NTPase (CG3059, also called NTPDase6) is the only GDA1/CD39-family nucleoside triphosphate diphosphohydrolase gene in Drosophila melanogaster. Its full-length products are type II membrane proteins with one N-terminal transmembrane segment and a large catalytic domain containing the apyrase conserved regions. The characterised 461-residue product hydrolyses UDP, GDP and IDP about equally well and turns over other nucleoside di- and triphosphates poorly. In transfected S2 cells the protein sits mainly in the endoplasmic reticulum, and its activity is latent until the membranes are permeabilised with detergent, which places the catalytic domain on the luminal side and not at the cell surface. Oxidative cross-linking through a transmembrane cysteine and an exoplasmic cysteine produces disulfide-linked dimers and inactivates the enzyme. In the GDA1/CD39 tree it groups with the nematode ER-luminal UDP/GDPase UDA-1, not with the ATP-hydrolysing cell-surface NTPDases. Like UDA-1, it probably clears the nucleoside diphosphates released by glycosyltransferases in the secretory pathway, but no loss-of-function study in the fly has tested that role.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004382 GDP phosphatase activity | IDA PMID:19467631 The single NTPase gene of Drosophila melanogaster encodes an... | ACCEPT | Summary: GDP hydrolysis was shown directly for the cloned fly enzyme. Reason: In substrate comparisons with the cloned O76268-numbered cDNA, GDP was one of three equally good substrates. This is a core activity. The PANTHER subfamily co-member UDA-1 shares the same UDP/GDP preference, which supports it as an inherited property of the subfamily. Supporting Evidence: PMID:19467631 The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. |
| GO:0004382 GDP phosphatase activity | IEA GO_REF:0000117 | ACCEPT | Summary: Same claim as the IDA row; GDP is a directly demonstrated substrate. Reason: The electronic inference agrees with the direct enzymology. It is accepted on the strength of that evidence, not of the ARBA rule. Supporting Evidence: PMID:19467631 The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. |
| GO:0005576 extracellular region | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: No evidence that the fly enzyme is released; the characterised product is an ER membrane protein. Reason: Some vertebrate relatives, such as ENTPD5 and ENTPD6, are known to be shed or secreted, but they sit in other PANTHER subfamilies (SF35, SF99). The fly protein is in SF127 with nematode UDA-1, which is a luminal membrane enzyme. In S2 cells the expressed fly protein is mainly in the ER, and its activity is latent in intact cells. None of the four annotated isoforms has a cleavable signal peptide that would generate a soluble secreted form, and the 271-residue PC/PD product lacks the transmembrane segment entirely. The term is not impossible, but nothing specific to the fly supports it. Supporting Evidence: PMID:19467631 S2 cells stably transfected with the Drosophila NTPDase6 cDNA displayed strong UDPase activity only after addition of NP-40, indicating the intracellular location of the enzyme. PMID:19467631 Fluorescence microscopy revealed that the protein was located primarily in the ER. file:DROME/NTPase/NTPase-bioinformatics/RESULTS-rereview.md is an N-terminally truncated product identical to O76268 residues 191-461. |
| GO:0005737 cytoplasm | IDA PMID:19467631 The single NTPase gene of Drosophila melanogaster encodes an... | MODIFY | Summary: The direct localisation data support endoplasmic reticulum, a more specific part of the cytoplasm. Reason: The same paper that supports this IDA row reports that the protein is located mainly in the ER. Cytoplasm is correct because it includes the ER, but it is less specific than the evidence allows. Endoplasmic reticulum is the supported refinement. A membrane-level term would also fit the single predicted transmembrane helix and the detergent-dependent activity. Proposed replacements: endoplasmic reticulum Supporting Evidence: PMID:19467631 Fluorescence microscopy revealed that the protein was located primarily in the ER. |
| GO:0005794 Golgi apparatus | IEA GO_REF:0000117 | UNDECIDED | Summary: A minor Golgi or pre-Golgi pool is plausible but has not been shown. Reason: The fly enzyme was found mainly in the ER, a wording that does not exclude a minor pool elsewhere. The SF127 co-member UDA-1, expressed in yeast, fractionated with vesicles of density between ER and Golgi. The Golgi assignment itself comes from an ARBA rule that is driven by Golgi-resident family members such as yeast GDA1 and human ENTPD6, which are in other subfamilies. The evidence does not settle whether any of the fly protein resides in the Golgi. Supporting Evidence: PMID:19467631 Fluorescence microscopy revealed that the protein was located primarily in the ER. PMID:15102851 has a lumenal active site in vesicles displaying an intermediate density between those of the ER and GA when expressed in S. cerevisiae |
| GO:0005886 plasma membrane | IEA GO_REF:0000117 | REMOVE | Summary: The direct data argue against a cell-surface location for the fly enzyme. Reason: If the catalytic domain were at the cell surface, intact cells would hydrolyse extracellular UDP. In fact, transfected S2 cells show strong UDPase activity only after detergent permeabilisation, and microscopy places the protein mainly in the ER. Several inhibitors of the cell-surface NTPDases also fail to inhibit the fly enzyme, or inhibit it only partly. The fly protein belongs to the intracellular SF127 subfamily, so the plasma membrane location of the vertebrate ENTPD1-3 and ENTPD8 ectoenzymes should not be transferred to it. Supporting Evidence: PMID:19467631 S2 cells stably transfected with the Drosophila NTPDase6 cDNA displayed strong UDPase activity only after addition of NP-40, indicating the intracellular location of the enzyme. PMID:19467631 It was not or only partially inhibited by several modulators of the cell surface NTPDases |
| GO:0016787 hydrolase activity | IEA GO_REF:0000002 | MODIFY | Summary: The generic hydrolase term should be refined to the demonstrated nucleoside diphosphate phosphatase activity. Reason: The InterPro GDA1/CD39 signature is correct. The substrate data narrow the activity to hydrolysis of nucleoside diphosphates (GDP, UDP and IDP), a term this gene already carries. Proposed replacements: nucleoside diphosphate phosphatase activity Supporting Evidence: PMID:19467631 The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. |
| GO:0017110 nucleoside diphosphate phosphatase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 3.6.1.6 nucleoside diphosphate phosphatase is the core catalytic activity. Reason: The enzyme acts on a set of nucleoside diphosphates (UDP, GDP and IDP), so this term describes the catalytic activity as a whole. The substrate-specific child terms are also accepted. The historical NTPDase or apyrase label overstates its NTP activity, because nucleoside triphosphates were poor substrates. Supporting Evidence: PMID:19467631 The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. |
| GO:0036384 CDP phosphatase activity | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: CDP is not among the preferred substrates of the characterised enzyme. Reason: Direct substrate comparison found UDP, GDP and IDP to be efficient substrates and other nucleoside diphosphates, which includes CDP, to be poor ones. The SF127 co-member UDA-1 likewise hydrolyses UDP and GDP but no other nucleoside diphosphate. Any CDP turnover by the fly enzyme would be a minor side reaction, not its characteristic function. Supporting Evidence: PMID:19467631 The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. |
| GO:0045134 UDP phosphatase activity | IDA PMID:19467631 The single NTPase gene of Drosophila melanogaster encodes an... | ACCEPT | Summary: UDP hydrolysis was shown directly and is a core activity. Reason: UDPase activity appears in transfected S2 cells once membranes are permeabilised, and UDP is among the equally preferred substrates. UDA-1, in the same subfamily, is a UDP/GDPase, which supports UDP hydrolysis as the conserved activity of the subfamily. Supporting Evidence: PMID:19467631 The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. PMID:19467631 S2 cells stably transfected with the Drosophila NTPDase6 cDNA displayed strong UDPase activity only after addition of NP-40, indicating the intracellular location of the enzyme. |
| GO:0045134 UDP phosphatase activity | IEA GO_REF:0000117 | ACCEPT | Summary: Same claim as the IDA row; UDP hydrolysis was shown directly. Reason: The electronic inference agrees with the direct enzymology and is accepted on the strength of that evidence. Supporting Evidence: PMID:19467631 The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. |
| GO:1990003 IDP phosphatase activity | IEA GO_REF:0000117 | ACCEPT | Summary: IDP hydrolysis is supported by the direct substrate comparison. Reason: The cloned enzyme hydrolyses IDP as well as it hydrolyses GDP and UDP. The term is therefore correct despite its electronic provenance. IDP is unlikely to be a major physiological substrate in the ER lumen, but the activity itself is real. Supporting Evidence: PMID:19467631 The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. |
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Download this section (compressed HTML)Q: Does loss of NTPase alter ER UDP/GDP levels, nucleotide-sugar import, or N-glycan processing and reglucosylation in fly tissues such as the salivary gland, where it is strongly expressed?
Q: Is the N-terminally truncated 271-residue PC/PD product, which lacks the transmembrane segment and ACR1, translated and catalytically active, or is it a non-functional gene-model product?
Q: Is fly NTPase, like its nematode subfamily co-member uda-1, induced by the unfolded protein response through Ire1?
Experiment: Tag the endogenous protein to determine its localisation and topology in vivo, and use an NTPase null allele (for example the existing CRISPR sgRNA lines) with ER glycoproteomics and measurements of nucleotide and nucleotide-sugar pools.
Experiment: Express PA, PE, PF and PC/PD separately and compare their activity, localisation, and cross-linking-dependent dimerisation.
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