Ofut1 (O-fut1, neurotic) is the Drosophila GDP-fucose protein O-fucosyltransferase 1 (glycosyltransferase family 65), a soluble endoplasmic reticulum lumenal enzyme that transfers fucose from GDP-fucose onto serine/threonine residues within the C2-X(4,5)-[S/T]-C3 consensus of EGF-like repeats, most importantly on the Notch receptor extracellular domain. The O-fucose it adds is the substrate that Fringe elongates with GlcNAc, so O-fut1 is required both for Fringe-dependent modulation of Delta/Serrate binding and for Notch-ligand binding generally. Independently of its catalytic activity, O-fut1 binds the Notch extracellular domain and acts as a Notch-specific chaperone that promotes Notch folding, exit from the ER, delivery to the cell surface and constitutive endocytic trafficking to early endosomes. Loss of O-fut1 gives a maternal neurogenic phenotype and cell-autonomous loss of Notch signalling in the signal-receiving cell, while overexpression inhibits signalling. A catalytically dead knock-in (R245A) retains chaperone activity and supports most, but not all (temperature-sensitive and Fringe-dependent), Notch signalling events.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005112 Notch binding | IDA PMID:15692013 Chaperone activity of protein O-fucosyltransferase 1 promote... | ACCEPT | Summary: OFUT1 binds Notch in the ER; this binding underlies its chaperone function in Notch folding and ER exit. Reason: Direct binding of OFUT1 to Notch is shown in the cited paper and is the molecular basis of its catalytic-activity-independent Notch chaperone function. Notch binding is informative here because OFUT1 is a Notch-selective folding/trafficking factor, not a generic glycosylation enzyme-substrate encounter. Supporting Evidence: PMID:15692013 OFUT1 could bind to Notch, was required for the trafficking of wild-type Notch out of the endoplasmic reticulum, and could partially rescue defects in secretion and ligand binding associated with Notch point mutations. |
| GO:0005112 Notch binding | IDA PMID:17329366 The O-fucosyltransferase O-fut1 is an extracellular componen... | ACCEPT | Summary: O-fut1 forms a stable complex with the Notch extracellular domain, and this extracellular interaction is required for constitutive endocytic transport of Notch. Reason: Independent confirmation of direct O-fut1-Notch extracellular domain association, here linked to its role in Notch endocytic trafficking. Supporting Evidence: PMID:17329366 O-fut1 formed a stable complex with the extracellular domain of Notch. |
| GO:0005783 endoplasmic reticulum | IDA PMID:15692013 Chaperone activity of protein O-fucosyltransferase 1 promote... | ACCEPT | Summary: OFUT1 is an ER protein and ER localization is essential for its function in vivo. Reason: Directly observed ER localization, with functional requirement for ER retention, matches both the fucosyltransferase and chaperone roles. Supporting Evidence: PMID:15692013 OFUT1 is an endoplasmic reticulum protein, and its localization was essential for function in vivo. |
| GO:0005783 endoplasmic reticulum | ISS GO_REF:0000024 | ACCEPT | Summary: ER localization transferred from a POFUT1 ortholog; consistent with direct fly data. Reason: Consistent with the direct IDA evidence in Drosophila; redundant but correct. Supporting Evidence: PMID:15692013 OFUT1 is an endoplasmic reticulum protein, and its localization was essential for function in vivo. |
| GO:0005788 endoplasmic reticulum lumen | IDA PMID:15692013 Chaperone activity of protein O-fucosyltransferase 1 promote... | ACCEPT | Summary: OFUT1 is a soluble lumenal ER protein. Reason: Drosophila OFUT1 is a soluble ER protein (it carries a C-terminal ER-retention signal rather than the type II transmembrane anchor originally predicted for the mammalian enzyme), so ER lumen is the most precise location for the catalytic and chaperone activities. Supporting Evidence: PMID:18194540 OFUT1 is a soluble ER protein [10,11] and, at least in Drosophila, acts as a Notch chaperone, facilitating the folding and secretion of Notch[10]. file:DROME/O-fut1/O-fut1-deep-research-falcon.md Ofut1 is a **soluble endoplasmic reticulum (ER)-resident protein** and acts primarily in the ER lumen |
| GO:0005788 endoplasmic reticulum lumen | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping to ER lumen; consistent with experimental data. Reason: Correct and consistent with direct evidence. Supporting Evidence: PMID:18194540 OFUT1 is a soluble ER protein [10,11] and, at least in Drosophila, acts as a Notch chaperone, facilitating the folding and secretion of Notch[10]. |
| GO:0007219 Notch signaling pathway | IEA GO_REF:0000002 | ACCEPT | Summary: O-fut1 is a core, cell-autonomous component required in the signal-receiving cell for Notch activation by its ligands. Reason: Although electronic, the assignment is well supported: O-fut1 modifies and chaperones the receptor itself and is required upstream of Notch activation for all tested Notch signalling events. Supporting Evidence: PMID:12526814 Together, these results indicate that OFUT1 is a core component of the Notch pathway, which is required for the activation of Notch by its ligands |
| GO:0036066 protein O-linked glycosylation via fucose | IEA GO_REF:0000002 | ACCEPT | Summary: O-fut1 catalyses O-fucosylation of EGF repeats; InterPro-based assignment is correct. Reason: Correct and consistent with direct enzymatic evidence. Supporting Evidence: PMID:17329366 A Drosophila O-FucT-1 homolog, encoded by O-fut1, catalyzes the O-fucosylation of Notch, a modification essential for Notch signaling and ligand binding. |
| GO:0036066 protein O-linked glycosylation via fucose | IMP PMID:12526814 Regulation of notch signaling by o-linked fucose. | ACCEPT | Summary: RNAi knockdown of Ofut1 reduces O-linked fucose on Notch and blocks Notch signalling. Reason: O-fut1 is the enzyme that performs the O-fucosylation step, so this process annotation reflects direct participation. Supporting Evidence: PMID:12526814 Notch and its ligands are modified by a protein O-fucosyltransferase (OFUT1) that attaches fucose to a Serine or Threonine within EGF domains. |
| GO:0036066 protein O-linked glycosylation via fucose | ISS PMID:11524432 Modification of epidermal growth factor-like repeats with O-... | ACCEPT | Summary: Similarity-based transfer from human POFUT1, whose cloning identified the fly homolog. Reason: Consistent with direct fly evidence; redundant but correct. Supporting Evidence: PMID:11524432 Homologs were also found in Drosophila and Caenorhabditis elegans with 41.2 and 29.4% identity to human O-FucT-1, respectively. |
| GO:0045165 cell fate commitment | IMP PMID:21965616 Distinct levels of Notch activity for commitment and termina... | KEEP AS NON CORE | Summary: In the adult midgut, reducing Notch pathway glycosylation (Gmd, and by the curator's reading O-fut1) impairs commitment of intestinal stem cell daughters, which needs high Notch activity. Reason: The cached abstract is about Gmd and Notch signalling levels in stem cell commitment; the O-fut1 perturbation is presumably in the full text, which the curator read. The phenotype is a downstream consequence of reduced Notch signalling rather than a separate role of O-fut1, so it is kept as a non-core, context-specific process. Supporting Evidence: PMID:21965616 Here, we show that distinct requirements for Notch activity exist: commitment requires high Notch activity, whereas terminal differentiation can occur with lower Notch activity. |
| GO:0045747 positive regulation of Notch signaling pathway | IMP PMID:12909620 Modulation of notch-ligand binding by protein O-fucosyltrans... | ACCEPT | Summary: OFUT1 knockdown in Notch-expressing cells inhibits Delta-Notch and Serrate-Notch binding. Reason: O-fut1 is positively required for ligand binding by Notch and hence for signalling. Note that overexpression inhibits Delta-Notch binding, so O-fut1 levels modulate rather than simply enable signalling. Supporting Evidence: PMID:12909620 Down-regulation of OFUT1 by RNA interference in Notch-secreting cells inhibits both Delta-Notch and Serrate-Notch binding, demonstrating a requirement for O-linked fucose for efficient binding of Notch to its ligands. |
| GO:0045747 positive regulation of Notch signaling pathway | IMP PMID:12917292 neurotic, a novel maternal neurogenic gene, encodes an O-fuc... | ACCEPT | Summary: neurotic (O-fut1) is a maternal neurogenic gene essential for Notch signalling and Notch-Delta binding. Reason: Loss of function produces the neurogenic phenotype and epistasis places O-fut1 upstream of Notch activation, acting on full-length Notch. Supporting Evidence: PMID:12917292 neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. |
| GO:0045747 positive regulation of Notch signaling pathway | IMP PMID:15692013 Chaperone activity of protein O-fucosyltransferase 1 promote... | ACCEPT | Summary: OFUT1 chaperone activity is required for Notch trafficking out of the ER and ligand binding. Reason: Supports a positive, receptor-level requirement for O-fut1 in Notch signalling. Supporting Evidence: PMID:15692013 This ability of OFUT1 to facilitate folding of Notch did not require its fucosyltransferase activity. |
| GO:0045747 positive regulation of Notch signaling pathway | IMP PMID:21965616 Distinct levels of Notch activity for commitment and termina... | ACCEPT | Summary: Reduced Notch glycosylation lowers Notch reporter activity in intestinal stem cell lineages. Reason: Consistent with the well-established positive requirement for O-fut1 in Notch signalling; the O-fut1-specific experiment is not described in the cached abstract but is expected in the full text read by the curator. Supporting Evidence: PMID:21965616 Gmd loss resulted in aberrant, self-renewing stem cell divisions that generated extra ISC-like cells defective in Notch reporter activation |
| GO:0045747 positive regulation of Notch signaling pathway | IMP PMID:25378397 O-fucose monosaccharide of Drosophila Notch has a temperatur... | ACCEPT | Summary: Catalytically dead O-fut1 knock-in shows the O-fucose monosaccharide is required, temperature-sensitively and redundantly with Rumi O-glucose, for Notch signalling. Reason: Direct genetic evidence that O-fut1 enzymatic activity positively contributes to Notch signalling. Supporting Evidence: PMID:25378397 we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. |
| GO:0045807 positive regulation of endocytosis | IMP PMID:17329366 The O-fucosyltransferase O-fut1 is an extracellular componen... | KEEP AS NON CORE | Summary: O-fut1 is required for constitutive endocytic transport of Notch from the plasma membrane to early endosomes. Reason: The evidence concerns endocytic trafficking of a single cargo (Notch) that depends on O-fut1 binding to the Notch extracellular domain, not a general stimulation of endocytosis. The annotation is retained but as a non-core, cargo-specific trafficking role; it is better understood as a consequence of O-fut1 acting as a Notch chaperone. Supporting Evidence: PMID:17329366 O-fut1 was indispensable for the constitutive transportation of Notch from the plasma membrane to the early endosome, which we show was independent of the O-fucosyltransferase activity of O-fut1. |
| GO:0046922 peptide-O-fucosyltransferase activity | IDA PMID:12526814 Regulation of notch signaling by o-linked fucose. | ACCEPT | Summary: O-fut1 attaches fucose to Ser/Thr within EGF domains of Notch and its ligands. Reason: The defining catalytic activity of O-fut1 (EC 2.4.1.221). Supporting Evidence: PMID:12526814 Notch and its ligands are modified by a protein O-fucosyltransferase (OFUT1) that attaches fucose to a Serine or Threonine within EGF domains. |
| GO:0046922 peptide-O-fucosyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated assignment consistent with experimental data. Reason: Correct; duplicate of the IDA-supported activity. Supporting Evidence: PMID:17329366 A Drosophila O-FucT-1 homolog, encoded by O-fut1, catalyzes the O-fucosylation of Notch, a modification essential for Notch signaling and ligand binding. |
| GO:0046922 peptide-O-fucosyltransferase activity | ISS PMID:11524432 Modification of epidermal growth factor-like repeats with O-... | ACCEPT | Summary: Transfer from human POFUT1 (O-FucT-1), whose enzymatic activity was characterized. Reason: Consistent with direct fly evidence. Supporting Evidence: PMID:11524432 Expression of a soluble form of human O-FucT-1 in insect cells yielded a protein of the predicted molecular weight with O-FucT-1 kinetic and enzymatic properties similar to those of O-FucT-1 purified from CHO cells. |
| GO:2000010 positive regulation of protein localization to cell surface | IMP PMID:15692013 Chaperone activity of protein O-fucosyltransferase 1 promote... | KEEP AS NON CORE | Summary: OFUT1 is required for Notch to leave the ER and reach the cell surface. Reason: Accurate but cargo-specific (Notch); it describes a consequence of the Notch chaperone activity. Retained as non-core. Supporting Evidence: PMID:15692013 OFUT1 could bind to Notch, was required for the trafficking of wild-type Notch out of the endoplasmic reticulum PMID:25378397 We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. |
| GO:0044183 protein folding chaperone | IDA PMID:15692013 Chaperone activity of protein O-fucosyltransferase 1 promote... | NEW | Summary: OFUT1 has a Notch chaperone activity, separable from its fucosyltransferase activity, that facilitates folding and secretion of Notch. Reason: The chaperone activity is a distinct molecular function of O-fut1 (the catalytically dead R245A isoform retains it and rescues embryonic neurogenesis), and it is not captured by Notch binding or by the fucosyltransferase term. The activity is performed by O-fut1 itself on Notch. Evidence is cell-based (folding/secretion rescue of Notch point mutants) rather than an in vitro refolding assay, and it is Notch-selective; mammalian POFUT1 chaperone activity is less clear. Supporting Evidence: PMID:15692013 We found that in Drosophila, Protein O-fucosyltransferase 1 (OFUT1), an enzyme that glycosylates epidermal growth factor-like domains of Notch, also has a distinct Notch chaperone activity. PMID:18194540 Our results establish that Notch does not need to be O-fucosylated for fringe-independent Notch signaling in Drosophila; the chaperone activity of OFUT1 is sufficient for the generation of functional Notch. file:DROME/O-fut1/O-fut1-deep-research-falcon.md Independent of its enzymatic activity, Ofut1 functions as an ER-resident molecular chaperone that promotes proper Notch folding, maturation, and quality-control passage through the secretory pathway |
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Download this section (compressed HTML)Q: Is the Notch chaperone activity of O-fut1 conserved in vertebrate POFUT1, or is it a Drosophila-specific feature that makes the fly enzyme catalytically dispensable for Fringe-independent signalling?
Experiment: Compare Notch folding/ER exit and signalling in flies carrying the catalytically dead O-fut1 R245A knock-in versus a chaperone-deficient but catalytically active allele (if separable), across temperatures and in rumi mutant backgrounds.
Type: in vivo genetic separation-of-function
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Download this section (compressed HTML)O-fucosyltransferase 1 (Ofut1) encodes an endoplasmic reticulum enzyme that catalyzes the attachment of &bgr;-L-fucose to the hydroxyl group of serine or threonine residues in protein EGF-like repeats via an &agr;-glycosidic bond. It is involved in the synthesis of the O-fucosylated glycans on the N protein, which regulates N binding to its ligands encoded by Delta and Ser.
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