fath-1 (C25A1.5) encodes FATH-1, the sole Caenorhabditis elegans ortholog of mammalian fatty acid 2-hydroxylase (FA2H) and yeast Scs7. It is a 316-residue membrane enzyme of the sterol desaturase family (SCS7 subfamily) with an N-terminal cytochrome b5 heme-binding domain and a C-terminal fatty acid hydroxylase domain carrying four predicted transmembrane helices and the conserved histidine motifs of a di-metal (diiron/zinc) catalytic centre. By homology it resides in the endoplasmic reticulum membrane and hydroxylates fatty acyl chains at C-2 to produce (R)-2-hydroxy fatty acids that are incorporated into ceramides; C. elegans glucosylceramides are invariably 2-hydroxylated. In vivo, the growth defect of fath-1 RNAi animals is rescued by (R)- but not (S)-2-hydroxy palmitate. FATH-1 is expressed in neurons, intestine and epidermis from embryo to adult, and is required mainly in the intestine. Loss of fath-1 causes larval arrest or growth retardation, shortened lifespan, loss of intestinal apicobasal polarity, reduced lipid droplets and aggregation of peroxisomes and apical endosomes, consistent with a requirement for hydroxylated glucosylceramide in intestinal membrane organisation and postembryonic development.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:14992718 Systematic interactome mapping and genetic perturbation anal... | REMOVE | Summary: Yeast two-hybrid interaction between FATH-1 and DAF-3/SMAD4 from a large-scale interactome map of the DAF-7/TGF-beta network. Reason: Bare protein binding is uninformative and is removed under the protein-binding policy; this does not deny that the Y2H interaction was observed. The interaction comes from a systematic Y2H screen (abstract only available), and no more informative molecular function follows from it. FATH-1 is a polytopic ER-membrane hydroxylase whereas DAF-3 is a SMAD transcription factor, so the physiological relevance of a direct interaction is uncertain. Supporting Evidence: PMID:14992718 Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. PMID:30184537 It was proposed to physically interact with DAF-3/SMAD4 and repress dauer formation |
| GO:0005783 endoplasmic reticulum | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that FATH-1 acts in the endoplasmic reticulum, from the PTN000304948 node seeded by Arabidopsis FAH1/FAH2 and mouse Fa2h ER localisation. Reason: Root organelle term; kept under the organelle policy. The IBA node is the fatty acid 2-hydroxylase (SCS7) clade, which FATH-1 belongs to (PIRSF005149, IPR014430, PTHR12863:SF1), and its experimental seeds are ER-resident orthologs. FATH-1 conserves the four transmembrane helices of the fatty acid hydroxylase domain and there is no evidence of divergence in the worm protein. The only worm localisation data, a GFP::FATH-1 reporter that appeared uniformly cytoplasmic with some nuclear signal, did not use an ER marker and cannot resolve the ER network from cytoplasm, so it does not contradict the IBA. Supporting Evidence: file:worm/fath-1/fath-1-uniprot.txt Belongs to the sterol desaturase family. SCS7 subfamily. file:worm/fath-1/fath-1-uniprot.txt TRANSMEM 128..149 PMID:30184537 Close-up images of the intestinal cells revealed that most FATH-1 proteins were uniformly distributed in the cytoplasm, with moderate enrichment in the nuclei |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic ER membrane localisation from the UniRule family rule and subcellular-location mapping. Reason: Two independent lines agree on the ER membrane sub-compartment, membership in the FA2H/SCS7 family whose characterised members are ER membrane enzymes, and four predicted transmembrane helices in the hydroxylase domain (Phobius). The catalytic histidine motifs lie in the membrane domain, as expected for a membrane-embedded desaturase-like hydroxylase. This is the core location of the enzyme activity. Supporting Evidence: file:worm/fath-1/fath-1-uniprot.txt Belongs to the sterol desaturase family. SCS7 subfamily. file:worm/fath-1/fath-1-uniprot.txt TRANSMEM 219..237 |
| GO:0006629 lipid metabolic process | IEA GO_REF:0000120 | MODIFY | Summary: Electronic lipid metabolic process annotation from the Scs7 InterPro entry and an ARBA rule. Reason: Correct but too general. FATH-1 2-hydroxylates fatty acyl chains, which is fatty acid metabolism, and the products are the 2-hydroxy acyl chains of ceramides; both more specific terms are supported (ceramide biosynthetic process is already carried by this gene through IBA). Proposed replacements: fatty acid metabolic process ceramide biosynthetic process Supporting Evidence: PMID:30184537 The results showed that (R)-2-OH FA supplement nicely rescued the growth defect caused by fath-1 RNAi, while (S)-2-OH FA did not PMID:21926990 Finally, our MS analysis characterized GlcCer as invariably hydroxylated, consistent with the identified sphingolipid fatty-acid hydroxylase, C25A1.5 |
| GO:0016020 membrane | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO membrane annotation from the Scs7 family signature. Reason: Correct but too general; FATH-1 is an integral membrane enzyme of the ER membrane, a term this gene already carries (IEA) and which is supported by the predicted transmembrane helices and family membership. Proposed replacements: endoplasmic reticulum membrane Supporting Evidence: file:worm/fath-1/fath-1-uniprot.txt TRANSMEM 243..267 |
| GO:0046513 ceramide biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that FATH-1 participates in ceramide biosynthesis, from the FA2H/Scs7 node seeded by yeast Scs7 and mammalian FA2H. Reason: FATH-1 performs a chemical step of the pathway, the C-2 hydroxylation that generates the 2-hydroxy acyl chain of 2-hydroxyceramides, so it does some of the work of ceramide biosynthesis rather than merely being required for it. FATH-1 lies within the PTN000304948 clade with no evidence of divergence. In worm, glucosylceramide is invariably 2-hydroxylated, fath-1 knockdown phenocopies loss of the glucosylceramide synthases cgt-1/cgt-3 (polarity conversion and L1 arrest), and the fath-1 arrest is not bypassed by the sphingoid base d17iso-sphinganine, placing FATH-1 in the ceramide/GlcCer branch. Supporting Evidence: PMID:21926990 Finally, our MS analysis characterized GlcCer as invariably hydroxylated, consistent with the identified sphingolipid fatty-acid hydroxylase, C25A1.5 PMID:21926990 The phenotype was found in the single knockdown of a predicted sphingolipid-fatty-acid hydroxylase, C25A1.5, and the double knockdown of the CGT orthologs cgt-1 and cgt-3 PMID:23705068 As shown above, RNAi of fath-1, or mutating both cgt-1 and cgt-3, caused larval arrest that could not be rescued by feeding with either mmBCFAs or d17iso-SPA |
| GO:0080132 fatty acid 2-hydroxylase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of fatty acid 2-hydroxylase activity from the FA2H/Scs7 clade, seeded by experimentally characterised plant, fly, yeast and mammalian orthologs. Reason: Core molecular function. FATH-1 is the sole C. elegans member of the FA2H/SCS7 subfamily and retains the cytochrome b5 heme-binding domain and the full set of histidine di-metal ligands of the catalytic centre. Worm in vivo evidence is consistent with the activity, since the fath-1 RNAi growth defect is rescued stereospecifically by the (R)-2-hydroxy fatty acid, the product of FA2H-type enzymes. No direct enzyme assay of FATH-1 has been reported. Kept at the parent term rather than free fatty acid 2-hydroxylase activity because the substrate form (free versus ceramide-bound acyl chain) has not been tested for the worm enzyme. Supporting Evidence: file:worm/fath-1/fath-1-uniprot.txt Catalyzes stereospecific hydroxylation of free fatty acids at the C-2 position to produce (R)-2-hydroxy fatty acids file:worm/fath-1/fath-1-uniprot.txt Binds 2 Zn(2+) ions per subunit that likely form a catalytic dimetal center. PMID:30184537 The results showed that (R)-2-OH FA supplement nicely rescued the growth defect caused by fath-1 RNAi, while (S)-2-OH FA did not PMID:30184537 Fatty acid two-hydroxylase 1 (FATH-1) is the sole C. elegans homolog for mammalian FA2H, whose corresponding gene number is C25A1.5 |
| GO:0080132 fatty acid 2-hydroxylase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic fatty acid 2-hydroxylase activity from the Scs7 InterPro entry and UniRule. Reason: Consistent with the IBA row for the same term; family membership and conserved catalytic residues support the activity, and the stereospecific (R)-2-hydroxy fatty acid rescue in worm is consistent with it. Supporting Evidence: file:worm/fath-1/fath-1-uniprot.txt Belongs to the sterol desaturase family. SCS7 subfamily. PMID:30184537 These observations suggested that FATH-1 primarily function through its catalytic product (R)-2-OH FAs in C. elegans, similar as its mammalian counterpart. |
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Download this section (compressed HTML)Q: Does C. elegans FATH-1 hydroxylate free fatty acids (like mammalian FA2H, supporting free fatty acid 2-hydroxylase activity) or ceramide-bound acyl chains (like yeast Scs7)?
Q: Is the loss of 2-hydroxylated glucosylceramide the cause of the fath-1 RNAi larval arrest and polarity defects, and should glucosylceramide biosynthetic process be asserted once 2-OH GlcCer depletion is shown in fath-1 animals?
Q: How does loss of FATH-1 reduce Delta10-C17:1 fatty acid, for example via alpha-oxidation of 2-hydroxy C18 fatty acids?
Q: Is the reported Y2H interaction with DAF-3/SMAD4 physiologically meaningful for an ER-membrane enzyme?
Experiment: Express FATH-1 in yeast scs7 mutants or insect/mammalian microsomes and assay 2-hydroxylation of free fatty acids versus ceramide-bound acyl chains by GC-MS, including stereochemistry of the product.
Experiment: Lipidomic profiling of fath-1 deletion or RNAi animals to quantify 2-hydroxy and non-hydroxy ceramides and glucosylceramides.
Experiment: Localise endogenously tagged FATH-1 with an ER marker (e.g. TRAM or SP12) in the intestine to confirm ER-membrane residence.
Experiment: Introduce CRISPR point mutations in the conserved histidine motif (e.g. His183/His188) to test whether catalytic activity is required for development and intestinal polarity.
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