is a major in vivo function of ATG4D - it is the primary delipidating enzyme for LC3B in cells, more important than ATG4B for this function. Reason: Core molecular function directly demonstrated. ATG4D constitutes the major protein for delipidation activity while having weak priming activity (PMID:29458288). Supporting Evidence: PMID:29458288 all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates... We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs
GO:0034727 piecemeal microautophagy of the nucleus	IBA GO_REF:0000033	KEEP AS NON CORE	Summary: This IBA annotation suggests ATG4D involvement in piecemeal microautophagy of the nucleus based on phylogenetic inference. However, direct experimental evidence for ATG4D specifically in this process in humans is limited. Reason: Phylogenetically inferred function. While ATG4 family members are broadly involved in autophagy processes, piecemeal microautophagy of the nucleus is not a demonstrated core function of human ATG4D. The deep research review focuses on macroautophagy and mitophagy as the main functions.
GO:0035973 aggrephagy	IBA GO_REF:0000033	KEEP AS NON CORE	Summary: ATG4D is involved in aggrephagy (selective autophagy of protein aggregates) through its general role in autophagosome biogenesis and ATG8 processing. The SQSTM1/p62 pathway for aggregate clearance requires functional ATG8 lipidation/delipidation. Reason: This is a consequence of ATG4D's core autophagy function rather than a specific evolved role in aggrephagy. ATG4D enables general autophagy which includes aggrephagy, but is not specifically adapted for this selective autophagy pathway. Supporting Evidence: PMID:30661429 Cells lacking ATG4B also showed an accumulation of SQSTM1 and a reduced clearance of this protein upon Torin1 treatment, suggestive of an autophagy defect
GO:0005737 cytoplasm	IEA GO_REF:0000044	ACCEPT	Summary: IEA annotation confirming cytoplasmic localization based on UniProt subcellular location mapping. Consistent with the IBA annotation for the same term. Reason: Redundant with IBA annotation but correct. Cytoplasm is the primary location where ATG4D functions in ATG8 delipidation on autophagosomal membranes.
GO:0005759 mitochondrial matrix	IEA GO_REF:0000044	KEEP AS NON CORE	Summary: The caspase-3 cleaved form of ATG4D (DeltaN63) localizes to the mitochondrial matrix after cleavage at DEVD63 exposes a cryptic mitochondrial targeting sequence. This is not the primary localization of full-length ATG4D. Reason: Conditional localization dependent on caspase-3 cleavage during stress/apoptosis. The full-length protein is cytoplasmic; only the processed mitochondrial form enters the matrix. This represents a secondary, stress-induced localization.
GO:0006508 proteolysis	IEA GO_REF:0000043	ACCEPT	Summary: ATG4D is a cysteine protease that performs proteolytic cleavage of ATG8-family proteins. This is a broad parent term of the more specific peptidase activities. Reason: Correct but general. ATG4D performs proteolysis as part of its ATG8 priming and delipidation functions. More specific terms like cysteine-type peptidase activity are more informative. Supporting Evidence: PMID:21177865 The Atg4 cysteine proteases are required for processing Atg8 for the latter to be conjugated to phosphatidylethanolamine on autophagosomal membranes
GO:0006914 autophagy	IEA GO_REF:0000043	ACCEPT	Summary: ATG4D is a core autophagy protein involved in both priming and delipidation of ATG8-family proteins, essential steps in autophagosome biogenesis and maturation. Reason: Core function. ATG4D is an essential component of the autophagy machinery, with particular importance for delipidation activity in mammalian cells. Supporting Evidence: PMID:30661429 The cysteine protease Atg4 is thought to regulate Atg8 lipidation through 2 processing steps
GO:0006915 apoptotic process	IEA GO_REF:0000043	MARK AS OVER ANNOTATED	Summary: This annotation derives from the UniProt Apoptosis keyword, assigned because ATG4D is cleaved by caspase-3 during apoptosis and the cleaved form translocates to mitochondria. However, the core evolved function of ATG4D is autophagy, not apoptosis. Reason: OVER-ANNOTATION. ATG4D's core function is autophagy (LC3/GABARAP processing). The apoptosis connection is secondary - ATG4D is a substrate of caspase-3 rather than having an evolved apoptotic function. The keyword mapping has led to over-interpretation. Autophagy and apoptosis have crosstalk, but ATG4D's primary role is autophagosome biogenesis through ATG8 delipidation, not apoptosis execution.
GO:0008233 peptidase activity	IEA GO_REF:0000043	ACCEPT	Summary: ATG4D has peptidase activity as a member of the peptidase C54 family. This is a correct but general annotation; more specific terms exist. Reason: Correct parent term. ATG4D is a peptidase that cleaves ATG8-family proteins. More specific terms (cysteine-type endopeptidase activity) are more informative but this annotation is not incorrect. Supporting Evidence: PMID:21177865 The Atg4 cysteine proteases are required for processing Atg8
GO:0008234 cysteine-type peptidase activity	IEA GO_REF:0000120	ACCEPT	Summary: ATG4D is a cysteine-type peptidase of the C54 family. This activity is demonstrated by kinetic studies showing cleavage of ATG8 substrates. Reason: Core molecular function. ATG4D uses an active site cysteine (Cys144) for catalysis, characteristic of papain-like cysteine proteases. Supporting Evidence: PMID:21177865 The Atg4 cysteine proteases are required for processing Atg8
GO:0015031 protein transport	IEA GO_REF:0000043	MARK AS OVER ANNOTATED	Summary: This annotation likely derives from the UniProt Protein transport keyword. ATG4D has an indirect role in protein transport through autophagy-mediated processes, but this is not its primary function. Reason: Indirect and tangential. While autophagy involves membrane trafficking, ATG4D's function is specifically ATG8 processing, not general protein transport. The keyword mapping has led to a non-specific annotation that doesn't reflect the core molecular function of ATG4D.
GO:0016787 hydrolase activity	IEA GO_REF:0000043	ACCEPT	Summary: ATG4D is a hydrolase that cleaves peptide bonds in ATG8 proteins and hydrolyzes the amide bond between ATG8 and PE/PS during delipidation. Reason: Correct broad parent term. ATG4D performs hydrolysis of both peptide bonds (priming) and ATG8-lipid conjugates (delipidation). Supporting Evidence: PMID:30661429 Atg4 can hydrolyze the amide linkage between Atg8 and PE in a delipidation/deconjugation step that recycles free Atg8
GO:0019786 protein-phosphatidylethanolamide deconjugating activity	IEA GO_REF:0000002	ACCEPT	Summary: Duplicate annotation of delipidation activity from InterPro mapping. ATG4D removes PE from ATG8-PE conjugates as a major in vivo function. Reason: Core molecular function. This IEA annotation is redundant with the IBA annotation but both correctly capture ATG4D's delipidation activity, which is its predominant function in cells. Supporting Evidence: PMID:29458288 Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases
GO:0005739 mitochondrion	IDA GO_REF:0000052	KEEP AS NON CORE	Summary: Mitochondrial localization based on immunofluorescence data (HPA). The caspase-cleaved form of ATG4D (DeltaN63) translocates to mitochondria where it associates with cardiolipin and affects mitochondrial ultrastructure. Reason: Conditional localization. Full-length ATG4D is cytoplasmic; only the caspase-3 processed form localizes to mitochondria during stress conditions. This is a secondary function rather than the primary site of ATG4D action.
GO:0000423 mitophagy	IMP PMID:33773106 ATG4 family proteins drive phagophore growth independently o...	ACCEPT	Summary: Experimental evidence from mutant phenotype showing ATG4D involvement in mitophagy. ATG4D promotes phagophore growth during mitophagy by regulating ATG9A trafficking to mitochondria. Reason: Direct experimental evidence. ATG4D has a specific role in mitophagy beyond general autophagy, involving regulation of ATG9A vesicle trafficking to mitochondria and promoting phagophore-ER contacts during mitophagy. Supporting Evidence: PMID:33773106 The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy
GO:0006914 autophagy	IDA PMID:30661429 Redundancy of human ATG4 protease isoforms in autophagy and ...	ACCEPT	Summary: Direct experimental evidence for ATG4D role in autophagy from comprehensive analysis of ATG4 isoform redundancy. ATG4D contributes to LC3/GABARAP processing in cells. Reason: Core function with strong experimental support. ATG4D contributes to priming of GABARAP isoforms and is a major delipidating enzyme (PMID:30661429). Supporting Evidence: PMID:30661429 By further depletion of ATG4 isoforms, we discover that ATG4A, ATG4C and ATGD all contribute to the remaining processing activity and thus show overlapping redundancy in cells
GO:0008234 cysteine-type peptidase activity	IDA PMID:30661429 Redundancy of human ATG4 protease isoforms in autophagy and ...	ACCEPT	Summary: Direct experimental demonstration of ATG4D peptidase activity in cells. Loss of ATG4D (with other ATG4s) affects LC3/GABARAP priming. Reason: Core molecular function experimentally demonstrated. ATG4D contributes to LC3/GABARAP processing in cells, particularly GABARAPL1 and GABARAPL2. Supporting Evidence: PMID:30661429 All human ATG4 isoforms contribute to the priming of LC3/GABARAP isoforms, because complete loss of LC3/GABARAP priming and lipidation in cells could only be achieved upon loss of all ATG4 isoforms
GO:0034497 protein localization to phagophore assembly site	IMP PMID:33773106 ATG4 family proteins drive phagophore growth independently o...	ACCEPT	Summary: ATG4D promotes protein localization to the phagophore assembly site by regulating ATG9A vesicle trafficking. This is a protease-independent function. Reason: Experimentally demonstrated function. ATG4D has a non-canonical role in promoting ATG9A trafficking to the phagophore assembly site, independent of its protease activity (PMID:33773106). Supporting Evidence: PMID:33773106 ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria
GO:0051697 protein delipidation	IDA PMID:29458288 Delipidation of mammalian Atg8-family proteins by each of th...	ACCEPT	Summary: ATG4D catalyzes protein delipidation, removing ATG8-family proteins from membrane lipids. In reconstituted delipidation assays, all four ATG4 proteases can be activated to similar activities on lipid-attached substrates. Reason: Core molecular function. ATG4D is the predominant delipidating enzyme for LC3B in cells, more important for delipidation than for priming (PMID:29458288). Supporting Evidence: PMID:29458288 In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates
GO:0006508 proteolysis	IDA PMID:21177865 Kinetics comparisons of mammalian Atg4 homologues indicate s...	ACCEPT	Summary: ATG4D performs proteolysis of ATG8 substrates. Kinetic analysis demonstrated proteolytic activity against LC3B and GABARAPL2, though with lower activity than ATG4B. Reason: Core function directly demonstrated. ATG4D cleaves ATG8 substrates, with Km values of 13.1 uM for MAP1LC3B and 7.2 uM for GABARAPL2 (PMID:21177865). Supporting Evidence: PMID:21177865 The present study provided the first detailed kinetics analysis of all four Atg4 homologues against four representative Atg8 homologues
GO:0008234 cysteine-type peptidase activity	IDA PMID:21177865 Kinetics comparisons of mammalian Atg4 homologues indicate s...	ACCEPT	Summary: ATG4D has cysteine-type peptidase activity demonstrated by kinetic analysis. Activity is inhibited by N-ethylmaleimide, confirming the cysteine-dependent mechanism. Reason: Core molecular function with biochemical characterization. ATG4D uses an active site cysteine for catalysis, characteristic of C54 family proteases (PMID:21177865). Supporting Evidence: PMID:21177865 The Atg4 cysteine proteases are required for processing Atg8 for the latter to be conjugated to phosphatidylethanolamine on autophagosomal membranes

Gene Description

Existing Annotations Review

Core Functions

ATG4D's primary molecular function is delipidation of ATG8-PE conjugates. While it has weak priming activity compared to ATG4B, ATG4D is the major delipidating enzyme in mammalian cells, particularly for LC3B.

References

Deep Research

Falcon

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