id: A0A9L0ILJ0 gene_symbol: GPAT2 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9793 label: Equus asinus description: GPAT2 is a mitochondrial glycerol-3-phosphate acyltransferase of the GPAT/DHAPAT family (PANTHER subfamily GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 2, MITOCHONDRIAL). Its characterised mouse ortholog transfers a fatty acyl group from acyl-CoA, preferring arachidonoyl-CoA, to sn-glycerol 3-phosphate to form lysophosphatidic acid, the first step of de novo glycerolipid synthesis, and its overexpression raises arachidonate-rich triacylglycerol. The protein spans the mitochondrial outer membrane twice with its N- and C-terminal regions in the cytosol. Expression is largely restricted to spermatocytes, where the mouse protein binds the PIWI protein MILI/PIWIL2 and is required for primary piRNA biogenesis by a mechanism that does not need its acyltransferase catalytic motif. This donkey entry is one of three gene models from the same locus; it keeps the acyltransferase domain, the HXXXXD motif and both transmembrane segments but ends in a C-terminal tail of about 85 residues that differs from the conserved GPAT2 C-terminus present in the full-length 800-residue model. existing_annotations: - term: id: GO:0004366 label: glycerol-3-phosphate O-acyltransferase activity evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: enables supporting_entities: - PANTHER:PTN008998243 review: summary: TreeGrafter transfer from PANTHER node PTN008998243 within the GPAT2 subfamily (PTHR12563:SF15), the subfamily that also contains mouse Gpat2 Q14DK4 and human GPAT2 Q6NUI2. PAINT placed GPAT activity at the ancestral GPAT1/GPAT2 node PTN000970957 with mouse Gpat2 and human GPAT2 among the seeds. action: ACCEPT reason: Mouse Gpat2, in the same PANTHER subfamily, has measured GPAT activity with arachidonoyl-CoA after overexpression, and the donkey model retains the acyltransferase domain and the HXXXXD catalytic motif at the same positions. This is the core molecular activity of the subfamily. Activity of the donkey protein itself is not measured. supported_by: - reference_id: PMID:22905194 supporting_text: GPAT2 overexpression significantly increased both GPAT and AGPAT activities only when arachidonoyl-CoA was used as a substrate - reference_id: PMID:17689486 supporting_text: When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. - reference_id: file:EQUAS/GPAT2/GPAT2-bioinformatics/RESULTS-rereview.md supporting_text: The benchmark A0A9L0ILJ0 matches A0A8C4KXU5 through the acyltransferase domain, the HXXXXD motif - term: id: GO:0006072 label: glycerol-3-phosphate metabolic process evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN008998243 review: summary: TreeGrafter glycerol-3-phosphate metabolic process from PTN008998243; mouse Gpat2 carries the same term by IDA (MGI). action: KEEP_AS_NON_CORE reason: GPAT2 acylates glycerol 3-phosphate, so it does perform a step that consumes this metabolite, and the term is true. The informative process for the reaction is glycerolipid (phospholipid and triacylglycerol) synthesis, which is captured separately; this substrate-centred view is kept as non-core rather than as the main process. supported_by: - reference_id: PMID:22905194 supporting_text: De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). - term: id: GO:0006629 label: lipid metabolic process evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in supporting_entities: - InterPro:IPR022284 review: summary: InterPro2GO lipid metabolic process from the GPAT/DHAPAT family entry IPR022284. action: TOO_GENERAL reason: True but uninformative; entailed by the accepted phospholipid biosynthetic process. - term: id: GO:0006631 label: fatty acid metabolic process evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN008998243 review: summary: TreeGrafter fatty acid metabolic process from PTN008998243. In the cached PAINT data this term is placed only at a bacterial PlsB node (PTN000285870), not at the eukaryotic GPAT node. action: MARK_AS_OVER_ANNOTATED reason: The fatty acyl-CoA is a co-substrate that is esterified onto glycerol 3-phosphate; GPAT2 does not synthesise, degrade or modify fatty acids, so fatty acid metabolism overstates its role. The acyl-chain preference for arachidonoyl-CoA is better described by the glycerolipid synthesis terms. Not removed because acyl-CoA is consumed by the reaction. supported_by: - reference_id: PMID:22905194 supporting_text: GPAT2 overexpression increased AGPAT activity ∼50% with oleoyl-CoA and 2- fold when arachidonoyl-CoA was used as substrate, confirming the preference of GPAT2 for this fatty acid. - term: id: GO:0008654 label: phospholipid biosynthetic process evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN008998243 review: summary: TreeGrafter phospholipid biosynthetic process from PTN008998243. action: ACCEPT reason: Lysophosphatidic acid made by GPAT is the precursor of phosphatidic acid and the glycerophospholipids, so GPAT2 catalyses a step of this process. PAINT placed the related glycerophospholipid metabolic process (GO:0006650) at the ancestral GPAT1/GPAT2 node PTN000970957. In cell overexpression GPAT2 drove label mainly into triacylglycerol rather than phospholipid, so the phospholipid output in germ cells is not quantified. supported_by: - reference_id: PMID:22905194 supporting_text: The GPAT product, lysophosphatidic acid (LPA) is subsequently acylated to phosphatidic acid (PA) by 1-acylglycerol-3-phosphate acyltransferase (AGPAT), which also consists of several isoforms. - term: id: GO:0016020 label: membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0162 review: summary: Membrane, from the UniProt subcellular-location mapping (ARBA-based on this record). action: MODIFY reason: Correct but general. Mouse Gpat2 is an integral membrane protein of the mitochondrial outer membrane with two transmembrane segments, both conserved in this donkey model; the specific term is mitochondrial outer membrane, which this review also proposes for the mitochondrial membrane row. proposed_replacement_terms: - id: GO:0005741 label: mitochondrial outer membrane supported_by: - reference_id: PMID:22285183 supporting_text: These results suggest that the GPAT2 protein has two transmembrane segments and that the N-terminal and C-terminal regions of this protein face the cytoplasm. - reference_id: PMID:23611983 supporting_text: GPAT2 is an enzyme of the mitochondrial outer membrane - term: id: GO:0016746 label: acyltransferase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR002123 review: summary: InterPro2GO acyltransferase activity from the phospholipid/glycerol acyltransferase domain. action: TOO_GENERAL reason: True but entailed by the accepted glycerol-3-phosphate O-acyltransferase activity. - term: id: GO:0016747 label: acyltransferase activity, transferring groups other than amino-acyl groups evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR022284 review: summary: InterPro2GO acyltransferase activity, transferring groups other than amino-acyl groups, from IPR022284. action: TOO_GENERAL reason: True but entailed by the accepted glycerol-3-phosphate O-acyltransferase activity. - term: id: GO:0019432 label: triglyceride biosynthetic process evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN008998243 review: summary: TreeGrafter triglyceride biosynthetic process from PTN008998243; PAINT placed this term at the ancestral GPAT1/GPAT2 node PTN000970957. action: KEEP_AS_NON_CORE reason: GPAT catalyses the first step of triacylglycerol synthesis, and overexpressed mouse Gpat2 increased arachidonate incorporation into TAG; in testis, TAG arachidonate content tracks Gpat2 expression. The evidence is from overexpression and correlation rather than loss of function, and GPAT2's best-supported in vivo requirement is in piRNA biogenesis, so the term is kept as true but non-core. supported_by: - reference_id: PMID:22905194 supporting_text: Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. - reference_id: PMID:17689486 supporting_text: Incubation of GPAT2-transfected Cos-7 cells with trace (3 microM; 0.25 microCi) [1-(14)C]oleate for 6h increased incorporation of [(14)C]oleate into TAG 84%. - term: id: GO:0031966 label: mitochondrial membrane evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: located_in supporting_entities: - PANTHER:PTN008998243 review: summary: TreeGrafter mitochondrial membrane from PTN008998243; PAINT placed mitochondrion (GO:0005739) at the ancestral GPAT1/GPAT2 node. action: MODIFY reason: Correct, but the mouse ortholog is an integral protein of the mitochondrial outer membrane specifically, and both transmembrane segments are retained in this donkey model. proposed_replacement_terms: - id: GO:0005741 label: mitochondrial outer membrane supported_by: - reference_id: PMID:23611983 supporting_text: GPAT2 is an enzyme of the mitochondrial outer membrane - reference_id: PMID:17689486 supporting_text: Confocal microscopy confirmed a mitochondrial location. - reference_id: file:EQUAS/GPAT2/GPAT2-bioinformatics/RESULTS-rereview.md supporting_text: both predicted transmembrane segments - term: id: GO:0034587 label: piRNA processing evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN008998243 review: summary: TreeGrafter piRNA processing from the GPAT2-subfamily node PTN008998243; the term is not on the ancestral GPAT1/GPAT2 node, so it is a subfamily-specific placement. Mouse Gpat2 co-precipitates with MILI/PIWIL2 from testis, its knockdown in germline stem cells abolishes primary piRNA production, and HXXXXD-mutant Gpat2 still rescues. action: ACCEPT reason: The knockdown phenotype alone would only show necessity, but two further observations argue that GPAT2 itself takes part in the processing machinery rather than supplying a lipid metabolite upstream. Its requirement does not depend on the catalytic motif, and it binds the PIWI protein MILI on the mitochondrial outer membrane, the site of primary piRNA processing; the authors propose a scaffold role. The donkey model lies in the same subfamily that carries this term. The PIWIL2-binding region is not mapped, so the effect of this model's divergent C-terminus is unknown. supported_by: - reference_id: PMID:23611983 supporting_text: These data clearly show that GPAT2 plays a critical role in primary processing during piRNA production. - reference_id: PMID:23611983 supporting_text: These data suggest that the acyltransferase activity of GPAT2 is dispensable for the biogenesis of piRNA. - reference_id: PMID:23611983 supporting_text: Binding of MILI to GPAT2 was confirmed by coimmunoprecipitation analysis of testis samples references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000118 title: TreeGrafter-generated GO annotations findings: [] - id: PMID:23611983 title: GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached. Mouse germline stem cells; MILI co-IP, shRNA knockdown with piRNA deep sequencing, and rescue by HXXXXD-mutant Gpat2. Primary evidence for the piRNA processing row. - id: PMID:22905194 title: Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic acid into triacylglycerols. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached. GPAT and AGPAT activity with arachidonoyl-CoA after overexpression in CHO-K1 cells; TAG arachidonate; expression in rat and mouse spermatocytes. - id: PMID:17689486 title: Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2). findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Abstract only cached. Cloning of mouse Gpat2 from testis, mitochondrial location by confocal microscopy, modest increase in NEM-sensitive GPAT activity and oleate incorporation into TAG. - id: PMID:22285183 title: Membrane topology of murine glycerol-3-phosphate acyltransferase 2. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Abstract only cached. Epitope-protection topology in HEK293 membranes; two transmembrane segments with cytosolic N- and C-termini. - id: PMID:26268560 title: Methylation of the Gpat2 promoter regulates transient expression during mouse spermatogenesis. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Expression timing in pachytene spermatocytes and promoter methylation; context for the germline role, not used as support for any annotation. - id: PMID:17013544 title: Molecular cloning of a murine glycerol-3-phosphate acyltransferase-like protein 1 (xGPAT1). findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Abstract only cached. Independent cloning of the same mouse gene (UniProt Q14DK4 isoform 1) as xGPAT1 with GPAT activity in CHO-K1 cells; corroborating, not cited as support. - id: file:EQUAS/GPAT2/GPAT2-bioinformatics/RESULTS.md title: 'GPAT2: donkey-human sequence comparison' findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Original 2026-10-14 global alignment against human Q6NUI2. Superseded as support in the 2026-10-21 re-review by RESULTS-rereview.md, which compares all three donkey gene models with the experimentally characterised mouse protein; its findings are consistent with the newer analysis. - id: file:EQUAS/GPAT2/GPAT2-bioinformatics/human-Q6NUI2-uniprot.txt title: UniProtKB/Swiss-Prot record Q6NUI2 (human GPAT2), downloaded 2026-10-14 findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Reviewed human record whose FUNCTION, CATALYTIC ACTIVITY and location statements are by similarity to mouse Gpat2. No longer used as support in the re-review, which quotes the mouse primary papers directly. - id: file:EQUAS/GPAT2/GPAT2-bioinformatics/RESULTS-rereview.md title: 'GPAT2 (donkey): gene-model comparison against mouse Gpat2 (re-review, 2026-10-21)' findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Reproducible comparison (compare_models_rereview.py, models-vs-mouse-rereview.json) of the three donkey GPAT2 models from Ensembl gene ENSEASG00005002382 with mouse Q14DK4. core_functions: - description: Mitochondrial outer membrane glycerol-3-phosphate acyltransferase that esterifies a fatty acyl-CoA, preferentially arachidonoyl-CoA, onto glycerol 3-phosphate, forming lysophosphatidic acid for glycerolipid synthesis. molecular_function: id: GO:0004366 label: glycerol-3-phosphate O-acyltransferase activity directly_involved_in: - id: GO:0008654 label: phospholipid biosynthetic process locations: - id: GO:0005741 label: mitochondrial outer membrane supported_by: - reference_id: PMID:22905194 supporting_text: GPAT2 overexpression significantly increased both GPAT and AGPAT activities only when arachidonoyl-CoA was used as a substrate - reference_id: PMID:23611983 supporting_text: GPAT2 is an enzyme of the mitochondrial outer membrane - reference_id: file:EQUAS/GPAT2/GPAT2-bioinformatics/RESULTS-rereview.md supporting_text: The benchmark A0A9L0ILJ0 matches A0A8C4KXU5 through the acyltransferase domain, the HXXXXD motif - description: Germline factor that binds MILI/PIWIL2 at the mitochondrial outer membrane and is required for primary piRNA processing by a mechanism that does not need its acyltransferase catalytic motif. directly_involved_in: - id: GO:0034587 label: piRNA processing locations: - id: GO:0005741 label: mitochondrial outer membrane supported_by: - reference_id: PMID:23611983 supporting_text: These data clearly show that GPAT2 plays a critical role in primary processing during piRNA production. - reference_id: PMID:23611983 supporting_text: These data suggest that the acyltransferase activity of GPAT2 is dispensable for the biogenesis of piRNA. knowledge_gaps: - gap_statement: How GPAT2 promotes primary piRNA processing without its acyltransferase catalytic motif is unknown; a scaffold role tethering MILI and other factors to the mitochondrial outer membrane has been proposed but not tested. gap_kind: - BIOLOGY suggested_questions: - question: Is the alternative last exon of the 718-residue donkey GPAT2 model (A0A9L0ILJ0) expressed in donkey testis, or is the 800-residue model with the conserved C-terminus (A0A8C4KXU5) the only product? - question: Which region of GPAT2 binds MILI/PIWIL2, and does it lie in the C-terminal segment that differs between the donkey gene models? suggested_experiments: - description: RT-PCR or long-read sequencing of donkey testis RNA across the GPAT2 3' exons to determine which C-terminal exon is used. - description: Map the MILI/PIWIL2-binding region of GPAT2 by co-immunoprecipitation of truncation mutants, and test whether a catalytically dead GPAT2 lacking the conserved C-terminus still rescues primary piRNA production in Gpat2-knockdown germline stem cells.