Pld is the single conventional (HKD-type) phospholipase D of Drosophila melanogaster. It hydrolyzes membrane phosphatidylcholine to phosphatidic acid and choline through two HKD catalytic motifs, and carries an N-terminal PX domain, a PH domain and a polybasic PI(4,5)P2-interacting region that govern its membrane targeting and regulation. Its activity is stimulated by receptor signaling and by ARF GTPases. The phosphatidic acid it produces acts in membrane trafficking. During embryonic cellularization Pld is found on small cytoplasmic vesicles and is needed for normal Golgi morphology and for insertion of Golgi-derived secretory vesicles into the growing plasma membrane. In photoreceptors it sits in the cell body and at the base of the rhabdomere. There it supports light sensitivity and phosphoinositide (PIP2) replenishment, and during illumination it drives Arf1- and retromer-dependent recycling of endocytosed rhodopsin back to the rhabdomere membrane, protecting photoreceptors from light-dependent degeneration.
Summary: Catalytic activity is correct but uninformative. Pld is an experimentally demonstrated phospholipase D, so the specific enzyme term should replace the generic one.
Reason: The InterPro PLD/transphosphatidylase signature (IPR001736) maps only to generic catalytic activity. Pld has measured phospholipase D activity in transfected cells (PMID:17156430), and Pld-null flies make no phosphatidylethanol in vivo (PMID:27848911). The specific descendant GO:0004630 D-type glycerophospholipase activity, which the gene already carries by IDA, is the appropriate refinement.
IDA PMID:17156430 A role for Phospholipase D in Drosophila embryonic cellulari...
ACCEPT
Summary: Core enzymatic activity. Recombinant Pld expressed in HEK293 cells shows receptor- and ARF1-stimulated transphosphatidylation activity that the HKD2 point mutant H1095N lacks.
Reason: The paper assays PLD activity directly with the butanol transphosphatidylation assay of PC hydrolysis, and uses the catalytic-site mutant as a negative control. The independent in vivo result in PMID:27848911 (no phosphatidylethanol in the null; rescue only by catalytically active Pld) agrees. Both HKD motifs are retained in A4UZ54 (HxK(x4)D at 597 and 1077).
A small amount of activity was observed in cells overexpressing the inactive Pld-H1095N protein, which derived from the endogenous mammalian PLDs present in the HEK293 cells.
D. melanogaster Pld exhibits classic Pld activity, increases its activity in signaling contexts, and is inactivated by a point mutation to the conserved catalytic domain
Summary: The automated assignment agrees with the experimental IDA row for the same term and with the eukaryotic PLD family signature (IPR016555) and two retained HKD motifs.
Reason: Consistent with the IDA annotation for the same term. Membership in the eukaryotic PLD family and PAINT placement of fly Pld among the donors of the PLD-activity IBA (node PTN004499769, which includes human PLD1 and yeast SPO14) support the activity independently of the ARBA rule.
Summary: Correct but very broad. The specific lipid-metabolic outcome of Pld action, production of phosphatidic acid from phosphatidylcholine, is already captured by a descendant term on this gene.
Reason: GO:0006654 phosphatidic acid biosynthetic process is an is_a descendant of lipid metabolic process and is already carried by this gene. Pld-null retinae have reduced PA with unchanged PC, which shows Pld contributes to the cellular PA pool.
These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD.
Summary: PC hydrolysis by Pld directly generates phosphatidic acid. Loss of Pld lowers retinal PA, and catalytically active Pld restores it.
Reason: The enzyme catalyzes the step that produces PA, so this process is the direct chemical consequence of its activity rather than an indirect effect. Lipidomics in Pld-null retina gives gene-specific support for the InterPro-based inference.
These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD.
IMP PMID:17156430 A role for Phospholipase D in Drosophila embryonic cellulari...
KEEP AS NON CORE
Summary: Pld-null cellularizing embryos have enlarged Lva-marked Golgi or Golgi-derived vesicles. The phenotype is real, but it reads out a defect in Golgi-derived vesicle trafficking rather than a dedicated role in organizing the Golgi.
Reason: The experimental phenotype supports the annotation, so it is retained. However, Pld does not localize to the Golgi at steady state, and the authors interpret the enlarged Golgi structures as a consequence of impaired production and trafficking of fusion-competent vesicles. The core process is captured by the vesicle-fusion regulation term, so Golgi organization is non-core.
IMP PMID:17156430 A role for Phospholipase D in Drosophila embryonic cellulari...
KEEP AS NON CORE
Summary: Pld-null embryos show delayed cellularization and defective insertion of Golgi-derived membrane into the extending plasma membrane. Pld contributes to this process by supplying vesicle trafficking, which is a developmental context for its core membrane-trafficking role.
Reason: Cellularization is a cellular membrane-growth process. Pld contributes directly by promoting delivery of Golgi-derived vesicles to the furrows, not only as a distant downstream phenotype, so the annotation is retained. It is a context-specific use of the core trafficking function, hence non-core.
IMP PMID:15883198 Regulation of phototransduction responsiveness and retinal d...
KEEP AS NON CORE
Summary: Pld-null eyes have strongly reduced light sensitivity, and Pld overexpression restores visual signaling in rdgB mutants. Pld is not itself activated by light, and it supports phototransduction by sustaining PIP2 replenishment and membrane turnover in the photoreceptor.
Reason: The curator's IMP is supported, so the row is retained. The authors state that Pld does not transduce the light signal directly but maintains PIP2 levels. Later work places its photoreceptor role in retromer-dependent rhodopsin recycling. Phototransduction is therefore a physiological context that Pld supports, not its core activity.
findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced,
Pld does not directly transduce light signals, but does morphologically rescue retinal degeneration in the
GO:0010004 gastrulation involving germ band extension
IMP PMID:17156430 A role for Phospholipase D in Drosophila embryonic cellulari...
MARK AS OVER ANNOTATED
Summary: Only a minority of Pld-null embryos (15 of 78) showed abnormal gastrulation with a thin, shortened germ band, and the authors attribute this to defects that began during cellularization. This is a downstream developmental phenotype, not evidence that Pld does work within gastrulation.
Reason: The paper offers no gastrulation-specific mechanism, and three of the fifteen affected embryos still hatched. The authors explicitly suggest the gastrulation defect could follow from the earlier cellularization abnormalities. Under the participation standard, this is a pleiotropic consequence of the trafficking defect. The row is experimental and is not contradicted, so it is marked over-annotated rather than removed.
Summary: Consistent with immunolocalization of Pld to small cytoplasmic vesicles in embryos and to vesicles and the plasma membrane when expressed in COS7 cells.
Reason: The UniProt subcellular location is an ARBA prediction, but gene-specific imaging independently supports an endomembrane localization. GO:0031410 cytoplasmic vesicle is not an is_a or part_of descendant of endomembrane system in GO, so this row is not a candidate for refinement to that term. The vesicle term is carried separately by IDA.
In cellularizing embryos, Pld localized to the cytoplasm and/or to cytosolic vesicles of uniform small size evenly distributed between apical and basal regions of the blastoderm (Fig. 3A–D).
IDA PMID:17156430 A role for Phospholipase D in Drosophila embryonic cellulari...
ACCEPT
Summary: Affinity-purified anti-Pld antiserum detects Pld on small cytoplasmic vesicles in cellularizing embryos. Recombinant Pld also labels cytoplasmic vesicles in COS7 cells.
Reason: Direct immunolocalization with a pre-immune control. The authors note that the staining may be partly cytoplasmic, but the vesicle pattern is reproducible. The PAINT IBA on the canonical isoform B7YZT5 also assigns cytoplasmic vesicle, from node PTN001051940 shared with human PLD1.
In cellularizing embryos, Pld localized to the cytoplasm and/or to cytosolic vesicles of uniform small size evenly distributed between apical and basal regions of the blastoderm (Fig. 3A–D).
Summary: The PX domain (residues 146-355) and the polybasic PI(4,5)P2-interacting motif make phosphoinositide binding plausible. GO:0035091 covers PtdIns and its phosphorylated derivatives, so the term fits the domain-based inference. No binding assay on fly Pld is published in the cached literature.
Reason: The term definition explicitly includes phosphorylated derivatives, so the earlier concern about parent PtdIns versus PIPs does not apply. The PX-domain InterPro2GO mapping is a sound family-level inference. Phosphoinositide binding is a regulatory and targeting feature, not the catalytic core, and no more specific lipid can be named from direct fly data. Note that A4UZ54 lacks an 18-residue segment near the C-terminal end of the PX domain (after residue 338) that is present in the 1278-residue cDNA product. Whether this alters lipid binding is unknown.
Summary: Pld produces the lipid second messenger PA and is stimulated by GPCR signaling and ARF1 in a heterologous assay. The physiological roles demonstrated in flies are membrane trafficking and photoreceptor membrane homeostasis rather than a defined intracellular signaling cascade.
Reason: The InterPro2GO mapping reflects the general second-messenger function of PLDs and is not wrong. In flies, though, the defined outputs of Pld-derived PA are vesicle trafficking and recycling. The paper also shows Pld is not activated by light in the photoreceptor. Retained as non-core.
Wild-type Drosophila Pld was activated by receptor signaling through carbachol stimulation and this was enhanced by overexpression of ARF1, as has been observed for mammalian PLD2 (Du et al., 2000).
GO:0048215 positive regulation of Golgi vesicle fusion to target membrane
IMP PMID:17156430 A role for Phospholipase D in Drosophila embryonic cellulari...
ACCEPT
Summary: In Pld-null embryos, Neurotactin-containing Golgi-derived vesicles reach the apical cortex but accumulate beside the plasma membrane without being inserted. This supports a role for Pld in promoting fusion competence of Golgi-derived secretory vesicles.
Reason: This term is the closest GO representation of the mechanism the authors propose. It is consistent with the PAINT-propagated regulation of vesicle-mediated transport (GO:0060627, an ancestor of this term) that fly Pld shares with human PLD1 at node PTN001051940. Whether the defect is at budding or at fusion is not fully resolved, so the evidence is genetic rather than mechanistic.
Our results suggest that Pld functions to promote trafficking of Golgi-derived fusion-competent vesicles during cellularization.
GO:2001137 positive regulation of endocytic recycling
IMP PMID:27848911 Phospholipase D activity couples plasma membrane endocytosis...
NEW
Summary: In illuminated photoreceptors, Pld loss causes accumulation of endocytosed Rab7-positive rhodopsin-containing vesicles and loss of rhabdomere membrane. Rescue requires catalytically active Pld. Pld overexpression clears these vesicles in a retromer- and Arf1-dependent manner.
Reason: The study combines loss of function, catalytic-dependent rescue, rescue by an independent PA source, and gain-of-function epistasis with retromer and Arf1. Together these show that Pld-derived PA promotes recycling of endocytosed membrane back to the plasma membrane. Pld performs a step in this process by supplying PA, so this is participation and not only necessity. The comparator check is consistent, since the lipid enzyme PLA2G4E carries this term by IMP in human and mouse. The term is a regulation term because PA modulates retromer-dependent sorting rather than forming part of the recycling machinery.
In wild-type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function.
These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD.
Core Functions
Phosphatidylcholine-hydrolyzing phospholipase D that generates the membrane lipid phosphatidic acid on cytoplasmic vesicle membranes.
no PEth could be detected in dPLD3.1 extracts under the equivalent conditions
Pld-generated phosphatidic acid promotes membrane trafficking. It is needed for fusion of Golgi-derived secretory vesicles with the plasma membrane during embryonic cellularization, and for Arf1- and retromer-dependent recycling of endocytosed rhodopsin to the rhabdomere in photoreceptors.
Q: Does the 18-residue segment near the end of the PX domain, absent from the A4UZ54 isoform but present in the 1278- and 1364-residue products, change phosphoinositide binding or membrane targeting?
Q: Is the embryonic requirement for Pld at budding of fusion-competent vesicles from the Golgi or at their fusion with the plasma membrane?
Suggested Experiments
Experiment: Compare liposome binding of recombinant PX domains from the 1260- and 1278-residue isoforms across a phosphoinositide panel, and test isoform-specific rescue of dPLD3.1 retinal degeneration.
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.
Both predictions are correct but less specific than annotations the Pld gene already carries. Lipid catabolic process is an ancestor of phospholipid catabolic process (IBA on the canonical product B7YZT5), and cytoplasm is an ancestor of the experimentally supported cytoplasmic-vesicle localization.
Review rationale: Pld hydrolyzes phosphatidylcholine to phosphatidic acid and choline, a lipid-catabolic reaction demonstrated for the Drosophila enzyme, which loses activity when a conserved catalytic residue is mutated. The prediction is therefore correct, but the same gene already carries the more specific is_a descendant GO:0009395 phospholipid catabolic process, by PAINT IBA on the canonical product B7YZT5 (node PTN004499769), and GO:0006654 phosphatidic acid biosynthetic process on this product. Lipid catabolic process adds no information beyond those annotations. Both annotated HKD catalytic regions are present in A4UZ54.
Supporting Evidence:
PMID:15883198: "D. melanogaster Pld exhibits classic Pld activity, increases its activity in signaling contexts, and is inactivated by a point mutation to the conserved catalytic domain"
PMID:17156430: "Phospholipase D (PLD), which hydrolyzes the membrane phospholipid phosphatidylcholine to yield the lipid second messenger phosphatidic acid"
PMID:27848911: "Phospholipase D (PLD) is an enzyme that hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid (PA)."
Review rationale: Immunolocalization in cellularizing embryos places Pld in the cytoplasm and on small cytoplasmic vesicles. The gene already carries GO:0031410 cytoplasmic vesicle by IDA, which is part_of cytoplasm, so the prediction is correct but less precise than the existing supported annotation rather than an erroneous cytosolic default.
Supporting Evidence:
PMID:17156430: "In cellularizing embryos, Pld localized to the cytoplasm and/or to cytosolic vesicles of uniform small size evenly distributed between apical and basal regions of the blastoderm (Fig. 3A–D)."