Cys-Gly metallodipeptidase of the peptidase M20A family (CNDP2/Dug1 family) in Saccharomyces cerevisiae. Dug1 is a cytoplasmic homodimer that hydrolyses L-cysteinylglycine to L-cysteine and glycine with high specificity, requiring Zn2+ or Mn2+, and is inactive toward tri- and tetrapeptides. It acts at the final step of glutathione degradation in both the gamma-glutamyl cycle (Cys-Gly released by the vacuolar gamma-glutamyltransferase Ecm38) and the fungus-specific DUG pathway, where the (Dug2-Dug3)2 glutamine amidotransferase first cleaves the gamma-glutamyl bond of glutathione. Loss of DUG1 causes Cys-Gly accumulation and prevents use of glutathione or Cys-Gly as sole sulfur source. The S. pombe orthologue and human CNDP2 also show Cys-Gly dipeptidase activity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | HDA PMID:11914276 Subcellular localization of the yeast proteome. | ACCEPT | Summary: Genome-wide epitope-tag immunolocalisation (Kumar et al. 2002) placed Dug1 in the cytoplasm; consistent with the GFP data of Ganguli et al. 2007 and with Dug1 acting on cytosolic Cys-Gly. Supporting Evidence: PMID:11914276 By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm, which suggested that all three proteins were localized to the cytoplasm |
| GO:0005737 cytoplasm | HDA PMID:14562095 Global analysis of protein localization in budding yeast. | ACCEPT | Summary: Huh et al. 2003 GFP-fusion localisation scored Dug1-GFP as cytoplasmic; UniProt cites this study for the cytoplasmic location. Supporting Evidence: PMID:14562095 We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories file:yeast/DUG1/DUG1-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005737 cytoplasm | IDA PMID:17179087 The alternative pathway of glutathione degradation is mediat... | ACCEPT | Summary: Ganguli et al. 2007 imaged functional C-terminal Dug1-GFP by confocal microscopy and observed uniform cytoplasmic fluorescence, the direct experimental basis for the cytoplasmic location of the Cys-Gly dipeptidase. Supporting Evidence: PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm, which suggested that all three proteins were localized to the cytoplasm |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location keyword mapping (SL-0086 Cytoplasm) derived from the experimentally supported UniProt location statement; correct for Dug1. Supporting Evidence: file:yeast/DUG1/DUG1-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005739 mitochondrion | HDA PMID:14562095 Global analysis of protein localization in budding yeast. | KEEP AS NON CORE | Summary: Huh et al. 2003 GFP survey assigned Dug1-GFP a secondary mitochondrial call in addition to cytoplasm. No follow-up study supports a mitochondrial pool, and the dedicated Dug1-GFP imaging of Ganguli et al. reports only uniform cytoplasmic fluorescence. Reason: Dug1 is an abundant soluble protein (~22,300 copies/cell) whose single-protein GFP and immunolocalisation studies show diffuse cytoplasmic signal; mitochondrial detection comes from organellar proteomics where abundant cytosolic proteins are frequent co-purifying contaminants. No mitochondrial targeting signal or mitochondrial function is described. Retained as non-core rather than removed because these are experimental HDA calls whose full data were not inspected. Supporting Evidence: PMID:14562095 We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm, which suggested that all three proteins were localized to the cytoplasm |
| GO:0005739 mitochondrion | HDA PMID:16823961 Toward the complete yeast mitochondrial proteome: multidimen... | KEEP AS NON CORE | Summary: Detection of Dug1 in the PROMITO mitochondrial proteome (Reinders et al. 2006). Mass-spectrometric detection in purified mitochondria does not distinguish matrix proteins from abundant cytosolic contaminants. Reason: Dug1 is an abundant soluble protein (~22,300 copies/cell) whose single-protein GFP and immunolocalisation studies show diffuse cytoplasmic signal; mitochondrial detection comes from organellar proteomics where abundant cytosolic proteins are frequent co-purifying contaminants. No mitochondrial targeting signal or mitochondrial function is described. Retained as non-core rather than removed because these are experimental HDA calls whose full data were not inspected. Supporting Evidence: PMID:16823961 A total of 851 different proteins (PROMITO dataset) were identified by use of multidimensional LC-MS/MS PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm, which suggested that all three proteins were localized to the cytoplasm |
| GO:0005739 mitochondrion | HDA PMID:24769239 Quantitative variations of the mitochondrial proteome and ph... | KEEP AS NON CORE | Summary: Detection of Dug1 in a quantitative proteome of isolated mitochondria grown on different carbon sources (Renvoise et al. 2014); same caveat as other organellar proteomics hits for a highly abundant cytosolic peptidase. Reason: Dug1 is an abundant soluble protein (~22,300 copies/cell) whose single-protein GFP and immunolocalisation studies show diffuse cytoplasmic signal; mitochondrial detection comes from organellar proteomics where abundant cytosolic proteins are frequent co-purifying contaminants. No mitochondrial targeting signal or mitochondrial function is described. Retained as non-core rather than removed because these are experimental HDA calls whose full data were not inspected. Supporting Evidence: PMID:24769239 we performed an overall quantitative proteomic and phosphoproteomic study of isolated mitochondria extracted from yeast grown on fermentative (glucose or galactose) and respiratory (lactate) media PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm, which suggested that all three proteins were localized to the cytoplasm |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt subcellular-location mapping (SL-0173 Mitochondrion) inherited from the PROMITO proteomics citation in the UniProt entry; it carries no evidence beyond the HDA rows. Reason: Dug1 is an abundant soluble protein (~22,300 copies/cell) whose single-protein GFP and immunolocalisation studies show diffuse cytoplasmic signal; mitochondrial detection comes from organellar proteomics where abundant cytosolic proteins are frequent co-purifying contaminants. No mitochondrial targeting signal or mitochondrial function is described. Retained as non-core rather than removed because these are experimental HDA calls whose full data were not inspected. Supporting Evidence: PMID:16823961 A total of 851 different proteins (PROMITO dataset) were identified by use of multidimensional LC-MS/MS |
| GO:0005829 cytosol | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways (PWY3O-114, superpathway of glutathione metabolism / truncated gamma-glutamyl cycle) places the Cys-Gly hydrolysis step in the cytosol. This matches the experimentally observed cytoplasmic Dug1 and its role in cleaving Cys-Gly produced by cytosolic Dug2-Dug3; cytosol is the more precise compartment. Supporting Evidence: PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm, which suggested that all three proteins were localized to the cytoplasm PMID:22277648 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0005829 cytosol | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways glutathione degradation pathway (PWYQT-4432, DUG pathway) places the Dug1 Cys-Gly dipeptidase step in the cytosol; consistent with GFP data. Supporting Evidence: PMID:17179087 all three proteins were observed to have uniform GFP fluorescence throughout the cytoplasm, which suggested that all three proteins were localized to the cytoplasm PMID:22277648 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0006508 proteolysis | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT IBA for proteolysis from the CNDP2/Dug1 node of the M20A peptidase family (mouse Cndp1/Cndp2, human CNDP2). Dug1 hydrolyses a free dipeptide (Cys-Gly) and has no activity on tri- or tetrapeptides; it does not degrade proteins. The broad proteolysis term is technically a parent of peptide-bond hydrolysis but conveys no information about Dug1 biology, which is better captured by glutathione catabolic process. Reason: Dug1 substrate is a dipeptide derived from glutathione, not protein; the informative BP is GO:0006751 glutathione catabolic process. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN001336067 · PTN001336067 SUPPORTS TRANSFER Peptidase function is correctly inherited across the CNDP2/Dug1 clade; the issue is that proteolysis is a poor BP for a free-dipeptide hydrolase. Supporting Evidence: PMID:19346245 We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. PMID:22277648 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0006749 glutathione metabolic process | RCA GO_REF:0000123 | TOO GENERAL | Summary: YeastPathways superpathway of glutathione metabolism (PWY3O-114) assignment. Correct but a broad parent of the specific glutathione catabolic process in which Dug1 catalyses the final step. Reason: Parent of the more informative GO:0006751 glutathione catabolic process. Supporting Evidence: PMID:22277648 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0006751 glutathione catabolic process | IMP PMID:17179087 The alternative pathway of glutathione degradation is mediat... | ACCEPT | Summary: dug1 mutants were isolated as unable to use glutathione as sole sulfur source (DUG = defective in utilization of glutathione), and DUG1 alone is needed to use Cys-Gly and Glu-Cys-Gly. Combined with the later biochemistry, Dug1 performs the Cys-Gly cleavage step of glutathione degradation. Supporting Evidence: PMID:17179087 Although dipeptides and tripeptides with a normal peptide bond such as cys-gly or glu-cys-gly required the presence of only a functional DUG1 gene that encoded a protein belonging to the M20A metallohydrolase family, 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 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0006751 glutathione catabolic process | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways glutathione degradation (DUG pathway, PWYQT-4432) assigns Dug1 the L-cysteinylglycine + H2O -> L-cysteine + glycine step (EC 3.4.13.18). This is the experimentally demonstrated Dug1 reaction. Supporting Evidence: PMID:19346245 We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. PMID:22277648 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0008233 peptidase activity | IBA GO_REF:0000033 | TOO GENERAL | Summary: PAINT IBA for generic peptidase activity at the CNDP2/Dug1 node (with/from includes DUG1 itself and DUG2). True for Dug1, but the specific activity (metallodipeptidase acting on Cys-Gly) is already annotated. Note that the Dug2 paralog in with/from has a catalytically inactive M20A domain (Kaur et al. 2012), so the node-level peptidase term should not be extended to Dug2. Reason: Correct but general parent of GO:0070573 metallodipeptidase activity. Supporting Evidence: PMID:19346245 We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. file:yeast/DUG1/DUG1-uniprot.txt SIMILARITY: Belongs to the peptidase M20A family. |
| GO:0008237 metallopeptidase activity | RCA GO_REF:0000123 | TOO GENERAL | Summary: YeastPathways (PWY3O-114) reaction-to-GO mapping for the Cys-Gly dipeptidase step gives metallopeptidase activity. Correct (Dug1 requires Zn2+ or Mn2+) but less specific than metallodipeptidase activity. Reason: Correct parent of GO:0070573. Supporting Evidence: PMID:19346245 This activity requires zinc or manganese ions. |
| GO:0008242 omega peptidase activity | IGI PMID:17179087 The alternative pathway of glutathione degradation is mediat... | MODIFY | Summary: Omega peptidase activity was inferred from the genetic requirement of DUG1 (together with ECM38 in the with/from) for growth on gamma-glutamyl compounds, when Dug1 was thought to be the catalytic subunit of a GSH degradosome. Later biochemistry showed that purified Dug1 cleaves only Cys-Gly and is inactive on glutathione and other tripeptides, and that the gamma-glutamyl (isopeptide) bond of GSH is cleaved by the (Dug2-Dug3)2 glutamine amidotransferase. Dug1 therefore does not perform an omega-peptidase reaction; its activity is a normal alpha-peptide-bond metallodipeptidase. Reason: The gamma-glutamyl bond cleavage attributed to the DUG pathway is catalysed by Dug2-Dug3; Dug1 acts downstream on Cys-Gly, an alpha-linked dipeptide. Proposed replacements: metallodipeptidase activity Supporting Evidence: PMID:19346245 We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. PMID:22277648 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0008242 omega peptidase activity | IGI PMID:17179087 The alternative pathway of glutathione degradation is mediat... | MODIFY | Summary: Same omega-peptidase inference from 17179087, here with MET17 (met15 genetic background) in with/from. The genetic data establish a requirement for DUG1 in using glutathione as sulfur source, not that Dug1 hydrolyses the gamma-glutamyl bond; Dug1 is a Cys-Gly-specific metallodipeptidase. Reason: Superseded by direct biochemistry showing Dug1 is a Cys-Gly (alpha-peptide) dipeptidase and Dug2-Dug3 performs the gamma-glutamyl cleavage. Proposed replacements: metallodipeptidase activity Supporting Evidence: PMID:19346245 We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. PMID:22277648 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0008270 zinc ion binding | RCA PMID:30358795 The cellular economy of the Saccharomyces cerevisiae zinc pr... | KEEP AS NON CORE | Summary: Bioinformatic zinc-proteome prediction (Wang et al. 2018) lists Dug1 as a zinc-binding protein via its M20A metallopeptidase domain. Purified Dug1 is active with Zn2+ or Mn2+, so metal binding is real but is a cofactor aspect of the catalytic activity. Reason: Cofactor binding supporting the metallodipeptidase activity; not a separate function. Supporting Evidence: PMID:30358795 The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified using a bioinformatics analysis that combined global domain searches with local motif searches. PMID:19346245 This activity requires zinc or manganese ions. file:yeast/DUG1/DUG1-uniprot.txt Name=Zn(2+); Xref=ChEBI:CHEBI:29105; |
| GO:0016787 hydrolase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro IPR002933 (peptidase M20) mapping to hydrolase activity; correct but uninformative relative to metallodipeptidase activity. Reason: Very general parent of the core MF. Supporting Evidence: file:yeast/DUG1/DUG1-uniprot.txt SIMILARITY: Belongs to the peptidase M20A family. |
| GO:0032991 protein-containing complex | RCA GO_REF:0000123 | MARK AS OVER ANNOTATED | Summary: YeastPathways DUG pathway (PWYQT-4432) models a Dug1-Dug2-Dug3 degradosomal complex, based on yeast two-hybrid interactions in Ganguli et al. 2007. Co-purification later found Dug1-Dug1 and Dug2-Dug2 homodimers with no Dug1-Dug2 heterodimer, and the active GSH-cleaving enzyme is (Dug2-Dug3)2; Dug1 acts separately on the released Cys-Gly. Dug1 is a homodimer, so membership of a protein-containing complex is trivially true, but the pathway-implied heteromeric complex is not supported. Reason: The DUG degradosome membership on which this RCA row is based was not confirmed biochemically; the only established Dug1 complex is its homodimer. Supporting Evidence: PMID:22277648 Co-expression and purification of both Dug1p and Dug2p, each of which have a single dimerization domain, reveals that the dominant population consists of Dug1p-Dug1p and Dug2p-Dug2p homodimer, with no detectable heterodimer. PMID:22277648 Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. |
| GO:0042802 identical protein binding | IPI PMID:22277648 Glutathione degradation by the alternative pathway (DUG path... | KEEP AS NON CORE | Summary: Co-purification and yeast two-hybrid data in Kaur et al. 2012 show Dug1 forms Dug1-Dug1 homodimers via its M20A dimerization domain. Correct, structural property of the enzyme rather than its function. Reason: Homodimerisation is a structural feature of the M20A dipeptidase. Supporting Evidence: PMID:22277648 Dug1p, an M20A peptidase, exists as a homodimer in solution PMID:22277648 Co-expression and purification of both Dug1p and Dug2p, each of which have a single dimerization domain, reveals that the dominant population consists of Dug1p-Dug1p and Dug2p-Dug2p homodimer, with no detectable heterodimer. |
| GO:0042802 identical protein binding | IPI PMID:31454312 The role of structural pleiotropy and regulatory evolution i... | KEEP AS NON CORE | Summary: High-throughput DHFR protein-fragment complementation survey of homomers and heteromers of paralogs (Marchant et al. 2019) detected Dug1 self-interaction; consistent with the known Dug1 homodimer. Reason: Structural homodimerisation; consistent with biochemical data. Supporting Evidence: PMID:31454312 Using yeast as a model, we find that heteromerization is frequent among duplicated homomers and correlates with functional similarity between paralogs. file:yeast/DUG1/DUG1-uniprot.txt SUBUNIT: Homodimer. |
| GO:0070573 metallodipeptidase activity | IDA PMID:19346245 Dug1p Is a Cys-Gly peptidase of the gamma-glutamyl cycle of ... | ACCEPT | Summary: Kaur et al. 2009 purified recombinant Dug1 and showed Zn2+/Mn2+-dependent dipeptidase activity highly specific for Cys-Gly (Km 0.4-0.8 mM), with no activity on tri- or tetrapeptides. This is the defining activity of Dug1. Supporting Evidence: PMID:19346245 We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. PMID:19346245 This activity requires zinc or manganese ions. |
| GO:0070573 metallodipeptidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: ARBA00085163/InterPro IPR017153 (CNDP2/Dug1 family) mapping to metallodipeptidase activity; matches the experimental activity. Supporting Evidence: file:yeast/DUG1/DUG1-uniprot.txt dipeptidase with high specificity for Cys-Gly and no activity toward PMID:19346245 Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. |
| GO:0070573 metallodipeptidase activity | IMP PMID:19346245 Dug1p Is a Cys-Gly peptidase of the gamma-glutamyl cycle of ... | ACCEPT | Summary: dug1 deletion cells accumulate Cys-Gly, showing that Dug1 is the principal in vivo Cys-Gly dipeptidase; supports the metallodipeptidase activity. Supporting Evidence: PMID:19346245 Yeast cells lacking Dug1p (dug1Delta) accumulate Cys-Gly. PMID:19346245 We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. |
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Download this section (compressed HTML)Q: Does Dug1 form any transient functional assembly with (Dug2-Dug3)2 in vivo (substrate channelling of Cys-Gly), given that yeast two-hybrid detected a weak Dug1-Dug2 interaction but co-purification did not?
Q: How does vacuolar Cys-Gly produced by Ecm38 reach cytosolic Dug1, or is there a separate vacuolar Cys-Gly dipeptidase?
Experiment: Measure vacuolar and cytosolic Cys-Gly pools in dug1, ecm38 and dug1 ecm38 strains under nitrogen starvation, using fractionation and LC-MS.
Hypothesis: Dug1 is the sole Cys-Gly dipeptidase acting on Cys-Gly derived from vacuolar glutathione turnover.
Cys-Gly metallo-di-peptidase; forms a complex with Dug2p and 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); human homolog CNDP2 can complement yeast dug1 mutant
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