Auxilin (DnaJ homolog subfamily C member 6, DNAJC6) is a neuronal J-domain protein co-chaperone that functions in the terminal step of clathrin-mediated endocytosis. The protein has a three-domain architecture consisting of an N-terminal PTEN-like phosphatase domain (which binds phosphoinositides and targets the protein to endocytic membranes), a C2 tensin-type domain (contributing to membrane association), and a C-terminal J domain (DnaJ domain) that recruits and stimulates the ATPase activity of HSC70 (HSPA8). Together with HSC70, auxilin catalyzes the ATP-dependent disassembly of clathrin coats from newly formed clathrin-coated vesicles, a step essential for synaptic vesicle recycling and sustained neurotransmission. Auxilin binds assembled clathrin lattices via its clathrin-binding domain and couples clathrin recognition to HSC70-mediated uncoating. The PTEN-like domain has only probable phosphatase activity and its primary role appears to be phosphoinositide-dependent membrane targeting rather than catalysis. In zebrafish, auxilin (zAux) is expressed predominantly in neural tissues -- hindbrain neurons, spinal cord neurons, and otic vesicles -- while its paralog GAK is more broadly expressed. Zebrafish auxilin can functionally substitute for Drosophila auxilin in rescuing clathrin-dependent Notch signaling defects, demonstrating evolutionary conservation of its core uncoating function. Loss-of-function mutations in human DNAJC6 cause PARK19, an autosomal recessive juvenile-onset form of Parkinson's disease, linking auxilin dysfunction to dopaminergic neurodegeneration.
Summary: The PTEN-like domain of auxilin is annotated as having phosphoprotein phosphatase activity based on UniRule transfer from homologous proteins. However, the phosphatase domain of auxilin/DNAJC6 has only "probable" phosphatase activity according to UniProt characterization of mammalian orthologs. The primary established role of this domain is phosphoinositide-dependent membrane targeting during clathrin-mediated endocytosis, not catalytic dephosphorylation. While the PTEN-like fold is present, there is no direct experimental evidence demonstrating phosphatase catalytic activity for auxilin itself. The annotation is not entirely wrong given the domain architecture, but it overstates what is functionally established for this protein.
Reason: The PTEN-like domain of auxilin contains the phosphatase fold but its primary role is membrane targeting via phosphoinositide binding, not catalytic phosphatase activity. UniProt itself describes this as only "probable" activity. This IEA annotation based on sequence features overstates the functional evidence.
Summary: This generic hydrolase activity annotation is a parent of GO:0004721 (phosphoprotein phosphatase activity) and suffers from the same issue: it is based on the PTEN-like domain fold, but auxilin's phosphatase activity is only "probable" and its primary role is as a co-chaperone for clathrin uncoating, not as an enzyme. This broad term provides no informative annotation beyond what GO:0004721 already captures (which is itself an over-annotation).
Reason: Uninformative parent term that adds nothing beyond the already questionable phosphoprotein phosphatase annotation. Auxilin's primary molecular function is as a co-chaperone, not a hydrolase. Retaining this generic term would misrepresent the protein's function.
Summary: Auxilin localizes to clathrin-coated vesicles as part of its core function in clathrin coat disassembly. The protein binds assembled clathrin lattices on newly formed vesicles via its clathrin-binding domain, recruiting HSC70 to catalyze uncoating. This localization is well supported by UniProt subcellular location data and is consistent with the extensive literature on auxilin function across vertebrate species.
Summary: Auxilin is directly involved in clathrin-dependent endocytosis, specifically at the terminal uncoating step. It recruits HSC70 to disassemble clathrin coats from newly formed clathrin-coated vesicles, which is essential for recycling clathrin and freeing vesicles for fusion with target membranes. This is a core annotation. The Bai et al. 2010 zebrafish study confirmed that both zAux and zGAK can functionally rescue Drosophila auxilin mutants in clathrin-dependent Notch ligand endocytosis, and the J-domain is essential for this function.
IDA PMID:20082716 Disruption of zebrafish cyclin G-associated kinase (GAK) fun...
ACCEPT
Summary: This IDA annotation is from Bai et al. 2010, which studied subcellular localization of GFP-tagged zebrafish auxilin (zAux) expressed in HeLa cells. The paper reports that at low expression levels, GFP-zAux signals were "mostly cytosolic and slightly enriched near the perinuclear regions." While this was observed in heterologous overexpression rather than endogenous zebrafish neurons, cytosolic localization is consistent with the known biology of auxilin as a soluble protein that is recruited to clathrin-coated vesicles from the cytosol. This is an IDA annotation from ZFIN curators who reviewed the full paper.
IDA PMID:20082716 Disruption of zebrafish cyclin G-associated kinase (GAK) fun...
ACCEPT
Summary: This IDA annotation is also from Bai et al. 2010, based on the observation that GFP-tagged zAux showed enrichment near perinuclear regions in HeLa cells at low expression levels. The paper notes these perinuclear structures "showed overlaps with clathrin, most likely representing the TGN." This localization is consistent with the known association of auxilin family proteins with clathrin-coated structures at the trans-Golgi network. The annotation was made by ZFIN curators who reviewed the full paper.
...GFP signals were mostly cytosolic and slightly enriched near the perinuclear regions. These perinuclear zGAK- and zAux-positive structures showed overlaps with clathrin, most likely representing the TGN...
Summary: NEW (proposed). As a J-domain co-chaperone (auxilin/dnajc6), the primary molecular function is binding and recruiting the HSC70/HSPA8 chaperone to clathrin-coated structures and stimulating its ATPase activity via the conserved His-Pro-Asp (HPD) J-domain motif. Hsp70 protein binding is the specific molecular function underlying the co-chaperone role and is not otherwise represented in the GOA set.
Reason: The co-chaperone activity is defined by binding HSC70/HSPA8; this specific Hsp70 protein binding term (not the uninformative generic protein binding) captures the auxilin J-domain function and grounds the core molecular function.
IEA PMID:20082716 Disruption of zebrafish cyclin G-associated kinase (GAK) fun...
NEW
Summary: NEW (proposed). Auxilin/dnajc6 catalyzes the terminal ATP-dependent disassembly (uncoating) of clathrin coats from newly formed clathrin-coated vesicles by recruiting HSC70, the essential final step of clathrin-mediated endocytosis. Clathrin coat disassembly is the specific biological process for this co-chaperone and is more informative than the broader clathrin-dependent endocytosis term already annotated.
Reason: Clathrin coat disassembly is the specific, well-characterized process auxilin mediates via HSC70 recruitment; it grounds the clathrin-uncoating core function and is supported by direct in-vitro disassembly evidence.
Summary: NEW (proposed). Through its conserved J-domain His-Pro-Asp (HPD) motif, auxilin/dnajc6 stimulates the ATPase activity of the recruited HSC70/HSPA8 chaperone, the step that powers ATP-dependent clathrin-coat disassembly. ATPase activator activity captures this mechanistic aspect of the co-chaperone function alongside Hsp70 protein binding.
Reason: The HPD J-domain motif of auxilin activates HSC70 ATP hydrolysis; ATPase activator activity is the specific molecular function for this stimulation and complements the Hsp70 protein binding annotation, inferred from conserved auxilin co-chaperone mechanism.
auxilin recruits HSC70 to clathrin-coated structures and stimulates HSC70's ATPase activity
Core Functions
Co-chaperone that binds clathrin-coated vesicles and recruits HSC70/HSPA8 via its J domain to catalyze ATP-dependent clathrin coat disassembly, the terminal step of clathrin-mediated endocytosis essential for synaptic vesicle recycling
The primary molecular function is not enzymatic in itself, but rather regulatory and targeting: auxilin recruits HSC70 to clathrin-coated structures and stimulates HSC70's ATPase activity through its conserved J-domain containing the essential His-Pro-Asp (HPD) motif.
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.
Prediction method: ProtNLM2 · Version: UniProt 2024_06 pilot
PSEUDOENZYME OVERANNOTATION
Review rationale: The target has the PTEN-like, C2, and J-domain architecture of auxilin/DNAJC6, but the PTEN-like region is a conserved pseudophosphatase rather than an active phosphatase. Structural work on bovine auxilin shows that this region lacks phosphatase activity and instead participates in membrane binding (PMID:20826345), and the focused sequence comparison found the dead C-X3-R P-loop across human, mouse, and zebrafish DNAJC6 instead of PTEN's active C-X5-R loop. The predicted dephosphorylation biological-process term is an over-propagation from a catalytic PTEN-like ancestor: no evidence establishes that dnajc6 itself catalyzes or directly executes a dephosphorylation step.
Supporting Evidence:
file:DANRE/A0A8M9QG43/A0A8M9QG43-uniprot.txt: "ID A0A8M9QG43_DANRE Unreviewed; 974 AA. ... DR InterPro; IPR029023; Tensin_phosphatase. ... DR PANTHER; PTHR23172:SF4; TYROSINE-PROTEIN PHOSPHATASE AUXILIN-RELATED; 1. ... FT DOMAIN 109..276 ... FT /note="Phosphatase tensin-type" ... FT DOMAIN 282..417 ... FT /note="C2 tensin-type" ... FT DOMAIN 910..974 ... FT /note="J""
PMID:20826345: "A change in the structure of the P loop accounts for the lack of phosphatase activity"