GPAT2 is a mitochondrial glycerol-3-phosphate acyltransferase of the GPAT/DHAPAT family (PANTHER subfamily GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 2, MITOCHONDRIAL), distinct from the GPAT1 (GPAM) subfamily. Its experimentally 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 synthesis of phosphatidic acid, glycerophospholipids and triacylglycerol; membranes from cells overexpressing it also show increased lysophosphatidic acid acyltransferase activity. The mouse protein is an integral protein of the mitochondrial outer membrane with two transmembrane segments and cytosolic N- and C-termini. Its expression is largely restricted to primary spermatocytes, and in germline stem cells it binds the PIWI protein MILI/PIWIL2 and is required for primary piRNA biogenesis by a mechanism that does not need its HXXXXD catalytic motif, possibly as a scaffold on the mitochondrial surface. This 800-residue donkey protein is the full-length model of the gene, covering the acyltransferase domain, the HXXXXD motif, both transmembrane segments and the conserved C-terminus of the mouse protein. None of these functions has been measured in donkey.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003841 1-acylglycerol-3-phosphate O-acyltransferase activity | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Ensembl Compara transfer from mouse Gpat2 Q14DK4, which carries this term by IDA (PMID:22905194). The mouse evidence is a roughly two-fold rise in lysophosphatidic acid acyltransferase (AGPAT) activity in total membranes from CHO-K1 cells overexpressing Gpat2, with arachidonoyl-CoA. Reason: The donkey protein is in the same PANTHER subfamily as mouse Gpat2 (PTHR12563:SF15) and the Ensembl Compara transfer rests on a curated experimental annotation, so it is not removed. The evidence is weaker than for GPAT activity. It is an activity measured in crude membranes, where endogenous AGPATs could account for part of it, and the authors leave open whether GPAT2 has two activities. PAINT placed only GPAT activity, not AGPAT activity, at the ancestral GPAT1/GPAT2 node PTN000970957. The term is kept as a possible secondary activity; it is not part of the core function. Supporting Evidence: PMID:22905194 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. PMID:22905194 These findings suggest that the increase in PA may be due to an AGPAT activity for this heterologously-expressed protein, but we cannot exclude the possibility that the protein has dual enzymatic activities. |
| GO:0004366 glycerol-3-phosphate O-acyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined Ensembl Compara and TreeGrafter transfer. PAINT placed GPAT activity at the ancestral GPAT1/GPAT2 node PTN000970957 with mouse Gpat2 among the seeds, and mouse Gpat2 has IDA and EXP annotations for it. Reason: This is the core catalytic activity of the GPAT2 subfamily. Mouse Gpat2, in the same PANTHER subfamily, raised NEM-sensitive GPAT activity when expressed in Cos-7 cells, and in CHO-K1 cells increased GPAT activity with arachidonoyl-CoA. The donkey protein retains the HXXXXD motif (HKSLLD) at the same positions as mouse residues 205-210. The activity of the donkey protein has not been measured. Supporting Evidence: PMID:22905194 GPAT2 overexpression significantly increased both GPAT and AGPAT activities only when arachidonoyl-CoA was used as a substrate PMID:17689486 When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. file:EQUAS/GPAT2__A0A8C4KXU5/GPAT2__A0A8C4KXU5-bioinformatics/RESULTS.md The donkey HXXXXD acyltransferase motif is HKSLLD at residues 205-210 |
| GO:0005739 mitochondrion | IEA GO_REF:0000107 | MODIFY | Summary: Ensembl Compara transfer of the mouse Gpat2 mitochondrion IDA annotations; PAINT also placed mitochondrion at the ancestral GPAT1/GPAT2 node. Reason: Correct but general. Mouse Gpat2 is a membrane-integral protein of the mitochondrial outer membrane with two transmembrane segments, and both segments are conserved in this donkey model. Mitochondrial outer membrane is the specific supported location. Proposed replacements: mitochondrial outer membrane Supporting Evidence: PMID:17689486 Confocal microscopy confirmed a mitochondrial location. PMID:23611983 GPAT2 is an enzyme of the mitochondrial outer membrane |
| GO:0006072 glycerol-3-phosphate metabolic process | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: TreeGrafter transfer; mouse Gpat2 carries this term by IDA (MGI). Reason: GPAT2 acylates glycerol 3-phosphate, so it catalyses a step that consumes this metabolite and the term is true. The informative process for the reaction is glycerolipid synthesis (phosphatidic acid and triacylglycerol), which other rows capture; this substrate-centred term is kept as non-core. Supporting Evidence: PMID:22905194 De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). |
| GO:0006629 lipid metabolic process | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro2GO transfer from the GPAT/DHAPAT family entry IPR022284. Reason: True but uninformative; entailed by the accepted phosphatidic acid biosynthetic process. |
| GO:0006631 fatty acid metabolic process | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: TreeGrafter transfer. In the cached PAINT data for PTHR12563 this term is placed only at the bacterial PlsB node PTN000285870 (seeded by E. coli P0A7A7), not at the eukaryotic GPAT1/GPAT2 node, and mouse Gpat2 does not carry it. Reason: GPAT2 esterifies a fatty acyl-CoA onto glycerol 3-phosphate; it does not synthesise, degrade or modify fatty acids, so fatty acid metabolism overstates its role. Its acyl-CoA preference is better expressed by the glycerolipid synthesis terms. Not removed, because acyl-CoA is a substrate of the reaction. Supporting Evidence: PMID:22905194 we have identified mitochondrial GPAT2 as an acyltransferase that prefers arachidonoyl-CoA |
| GO:0006654 phosphatidic acid biosynthetic process | IEA GO_REF:0000107 | ACCEPT | Summary: Ensembl Compara transfer of the mouse Gpat2 IMP annotation from PMID:22905194, in which Gpat2 overexpression increased phosphatidic acid formation with arachidonoyl-CoA. Reason: GPAT catalyses the first step of de novo phosphatidic acid synthesis (glycerol 3-phosphate to lysophosphatidic acid), so GPAT2 performs a step of this process, and the mouse protein increased PA production from arachidonoyl-CoA. This is the most specific process term for its catalytic role that the gene carries; it is a descendant of phospholipid and glycerophospholipid biosynthesis. The donkey protein is in the same PANTHER subfamily and keeps the catalytic motif. Supporting Evidence: PMID:22905194 We did not detect LPA as a product of the GPAT reaction with overexpressed GPAT2, but we found enhanced PA production when arachidonoyl-CoA was used as substrate, and both GPAT and AGPAT activities were higher in vitro. PMID:22905194 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. |
| GO:0008654 phospholipid biosynthetic process | IEA GO_REF:0000118 | TOO GENERAL | Summary: TreeGrafter transfer; PAINT placed the related glycerophospholipid metabolic process (GO:0006650) at the ancestral GPAT1/GPAT2 node PTN000970957. Reason: True, since lysophosphatidic acid made by GPAT is the precursor of phosphatidic acid and the glycerophospholipids, but entailed by the accepted phosphatidic acid biosynthetic process (GO:0006654 is_a GO:0008654). In overexpressing Cos-7 cells mouse Gpat2 drove oleate mainly into triacylglycerol, with less into phospholipid, so the phospholipid output in germ cells has not been quantified. Supporting Evidence: PMID:17689486 In contrast, incorporation into phospholipid species was lower than in control cells. |
| GO:0016020 membrane | IEA GO_REF:0000044 | MODIFY | Summary: UniProt subcellular-location mapping; on this unreviewed record the location rests on an ARBA rule. Reason: Correct but general. Mouse Gpat2 is a carbonate-resistant integral membrane protein with two transmembrane segments, located in the mitochondrial outer membrane; both segments are conserved in this donkey model. Proposed replacements: mitochondrial outer membrane Supporting Evidence: PMID:22285183 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. PMID:23611983 GPAT2 is an enzyme of the mitochondrial outer membrane file:EQUAS/GPAT2__A0A8C4KXU5/GPAT2__A0A8C4KXU5-bioinformatics/RESULTS.md both hydrophobic and gap-free |
| GO:0016746 acyltransferase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro2GO transfer from the phospholipid/glycerol acyltransferase domain (IPR002123). Reason: True but entailed by the accepted glycerol-3-phosphate O-acyltransferase activity. |
| GO:0016747 acyltransferase activity, transferring groups other than amino-acyl groups | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro2GO transfer from the GPAT/DHAPAT family entry IPR022284. Reason: True but entailed by the accepted glycerol-3-phosphate O-acyltransferase activity. |
| GO:0019432 triglyceride biosynthetic process | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: TreeGrafter transfer; PAINT placed this term at the ancestral GPAT1/GPAT2 node PTN000970957, and mouse Gpat2 carries it by IDA. Reason: GPAT catalyses the first step of triacylglycerol synthesis, and overexpressed mouse Gpat2 increased oleate incorporation into TAG and arachidonate content of TAG. The evidence is from overexpression rather than loss of function, and GPAT2's best-supported requirement in germ cells is in piRNA biogenesis, so the term is kept as true but non-core. Supporting Evidence: PMID:22905194 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. PMID:17689486 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%. |
| GO:0031966 mitochondrial membrane | IEA GO_REF:0000118 | MODIFY | Summary: TreeGrafter transfer; PAINT placed mitochondrion (GO:0005739) at the ancestral GPAT1/GPAT2 node. Reason: Correct, but mouse Gpat2 is in the mitochondrial outer membrane specifically, and both transmembrane segments are retained in this donkey model. Proposed replacements: mitochondrial outer membrane Supporting Evidence: PMID:23611983 GPAT2 is an enzyme of the mitochondrial outer membrane PMID:22285183 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. |
| GO:0034587 piRNA processing | IEA GO_REF:0000120 | ACCEPT | Summary: Combined Ensembl Compara and TreeGrafter transfer of the mouse Gpat2 IMP annotation (PMID:23611983). Mouse Gpat2 co-precipitates with MILI/PIWIL2 from testis, its knockdown in MILI-rescued germline stem cells abolishes primary piRNA production, and HXXXXD-mutant Gpat2 still rescues. PAINT did not place this term at any node of PTHR12563 in the cached data, including the GPAT1/GPAT2 node, so it is not asserted as an ancestral function of the family. Reason: The PAINT omission is consistent with this being a GPAT2-lineage function rather than one shared with GPAT1 (subfamily SF16), and this donkey protein is inside the GPAT2 subfamily (SF15) with the mouse donor, so the transfer stays within the clade that has the experimental evidence. Knockdown alone would show only necessity, but two further observations argue that GPAT2 does part of the work of primary processing rather than supplying a lipid upstream. Its requirement does not depend on the catalytic motif, and it binds MILI at the mitochondrial outer membrane, where the authors propose it acts as a scaffold for the processing machinery. The evidence is from one study in mouse germline stem cells, and nothing is known of GPAT2 in donkey germ cells. Supporting Evidence: PMID:23611983 These data clearly show that GPAT2 plays a critical role in primary processing during piRNA production. PMID:23611983 These data suggest that the acyltransferase activity of GPAT2 is dispensable for the biogenesis of piRNA. PMID:23611983 Binding of MILI to GPAT2 was confirmed by coimmunoprecipitation analysis of testis samples |
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Download this section (compressed HTML)Q: Is the lysophosphatidic acid acyltransferase activity seen in membranes of Gpat2-overexpressing cells an intrinsic activity of GPAT2, or does it come from endogenous AGPATs acting on GPAT2-made product?
Q: Is GPAT2 expressed in donkey testis, and is the 800-residue model (A0A8C4KXU5) with the conserved C-terminus the main product there?
Experiment: Assay purified or reconstituted GPAT2 for GPAT and AGPAT activity with arachidonoyl-CoA, alongside an HXXXXD mutant, to separate intrinsic from host-membrane activity.
Experiment: Map the MILI/PIWIL2-binding region of GPAT2 by co-immunoprecipitation of truncation mutants and test which regions are needed to rescue primary piRNA production in Gpat2-knockdown germline stem cells.
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