apl2 encodes the beta1 (large) subunit of the fission yeast AP-1 clathrin adaptor complex, a heterotetramer with the gamma subunit apl4, the mu1 subunit apm1 and the sigma1 subunit aps1. The 683-residue protein consists of the Adaptin_N trunk domain followed by a short hinge carrying a clathrin-box motif (LLDLD); unlike metazoan beta adaptins it has no C-terminal appendage domain. In pull-down assays Apl2 binds Apm1 even without the other two subunits, and all four subunits are needed for the complex to assemble and associate with membranes. GFP-Apl2 localizes to punctate Golgi/endosomal compartments, and this localization depends on the p200/Laa1-family accessory protein Sip1. Deletion of apl2 causes temperature and immunosuppressant sensitivity, more severe than loss of the gamma or sigma subunits, together with endosomal accumulation of the v-SNARE Syb1. This indicates that AP-1 is required for cargo exit from endosomes toward the Golgi and for recycling. Apl2 also binds the Rho GTPase Rho3 in vitro, and Rho3 overexpression suppresses the deletion phenotypes. Apl2 is therefore a structural core subunit of the clathrin-associated AP-1 coat that sorts membrane cargo at the trans-Golgi network and endosomes. Its direct binding to clathrin has not been tested in S. pombe and is inferred from the conserved clathrin box.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning assignment of cytoplasm. Apl2 is a peripheral, cytoplasmic-face coat subunit, so the location is true but uninformative. Reason: Correct but general. Experimental data place Apl2 on Golgi/endosomal compartments, both of which are part of the cytoplasm, and the gene already carries endosome (IDA) and trans-Golgi network (IBA). The specific terms are proposed as the replacements. Proposed replacements: trans-Golgi network endosome Supporting Evidence: PMID:23028933 the dot-like fluorescence of Apm1-GFP, GFP-Apl2, Aps1-GFP and GFP-Apl4 co-localized with FM4-64-positive structures in wild-type cells, suggesting that the four adaptin subunits of the AP-1 complex are localized to the Golgi/endosomes |
| GO:0005768 endosome | IDA PMID:19624755 Deletion mutants of AP-1 adaptin subunits display distinct p... | ACCEPT | Summary: PomBase IDA from the AP-1 subunit deletion study. Subunits localize to endosomes, and deleting any subunit dissociates the complex from endosomes. An independent study shows GFP-Apl2 on FM4-64-positive Golgi/endosomes. Reason: Endosomal association of AP-1 is shown directly in S. pombe and is where the complex acts in endosomal exit of Syb1. Core location. Supporting Evidence: PMID:19624755 the deletion of any subunit generally caused the disassociation of the heterotetrameric complex from endosomes PMID:23028933 suggesting that the four adaptin subunits of the AP-1 complex are localized to the Golgi/endosomes |
| GO:0005794 Golgi apparatus | IDA PMID:19624755 Deletion mutants of AP-1 adaptin subunits display distinct p... | MODIFY | Summary: PomBase IDA for Golgi localization. The fluorescence data show Apl2 on FM4-64-positive Golgi/endosome structures. Reason: Correct but more general than the biology warrants. AP-1 acts on the trans-Golgi network, not on the Golgi as a whole. The gene already carries trans-Golgi network (IBA from PTN000123671), and the Golgi signal reported for AP-1 in S. pombe is post-Golgi/TGN-endosomal. For the related accessory protein Sip1, partial colocalization was with the trans-Golgi marker Sec72 and not with the cis-Golgi marker Anp1. The cited abstract does not resolve the cisterna, so this refinement relies on the IBA and the conserved AP-1 biology rather than on a direct S. pombe TGN marker for Apl2. Proposed replacements: trans-Golgi network Supporting Evidence: PMID:23028933 suggesting that the four adaptin subunits of the AP-1 complex are localized to the Golgi/endosomes |
| GO:0005802 trans-Golgi network | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN000123671, a deep beta-adaptin node. TGN association is an ancestral property of the AP-1/AP-4 beta lineages. The S. pombe localization data (Golgi/endosome puncta) agree with it. Reason: S. pombe apl2 falls inside the fungal AP-1 beta clade, and AP-1 is the canonical TGN/endosome clathrin adaptor. The IBA agrees with the Golgi/endosome IDA data, so it is accepted as the specific Golgi-side location. Supporting Evidence: file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md S. pombe apl2, the AP-1 beta subunit (96/100) PMID:23028933 suggesting that the four adaptin subunits of the AP-1 complex are localized to the Golgi/endosomes |
| GO:0005829 cytosol | IEA GO_REF:0000108 | KEEP AS NON CORE | Summary: Inter-ontology inference from retrograde endosome-to-Golgi transport. Adaptor complexes cycle between a soluble cytosolic pool and membranes. In sip1-i4 cells, Apl2 is diffusely cytoplasmic. Reason: The soluble cytosolic pool of AP-1 is real but is not where the complex does its work. Kept as a non-core location. Supporting Evidence: PMID:23028933 Instead, they localized diffusely in the cytoplasm and were enriched in the nucleus. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | REMOVE | Summary: UniProt SubCell mapping of "Cell membrane" for this entry. The underlying UniProt statement is by-similarity (ECO:0000250) family boilerplate describing coat components of coated pits at the plasma membrane, which is the AP-2 location. Reason: Every S. pombe localization study puts Apl2 and the other AP-1 subunits on Golgi/endosomal puncta, and none reports it at the plasma membrane. The plasma-membrane coated-pit location belongs to the AP-2 complex, whose beta subunit in fission yeast is the separate paralog apl1 (a distinct clade in the beta adaptin tree). The by-similarity text was transferred across the AP-1/AP-2 paralog boundary, so the SubCell-derived IEA is wrong for this AP-1 subunit. Supporting Evidence: file:SCHPO/apl2/apl2-uniprot.txt SUBCELLULAR LOCATION: Cell membrane {ECO:0000250}. file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md S. pombe apl1 and S. cerevisiae PMID:23028933 suggesting that the four adaptin subunits of the AP-1 complex are localized to the Golgi/endosomes |
| GO:0006886 intracellular protein transport | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO from the Adaptin_N trunk domain. True, since AP-1 sorts membrane protein cargo, but general. Reason: Correct but general. The specific S. pombe process supported by experiment is endosomal exit and recycling of the v-SNARE Syb1, which PomBase captures as endosome to plasma membrane protein transport (GO:0099638, is_a intracellular protein transport). The gene already carries this term. Proposed replacements: endosome to plasma membrane protein transport Supporting Evidence: PMID:19624755 the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1 |
| GO:0006897 endocytosis | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from the deep beta-adaptin node PTN000123671. Its endocytosis evidence comes only from metazoan beta proteins (human AP2B1 P63010 and rodent, fly, worm and fish beta1/2 orthologs), where AP-2 beta, and in metazoa a partly redundant beta1, act at the plasma membrane. Reason: The node placement is sound for the metazoan beta1/beta2 lineage, but fungi have a separate AP-2 beta. In the beta-adaptin tree, S. pombe apl1 groups with the AP-2 beta clade, while apl2 sits in the fungal AP-1 beta clade. Plasma-membrane endocytosis is the AP-2 role. Apl2 also lacks the appendage domain that metazoan beta1/2 use to recruit endocytic accessory proteins. All S. pombe data on Apl2 concern Golgi/endosome traffic, and none shows a role in endocytosis. The PAINT curators placed the fungal AP-1 complex assertion at a separate node, PTN000123672, seeded by apl2 itself. No S. pombe experiment excludes a contribution to endocytosis, so the term is marked as over-annotated rather than removed. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN000123671 Β· PTN000123671 SUPPORTS SOURCE BUT NOT TARGET Deep beta-adaptin node whose endocytosis evidence comes only from metazoan beta1/beta2 members, led by AP-2 beta. Fungi split off a separate AP-2 beta (apl1); apl2 is in the AP-1 beta clade, which has no endocytosis evidence. Supporting Evidence: file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md S. pombe apl1 and S. cerevisiae file:SCHPO/apl2/apl2-bioinformatics/RESULTS.md the metazoan beta1/beta2 appendage that recruits endocytic accessory proteins is absent. PMID:19624755 for the AP-1 function in exit transport from endosomes |
| GO:0015031 protein transport | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO from the AP complex beta 1/2/4 signature. True but very general. Reason: Correct but general. The specific supported process, which the gene already carries, is endosome to plasma membrane protein transport (GO:0099638, is_a protein transport), based on the Syb1 endosomal accumulation phenotype. Proposed replacements: endosome to plasma membrane protein transport Supporting Evidence: PMID:19624755 the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1 |
| GO:0016020 membrane | IEA GO_REF:0000044 | MODIFY | Summary: UniProt SubCell mapping to membrane. Apl2 is a peripheral membrane protein on the cytoplasmic face of coated membranes, so the term is true but uninformative. Reason: Correct but general. The specific membrane-associated structure is the AP-1 adaptor complex (GO:0030121, part_of membrane via coated vesicle membrane), which the gene carries by IDA and IBA. Proposed replacements: AP-1 adaptor complex Supporting Evidence: PMID:19624755 the four subunits are all essential for the heterotetrameric complex formation |
| GO:0016192 vesicle-mediated transport | IEA GO_REF:0000120 | MODIFY | Summary: Combined IEA for vesicle-mediated transport. True, since AP-1 is a vesicle coat adaptor, but general. Reason: Correct but general. The specific vesicle-mediated processes supported in S. pombe are retrograde transport, endosome to Golgi (GO:0042147) and endosome to plasma membrane protein transport (GO:0099638). Both are is_a descendants of vesicle-mediated transport, and both are already carried by IDA. Proposed replacements: retrograde transport, endosome to Golgi endosome to plasma membrane protein transport Supporting Evidence: PMID:19624755 for the AP-1 function in exit transport from endosomes |
| GO:0030117 membrane coat | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO from the clathrin/coatomer adaptor N-terminal domain, which is shared by AP complexes and COPI. True but general. Reason: Correct but general. The specific coat complex is the AP-1 adaptor complex (GO:0030121, is_a membrane coat via AP-type membrane coat adaptor complex), which is established for Apl2 by pull-down and deletion data. Proposed replacements: AP-1 adaptor complex Supporting Evidence: PMID:19624755 Apm1 binds Apl2 even in the absence of Aps1 and Apl4 |
| GO:0030121 AP-1 adaptor complex | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from the AP-1 beta node PTN000123672, whose seeds include apl2 itself (PomBase SPBC947.02), S. cerevisiae APL2 and Dictyostelium ap1b1. Reason: Apl2 is inside the AP-1 beta clade. Its own experimental annotation is one of the seeds of the IBD, as expected, and is not circular. The IBA confirms that AP-1 membership is inherited across eukaryotes. Supporting Evidence: PMID:19624755 the four subunits are all essential for the heterotetrameric complex formation file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md S. pombe apl2, the AP-1 beta subunit (96/100) |
| GO:0030121 AP-1 adaptor complex | IDA PMID:19624755 Deletion mutants of AP-1 adaptin subunits display distinct p... | ACCEPT | Summary: Pull-down shows Apm1 binds Apl2 even without the other subunits, and deletion of any subunit disrupts the heterotetramer. Reason: Direct S. pombe evidence for Apl2 as the beta subunit of AP-1. Core. Supporting Evidence: PMID:19624755 In pull-down assay, Apm1 binds Apl2 even in the absence of Aps1 and Apl4 PMID:19624755 the four subunits are all essential for the heterotetrameric complex formation |
| GO:0030121 AP-1 adaptor complex | NAS PMID:19624755 Deletion mutants of AP-1 adaptin subunits display distinct p... | ACCEPT | Summary: Author statement (ComplexPortal-style) that Apl2 is the beta subunit of AP-1. Reason: Consistent with the IDA and IBA rows for the same term. Supporting Evidence: PMID:19624755 the deletion strains of each individual subunit of the AP-1 complex [Apl2 (beta), Apl4 (gamma), Apm1 (mu) and Aps1 (sigma)] |
| GO:0030276 clathrin binding | IC PMID:19624755 Deletion mutants of AP-1 adaptin subunits display distinct p... | ACCEPT | Summary: PomBase curator inference from AP-1 complex membership. In metazoa, the beta subunit binds clathrin through a hinge clathrin box and the appendage. Apl2 has no appendage but keeps a hinge clathrin box, LLDLD at 623-627. Reason: Clathrin binding by the beta subunit is a conserved property of AP-1/AP-2 beta adaptins, and the clathrin-box motif is conserved in Apl2, so the curator inference is reasonable. No S. pombe experiment has tested it, so this is an inferred, not experimentally established, function. Supporting Evidence: file:SCHPO/apl2/apl2-bioinformatics/RESULTS.md One clathrin-box consensus match L[LI][DEN][LF][DE]: LLDLD at 623-627 file:SCHPO/apl2/apl2-uniprot.txt The AP-1 complex interacts directly with clathrin (By similarity). |
| GO:0030276 clathrin binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO from the AP complex beta 1/2/4 signature (IPR016342). Reason: Consistent with the IC row and with the conserved clathrin box in the Apl2 hinge. This is a sequence-based inference. Supporting Evidence: file:SCHPO/apl2/apl2-bioinformatics/RESULTS.md One clathrin-box consensus match L[LI][DEN][LF][DE]: LLDLD at 623-627 |
| GO:0035615 clathrin-cargo adaptor activity | IC GO_REF:0000111 | ACCEPT | Summary: Curator inference that, as an AP-1 subunit, Apl2 contributes to the clathrin-cargo adaptor activity of the complex. Reason: The contributes_to qualifier is appropriate for a heterotetramer subunit. This is the core molecular function of AP-1, linking membrane cargo to clathrin. Cargo recognition itself is mainly by mu1 and the gamma/sigma1 hemicomplex, and Apl2 supplies the trunk scaffold and the clathrin-binding hinge. In S. pombe the activity is inferred from complex membership and the Syb1 sorting phenotype; it has not been reconstituted. Supporting Evidence: PMID:19624755 the four subunits are all essential for the heterotetrameric complex formation and for the AP-1 function in exit transport from endosomes file:SCHPO/apl2/apl2-uniprot.txt Adaptins are components of the adaptor complexes which link |
| GO:0042147 retrograde transport, endosome to Golgi | IDA PMID:19624755 Deletion mutants of AP-1 adaptin subunits display distinct p... | ACCEPT | Summary: PomBase IDA. Deletion of AP-1 subunits, including Apl2, causes endosomal accumulation of the v-SNARE Syb1, indicating that AP-1 is required for exit transport from endosomes. Reason: The cached abstract reports an endosomal-exit defect, and the curator, who read the full text, assigned endosome-to-Golgi retrograde transport. This is consistent with the conserved AP-1 role in endosome-TGN cargo cycling. As a structural coat subunit, Apl2 participates directly in forming the transport carrier, so it passes the participation test. Core process. Supporting Evidence: PMID:19624755 the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1 PMID:19624755 for the AP-1 function in exit transport from endosomes |
| GO:0099638 obsolete endosome to plasma membrane protein transport | IDA PMID:19624755 Deletion mutants of AP-1 adaptin subunits display distinct p... | ACCEPT | Summary: PomBase IDA. This rests on the same Syb1 endosomal-accumulation observation. Syb1 is an exocytic v-SNARE that recycles from endosomes back to the cell surface. The label in the seed carries an "obsolete" prefix from GOA, but OLS currently lists GO:0099638 as an active term ("The directed movement of proteins from the endosome to the plasma membrane in transport vesicles"). Reason: The curator, who read the full text, captured the recycling of Syb1 out of endosomes. The id is retained as supplied by GOA. The Syb1 phenotype does not on its own distinguish direct endosome-to-PM recycling from recycling via the TGN, so this process is accepted as a reasonable but less certain complement to GO:0042147 (see suggested questions). Supporting Evidence: PMID:19624755 the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1 |
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Download this section (compressed HTML)Q: Does Apl2 bind clathrin directly in S. pombe through its LLDLD hinge motif, given that it lacks the appendage domain that provides the second clathrin contact in metazoan beta adaptins?
Q: Does AP-1 return Syb1 from endosomes to the TGN, or recycle it directly to the plasma membrane? In other words, which of GO:0042147 and GO:0099638 describes the Apl2-dependent step?
Q: Does fission yeast AP-1 contribute at all to plasma-membrane endocytosis, or is that role restricted to AP-2 (apl1-containing) complexes as the separate fungal AP-2 beta clade suggests?
Experiment: In vitro binding of recombinant Apl2 hinge (wild type and an LLDLD-to-AAAAA mutant) to the S. pombe clathrin heavy-chain terminal domain, plus complementation of Ξapl2 with the mutant to test the role of the clathrin box in Syb1 sorting.
Experiment: Time-resolved imaging of Syb1 and an endosome-to-TGN cargo in Ξapl2 cells co-labelled with TGN (Sec72) and early endosome markers, to place the trafficking block.
Experiment: FM4-64 and Lucifer Yellow uptake assays in Ξapl2 versus Ξapl1 cells, to test whether AP-1 beta contributes to endocytosis.
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