Use of the ND evidence code for Gene Ontology (GO) terms

SGD applied the ND (No biological Data available) evidence code to FSH3, formally recording that no experimental data supported a GO assignment in the affected aspect at the time of curation. The later peroxisomal IDA and the activity-based serine-hydrolase proteomics supersede this for the CC and MF aspects; the biological process remains the genuinely open one.

Annotation inferences using phylogenetic trees

PAINT/IBA annotations propagated to FSH3 from the OVCA2/FSH family node include a nuclear localization that conflicts with the yeast-specific experimental evidence: FSH3 carries a functional PTS1 and is a confirmed peroxisomal matrix protein. The family-node location is therefore treated here as over-propagated for this gene rather than as evidence for a second compartment.

Synergistic computational and experimental proteomics approaches for more accurate detection of active serine hydrolases in yeast.

FSH3 was identified as a member of a newly defined eukaryotic serine hydrolase family (Fsh), whose other members include the human candidate tumour suppressor OVCA2 and a Schizosaccharomyces pombe dihydrofolate reductase. This is the origin of the family name and of the UniProt FUNCTION "serine hydrolase of unknown specificity".

"Three of the previously uncharacterized proteins are members of a eukaryotic serine hydrolase family, designated as Fsh (family of serine hydrolase), identified here for the first time."
The identification combined computational active-site structure analysis with chemical labelling of the reactive active-site serine in complex mixtures, so it reports an expressed protein with a chemically reactive catalytic serine rather than a sequence motif alone. The paper frames this explicitly as a caution about annotation transfer, which is why no specific substrate follows from it.

"The chemical proteomics technology indicates that a functional protein is being expressed in the cell, while the computational proteomics technology adds details about the specific type of function and residue that is likely being labeled."

PMID:15494396

Identification of new genes regulated by the Crt1 transcription factor, an effector of the DNA damage checkpoint pathway in Saccharomyces cerevisiae.

FSH3 transcription is under the control of the DNA-damage-checkpoint effector Crt1p (RFX1), confirmed by RT-PCR in wild-type versus crt1-delta strains. This is a regulatory relationship - the source of the UniProt INDUCTION note - and not evidence of a molecular function or a DNA-damage-response role for the FSH3 protein itself.

"Our results indicated that FSH3, YLR345W, and NTH2 are indeed under the regulation of Crt1p."
The paper's proposed link between FSH3 and folate metabolism / dihydrofolate reductase is explicitly sequence-similarity speculation, with no supporting assay, and must not be used to annotate folate metabolism for FSH3.

"Sequence analysis of the FSH3p indicated that this protein may be involved in folate metabolism either by carrying serine hydrolase activity required for the novel metabolic pathway involving dihydrofolate reductase (DHFR) or by directly interacting with the DHFR enzyme."

PMID:36164978

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.

Fsh3 is an experimentally confirmed peroxisomal matrix protein: it carries an unusual but functional PTS1 (G at position -3), and its last 10 residues are sufficient to target GFP to peroxisomes in a Pex5-dependent manner. This is the direct evidence behind the IDA C:peroxisome annotation.

"We confirmed that Fsh3, which contains a unique PTS1 sequence (G in position −3), is indeed a matrix protein (Fig 2E), and that its C′ motif functions as a PTS1 – its last 10 amino acids mediate GFP targeting to peroxisomes in a Pex5‐dependent manner (Appendix Fig S7B)."
A loss-of-function genetic interaction places FSH3 in peroxisomal fatty-acid beta-oxidation: the fsh3/lpx1 double deletion, but neither single deletion, has significantly reduced beta-oxidation activity, indicating a role overlapping and redundant with the peroxisomal lipase Lpx1.

"A β‐oxidation activity assay of Δlpx1, Δfsh3, and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon‐ or 18 carbon‐free fatty acids in media supplemented with 0.5% glucose shows a significant reduction in β‐oxidation activity compared to the control strain and the two single mutants, suggesting an overlapping role for Fsh3 with Lpx1."
Among a panel of dark peroxisomal proteins, FSH3 overexpression produced the largest lipidome change, a reduction of phosphatidylcholine. This is an overexpression phenotype, consistent with but not proof of a direct glycerophospholipid-hydrolase activity.

"cells overexpressing FSH3 had the most significant change in all conditions compared to the control strain, with a reduction of PC"

PMID:30776074

FSH3 mediated cell death is dependent on NUC1 in Saccharomyces cerevisiae.

FSH3 mRNA and protein are induced by hydrogen peroxide, yet fsh3 deletion improves growth under H2O2 - FSH3 is dispensable under oxidative stress and its loss is advantageous, not deleterious.

"In this study, hydrogen peroxide (H2O2) exposure increased the expression of both mRNA and protein levels of FSH3 in wild-type (WT) yeast cells. The deletion of FSH3 improved the yeast growth rate under H2O2-induction as compared to WT control cells."
FSH3 overexpression produces an apoptotic phenotype (ROS accumulation, reduced viability, cell death) that requires NUC1. Because this is a gain-of-function phenotype and the single deletion is viable, it constrains a downstream genetic dependency rather than establishing a native pro-apoptotic role for FSH3.

"Moreover, the overexpression of FSH3 in cells lacking NUC1 did not cause any growth defect in the presence or absence of H2O2. Our results suggest that FSH3 induced apoptosis of yeast in a NUC1 dependent manner."
All three pairwise fsh double deletions (fsh1/fsh2, fsh1/fsh3, fsh2/fsh3) grow better than wild type, indicating that the three FSH paralogs are at least partly functionally overlapping and that none is individually required.

"The double deletions fsh1Δ fsh2Δ, fsh1Δ fsh3Δ and fsh2Δ fsh3Δ displayed increased growth compared to WT cells."

PMID:24187129

Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

Systematic screening of yeast GXSXG serine-hydrolase candidates confirmed the peroxisomal Lpx1p as a lipase acting in vivo and linked peroxisomal lipolysis to fatty-acid utilization. FSH3 itself is not assayed here; the reference is contextual, establishing that peroxisomal serine hydrolases of this class do perform lipid mobilization in yeast.

"These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo."