GGP1 (gamma-glutamyl peptidase 1, At4g30530) is a cytosolic class-I glutamine amidotransferase-like (peptidase C26 family) cysteine hydrolase that cleaves the gamma-glutamyl isopeptide bond of glutathione (GSH) and of GSH S-conjugates, releasing L-glutamate and the corresponding cysteinylglycine species. In the core glucosinolate pathway it processes the S-(alkyl/aryl-acetohydroximoyl)-GSH conjugates formed after GSH donates the thioglucoside sulfur, producing Cys-Gly conjugates upstream of the C-S lyase SUR1; the exact further processing and substrate handoff are not fully resolved; it acts redundantly with the tandem paralog GGP3 on aliphatic, indolic and aromatic conjugates. It performs the same reaction on GS-IAN in biosynthesis of the indole phytoalexin camalexin. GGP1 also hydrolyzes free reduced GSH (but not GSSG) with a Km in the physiological cytosolic range and is highly expressed in rosette leaves, so it contributes to cytosolic GSH turnover and sulfur reallocation. Crystal structures show a Cys100/His192 catalytic dyad that forms a covalent gamma-glutamyl-enzyme intermediate.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | ISM GO_REF:0000122 | MODIFY | Summary: The supported cytoplasmic compartment is the cytosol. Reason: Refine the true general cytoplasm location to cytosol, already supported elsewhere in this review. This refinement does not exclude distinct secondary compartments reported under other terms. Proposed replacements: cytosol Supporting Evidence: PMID:21712415 which showed that both protein fusions were cytosolic |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of cytosolic activity, grounded in the IDA data for GGP1 itself and its paralog GGP3 (the target appearing among its own donors is expected, not circular). Reason: Both GGP1 and GGP3 are cytosolic by YFP fusion, and the cytosol is where the glucosinolate/camalexin GSH-conjugate processing and GSH turnover take place. Supporting Evidence: PMID:21712415 GGP1 and GGP3 are cytosolic enzymes metabolizing GSH conjugates in the biosynthesis of both glucosinolates and camalexin in Arabidopsis |
| GO:0005829 cytosol | IDA PMID:21712415 Cytosolic Ξ³-glutamyl peptidases process glutathione conjugat... | ACCEPT | Summary: C-terminal YFP fusions of GGP1 and GGP3 transiently expressed in N. benthamiana leaves were cytosolic by confocal microscopy. Reason: Direct experimental localization; core location of GGP1 activity. Supporting Evidence: PMID:21712415 The subcellular localization of GGP1 and GGP3 was investigated using C-terminal yellow fluorescent protein (YFP) fusions transiently expressed in leaves of N. benthamiana using Agrobacterium . The YFP fluorescence was visualized 7 d after infiltration using confocal laser scanning microscopy (CLSM), which showed that both protein fusions were cytosolic |
| GO:0005829 cytosol | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic cytosol annotation, consistent with experimental evidence. Reason: Agrees with the IDA cytosol annotation from PMID:21712415. Supporting Evidence: PMID:21712415 which showed that both protein fusions were cytosolic |
| GO:0005886 plasma membrane | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | MARK AS OVER ANNOTATED | Summary: The microsome experiment does not independently establish plasma membrane localization. Reason: Full-text inspection of PMID:28887381 shows that washed microsomes were profiled by chromatography and sucrose gradients, while subcellular labels were taken from SUBAcon and prior GFP data. The authors explicitly report that no organelle occupied a single gradient cluster. Detection or membrane association is meaningful, but assigning GGP1 specifically to plasma membrane exceeds the spatial resolution of this experiment. This is not a claim that the detected protein is absent or that all additional localization is impossible; the unsupported compartment assignment, rather than the proteomic observation, is marked as over-annotated. Supporting Evidence: PMID:28887381 Over 150 proteins had a complicated localization pattern, and were clearly partitioned between cytosolic and membrane-associated pools. PMID:21712415 which showed that both protein fusions were cytosolic PMID:28887381 The proteins with known localizations had scattered distributions across the entire gradient and no organelle fell in a single cluster. PMID:28887381 We used SUBAcon localization predictions to analyze oligomerization as a function of subcellular localization (Fig. 7C). |
| GO:0008233 peptidase activity | IDA PMID:21712415 Cytosolic Ξ³-glutamyl peptidases process glutathione conjugat... | MODIFY | Summary: Purified recombinant His-tagged GGP1 hydrolyzed nine of ten glucosinolate-related GSH conjugates and GS-IAN to the corresponding Cys-Gly conjugates in vitro. Reason: The activity demonstrated is specifically cleavage of the gamma-glutamyl isopeptide bond of GSH conjugates, which is exactly GO:0034722 gamma-glutamyl-peptidase activity; the generic parent is correct but uninformative for an IDA of this precision. Proposed replacements: gamma-glutamyl-peptidase activity Supporting Evidence: PMID:21712415 The assays with the extracts showed that both GGP1 and GGP3 were able to metabolize nine out of 10 GSH conjugates to the corresponding enzymatic products (Cys-Gly conjugates) |
| GO:0008233 peptidase activity | IEA GO_REF:0000117 | MODIFY | Summary: ARBA electronic peptidase annotation. Correct at a general level. Reason: GGP1 is a peptidase C26-family hydrolase whose demonstrated activity is specifically gamma-glutamyl isopeptide bond cleavage; for consistency with the IDA row, the informative child term GO:0034722 is proposed. Proposed replacements: gamma-glutamyl-peptidase activity Supporting Evidence: PMID:41176694 During glutathione (GSH) turnover, GGP1 hydrolyzes the isopeptide bond of GSH to degrade the tripeptide into glutamate and cysteinylglycine. |
| GO:0010120 camalexin biosynthetic process | IMP PMID:23449503 Assigning gene function in biosynthetic pathways: camalexin ... | ACCEPT | Summary: ggp1 knockdown leaves make less camalexin and accumulate GS-IAN, and recombinant GGP1 converts GS-IAN to Cys(IAN)-Gly; GGP1 catalyzes the gamma-glutamyl cleavage step of the camalexin pathway in the cytosol. Reason: GGP1 performs a catalytic step of camalexin biosynthesis (not merely a necessity phenotype), with both mutant and in vitro enzyme evidence. It is a real but secondary role relative to glucosinolate biosynthesis and GSH turnover. Supporting Evidence: PMID:23449503 ggp1 mutants have reduced end-product (camalexin) levels and accumulate the expected intermediates PMID:21712415 we conclude that GGP1 and GGP3 are enzymes metabolizing GS-IAN in the camalexin pathway |
| GO:0010494 cytoplasmic stress granule | IDA PMID:30664249 Protein and metabolite composition of Arabidopsis stress gra... | KEEP AS NON CORE | Summary: GGP1 was among proteins identified by proteomics of Rbp47b-GFP affinity-purified stress granules; the study reports that many metabolic enzymes are sequestered in stress granules under stress. Reason: Stress-condition sequestration of a cytosolic enzyme in stress granules is plausible but does not reflect where GGP1 acts; abstract-only cache, so the annotation is kept as non-core rather than challenged. Supporting Evidence: PMID:30664249 the remaining proteins were enriched in key enzymes and regulators |
| GO:0019760 glucosinolate metabolic process | IDA PMID:19483696 Glucosinolate engineering identifies a gamma-glutamyl peptid... | MODIFY | Summary: In engineered benzylglucosinolate production in N. benthamiana, co-expression of GGP1 metabolized the accumulating GSH conjugate and substantially increased glucosinolate yield; recombinant GGP1 hydrolyzed phenylacetohydroximoyl-GSH. Reason: GGP1 catalyzes a biosynthetic step (GSH-conjugate processing upstream of the C-S lyase SUR1), so the more specific glucosinolate biosynthetic process is the appropriate term. Proposed replacements: glucosinolate biosynthetic process Supporting Evidence: PMID:19483696 gamma-glutamyl peptidase 1 (GGP1), which substantially increased glucosinolate production by metabolizing an accumulating glutathione conjugate |
| GO:0019760 glucosinolate metabolic process | IEA GO_REF:0000117 | MODIFY | Summary: ARBA electronic annotation to glucosinolate metabolism; correct at a general level. Reason: GGP1 catalyzes a biosynthetic step, so the child term glucosinolate biosynthetic process is proposed, consistent with the experimental rows. Proposed replacements: glucosinolate biosynthetic process Supporting Evidence: PMID:21712415 GGP1 and GGP3 are cytosolic enzymes metabolizing GSH conjugates in the biosynthesis of both glucosinolates and camalexin in Arabidopsis |
| GO:0019760 glucosinolate metabolic process | IMP PMID:21712415 Cytosolic Ξ³-glutamyl peptidases process glutathione conjugat... | MODIFY | Summary: The ggp1-1 ggp3-1 double knockdown has an altered glucosinolate profile and accumulates up to ten glucosinolate-related GSH conjugates, the expected GGP substrates. Reason: Accumulation of pathway intermediates identifies GGP1 as a biosynthetic enzyme; glucosinolate biosynthetic process is the more precise term. Proposed replacements: glucosinolate biosynthetic process Supporting Evidence: PMID:21712415 an Arabidopsis thaliana mutant impaired in the production of the Ξ³-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates |
| GO:0034722 gamma-glutamyl-peptidase activity | IDA PMID:19483696 Glucosinolate engineering identifies a gamma-glutamyl peptid... | ACCEPT | Summary: Core molecular function. GGP1 hydrolyzes the gamma-glutamyl isopeptide bond of GSH conjugates (Km 37 uM for phenylacetohydroximoyl-GSH per UniProt) and of free GSH, releasing glutamate and the Cys-Gly species. Reason: Directly demonstrated for the benzylglucosinolate GSH conjugate, extended to aliphatic/indolic conjugates, GS-IAN and GSH in later studies; structural data show a Cys100 covalent gamma-glutamyl intermediate. Supporting Evidence: PMID:19483696 gamma-glutamyl peptidase 1 (GGP1), which substantially increased glucosinolate production by metabolizing an accumulating glutathione conjugate PMID:41176694 The intermediate structure, in which Ξ³-Glu is covalently linked to the catalytic nucleophile cysteine (C100), was trapped by mutating the catalytic histidine to asparagine (H192N). |
| GO:0099503 secretory vesicle | HDA PMID:25293756 A proteomic strategy for global analysis of plant protein co... | MARK AS OVER ANNOTATED | Summary: Soluble-complex profiling does not establish secretory-vesicle localization. Reason: The full text of PMID:25293756 describes size-exclusion profiling of a crude cytosolic fraction to measure native protein-complex sizes, not purification of secretory vesicles or target-specific vesicle imaging. Independent GGP1/GGP3 fusion imaging in PMID:21712415 is cytosolic. Consequently, the secretory-vesicle assignment exceeds what these experiments establish; possible transient membrane recruitment is not excluded, and the detected protein is not declared a contaminant. Supporting Evidence: PMID:25293756 Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins PMID:21712415 which showed that both protein fusions were cytosolic |
| GO:0006751 glutathione catabolic process | IDA PMID:35932489 Glutathione degradation activity of Ξ³-glutamyl peptidase 1 m... | NEW | Summary: Proposed new annotation. GGP1 complements a yeast GSH-degradation assay, recombinant GGP1 hydrolyzes reduced GSH (not GSSG) with a Km (5 mM) matching cytosolic GSH concentrations, and ggp1 mutants constantly accumulate more GSH. Reason: GGP1 itself catalyzes the committing hydrolysis of GSH to glutamate and Cys-Gly, so it performs a step of GSH catabolism (participation test passes, not merely necessity). Comparator check - the other Arabidopsis enzymes that cleave GSH in its degradation (GGCT2;1, GGCT2;2, GGCT2;3, GGT1-GGT4) carry GO:0006751 in QuickGO; This direct catalytic evidence supports the existing proposal without assuming why GOA lacks the term. Supporting Evidence: PMID:35932489 Recombinant proteins of GGP1, as well as GGP3, showed high degradation activity of GSH, but not of oxidized glutathione (GSSG), in vitro. Notably, the GGP1 transcripts were highly abundant in rosette leaves, in agreement with the ggp1 mutants constantly accumulating more GSH regardless of nutritional conditions. PMID:41176694 During glutathione (GSH) turnover, GGP1 hydrolyzes the isopeptide bond of GSH to degrade the tripeptide into glutamate and cysteinylglycine. |
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Download this section (compressed HTML)Q: In vivo, which fraction of GGP1 flux goes to free GSH turnover versus glucosinolate/camalexin GSH conjugates, and does substrate competition in the cytosol (millimolar GSH vs micromolar conjugates) limit glucosinolate throughput?
Q: Which carboxypeptidase (if any) removes glycine from the Cys-Gly conjugates in the glucosinolate and camalexin pathways, or does SUR1 act directly on Cys-Gly conjugates?
Q: Does the redox-sensitive Cys100-Cys154 disulfide observed in crystals regulate GGP1 activity in planta under oxidative stress?
Experiment: Quantify glucosinolate-related GSH conjugates, Cys-Gly conjugates and free GSH in ggp1, ggp3 and ggp1 ggp3 null (CRISPR) lines across sulfur supply to partition GGP1 flux between primary and secondary sulfur metabolism.
Experiment: Test recombinant SUR1 with Cys-Gly versus Cys conjugates of glucosinolate precursors to determine whether a carboxypeptidase step is needed between GGP1 and SUR1.
Experiment: Express the C154S GGP1 variant in ggp1 plants and assay activity and GSH levels under oxidative stress to test redox regulation via the Cys100-Cys154 disulfide.
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