GstO1 (CG6662) is one of four Omega-class cytosolic glutathione S-transferases encoded in a cluster on chromosome 3L of Drosophila melanogaster, alongside sepia, GstO2 and GstO3. It has the thioredoxin-like GST N-terminal domain with the Omega-class N-terminal extension and an active-site cysteine (Cys30, CPYA motif), and an all-alpha GST C-terminal domain. Like other Omega GSTs, recombinant GstO1 has glutathione-dependent thiol transferase and dehydroascorbate reductase activities and only weak activity toward 1-chloro-2,4-dinitrobenzene, and unlike its paralog sepia it lacks pyrimidodiazepine synthase activity. In larval body-wall muscle GstO1 associates with ER and early/recycling endosome membranes, binds the BMP ligand Glass bottom boat (Gbb) and promotes S-glutathionylation of Gbb at Cys354 and Cys420, which favours Ctrip-dependent ubiquitination and proteasomal degradation of Gbb; loss of GstO1 raises Gbb levels and BMP signalling and causes neuromuscular junction overgrowth. Loss-of-function flies are also hypersensitive to hydrogen peroxide and to ethanol sedation, with neuronal GstO1 required for both responses. Note that the name DmGSTO1 used in some earlier work (e.g. Kim et al. 2012) refers to CG6673, now GstO2.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004364 glutathione transferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference placing glutathione transferase activity at the Omega-class GST node (PTN000883725), seeded by fly and human GSTO experimental annotations including GstO1 itself. Reason: GstO1 is an Omega-class GST (IPR005442) with the Omega N-terminal extension and active-site Cys30 in a CPYA motif. Recombinant CG6662 shows GSH-dependent thiol transferase activity and weak CDNB conjugation, so the inherited activity is confirmed on the target. GstO1 appearing among the IBA sources reflects its own experimental grounding. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro file:DROME/GstO1/GstO1-uniprot.txt DR InterPro; IPR005442; GST_omega. |
| GO:0004364 glutathione transferase activity | IDA PMID:16712527 Identification and characteristics of the structural gene fo... | ACCEPT | Summary: Recombinant CG6662 (one of five Omega GST candidates from the sepia locus) was assayed and had GSH-dependent thiol transferase and DHA reductase activities and low CDNB activity. Reason: Direct enzymatic evidence on the purified GstO1 protein. GO:0004364 covers the GSH-dependent transferase chemistry of Omega GSTs (thiol transferase, CDNB conjugation). Supporting Evidence: PMID:16712527 we identified five candidates, the products of CG6776, CG6781, CG6673A, CG6673B and CG6662 PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro |
| GO:0004364 glutathione transferase activity | IDA PMID:22082028 A preliminary characterization of the cytosolic glutathione ... | ACCEPT | Summary: Proteome-wide expression and kinetic characterisation of the 41 Drosophila cytosolic GSTs, including GstO1. Reason: Only the abstract is cached; FlyBase curators read the full text. The activity is independently confirmed by PMID:16712527, so the annotation is accepted and deferred to the curator. Supporting Evidence: PMID:22082028 The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character even compared with members from the same class. PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro |
| GO:0004364 glutathione transferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation from InterPro GST_omega (IPR005442), ARBA and EC 2.5.1.18 mapping. Reason: Consistent with direct assays of recombinant GstO1 and its Omega-class membership. Supporting Evidence: file:DROME/GstO1/GstO1-uniprot.txt DR InterPro; IPR005442; GST_omega. PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro |
| GO:0004734 pyrimidodiazepine synthase activity | IDA NOT PMID:16712527 Identification and characteristics of the structural gene fo... | ACCEPT | Summary: NOT annotation: recombinant CG6662 did not synthesise PDA (pyrimidodiazepine) from 6-PTP in vitro; only CG6781 (sepia) did. Reason: The negative result is explicit in the paper: all five candidates were assayed and only the CG6781 product made PDA. The NOT annotation usefully separates GstO1 from its paralog sepia. Supporting Evidence: PMID:16712527 Interestingly, only the product of CG6781 had this activity ( Table 2 and Figure 3 C), despite the high level of homology between the primary sequences of the five enzymes. PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that GstO1, like other Omega GSTs, is active in the cytoplasm. Reason: GstO1 is a soluble Omega GST with no predicted signal peptide or transmembrane segment. In larval muscle the endogenous protein is seen on ER and early/recycling endosome membranes, which are cytoplasmic structures, consistent with a cytoplasmic protein associating with their cytosolic face; its substrate Gbb is a secretory-pathway protein. Cytoplasm is therefore correct and as precise as the combined evidence allows. Supporting Evidence: file:DROME/GstO1/GstO1-uniprot.txt DR InterPro; IPR005442; GST_omega. PMID:37389657 These results together show that GstO1 is localized at ER and early/recycling endosomes, but not at late endosomes. |
| GO:0005737 cytoplasm | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from GST_omega (IPR005442) to cytoplasm. Reason: GstO1 is a soluble Omega GST with no predicted signal peptide or transmembrane segment. In larval muscle the endogenous protein is seen on ER and early/recycling endosome membranes, which are cytoplasmic structures, consistent with a cytoplasmic protein associating with their cytosolic face; its substrate Gbb is a secretory-pathway protein. Cytoplasm is therefore correct and as precise as the combined evidence allows. Supporting Evidence: file:DROME/GstO1/GstO1-uniprot.txt DR InterPro; IPR005442; GST_omega. PMID:37389657 These results together show that GstO1 is localized at ER and early/recycling endosomes, but not at late endosomes. |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | ACCEPT | Summary: Transfer from rat Gsto1 (RGD:70952), a cytosolic Omega GST. Reason: GstO1 is a soluble Omega GST with no predicted signal peptide or transmembrane segment. In larval muscle the endogenous protein is seen on ER and early/recycling endosome membranes, which are cytoplasmic structures, consistent with a cytoplasmic protein associating with their cytosolic face; its substrate Gbb is a secretory-pathway protein. Cytoplasm is therefore correct and as precise as the combined evidence allows. Supporting Evidence: file:DROME/GstO1/GstO1-uniprot.txt DR InterPro; IPR005442; GST_omega. PMID:37389657 These results together show that GstO1 is localized at ER and early/recycling endosomes, but not at late endosomes. |
| GO:0006749 glutathione metabolic process | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference of involvement in glutathione metabolism. Reason: GstO1 uses and oxidises GSH in its DHA reductase and thiol transferase reactions, and GstO1 mutant muscle has an increased GSH/GSSG ratio, so involvement in glutathione metabolism is reasonable. GSH is a co-substrate rather than the product of a GstO1-dependent pathway, so this broad metabolic term is peripheral to its core roles in ascorbate recycling and protein glutathionylation. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro PMID:37389657 Increased ratio of GSH/GSSG in GstO1 mutants (GstO16-11/Def) and rescued by postsynaptic overexpression of GstO1. |
| GO:0006749 glutathione metabolic process | IDA PMID:22082028 A preliminary characterization of the cytosolic glutathione ... | KEEP AS NON CORE | Summary: FlyBase curation from the Drosophila cytosolic GST proteome characterisation (abstract only cached). Reason: GstO1 uses and oxidises GSH in its DHA reductase and thiol transferase reactions, and GstO1 mutant muscle has an increased GSH/GSSG ratio, so involvement in glutathione metabolism is reasonable. GSH is a co-substrate rather than the product of a GstO1-dependent pathway, so this broad metabolic term is peripheral to its core roles in ascorbate recycling and protein glutathionylation. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro PMID:37389657 Increased ratio of GSH/GSSG in GstO1 mutants (GstO16-11/Def) and rescued by postsynaptic overexpression of GstO1. PMID:22082028 The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character even compared with members from the same class. |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of glutathione-dependent dehydroascorbate reductase activity at the Omega GST node. Reason: DHAR activity is a hallmark of Omega GSTs and was measured directly for recombinant CG6662. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro PMID:29435097 Furthermore, all isoforms of GSTOs have high thiol transferase and DHA reductase activities, characteristic of GSTOs, and low activity toward 1-chloro-2,4-dinitrobenzene (CDNB), a general GST substrate [27]. |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IDA PMID:16712527 Identification and characteristics of the structural gene fo... | ACCEPT | Summary: Recombinant CG6662 had glutathione-dependent dehydroascorbate reductase activity (DHA reduction followed at 265 nm with GSH). Reason: Direct assay of purified GstO1. Its relative DHAR rate compared with GstO2B (CG6673B) is not available in the cached text, but the activity was clearly detected. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro PMID:29435097 Furthermore, all isoforms of GSTOs have high thiol transferase and DHA reductase activities, characteristic of GSTOs, and low activity toward 1-chloro-2,4-dinitrobenzene (CDNB), a general GST substrate [27]. |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation via ARBA and EC 1.8.5.1 mapping. Reason: Consistent with direct DHAR assays of recombinant GstO1. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro |
| GO:0098869 cellular oxidant detoxification | IEA GO_REF:0000108 | KEEP AS NON CORE | Summary: Inferred from the DHAR molecular function via inter-ontology links. Reason: Regenerating ascorbate supports antioxidant capacity, and GstO1 loss-of-function flies are hypersensitive to H2O2 with neuronal rescue, so a role in oxidant defense is plausible. GstO1 is not shown to reduce H2O2 or other oxidants itself, so this is a peripheral, indirect role. Supporting Evidence: PMID:26024591 Furthermore, we show that GstO1 loss-of-function mutant flies display reduced survival than the control flies when subjected to H2O2 treatment. PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro |
| GO:0010731 protein glutathionylation | IMP PMID:37389657 Gbb glutathionylation promotes its proteasome-mediated degra... | NEW | Summary: GstO1 binds the BMP ligand Gbb in larval muscle and is required and sufficient for Gbb S-glutathionylation at Cys354/Cys420; no Gbb-SSG is detected in GstO1 nulls and overexpression increases it. Reason: GstO1 is the proposed enzyme performing the modification step (a GSH-binding Omega GST that physically associates with its substrate), not merely a required upstream factor; Grx1 acts as the opposing deglutathionylase. The comparator check is satisfied: the paralog GstO2 is annotated to protein glutathionylation for ATP synthase beta. A cell-free assay with recombinant GstO1 was not reported, so the evidence code is IMP rather than IDA. Supporting Evidence: PMID:37389657 We found that Gbb was glutathionylated in WT (Fig. 6 A), but no glutathionylation of Gbb was detected in the absence of GstO1; conversely, Gbb glutathionylation was elevated when GstO1 was overexpressed in postsynaptic muscles compared with WT (Fig. 6 A). PMID:37389657 In third-instar muscles, Gbb was co-IPed with Flag-tagged endogenous GstO1 by anti-Flag but was absent in mouse IgG IPed controls PMID:37389657 These results indicate that GstO1-mediated Gbb glutathionylation promotes its degradation through the proteasomal pathway. file:DROME/GstO1/GstO1-deep-research-manual.md Note that a cell-free assay with recombinant GstO1 was not reported |
| GO:0005783 endoplasmic reticulum | IDA PMID:37389657 Gbb glutathionylation promotes its proteasome-mediated degra... | NEW | Summary: Endogenous GstO1 shows near-perfect co-localisation with the ER marker KDEL in third-instar larval muscle, with partial overlap with Rab5/Rab11 endosomes. Reason: Direct immunolocalisation of the endogenous protein. Only whole-organelle co-staining is available, so the root organelle term is used; the lack of a signal peptide or TM segment suggests association with the cytosolic face, but no assay resolves the sub-compartment. Non-core location: it places the enzyme where its secretory substrate Gbb is trafficked. Supporting Evidence: PMID:37389657 We observed a near-perfect colocalization of GstO1 with the ER marker KDEL, and substantial overlap with the early endosomal marker Rab5 and the recycling endosomal marker Rab11 in third-instar larval muscle cells PMID:37389657 These results together show that GstO1 is localized at ER and early/recycling endosomes, but not at late endosomes. |
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Download this section (compressed HTML)Q: Should GstO1 be annotated to negative regulation of BMP signaling pathway or negative regulation of synapse growth at the neuromuscular junction, given that it acts on the BMP ligand Gbb itself (glutathionylation leading to degradation) rather than on the signalling machinery?
Q: Is the in vitro DHAR and thiol transferase activity of GstO1 comparable to GstO2B, and which of the four Drosophila Omega GSTs carries most DHAR flux in ovary and testis where GstO1 is reported to be highly expressed?
Q: Does Kim et al. 2012 (PMID:22219196) need a FlyBase/UniProt synonym note, since its "DmGSTO1" (CG6673/GstO2) is easily confused with FlyBase GstO1 (CG6662), and GstO1 UniProt synonyms also list DmGSTO1?
Experiment: Incubate purified recombinant GstO1, a C30A active-site mutant and the Gbb C-terminal fragment with GSH (with or without GSSG) and measure Gbb-SSG formation at Cys354/Cys420 by LC-MS/MS.
Hypothesis: GstO1 directly catalyses Gbb S-glutathionylation via its active-site Cys30.
Experiment: Fractionate larval muscle and use protease protection with ER-luminal and cytosolic markers to determine whether GstO1 faces the cytosol or the ER lumen.
Hypothesis: GstO1 is a peripheral protein on the cytosolic face of ER and endosomal membranes.
Glutathione S transferase O1 (GstO1) encodes a transferase involved in glutathione metabolism.
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