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.
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.
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.
Summary: TreeGrafter glycerol-3-phosphate metabolic process from PTN008998243; mouse Gpat2 carries the same term by IDA (MGI).
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.
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.
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.
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.
Summary: TreeGrafter phospholipid biosynthetic process from PTN008998243.
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.
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.
Summary: Membrane, from the UniProt subcellular-location mapping (ARBA-based on this record).
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.
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.
Summary: TreeGrafter triglyceride biosynthetic process from PTN008998243; PAINT placed this term at the ancestral GPAT1/GPAT2 node PTN000970957.
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.
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.
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%.
Summary: TreeGrafter mitochondrial membrane from PTN008998243; PAINT placed mitochondrion (GO:0005739) at the ancestral GPAT1/GPAT2 node.
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.
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.
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.
The benchmark A0A9L0ILJ0 matches A0A8C4KXU5 through the acyltransferase domain, the HXXXXD motif
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.
Q: 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?
Q: 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
Experiment: RT-PCR or long-read sequencing of donkey testis RNA across the GPAT2 3' exons to determine which C-terminal exon is used.
Experiment: 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.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Assessment of the single ProtNLM2 GO prediction for donkey GPAT2 (A0A9L0ILJ0, DONKEY40 cohort). The gene review accepts glycerol-3-phosphate O-acyltransferase activity (GO:0004366) as the core activity, transferred from the GPAT2 PANTHER subfamily (PTHR12563:SF15) on the basis of measured GPAT activity of mouse Gpat2, and the donkey model retains the acyltransferase domain and the HXXXXD catalytic motif. A PubMed search found no experimental study of GPAT2 in equids. The predicted GO:0008374 O-acyltransferase activity is an obsolete grouping class (replaced by GO:0016747); the activity it names is correct but less precise than glycerol-3-phosphate O-acyltransferase activity, which donkey GOA already has from TreeGrafter and which the ProtNLM2 evidence record cites (LSP). Non-GO ProtNLM2 output for this accession, not scored here: a function statement, "Esterifies acyl-group from acyl-ACP to the sn-1 position of glycerol-3-phosphate, an essential step in glycerolipids biosynthesis such as triglycerides, phosphatidic acids and lysophosphatidic acids" (supported by a phmmer match to human GPAT1/GPAM Q9HCL2 rather than GPAT2; the acyl-ACP wording follows UniProt text, whereas the mammalian catalytic-activity lines use acyl-CoA). No subcellular-location statement was emitted.
LSP — Less precise than existing annotation Review score: 2/2
Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-10-14
Review rationale: The GPAT2 subfamily esterifies acyl-CoA onto the sn-1 hydroxyl of glycerol 3-phosphate, an O-acyl transfer; overexpressed mouse Gpat2 raises GPAT activity with arachidonoyl-CoA, and the donkey model conserves the acyltransferase domain and HXXXXD motif, so the predicted activity is correct in substance. Scored for this product, A0A9L0ILJ0 (718 aa) keeps the acyltransferase domain (199-333), the HXXXXD motif and both transmembrane segments of the full-length 800-residue model A0A8C4KXU5, and lacks only the conserved GPAT2 C-terminal region (about 85 residues replaced by a different tail), which is not the catalytic region, so the activity does not depend on a feature this product lacks. The term is obsolete in the current GO release (an unnecessary grouping class, replaced by GO:0016747, which is also in donkey GOA from InterPro2GO). Donkey GOA already carries the specific glycerol-3-phosphate O-acyltransferase activity (TreeGrafter), accepted as core in the gene review, so the prediction is less precise than existing annotation. The obsolete identifier is a defect of the emitted term, not of the biology. Training-set membership is unknown.
Supporting Evidence:
PMID:22905194: "GPAT2 overexpression significantly increased both GPAT and AGPAT activities only when arachidonoyl-CoA was used as a substrate"