NCU09721 encodes the beta subunit (beta1-adaptin, an Apl2 ortholog) of the heterotetrameric AP-1 adaptor complex of Neurospora crassa. The 749-residue protein consists of an N-terminal adaptin trunk (Adaptin_N/ARM-like repeats), which assembles with the gamma, mu1 and sigma1 subunits, followed by a flexible Pro/Gly-rich tail. Like other Dikarya beta1 subunits, it lacks the C-terminal beta-appendage domain. Phylogenetically it belongs to the fungal AP-1 beta clade, with the AP-1 beta subunits of Aspergillus nidulans, Fusarium graminearum and Schizosaccharomyces pombe; its paralog NCU05232 is the AP-2 beta subunit. In related filamentous fungi, the AP-1 complex is recruited to the cytoplasmic face of the trans-Golgi network and endosomes. There it selects membrane cargo for post-Golgi carriers that deliver polarized cargo to the hyphal apex and that recycle endosomal cargo. The A. nidulans complex is essential, and its traffic is clathrin-dependent. NCU09721 itself has not been characterized experimentally. Its cargoes are unknown, as is whether it binds clathrin directly through short tail motifs (it retains an LLDID motif).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | MODIFY | Summary: Correct but very general. AP-1 subunits are cytosolic proteins recruited to the cytoplasmic face of the trans-Golgi network and endosomes. Reason: The term is true, because AP-1 assembles on the cytoplasmic surface of the TGN. However, the gene's own IBA, together with the TGN localization of the orthologous Fusarium AP-1 beta, supports the more specific descendant trans-Golgi network (GO:0005802), which the gene already carries. This follows the rule for refining general location terms. The ARBA source alone is not evidence. Proposed replacements: trans-Golgi network Supporting Evidence: PMID:36836259 Our results showed that FgAP1Ξ²-GFP, FgAP1Ξ³-GFP, and FgAP1ΞΌ-GFP all colocalized with FgKex2-mCherry at the Golgi apparatus (Figure 6) file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md NCU09721 is the N. crassa AP-1 beta (Apl2 ortholog) and not the AP-2 beta, which is NCU05232. |
| GO:0005802 trans-Golgi network | IBA GO_REF:0000033 | ACCEPT | Summary: The trans-Golgi network is the principal site of AP-1 action. The orthologous Fusarium AP-1 beta (FgAP1beta) colocalizes with the late-Golgi/TGN marker Kex2. Reason: The IBA is placed at the deep beta-adaptin node PTN000123671. Here it is independently corroborated within the target's own clade. NCU09721 groups with the Fusarium and Aspergillus AP-1 beta subunits (100% bootstrap). The Fusarium AP-1 beta, gamma and mu subunits all colocalize with FgKex2 at the Golgi/TGN. The A. nidulans AP-1 complex sorts secretory vesicles from the trans-Golgi. Supporting Evidence: PMID:36836259 Our results showed that FgAP1Ξ²-GFP, FgAP1Ξ³-GFP, and FgAP1ΞΌ-GFP all colocalized with FgKex2-mCherry at the Golgi apparatus (Figure 6) PMID:29925567 The AP-1 complex is essential for membrane protein traffic via its role in the pinching-off and sorting of secretory vesicles (SVs) from the trans-Golgi and/or endosomes. file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md NCU09721 clusters with F. graminearum V6RKE4 (100/100) and A. nidulans AN3029 (100/100) |
| GO:0006886 intracellular protein transport | IEA GO_REF:0000002 | ACCEPT | Summary: AP-1 selects transmembrane cargo proteins for post-Golgi and endosome-recycling carriers. Selecting and transporting membrane proteins between compartments is intracellular protein transport. Reason: Within the protein-transport branch, this term is as precise as the evidence allows. Fungal AP-1 cargoes are membrane proteins (polar apical cargoes, the v-SNARE Snc1) moving between the TGN, endosomes and the plasma membrane. No specific protein-targeting descendant has been shown for the N. crassa protein. The process is inherited by orthology within the fungal AP-1 beta clade. Supporting Evidence: PMID:29925567 show that it is absolutely essential for growth due to its role in clathrin-dependent maintenance of polar traffic of specific membrane cargoes toward the apex of growing hyphae. PMID:36836259 the loss of FgAP1Ο blocks the transportation of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane |
| GO:0006897 endocytosis | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: The endocytosis IBA is inherited from the deep beta-adaptin node PTN000123671. Its experimental support comes from AP-2 beta, or from metazoan beta subunits shared between AP-1 and AP-2. NCU09721 is the fungal AP-1-specific beta paralog, and in filamentous fungi endocytosis is carried out by AP-2, whose beta subunit is NCU05232. Reason: The donors listed (human AP2B1 P63010, rat Ap2b1, Drosophila and C. elegans beta subunits shared by AP-1 and AP-2, and mouse AP-2 beta) support plasma-membrane uptake by AP-2. Dikarya have separate AP-1 beta and AP-2 beta genes. NCU09721 falls in the AP-1 beta clade with S. pombe apl2 (96% bootstrap), and NCU05232 falls with the AP-2 beta proteins. In A. nidulans, AP-2 has a specialized role in apical endocytosis. The previous KEEP_AS_NON_CORE rested on Fusarium sigma1 deletion delaying FM4-64 internalization into the vacuole. That reflects disturbed endosomal recycling and delivery to the vacuole, an indirect effect, not participation in plasma-membrane uptake. The term is not refuted outright, because AP-1 acts on the downstream endosomal itinerary, so it is marked as an over-annotation rather than removed. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN000123671 Β· PTN000123671 SUPPORTS SOURCE BUT NOT TARGET Deep beta-adaptin node whose endocytosis evidence comes from AP-2 beta and from metazoan beta subunits shared by AP-1 and AP-2. The fungal AP-1-specific beta clade containing NCU09721 does not carry the AP-2 plasma-membrane uptake role; in Dikarya that role belongs to the separate AP-2 beta paralog (NCU05232 in N. crassa). Supporting Evidence: PMID:28220754 The experimental support of a clathrin-independent role of AP-2 in endocytosis was in good agreement with the in silico observation that the Ξ²2 subunit of AP-2 of all higher fungi lacks the entire C-terminal Ξ² appendage file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md NCU05232 instead clusters with A. nidulans AN5950, F. graminearum V6RQ48, S. pombe apl1 and S. cerevisiae APL1 (the AP-2 beta clade). |
| GO:0012505 endomembrane system | IEA GO_REF:0000044 | MODIFY | Summary: Correct but general; the specific compartment supported for fungal AP-1 is the trans-Golgi network. Reason: The UniProt subcellular-location mapping rests on an ARBA rule and is not itself evidence. The term is true, and the gene already carries its descendant trans-Golgi network (GO:0005802), which is supported by orthologous Fusarium AP-1 localization. Proposed replacements: trans-Golgi network Supporting Evidence: PMID:36836259 Our results showed that FgAP1Ξ²-GFP, FgAP1Ξ³-GFP, and FgAP1ΞΌ-GFP all colocalized with FgKex2-mCherry at the Golgi apparatus (Figure 6) |
| GO:0015031 protein transport | IEA GO_REF:0000002 | MODIFY | Summary: Correct but general. The gene also carries the descendant intracellular protein transport. Reason: AP-1 transports membrane-protein cargo between intracellular compartments. The descendant intracellular protein transport (GO:0006886), already annotated to this gene, is the precise term the evidence supports. Proposed replacements: intracellular protein transport Supporting Evidence: PMID:36836259 the loss of FgAP1Ο blocks the transportation of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane |
| GO:0016192 vesicle-mediated transport | IEA GO_REF:0000002 | MODIFY | Summary: Correct but general. Fungal AP-1 acts specifically in post-Golgi vesicle traffic, sorting secretory vesicles at the trans-Golgi and recycling endosomal cargo. Reason: The IPR002553/IPR026739 domain mapping applies to every AP and COPI large subunit, so it cannot place the protein at a specific trafficking step. Once NCU09721 is established as an AP-1 beta (fungal AP-1 beta clade), the orthologous A. nidulans and F. graminearum AP-1 data support the descendant post-Golgi vesicle-mediated transport (GO:0006892): sorting of secretory vesicles from the trans-Golgi to the hyphal apex, and Golgi-to-plasma-membrane delivery of Snc1. Proposed replacements: post-Golgi vesicle-mediated transport Supporting Evidence: PMID:29925567 We provide evidence that AP-1 is involved in both anterograde sorting of RabERab11-labeled SVs and RabA/BRab5-dependent endosome recycling. PMID:36836259 the loss of FgAP1Ο blocks the transportation of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane |
| GO:0030117 membrane coat | IEA GO_REF:0000002 | MODIFY | Summary: Correct but general. The specific coat complex is AP-1, which is a descendant of membrane coat and is already annotated by IBA. Reason: The Adaptin_N domain mapping covers AP-1 to AP-4 and COPI. Phylogenetic placement and orthologous Fusarium subunit interactions support the descendant AP-1 adaptor complex (GO:0030121), which the gene already carries. Proposed replacements: AP-1 adaptor complex Supporting Evidence: PMID:36836259 FgAP1Ξ² interacts with FgAP1Ο, FgAP1Ξ³, and FgAP1ΞΌ |
| GO:0030121 AP-1 adaptor complex | IBA GO_REF:0000033 | ACCEPT | Summary: Core. The IBD at PTN000123672 is seeded by the AP-1 beta subunits S. pombe apl2 (SPBC947.02), S. cerevisiae APL2 (S000001618) and Dictyostelium ap1b1. NCU09721 is their ortholog by phylogeny. Reason: In an independent MAFFT/NJ tree, NCU09721 groups with S. pombe apl2 (96/100 bootstrap) and with the Aspergillus and Fusarium AP-1 beta subunits (100/100). The orthologous Fusarium AP1 beta binds the sigma1, gamma and mu1 subunits. Family note, not a gene issue. In GOA the same PTN000123672 IBA also reaches NCU05232, which is the AP-2 beta paralog by the same tree. That suggests the node sits above the fungal AP-1/AP-2 beta split. That concern applies to NCU05232, not to NCU09721. Supporting Evidence: file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md NCU09721 clusters with F. graminearum V6RKE4 (100/100) and A. nidulans AN3029 (100/100), then with S. pombe apl2, the AP-1 beta subunit (96/100). PMID:36836259 FgAP1Ξ² interacts with FgAP1Ο, FgAP1Ξ³, and FgAP1ΞΌ |
| GO:0030276 clathrin binding | IEA GO_REF:0000002 | UNDECIDED | Summary: Unresolved. Dikarya beta1 subunits lack the canonical clathrin-binding appendage. In A. nidulans, AP-1 beta associates with clathrin through short C-terminal motifs (LLDID, LLNGF). NCU09721 retains LLDID (629-633) but lacks LLNGF. Reason: The IPR016342 mapping assumes a metazoan-type beta subunit with a clathrin box and appendage. Dikarya beta1 subunits lack these. Aspergillus AP-1 traffic is clathrin-dependent, and its full text (not in the local cache) reports motif-dependent clathrin association. NCU09721 retains only one of the two motifs (LLDID), and only short-motif similarity is available, with no binding assay for the N. crassa protein. Whether this subunit binds clathrin directly therefore remains open. Supporting Evidence: PMID:28220754 clathrin binding domains are also missing from the AP-1 Ξ² subunit (Ξ²1) of all Dikarya file:NEUCR/NCU09721/NCU09721-bioinformatics/RESULTS-rereview.md NCU09721 retains LLDID (629-633) and has a terminal LLGLF (745-749), but has no LLNGF. |
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Download this section (compressed HTML)Q: Does PTN000123672 (the AP-1 adaptor complex IBD) sit above the fungal AP-1 beta / AP-2 beta split? Both NCU09721 (AP-1 beta) and its paralog NCU05232 (AP-2 beta clade) receive the AP-1 complex IBA.
Q: Does the retained LLDID tail motif of NCU09721 bind the clathrin heavy-chain terminal domain, or does N. crassa AP-1 recruit clathrin only through other subunits or accessory proteins?
Experiment: Tag NCU09721 at its native locus with GFP and image it with N. crassa TGN (Kex2/Sec7) and endosome (Rab5) markers. Test whether an LLDID-to-AAAAA mutant changes clathrin light-chain recruitment or apical delivery of secretory-vesicle cargo.
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