apa-2 (formerly apt-4) encodes the alpha-adaptin large subunit of the AP-2 clathrin adaptor complex of Caenorhabditis elegans. Alpha-adaptin forms the alpha/sigma2 half of the heterotetrameric adaptor: its trunk contributes phosphatidylinositol-4,5-bisphosphate binding that docks AP-2 on the plasma membrane and, with sigma2, the pocket that reads dileucine sorting signals, while its hinge and appendage ('ear') domain recruits accessory endocytic proteins such as the RAB-5 exchange factor RME-6, the RAB-35 regulator RME-4, the clathrin-associated sorting protein DAB-1 and the engulfment adaptor CED-6. APA-2 is expressed in most cells, is highest in neurons, and marks plasma-membrane clathrin-coated pits in coelomocytes, the presynaptic periactive zone of motor neurons and phagocytic cups around apoptotic corpses. It functions in clathrin-mediated endocytosis of yolk by oocytes, synaptic vesicle retrieval at neuromuscular junctions, and CED-1-dependent apoptotic-cell engulfment. Null mutants are viable but dumpy, egg-laying defective and mildly uncoordinated, with a 30% reduction in synaptic vesicles; combined loss of alpha and mu2 removes essentially all AP-2 function and is nearly lethal.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:15895077 Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 a... | REMOVE | Summary: IPI with RME-6 (WBGene00004377/Q9GYH7): alpha-adaptin co-immunoprecipitates with GFP::RME-6 from worm lysates; RME-6 carries a WxxF alpha-ear-binding motif. Reason: Generic protein binding conveys no functional information; the interaction is a genuine alpha-appendage/accessory-protein contact but GO has no term for endocytic accessory-protein recruitment, and the biology is captured by the clathrin-cargo adaptor and endocytosis rows. Removal does not dispute the interaction. Supporting Evidence: PMID:15895077 We found that Ξ±-adaptin did indeed coimmunoprecipitate with GFP::RME-6, showing a physical link of RME-6 to clathrin-coated pits. |
| GO:0005515 protein binding | IPI PMID:16497226 Clathrin interaction and subcellular localization of Ce-DAB-... | REMOVE | Summary: IPI with DAB-1 (abstract only): Ce-DAB-1 binds clathrin and APT-4/alpha-adaptin in vitro. Reason: Generic protein binding conveys no functional information; the interaction is a genuine alpha-appendage/accessory-protein contact but GO has no term for endocytic accessory-protein recruitment, and the biology is captured by the clathrin-cargo adaptor and endocytosis rows. Removal does not dispute the interaction. Supporting Evidence: PMID:16497226 Ce-DAB-1 binds to clathrin and APT-4, the C. elegans homologue of the alpha-adaptin subunit of adaptor protein 2 (AP2) |
| GO:0005515 protein binding | IPI PMID:18354496 Regulation of endocytic recycling by C. elegans Rab35 and it... | REMOVE | Summary: IPI with RME-4 (abstract only): GFP-RME-4 physically interacts with AP-2 and is enriched on clathrin-coated pits. Reason: Generic protein binding conveys no functional information; the interaction is a genuine alpha-appendage/accessory-protein contact but GO has no term for endocytic accessory-protein recruitment, and the biology is captured by the clathrin-cargo adaptor and endocytosis rows. Removal does not dispute the interaction. Supporting Evidence: PMID:18354496 GFP-RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. |
| GO:0005515 protein binding | IPI PMID:23696751 Clathrin and AP2 are required for phagocytic receptor-mediat... | REMOVE | Summary: IPI with CED-6: 35S-APA-2 binds GST-CED-6 and GST-CED-1C directly, and APA-2::GFP co-immunoprecipitates with endogenous CED-1 and CED-6. Reason: Generic protein binding conveys no functional information; the interaction is a genuine alpha-appendage/accessory-protein contact but GO has no term for endocytic accessory-protein recruitment, and the biology is captured by the clathrin-cargo adaptor and endocytosis rows. Removal does not dispute the interaction. Supporting Evidence: PMID:23696751 GST-CED-6 directly interacted with 35S-labeled APA-2, APB-1 and DPY-23, and His6-tagged CHC-1C (Figure 4A). PMID:23696751 Importantly, APA-2::GFP was co-immunoprecipitated with endogenous CED-1 and CED-6 (Figure 4D and 4E) |
| GO:0005886 plasma membrane | IDA PMID:15895077 Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 a... | ACCEPT | Summary: mRFP1::alpha-adaptin (APT-4) is used as a plasma-membrane clathrin-coated-pit marker in coelomocytes, distributed unevenly on or near the plasma membrane with CHC-1. Reason: Directly observed; alpha-adaptin docks AP-2 on the cytoplasmic face of the plasma membrane, the core site of its activity. Supporting Evidence: PMID:15895077 In contrast, we found strong colocalization of GFP::RME-6 with mRFP1::CHC-1 and mRFP1::Ξ±-adaptin on or near the PM of coelomocytes. PMID:23482940 In apm-2 mutants, tagged Ξ±-adaptin is still localized to the synapse (Figure 6D) and the plasma membrane of coelomocytes. |
| GO:0006886 intracellular protein transport | IEA GO_REF:0000120 | ACCEPT | Summary: InterPro/UniRule mapping to intracellular protein transport; AP-2 internalises membrane proteins from the plasma membrane. Reason: Correct general parent of the endocytic transport rows. Supporting Evidence: PMID:23482940 These data suggest that recovery of di-leucine cargo depends on Ξ±-adaptin more than ΞΌ2-adaptin. |
| GO:0006898 receptor-mediated endocytosis | IMP PMID:10588660 Receptor-mediated endocytosis in the Caenorhabditis elegans ... | ACCEPT | Summary: Alpha-adaptin RNAi strongly inhibits uptake of YP170::GFP yolk into oocytes, with yolk accumulating in the pseudocoelom. Null mutants still take up yolk, showing that AP-2 assists the CLASP DAB-1 rather than being absolutely required. Reason: Direct genetic evidence for a role in receptor-mediated yolk endocytosis; the milder null phenotype qualifies but does not contradict it. Supporting Evidence: PMID:10588660 elegans clathrin heavy chain, Ξ±-adaptin, and Ξ²-adaptin RNAi all produced similar defects in oocytes and embryos. PMID:10588660 We found that RNAi of these CCP components strongly inhibited uptake of YP170::GFP into oocytes, resulting in high accumulation of YP170::GFP in the pseudocoelomic space and many fewer YP170::GFP-containing yolk granules in late-stage oocytes and embryos. PMID:23482940 Thus, the AP2 complex appears to assist Disabled for yolk endocytosis, and Ξ±- and ΞΌ2-adaptin do not contribute differentially to this process. |
| GO:0008104 intracellular protein localization | IMP PMID:17636000 C. elegans Disabled is required for cell-type specific endoc... | KEEP AS NON CORE | Summary: Abstract-only record stating that DAB-1 localisation in oocytes depends on clathrin and AP-2. AP-2's alpha appendage recruits DAB-1-type clathrin-associated sorting proteins to coated pits, so the effect is plausibly direct, but the term is very general. Reason: Supported by the abstract and mechanistically plausible (adaptor recruits the CLASP), but 'intracellular protein localization' is an unspecific consequence of AP-2's coat-assembly role; kept as non-core. Supporting Evidence: PMID:17636000 The localisation of DAB-1 in oocytes is itself dependent upon clathrin and AP2, consistent with DAB-1 acting as a clathrin-associated sorting protein during yolk protein endocytosis. |
| GO:0009792 embryo development ending in birth or egg hatching | IMP PMID:10588660 Receptor-mediated endocytosis in the Caenorhabditis elegans ... | REMOVE | Summary: Embryos of alpha-adaptin RNAi mothers were inviable in the yolk-uptake study. Subsequent analysis of two null alleles shows apa-2 mutants are viable as homozygous strains with embryonic lethality of 2.4% versus 1.1% in wild type (not significant), so alpha-adaptin is not required for embryogenesis; the RNAi lethality most likely reflects yolk deprivation of the oocytes. Reason: Contradicted by null-mutant data: viable apa-2 strains with near wild-type embryonic viability show the gene product is not involved in embryo development, and the RNAi effect is a maternal yolk-supply consequence rather than participation in embryogenesis. Supporting Evidence: PMID:10588660 In all of these cases, late-stage oocytes were smaller than in wild type, and embryos proved inviable. PMID:23482940 (A) Embryonic lethality (% total embryos) of AP2 mutants Β± SEM: wild type 1.13 Β± 0.25 n = 10, apa-2(ox422) 2.42 Β± 0.96 n = 9 (p=0.1902) PMID:23482940 Ξ±-adaptin mutants are viable and the remaining ΞΌ2/Ξ² hemicomplex retains some function. |
| GO:0015031 protein transport | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro AP-2 alpha subunit (IPR017104) mapping to protein transport. Reason: Correct general parent. Supporting Evidence: PMID:10588660 These results are consistent with the proposed roles of clathrin, Ξ±-adaptin, and Ξ²-adaptin in coated pit and coated vesicle formation. |
| GO:0016020 membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (coated pit, peripheral membrane protein) to membrane. Reason: Correct though general. Supporting Evidence: PMID:15895077 In particular we focused on two markers for PM clathin-coated pits: clathrin heavy chain (CHC-1) and Ξ±-adaptin (APT-4) |
| GO:0016192 vesicle-mediated transport | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro adaptin mapping to vesicle-mediated transport. Reason: Correct high-level parent of clathrin-dependent endocytosis. Supporting Evidence: PMID:10588660 These results are consistent with the proposed roles of clathrin, Ξ±-adaptin, and Ξ²-adaptin in coated pit and coated vesicle formation. |
| GO:0030117 membrane coat | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro mapping to membrane coat; AP-2 is the adaptor layer of the plasma-membrane clathrin coat. Reason: Correct; the adaptor complex terms are more specific. Supporting Evidence: PMID:15895077 In particular we focused on two markers for PM clathin-coated pits: clathrin heavy chain (CHC-1) and Ξ±-adaptin (APT-4) |
| GO:0030122 AP-2 adaptor complex | IBA GO_REF:0000033 | ACCEPT | Summary: IBA AP-2 adaptor complex from the alpha-adaptin node PTN000512892 (fly, mammalian, yeast, Dictyostelium alpha subunits). APA-2 is the worm alpha subunit; it forms the alpha/sigma2 hemicomplex and its loss is synthetic with mu2 loss. Reason: Defining complex membership with direct worm genetic and biochemical support. Supporting Evidence: PMID:23482940 elegans, Ξ±-adaptin is encoded by the apa-2 gene (Figure 1A). PMID:23482940 Conversely, in ΞΌ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. |
| GO:0030122 AP-2 adaptor complex | IEA GO_REF:0000120 | ACCEPT | Summary: InterPro/ARBA mapping of the AP-2 alpha subunit to the AP-2 adaptor complex. Reason: Correct. Supporting Evidence: PMID:23482940 elegans, Ξ±-adaptin is encoded by the apa-2 gene (Figure 1A). |
| GO:0030131 clathrin adaptor complex | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro mapping to clathrin adaptor complex (parent of AP-2 adaptor complex). Reason: Correct parent term. Supporting Evidence: PMID:23482940 elegans, Ξ±-adaptin is encoded by the apa-2 gene (Figure 1A). |
| GO:0030139 endocytic vesicle | IDA PMID:19043554 Profiling synaptic proteins identifies regulators of insulin... | ACCEPT | Summary: In the synaptic-protein imaging screen, APT-4::GFP is confined to synaptic puncta and is used as a marker of endocytic intermediates; its punctal abundance increases in synaptojanin and endophilin mutants that accumulate coated vesicles. Reason: AP-2 remains on clathrin-coated endocytic vesicles until uncoating, and the mutant profiles confirm the marker reports endocytic intermediates; the more specific clathrin-coated endocytic vesicle term is carried by another row. Supporting Evidence: PMID:19043554 We observed increased punctal fluorescence of APT-4 Ξ±2 adaptin and ITSN-1 intersectin in unc-26 synaptojanin and unc-57 endophilin A mutant synapses (Table S2), consistent with the accumulation of endocytic intermediates in these mutants PMID:19043554 APT-4 Ξ±2-adaptin associates with clathrin-coated vesicles, promoting recycling of SVs following fusion [45]. |
| GO:0032794 GTPase activating protein binding | IPI PMID:15895077 Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 a... | REMOVE | Summary: IPI 'GTPase activating protein binding' with RME-6. RME-6 is a Vps9-domain guanine-nucleotide exchange factor for RAB-5, not a GTPase-activating protein; the paper shows it binds GDP-RAB-5 and that rme-6 mutants have low RAB-5 activity. The alpha-adaptin/RME-6 co-immunoprecipitation is genuine but the term mischaracterises the partner. Reason: The GO term asserts binding to a GAP, whereas RME-6 is a RAB-5 GEF; GO has no guanyl-nucleotide-exchange-factor-binding term to move the annotation to, and the interaction is an alpha-appendage accessory-protein contact already described in the notes. The row is therefore removed as mislabelled rather than as false. RME-6 does carry an N-terminal RasGAP-related domain, which is probably why the term was chosen. The same paper does not report GAP activity for it, likens it to IQGAP proteins, which lack GAP activity toward Ras, and found no genetic or physical interaction with LET-60/Ras. A RasGAP-like domain does not make the partner a GTPase-activating protein, so the REMOVE stands. Supporting Evidence: PMID:15895077 This motif is also found in IQGAP proteins, which lack GAP activity toward Ras but are known to instead interact with Rac and Cdc42 PMID:15895077 Our preliminary analysis has failed to identify genetic or physical interactions between RME-6 and LET-60, the canonical C. elegans Ras PMID:15895077 Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to Caenorhabditis elegans RAB-5 in the GDP-bound conformation, and rme-6 mutants have phenotypes that indicate low RAB-5 activity. PMID:15895077 We found that Ξ±-adaptin did indeed coimmunoprecipitate with GFP::RME-6, showing a physical link of RME-6 to clathrin-coated pits. |
| GO:0035615 clathrin-cargo adaptor activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA clathrin-cargo adaptor activity from the alpha-adaptin node. Alpha contributes the PI(4,5)P2 docking site and, with sigma2, dileucine-motif recognition; in the worm, recovery of a CD4-dileucine reporter depends more on alpha than on mu2, and the alpha appendage recruits clathrin-associated accessory proteins. Reason: Core activity of the complex to which the alpha subunit contributes a defined part (membrane docking, dileucine cargo, accessory recruitment); consistent across the family and with worm cargo-specific phenotypes. Supporting Evidence: PMID:23482940 These data suggest that recovery of di-leucine cargo depends on Ξ±-adaptin more than ΞΌ2-adaptin. file:worm/apa-2/apa-2-deep-research-falcon.md An intestinal eGFPβCD4 reporter carrying an ExxxLL signal accumulated at the basolateral membrane in *apa-2(ox422)* animals, with fluorescence of 14,907 Β± 990 versus 1,998 Β± 275 in wild type (*p*<0.0001). PMID:23482940 A purified human Ξ±-Ο2 hemicomplex can bind di-leucine motifs, suggesting that hemicomplexes can be stable and exhibit appropriate biochemical interactions (Doray et al., 2007). |
| GO:0035615 clathrin-cargo adaptor activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro AP-2 alpha subunit mapping to clathrin-cargo adaptor activity. Reason: Correct family-level mapping, matching the IBA and the worm dileucine-cargo data. Supporting Evidence: PMID:23482940 These data suggest that recovery of di-leucine cargo depends on Ξ±-adaptin more than ΞΌ2-adaptin. |
| GO:0036062 presynaptic periactive zone | IDA PMID:22588719 Recruitment of sphingosine kinase to presynaptic terminals b... | ACCEPT | Summary: APT-4::GFP is used as a periactive-zone marker in DA motor neurons alongside synapsin and intersectin; its puncta are unchanged in sphk-1 mutants. Reason: Directly imaged localisation to the presynaptic periactive zone, the endocytic region where AP-2 retrieves synaptic vesicle membrane. Supporting Evidence: PMID:22588719 periactive zone proteins (SNN-1/synapsin, APT-4/adaptin, and ITSN-1/intersectin) in the DA class of motor neurons PMID:22588719 Periactive zones are regions of presynaptic membranes that contain proteins involved in endocytosis and actin regulation and may be specialized for SV recycling. |
| GO:0045202 synapse | IDA PMID:16049479 Systematic analysis of genes required for synapse structure ... | ACCEPT | Summary: Abstract-only record of the genome-scale synaptic screen in which 24 gene products were localised to presynaptic specialisations; apt-4/alpha-adaptin is among the genes required for acetylcholine secretion. Synaptic localisation of APT-4::GFP is confirmed by later full-text studies. Reason: Consistent with independent imaging of APT-4::GFP at synaptic puncta and of alpha-adaptin at synapses in mu2 mutants. Supporting Evidence: PMID:16049479 Twenty-four genes encoded proteins that were localized to presynaptic specializations. PMID:19043554 Under the conditions used for imaging, we determined that UNC-10::GFP, GFP::SYD-2 and APT-4::GFP were exclusively or predominantly localized to synaptic puncta PMID:23482940 In apm-2 mutants, tagged Ξ±-adaptin is still localized to the synapse (Figure 6D) and the plasma membrane of coelomocytes. |
| GO:0045334 clathrin-coated endocytic vesicle | IDA PMID:15895077 Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 a... | ACCEPT | Summary: mRFP1::alpha-adaptin colocalises with mRFP1::CHC-1 in punctate coated structures on or near the coelomocyte plasma membrane, used by the authors as clathrin-coated pit markers. Reason: AP-2 is a constituent of the clathrin coat from pit through budded vesicle until uncoating, so the coated endocytic vesicle term is appropriate for these structures. Supporting Evidence: PMID:15895077 In particular we focused on two markers for PM clathin-coated pits: clathrin heavy chain (CHC-1) and Ξ±-adaptin (APT-4) PMID:15895077 Interestingly, mRFP1::CHC-1 and mRFP1::Ξ±-adaptin as well as GFP::RME-6 were distributed unevenly around the PM, with regions of high and low labeling, reminiscent of the uneven distribution of endocytic pit structures that can be seen in coleomocytes by EM 26 . |
| GO:0048488 synaptic vesicle endocytosis | IDA PMID:23482940 AP2 hemicomplexes contribute independently to synaptic vesic... | ACCEPT | Summary: Electron microscopy and electrophysiology of apa-2 null mutants: synaptic vesicle number falls to 59-71% of wild type when alpha is absent from neurons, vesicles are slightly larger, evoked amplitude is reduced, and combined alpha/mu2 loss reduces vesicles by 70% with large vacuoles; neuronal rescue restores vesicle number. Reason: Direct demonstration that the alpha subunit of AP-2 participates in synaptic vesicle retrieval at the neuromuscular junction. Supporting Evidence: PMID:23482940 Mutations in either Ξ± or ΞΌ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both Ξ± and ΞΌ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. PMID:23482940 In mutants lacking apa-2 in the nervous system, synaptic vesicle numbers are reduced to 71% in acetylcholine neurons and 59% in GABA neurons (Figure 8B; Figure 8βfigure supplement 1). |
| GO:0048488 synaptic vesicle endocytosis | IMP PMID:23482940 AP2 hemicomplexes contribute independently to synaptic vesic... | ACCEPT | Summary: Same study, mutant-phenotype evidence: apa-2 null alleles reduce synaptic vesicle numbers and evoked release, the defect is rescued by neuronal apa-2 expression, and the apa-2 apm-2 double removes 70% of vesicles. Reason: Core process: AP-2 alpha is required for efficient synaptic vesicle endocytosis. Supporting Evidence: PMID:23482940 There is a 25% reduction in the amplitude of evoked release in skin-rescued apa-2 mutants, and this defect can be rescued by expressing apa-2 in neurons. PMID:23482940 In summary, loss of ΞΌ2 alone leads to a 31% decrease in synaptic vesicles in acetylcholine neurons (Gu et al., 2008), loss of Ξ±-adaptin alone leads to a 29% decrease, but a complete loss of AP2 leads to a 70% reduction in synaptic vesicle number. |
| GO:0072583 clathrin-dependent endocytosis | IBA GO_REF:0000033 | ACCEPT | Summary: IBA clathrin-dependent endocytosis from the alpha-adaptin node (mammalian Ap2a1/Ap2a2, fission yeast, and the worm gene itself). Alpha-adaptin mutants show defects in dileucine-cargo uptake, synaptic vesicle recycling and corpse engulfment. Reason: Core process with direct worm grounding; apa-2 among the node's sources reflects that grounding. Supporting Evidence: PMID:23482940 Cargo specific defects in Ξ±-adaptin vs ΞΌ2-adaptin mutants At a cellular level, Ξ±-adaptin mutants exhibit defects in endocytosis. |
| GO:0072583 clathrin-dependent endocytosis | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro AP-2 alpha subunit mapping to clathrin-dependent endocytosis. Reason: Correct. Supporting Evidence: PMID:23482940 Cargo specific defects in Ξ±-adaptin vs ΞΌ2-adaptin mutants At a cellular level, Ξ±-adaptin mutants exhibit defects in endocytosis. |
| GO:0072583 clathrin-dependent endocytosis | IMP PMID:25303366 The membrane-associated proteins FCHo and SGIP are allosteri... | ACCEPT | Summary: apa-2 mutants share the jowls/Dpy/Egl/Unc phenotype of fcho-1 and other AP-2 subunit mutants; the jowls screen recovered alpha-adaptin alleles, and fcho-1 bypass suppressors include alpha-solenoid residues that favour the open AP-2 conformation. Reason: Genetic evidence that alpha-adaptin is an essential part of the endocytic AP-2 complex whose activation FCHo controls. Supporting Evidence: PMID:25303366 We screened for mutants with the jowls phenotype and identified multiple mutations in three genes coding for alpha adaptin, mu2 adaptin and the nematode homolog of FCHo (Figure 1βfigure supplement 2). PMID:25303366 (3) residues in the alpha subunit that are found in the helical solenoid that lies across the top of the complex |
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Download this section (compressed HTML)Q: Which endogenous dileucine-motif cargoes depend on the APA-2/APS-2 hemicomplex in vivo, and does the alpha appendage's recruitment of RME-6 couple coated-pit formation to RAB-5 activation on nascent vesicles?
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Download this section (compressed HTML)apa-2 encodes an adaptin: specifically, it encodes an ortholog of the alpha subunit of adaptor protein complex 2 (AP2); loss of apa-2 activity via RNAi results in defects in receptor-mediated yolk endocytosis and thus, embryonic lethality.
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