FSH3 (YOR280C, "Family of Serine Hydrolases 3") is a 266-residue serine hydrolase of the alpha/beta-hydrolase fold (AB-hydrolase-3 / Pfam FSH1 family) in the budding yeast Saccharomyces cerevisiae. It carries a complete, canonical Ser-Asp-His catalytic triad, with the nucleophilic serine (Ser117) in a classic GXSXG "nucleophile elbow" motif, and was directly detected as a catalytically active serine hydrolase in yeast by activity-based protein profiling. Its physiological substrate and specific reaction are undetermined; UniProt assigns only the uncommitted esterase activity EC 3.1.-.- and describes it as a "serine hydrolase of unknown specificity". FSH3 is one of three paralogous S. cerevisiae FSH proteins (with FSH1 and FSH2) and is the yeast counterpart of the human candidate tumor-suppressor esterase OVCA2. It localizes to the peroxisome and to cytoplasmic pools; its expression is induced by the DNA-damage-checkpoint effector Crt1/RFX1 and by oxidative (H2O2) stress. Overexpression of FSH3 perturbs cellular lipid content (notably reducing phosphatidylcholine) and triggers a NUC1-dependent apoptotic phenotype. Single deletion causes no growth defect under standard conditions, but a double mutant lacking both FSH3 and the peroxisomal lipase Lpx1 has reduced peroxisomal fatty-acid beta-oxidation activity, implicating FSH3 (redundantly with Lpx1) in peroxisomal lipid/fatty-acid metabolism; its precise molecular contribution and physiological substrate remain unresolved.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetically inferred (IBA) nuclear localization propagated across the OVCA2/FSH family. It is not supported by the yeast-specific experimental evidence, which places FSH3 in the peroxisome (IDA), nor by any FSH3 assay. This is a weak, over-propagated location for this gene. Reason: IBA-only nuclear localization with no experimental support in yeast; the experimentally verified compartment is the peroxisome (GO:0005777, IDA). Family-wide phylogenetic propagation of a nuclear location is not substantiated for FSH3. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN000512658 SUPPORTS SOURCE BUT NOT TARGET The node carries a metazoan OVCA2-backed nuclear IBD, but yeast Fsh3 has direct peroxisomal matrix evidence. MGI:MGI:2179725 SUPPORTS SOURCE BUT NOT TARGET Nuclear OVCA2-family source member; does not override FSH3-specific peroxisomal localization. UniProtKB:Q8WZ82 Β· OVCA2 (human) SUPPORTS SOURCE BUT NOT TARGET PANTHER OVCA2 subfamily (PTHR48070:SF6) seed; a nuclear location for the family is not substantiated for yeast FSH3, whose experimental compartment is the peroxisome. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetically inferred (IBA) cytoplasmic localization. FSH3 has cytoplasmic/stress-granule associations (CD-CODE stress granule cross-ref) and a soluble alpha/beta-hydrolase is consistent with a cytoplasmic pool, so this general location is plausible, but it is non-core relative to the experimentally established peroxisomal localization. Reason: General, phylogenetically inferred location that is plausible for a soluble hydrolase but is not the informative, experimentally supported compartment; retained as non-core alongside the IDA peroxisome annotation. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN000512658 SUPPORTS TRANSFER The same OVCA2/FSH-family node also carries a broad cytoplasmic IBD. This transfer is biologically plausible for Fsh3 and includes FSH3 itself as an SGD seed, but it remains secondary to direct peroxisomal localization. SGD:S000005806 SUPPORTS TRANSFER FSH3 self-evidence contributes to this inherited cytoplasmic-location call. |
| GO:0016787 hydrolase activity | IBA GO_REF:0000033 | MODIFY | Summary: Correct but uninformatively general. FSH3 has a complete, canonical Ser-Asp-His catalytic triad (Ser117 in a GXSXG motif, Asp180, His209) on an alpha/beta-hydrolase scaffold, and was detected as a catalytically active serine hydrolase by activity-based proteomics. These support the more specific mechanism-defined term serine hydrolase activity (GO:0017171). Reason: The generic hydrolase activity term should be replaced by the mechanism-specific GO:0017171 (serine hydrolase activity), which is directly supported by the intact Ser-Asp-His triad, the diagnostic GXSXG nucleophile-elbow motif, and the activity-based proteomic detection of FSH3 as an active serine hydrolase. The specific substrate/reaction remains unknown (EC 3.1.-.-), so a more specific catalytic term is not yet justified. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000512658 SUPPORTS TRANSFER The OVCA2/FSH-family node correctly supports transfer of a generic hydrolase activity to catalytically intact FSH3, but the term should be refined to serine hydrolase activity for this target. SGD:S000001091 SUPPORTS TRANSFER Yeast FSH1 is a same-family serine hydrolase seed for the generic hydrolase IBD and supports the fold-level transfer. UniProtKB:Q8WZ82 Β· OVCA2 (human) SUPPORTS TRANSFER The IBA hydrolase-activity transfer from the OVCA2 esterase subfamily is correct in branch but too general; the intact Ser-Asp-His triad and GXSXG motif in FSH3 support the more specific serine hydrolase activity. Proposed replacements: serine hydrolase activity Supporting Evidence: PMID:14645503 Three of the previously uncharacterized proteins are members of a eukaryotic serine hydrolase family |
| GO:0005777 peroxisome | IDA PMID:36164978 Systematic multi-level analysis of an organelle proteome rev... | ACCEPT | Summary: Experimentally determined (IDA) peroxisomal localization from a systematic peroxisome-proteome analysis in which FSH3 is one of the newly identified peroxisomal proteins. This is the informative, experimentally supported compartment for FSH3 and is retained as a core localization. Reason: Direct experimental evidence (IDA) from a systematic peroxisome-proteome study; the curator had access to the full dataset. This is the strongest localization annotation for FSH3. Supporting Evidence: PMID:36164978 We confirmed that Fsh3, which contains a unique PTS1 sequence (G in position β3), is indeed a matrix protein (Fig 2E), and that its Cβ² motif functions as a PTS1 β its last 10 amino acids mediate GFP targeting to peroxisomes in a Pex5βdependent manner (Appendix Fig S7B). |
| GO:0003674 molecular_function | ND GO_REF:0000015 | REMOVE | Summary: Root-level placeholder (ND, "no biological data") reflecting the historical absence of a curated molecular function. It is superseded by the informative serine hydrolase activity now supported by the domain/triad analysis and the IBA hydrolase annotation. Reason: ND root placeholder is superseded by informative molecular-function evidence (serine hydrolase activity); root ND annotations should not be retained once a real function is available. NO_DATA re-check: Re-checked 2026-10-31 against core_functions; activity-based protein profiling in S. cerevisiae (PMID:14645503) detects FSH3 as a catalytically active serine hydrolase. Supporting Evidence: PMID:14645503 Three of the previously uncharacterized proteins are members of a eukaryotic serine hydrolase family |
| GO:0008150 biological_process | ND GO_REF:0000015 | ACCEPT | Summary: Root-level placeholder (ND) for biological process. FSH3's native biological process is genuinely unresolved (no deletion phenotype under standard conditions), so this remains a real knowledge gap rather than a curatable term. Reason: ACCEPT under the ND acceptance rule (projects/NO_DATA.md). The evidence this review cites for the biological process is inferred (from orthologs, family membership or sequence features) or comes from other species; none of it is literature on this gene product in this species. This was judged from the review's own content during the NO_DATA re-action pass, not from a new literature search. Earlier action: REMOVE. Possibly stale: the 2026-11-06 ND citation pass found literature on this gene in this species that may identify a biological process; see FSH3-notes.md, section "ND literature-search citations (2026-11-06)". A curator should check it before this ACCEPT stands. |
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Download this section (compressed HTML)Q: What is the natural substrate of FSH3, and does the S117A catalytic-dead mutant lose the overexpression phenotypes (apoptosis, PC reduction)?
Q: Is FSH3 functionally redundant with FSH2 (its OVCA2-subfamily paralog) and/or FSH1, and does a triple fsh deletion reveal a phenotype absent in single mutants?
Q: Does FSH3 act in peroxisomal glycerophospholipid metabolism, and is the OVCA2 human ortholog's function conserved?
Experiment: Compare wild-type FSH3, catalytic-dead FSH3(S117A), and empty-vector strains for the overexpression phenotypes (ROS accumulation, viability, PC levels). Loss of phenotype in S117A would establish that they depend on catalytic serine-hydrolase activity.
Hypothesis: FSH3 is a catalytically active serine hydrolase whose in-cell phenotypes require the Ser117 nucleophile.
Experiment: Purify recombinant FSH3 and assay activity against fluorogenic ester panels and defined lipids (including phosphatidylcholine) in vitro; combine with untargeted lipidomics/metabolomics of fsh3-delta vs wild type to identify accumulating candidate substrates.
Hypothesis: FSH3 hydrolyzes a peroxisomal glycerophospholipid or ester substrate.
Experiment: Construct single, double and triple fsh deletions and phenotype across oxidative, DNA-damage and oleate (peroxisome-inducing) conditions, with epistasis to NUC1.
Hypothesis: Single-deletion silence of FSH3 is due to redundancy with FSH1/FSH2.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The physiological substrate of FSH3 and the specific reaction it catalyzes are undetermined. Only an uncommitted esterase activity (EC 3.1.-.-) is assigned, and UniProt describes FSH3 as a "serine hydrolase of unknown specificity".
OPEN BIOLOGY MF_DARK
What is known: FSH3 has a complete, canonical Ser-Asp-His serine-hydrolase catalytic triad (Ser117 in a GXSXG motif, Asp180, His209) on an alpha/beta-hydrolase fold and was detected as a catalytically active serine hydrolase by activity-based proteomics in yeast; the class of chemistry (serine-nucleophile ester/amide hydrolysis) is therefore known, but no natural substrate has been identified.
Significance: FSH3 is the yeast counterpart of the human candidate tumor-suppressor esterase OVCA2; identifying its substrate would define the molecular function of a conserved but functionally dark eukaryotic serine-hydrolase family.
What would resolve it: In-vitro substrate profiling (e.g. fluorogenic ester/lipid panels, competitive activity-based protein profiling with FP-probes) of recombinant FSH3 and a catalytic-serine (S117A) mutant, paired with untargeted metabolomics/lipidomics of fsh3-delta vs wild type to identify accumulating candidate substrates.
Provenance (the field's own admissions):
Gap: The precise biological role/pathway of FSH3 is not fully resolved. Its single deletion causes no growth defect under standard conditions, and its overexpression phenotypes (PC reduction, NUC1-dependent apoptosis) cannot be read as its native role. A loss-of-function clue does exist: a double mutant lacking both FSH3 and the peroxisomal lipase Lpx1 (but neither single mutant) has reduced peroxisomal fatty-acid beta-oxidation activity, implicating FSH3 in peroxisomal fatty-acid beta-oxidation (GO:0006635; peroxisomal in yeast) redundantly with Lpx1 -- but FSH3's specific molecular step in that pathway is undetermined.
NARROWING BIOLOGY BP_DARK
What is known: FSH3 localizes to the peroxisome, is induced by the DNA-damage-checkpoint effector Crt1/RFX1 and by H2O2, its overexpression reduces phosphatidylcholine and triggers NUC1-dependent apoptosis, and a double deletion of FSH3 with LPX1 shows a significant reduction in beta-oxidation activity relative to control and both single mutants (an overlapping role for Fsh3 with Lpx1). Single-deletion growth is silent, so the specific pathway step and whether the beta-oxidation contribution is direct (an acyl-ester hydrolase step) or indirect remain open.
Significance: The Fsh3/Lpx1 double-mutant beta-oxidation defect provides a candidate pathway (peroxisomal fatty-acid beta-oxidation / lipid mobilization) and explains why single deletions look silent (redundancy with Lpx1); resolving FSH3's specific role there would move it from BP-dark to an annotatable biological process (candidate GO:0006635 fatty acid beta-oxidation), and let the overexpression apoptosis phenotype be interpreted.
What would resolve it: Condition-specific phenotyping of fsh3-delta and fsh3/lpx1 combinations on fatty-acid (oleate) growth and direct beta-oxidation-flux assays, epistasis with FSH1/FSH2 and NUC1, to define FSH3's step in peroxisomal fatty-acid metabolism and justify a GO:0006635 (fatty acid beta-oxidation) annotation.
Provenance (the field's own admissions):
Gap: The degree and direction of functional redundancy among the three paralogous S. cerevisiae serine hydrolases FSH1, FSH2 and FSH3 is unresolved; whether they share substrates or act in overlapping/opposing pathways is unknown.
OPEN BIOLOGY MF_DARK
What is known: All three are members of the Fsh serine-hydrolase family sharing conservation with human OVCA2, and fsh double deletions (fsh1 fsh2, fsh1 fsh3, fsh2 fsh3) grow faster than wild type, indicating some shared or overlapping influence on growth; but no shared molecular substrate or pathway has been established.
Significance: Redundancy among paralogs is the most likely reason single deletions are silent; resolving it is a prerequisite for detecting loss-of-function phenotypes and for assigning family-level function.
What would resolve it: Comparative biochemistry of purified FSH1/FSH2/FSH3 on shared substrate panels and phenotyping of single, double, and triple fsh deletions across stress conditions.
Provenance (the field's own admissions):
Gap: It is unknown whether peroxisomal FSH3 acts directly on a glycerophospholipid substrate (e.g. phosphatidylcholine) or whether the phosphatidylcholine reduction seen on FSH3 overexpression is an indirect consequence of a different activity.
OPEN BIOLOGY MF_DARK
What is known: FSH3 overexpression produces the strongest lipidome change (PC reduction) among a panel of dark peroxisomal proteins, and FSH3 has the catalytic machinery of a serine ester hydrolase; but no in-vitro assay has shown FSH3 hydrolyzing PC or any defined lipid.
Significance: A direct link between FSH3 and glycerophospholipid turnover would give the family a concrete molecular function and connect it to peroxisomal lipid metabolism.
What would resolve it: Direct in-vitro lipase/phospholipase assays on recombinant FSH3 (and S117A) against PC and related glycerophospholipids, with lipidomics of fsh3-delta to test for the reciprocal (accumulation) effect.
Provenance (the field's own admissions):
Putative serine hydrolase; likely target of Cyc8p-Tup1p-Rfx1p transcriptional regulation; localizes to cytosol and peroxisomes; similar to S. cerevisiae Fsh1p and Fsh2p and the human candidate tumor suppressor OVCA2
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