Dug2 (YBR281C) is the non-catalytic scaffold subunit of the fungal-specific Dug2/Dug3 glutathione hydrolase of Saccharomyces cerevisiae (the DUG, "defective in utilization of glutathione", pathway). It has an N-terminal WD40 repeat region that binds the glutamine amidotransferase-type subunit Dug3 and a C-terminal M20A peptidase-like domain that mediates homodimerization but has no peptidase activity. The cytosolic (Dug2-Dug3)2 complex cleaves the gamma-glutamyl bond of glutathione (and gamma-Glu-Cys) to glutamate and Cys-Gly, which the Dug1 dipeptidase then hydrolyses, allowing glutathione to be used as a sulfur and nitrogen source. DUG2 is derepressed under sulfur limitation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:11283351 A comprehensive two-hybrid analysis to explore the yeast pro... | REMOVE | Summary: Ito et al. comprehensive yeast two-hybrid screen detected the Dug2-Dug3 interaction. The interaction is real and functionally essential - Dug2 binds Dug3 through its WD40 domain to form the (Dug2-Dug3)2 glutathione hydrolase - but protein binding is uninformative; the relationship is better captured by part_of GO:0061672 glutathione hydrolase complex and contributes_to GO:0036374. Reason: Bare protein binding carries no functional information; removal does not dispute the interaction, which is represented by part_of GO:0061672 and contributes_to GO:0036374. Supporting Evidence: PMID:22277648 The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. PMID:22277648 Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. |
| GO:0005515 protein binding | IPI PMID:16429126 Proteome survey reveals modularity of the yeast cell machine... | REMOVE | Summary: Gavin et al. TAP-MS proteome survey (abstract-only cache) detected the Dug2-Dug3 interaction. The interaction is real and functionally essential - Dug2 binds Dug3 through its WD40 domain to form the (Dug2-Dug3)2 glutathione hydrolase - but protein binding is uninformative; the relationship is better captured by part_of GO:0061672 glutathione hydrolase complex and contributes_to GO:0036374. Reason: Bare protein binding carries no functional information; removal does not dispute the interaction, which is represented by part_of GO:0061672 and contributes_to GO:0036374. Supporting Evidence: PMID:22277648 The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. PMID:22277648 Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. |
| GO:0005515 protein binding | IPI PMID:22277648 Glutathione degradation by the alternative pathway (DUG path... | REMOVE | Summary: Kaur et al. (two-hybrid domain mapping and co-purification) detected the Dug2-Dug3 interaction. The interaction is real and functionally essential - Dug2 binds Dug3 through its WD40 domain to form the (Dug2-Dug3)2 glutathione hydrolase - but protein binding is uninformative; the relationship is better captured by part_of GO:0061672 glutathione hydrolase complex and contributes_to GO:0036374. Reason: Bare protein binding carries no functional information; removal does not dispute the interaction, which is represented by part_of GO:0061672 and contributes_to GO:0036374. Supporting Evidence: PMID:22277648 The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. PMID:22277648 Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. |
| GO:0005515 protein binding | IPI PMID:37968396 The social and structural architecture of the yeast protein ... | REMOVE | Summary: The 2023 yeast interactome study (Michaelis et al.) detected the Dug2-Dug3 interaction. The interaction is real and functionally essential - Dug2 binds Dug3 through its WD40 domain to form the (Dug2-Dug3)2 glutathione hydrolase - but protein binding is uninformative; the relationship is better captured by part_of GO:0061672 glutathione hydrolase complex and contributes_to GO:0036374. Reason: Bare protein binding carries no functional information; removal does not dispute the interaction, which is represented by part_of GO:0061672 and contributes_to GO:0036374. Supporting Evidence: PMID:22277648 The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. PMID:22277648 Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. |
| GO:0005634 nucleus | HDA PMID:14562095 Global analysis of protein localization in budding yeast. | KEEP AS NON CORE | Summary: Huh GFP survey reported cytoplasmic and nuclear signal. Dedicated localization (Ganguli et al.) showed uniform cytoplasmic fluorescence; no nuclear role is known for Dug2. Supporting Evidence: file:yeast/DUG2/DUG2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Nucleus. PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt SubCell mapping (nucleus) from the Huh survey; retained as non-core. Supporting Evidence: file:yeast/DUG2/DUG2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Nucleus. |
| GO:0005737 cytoplasm | HDA PMID:14562095 Global analysis of protein localization in budding yeast. | ACCEPT | Summary: Huh GFP survey: cytoplasm; consistent with dedicated localization and cytosolic GSH degradation. Supporting Evidence: file:yeast/DUG2/DUG2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Nucleus. |
| GO:0005737 cytoplasm | IDA PMID:17179087 The alternative pathway of glutathione degradation is mediat... | ACCEPT | Summary: Ganguli et al. imaged Dug2-GFP (and Dug1/Dug3-GFP) with uniform cytoplasmic fluorescence. Supporting Evidence: PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SubCell mapping (cytoplasm). Supporting Evidence: file:yeast/DUG2/DUG2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Nucleus. |
| GO:0006508 proteolysis | IBA GO_REF:0000033 | REMOVE | Summary: IBA from a PANTHER node (PTN001336067) of active M20 dipeptidases (mouse/human CNDP2-type donors). Purified Dug2 has no peptidase activity; the Dug2/Dug3 complex hydrolyses the gamma-glutamyl bond of the small-molecule thiol glutathione, which is not proteolysis. Reason: Peptidase activity of the M20A domain is lost in Dug2 (direct assay), and the complex substrate is not a protein. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN001336067 SUPPORTS SOURCE BUT NOT TARGET Donors are catalytically active M20 dipeptidases; Dug2 M20A domain is catalytically inactive and serves for dimerization. Supporting Evidence: PMID:22277648 Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. PMID:22277648 purified Dug2p showed negligible cleavage of Cys-Gly peptide (only 4%) |
| GO:0006751 glutathione catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN005154692 (fungal Dug2 clade) with Dug2 itself as donor. Core process. Supporting Evidence: PMID:17179087 the presence of an unusual peptide bond such as in the dipeptide, gamma-glu-cys, or in GSH, required the participation of the DUG2 and DUG3 gene products as well. PMID:22277648 In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. |
| GO:0006751 glutathione catabolic process | IDA PMID:22277648 Glutathione degradation by the alternative pathway (DUG path... | ACCEPT | Summary: In vitro reconstitution shows Dug2 is required with Dug3 for cleavage of glutathione to glutamate and Cys-Gly. Dug2 is a structural component of the hydrolase complex, so it does part of the work of the process. Supporting Evidence: PMID:22277648 In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. |
| GO:0006751 glutathione catabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro IPR017149 (GSH degradosome Dug2) mapping; family-specific and correct. Supporting Evidence: PMID:17179087 the presence of an unusual peptide bond such as in the dipeptide, gamma-glu-cys, or in GSH, required the participation of the DUG2 and DUG3 gene products as well. |
| GO:0006751 glutathione catabolic process | IMP PMID:17179087 The alternative pathway of glutathione degradation is mediat... | ACCEPT | Summary: dug2 mutants cannot use glutathione or gamma-Glu-Cys as sulfur source while normal-bond peptides still require only DUG1. Supporting Evidence: PMID:17179087 the presence of an unusual peptide bond such as in the dipeptide, gamma-glu-cys, or in GSH, required the participation of the DUG2 and DUG3 gene products as well. |
| GO:0006751 glutathione catabolic process | IMP PMID:22277648 Glutathione degradation by the alternative pathway (DUG path... | ACCEPT | Summary: Kaur et al. mutational and genetic analysis of DUG2 domains in GSH utilization (WD40 required for Dug3 binding). Supporting Evidence: PMID:22277648 The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. PMID:22277648 In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. |
| GO:0008233 peptidase activity | IBA GO_REF:0000033 | REMOVE | Summary: IBA from PANTHER M20 dipeptidase node (PTN001336067). Directly contradicted: purified Dug2 shows negligible Cys-Gly cleavage and "has no peptidase activity". Reason: Catalytic loss of the M20A domain in Dug2 demonstrated biochemically. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS Sources checked: PANTHER:PTN001336067 SUPPORTS SOURCE BUT NOT TARGET Active CNDP2/Dug1-type dipeptidase donors; the Dug2 M20A domain is inactive. Supporting Evidence: PMID:22277648 Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. PMID:22277648 purified Dug2p showed negligible cleavage of Cys-Gly peptide (only 4%) |
| GO:0008233 peptidase activity | IEA GO_REF:0000117 | REMOVE | Summary: ARBA machine-learning rule assigns peptidase activity from the M20 domain signature; contradicted by direct assay of purified Dug2. Reason: Domain-based transfer to a catalytically inactive M20A domain. Supporting Evidence: PMID:22277648 Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. PMID:22277648 purified Dug2p showed negligible cleavage of Cys-Gly peptide (only 4%) |
| GO:0008233 peptidase activity | IGI PMID:17179087 The alternative pathway of glutathione degradation is mediat... | MODIFY | Summary: IGI (with MET17 sulfur-auxotroph background) from the GSH utilization screen. The genetic result supports a role in GSH cleavage, but Dug2 by itself has no peptidase activity; it contributes to the glutathione gamma-glutamate hydrolase activity of the Dug2/Dug3 complex. The replacement should carry the contributes_to qualifier. Reason: Generic peptidase activity attributed to a non-catalytic subunit; later biochemistry localises catalysis to the Dug2/Dug3 complex (Dug3 Cys2). Proposed replacements: glutathione gamma-glutamate hydrolase Supporting Evidence: PMID:17179087 the presence of an unusual peptide bond such as in the dipeptide, gamma-glu-cys, or in GSH, required the participation of the DUG2 and DUG3 gene products as well. PMID:22277648 In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. PMID:22277648 Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. |
| GO:0008242 omega peptidase activity | IGI PMID:17179087 The alternative pathway of glutathione degradation is mediat... | MODIFY | Summary: IGI with ECM38 (GGT): in ecm38 cells, GSH utilization depends on DUG2. The gamma-glutamyl bond cleavage is real but is an activity of the Dug2/Dug3 complex, to which Dug2 contributes; the specific term is GO:0036374 (a child of omega peptidase), with contributes_to qualifier. Reason: Dug2 alone lacks peptidase activity; replace with the specific complex activity as contributes_to. Proposed replacements: glutathione gamma-glutamate hydrolase Supporting Evidence: PMID:17179087 the presence of an unusual peptide bond such as in the dipeptide, gamma-glu-cys, or in GSH, required the participation of the DUG2 and DUG3 gene products as well. PMID:22277648 In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. PMID:22277648 Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. |
| GO:0008242 omega peptidase activity | IGI PMID:17179087 The alternative pathway of glutathione degradation is mediat... | MODIFY | Summary: IGI with MET17 (sulfur auxotrophy background for GSH-as-sulfur assays). Same reasoning: activity belongs to the Dug2/Dug3 complex; Dug2 contributes. Reason: Dug2 alone lacks peptidase activity; replace with the specific complex activity as contributes_to. Proposed replacements: glutathione gamma-glutamate hydrolase Supporting Evidence: PMID:17179087 the presence of an unusual peptide bond such as in the dipeptide, gamma-glu-cys, or in GSH, required the participation of the DUG2 and DUG3 gene products as well. PMID:22277648 In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. PMID:22277648 Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. |
| GO:0008270 zinc ion binding | RCA PMID:30358795 The cellular economy of the Saccharomyces cerevisiae zinc pr... | UNDECIDED | Summary: Zinc proteome RCA (bioinformatic prediction from the M20 domain; abstract-only cache). UniProt annotates the M20 Zn ligands by similarity, but Dug2 M20A domain is catalytically inactive and metal binding has not been tested; cannot judge whether the metal sites are occupied. Supporting Evidence: file:yeast/DUG2/DUG2-uniprot.txt Note=Binds 2 Zn(2+) ions per subunit. {ECO:0000250}; |
| GO:0016787 hydrolase activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro IPR002933 (Peptidase M20) mapping to hydrolase activity; the Dug2 M20A domain has no peptidase activity. The hydrolase activity of the complex is captured by contributes_to GO:0036374. Reason: Catalytically inactive M20A domain in Dug2. Supporting Evidence: PMID:22277648 Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. |
| GO:0034399 nuclear periphery | IDA PMID:25817432 Cmr1/WDR76 defines a nuclear genotoxic stress body linking g... | KEEP AS NON CORE | Summary: Gallina et al. screened GFP-fusion proteins for relocalization to the Cmr1 nuclear-periphery stress body (INQ) under genotoxic stress; Dug2 is not discussed in the cached main text, presumably a screen hit. Stress-induced relocalization unrelated to GSH degradation; retained as non-core, deferring to the curator. |
| GO:0036374 glutathione gamma-glutamate hydrolase | IDA PMID:22277648 Glutathione degradation by the alternative pathway (DUG path... | ACCEPT | Summary: Core activity (contributes_to). Purified Dug2 and Dug3 together, but neither alone, cleave GSH into glutamate and Cys-Gly; the (Dug2-Dug3)2 complex has Km 1.2 mM for GSH. Dug3 Cys2 is the nucleophile; Dug2 is the essential partner. Supporting Evidence: PMID:22277648 In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. PMID:22277648 we could also demonstrate that the products of the reaction were glutamate and Cys-Gly. |
| GO:0042802 identical protein binding | IPI PMID:22277648 Glutathione degradation by the alternative pathway (DUG path... | KEEP AS NON CORE | Summary: Dug2 homodimerizes through its M20A domain, underlying the (Dug2-Dug3)2 stoichiometry. Real but uninformative as an MF. Supporting Evidence: PMID:22277648 Dug2p was also shown to be able to homodimerize, and this was mediated by its M20A peptidase domain. |
| GO:0061672 glutathione hydrolase complex | IDA PMID:22277648 Glutathione degradation by the alternative pathway (DUG path... | ACCEPT | Summary: Gel filtration purified a Dug2-Dug3 complex ((Dug2-Dug3)2 and higher oligomers) that cleaves GSH; this is the defining complex of GO:0061672. Supporting Evidence: PMID:22277648 Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. PMID:22277648 In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. |
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Download this section (compressed HTML)Q: What structural role does the inactive M20A domain of Dug2 play beyond dimerization - does it bind GSH or position the gamma-glutamyl bond for the Dug3 nucleophile?
Q: Does the Dug1-Dug2 interaction channel Cys-Gly from the Dug2/Dug3 complex to Dug1 in vivo?
Experiment: Solve a cryo-EM structure of (Dug2-Dug3)2 with GSH or a non-hydrolysable analogue, and test M20A pocket mutants for Km effects.
Hypothesis: The Dug2 M20A domain contributes substrate binding rather than catalysis.
Component of glutamine amidotransferase (GATase II); forms a complex with Dug3p to degrade glutathione (GSH) and other peptides containing a gamma-glu-X bond in an alternative pathway to GSH degradation by gamma-glutamyl transpeptidase (Ecm38p)
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