GSTU20 (At1g78370; also FIP1, FIN219-interacting protein 1) is a 217-residue tau-class glutathione S-transferase of Arabidopsis. It forms a canonical homodimeric GST fold with glutathione bound in the G-site and catalyses conjugation of glutathione to electrophiles such as 1-chloro-2,4-dinitrobenzene (Km 0.47 mM for GSH). It is a soluble protein found in the cytosol and, as a GUS fusion, also in the nucleus, and is expressed mainly in vascular tissues. Two functional contexts are documented. First, FIP1 binds the jasmonoyl-amino acid synthetase FIN219/JAR1 (GH3.11); the crystal structure of the complex shows that FIP1 binding reorganises the FIN219 C-terminal domain and roughly doubles its adenylation activity, and fip1 gain- and loss-of-function lines are hyposensitive to continuous far-red light and late flowering, linking FIP1 to the interplay of phytochrome A and jasmonate signalling. Second, GSTU20 is co-expressed with and regulated by MYB28 together with the aliphatic glucosinolate core pathway, and CRISPR gstu20 knockouts lose most methionine-derived (aliphatic) glucosinolates in leaves and seeds without affecting indolic glucosinolates, more strongly than gstf11 and additively with it. It is therefore considered the main candidate enzyme for glutathione conjugation of the CYP83A1-activated aldoxime, although catalysis on that native intermediate has not been demonstrated biochemically.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004364 glutathione transferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (PAINT) inference of glutathione transferase activity for the tau-class GST clade (PTN001680376). GSTU20 itself is among the experimentally characterised members of the node (AT1G78370 in WITH/FROM), which is expected, not circular. Reason: GSTU20 is a canonical tau GST with direct enzymatic evidence (CDNB conjugation, GSH-bound crystal structure) [PMID:17220357, PMID:28223489]. The IBA correctly captures the core catalytic activity of the clade. Supporting Evidence: PMID:17220357 The result showed that FIP1 can use GSH and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates PMID:28223489 FIP1 showed lower GST activity than Schistosoma japonicum GST (SjGST) |
| GO:0004364 glutathione transferase activity | IDA PMID:28223489 Structural basis of jasmonate-amido synthetase FIN219 in com... | ACCEPT | Summary: Recombinant FIP1/GSTU20 was assayed for formation of the GS-DNB conjugate and showed GST activity (lower than the Schistosoma GST control); the crystal structure shows GSH bound in the G-site. Reason: Direct in vitro assay of the purified protein; core molecular function. Supporting Evidence: PMID:28223489 we performed a transferase assay to detect the product formation of [S-(2,4-dinitrobenzyl)glutathione (GS–DNB)] at A 340 . FIP1 showed lower GST activity than Schistosoma japonicum GST (SjGST) |
| GO:0004364 glutathione transferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of EC 2.5.1.18 / glutathione transferase activity from the tau-class GST C-terminal domain (IPR045074) and the UniProt catalytic-activity record. Reason: Consistent with the experimental enzyme assays [PMID:17220357, PMID:28223489]. Supporting Evidence: PMID:17220357 The result showed that FIP1 can use GSH and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates |
| GO:0004364 glutathione transferase activity | IMP PMID:17220357 Glutathione S-transferase interacting with far-red insensiti... | ACCEPT | Summary: Chen et al. 2007 expressed FIP1 in E. coli and measured GSH/CDNB conjugation kinetics (Km 0.467 mM GSH, 1.794 mM CDNB). The IMP code is unusual for a recombinant enzyme assay (IDA would be expected) but the activity itself is directly demonstrated. Reason: The function is correct and experimentally shown in this paper; the evidence code choice does not change the validity of the assertion. Supporting Evidence: PMID:17220357 we expressed it in E. coli and obtained the recombinant at about 95% purity for activity assay PMID:17220357 The result showed that FIP1 can use GSH and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates |
| GO:0005634 nucleus | IDA PMID:17220357 Glutathione S-transferase interacting with far-red insensiti... | KEEP AS NON CORE | Summary: A GUS-FIP1 fusion transiently expressed in onion epidermal cells was seen in the cytoplasm and the nucleus. Reason: Direct but heterologous (onion, large GUS fusion) observation. Nuclear presence is plausible for a small soluble GST, but its known partner FIN219/JAR1 and its catalytic role are cytosolic, so the nucleus is not the primary site of function. Supporting Evidence: PMID:17220357 bombarded cells showed the GUS-FIP1 fusion protein localized in the cytoplasm and the nucleus |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Mapping of the UniProt subcellular location (Nucleus), itself derived from the GUS-FIP1 localisation in PMID:17220357. Reason: Same evidence as the IDA nucleus annotation; retained as a non-core location. Supporting Evidence: PMID:17220357 bombarded cells showed the GUS-FIP1 fusion protein localized in the cytoplasm and the nucleus |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | 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. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN001680376 UNRESOLVED Proximate PAINT node traced from the original annotation. Its full ancestral assertion history has not been reconstructed in this audit; the target-specific assessment is grounded in the primary evidence explained in review.reason, not donor counts or self-inclusion. Proposed replacements: cytosol Supporting Evidence: PMID:17220357 bombarded cells showed the GUS-FIP1 fusion protein localized in the cytoplasm and the nucleus |
| GO:0005737 cytoplasm | IDA PMID:17220357 Glutathione S-transferase interacting with far-red insensiti... | 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:17220357 bombarded cells showed the GUS-FIP1 fusion protein localized in the cytoplasm and the nucleus |
| 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:17220357 bombarded cells showed the GUS-FIP1 fusion protein localized in the cytoplasm and the nucleus |
| GO:0005737 cytoplasm | NAS PMID:12090627 Probing the diversity of the Arabidopsis glutathione S-trans... | 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:17220357 bombarded cells showed the GUS-FIP1 fusion protein localized in the cytoplasm and the nucleus |
| GO:0005739 mitochondrion | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | MARK AS OVER ANNOTATED | Summary: The microsome experiment does not independently establish mitochondrion 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 GSTU20 specifically to mitochondrion 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 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:0005829 cytosol | HDA PMID:25293756 A proteomic strategy for global analysis of plant protein co... | ACCEPT | Summary: Detected in the soluble cytosolic fraction in a size-exclusion/LC-MS profiling study of leaf protein complexes. Reason: Consistent with the targeted localisation and the known cytosolic nature of tau GSTs. Supporting Evidence: PMID:17220357 bombarded cells showed the GUS-FIP1 fusion protein localized in the cytoplasm and the nucleus |
| GO:0005829 cytosol | IDA PMID:29877633 Proteome analysis of peroxisomes from dark-treated senescent... | ACCEPT | Summary: Peroxisome proteome of dark-treated senescent leaves, in which candidate proteins were checked by in vivo targeting; the cytosol annotation presumably reflects GSTU20 being found non-peroxisomal in that analysis. Cache is abstract-only. Reason: The curator saw the full text; a cytosolic result agrees with all other localisation evidence for GSTU20, so the annotation is accepted. The cached abstract has no GSTU20-specific sentence, so the supporting quote is the independent targeted localisation result. Supporting Evidence: PMID:17220357 Subcellular localization studies revealed that the beta-glucuronidase-FIP1 fusion protein was localized in the nucleus and cytoplasm. |
| GO:0005829 cytosol | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping of UniProt subcellular location "Cytoplasm, cytosol". Reason: Cytosol is the principal location of GSTU20 activity. Supporting Evidence: PMID:17220357 bombarded cells showed the GUS-FIP1 fusion protein localized in the cytoplasm and the nucleus |
| GO:0006749 glutathione metabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: Glutathione conjugation is the biochemical process performed by this GST. Reason: The enzyme uses glutathione in conjugate formation. This term is as precise as the established general GST chemistry permits without asserting a fully reconstructed native glucosinolate-intermediate reaction; pathway genetics is discussed separately. |
| GO:0006749 glutathione metabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: Glutathione conjugation is the biochemical process performed by this GST. Reason: The enzyme uses glutathione in conjugate formation. This term is as precise as the established general GST chemistry permits without asserting a fully reconstructed native glucosinolate-intermediate reaction; pathway genetics is discussed separately. Supporting Evidence: PMID:17220357 The result showed that FIP1 can use GSH and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates |
| GO:0009407 toxin catabolic process | IEA GO_REF:0000117 | UNDECIDED | Summary: A specific toxin-catabolic role is not resolved by the available evidence. Reason: Model-substrate conjugation and GST-family membership do not by themselves establish physiological toxin catabolism. The original broad family study is abstract-only in the cache and its gene-specific claim could not be checked; retain uncertainty consistently for the TAS and propagated row rather than infer detoxification from family reputation. Supporting Evidence: |
| GO:0009407 toxin catabolic process | TAS PMID:12090627 Probing the diversity of the Arabidopsis glutathione S-trans... | UNDECIDED | Summary: A specific toxin-catabolic role is not resolved by the available evidence. Reason: Model-substrate conjugation and GST-family membership do not by themselves establish physiological toxin catabolism. The original broad family study is abstract-only in the cache and its gene-specific claim could not be checked; retain uncertainty consistently for the TAS and propagated row rather than infer detoxification from family reputation. Supporting Evidence: |
| GO:0009507 chloroplast | HDA PMID:18431481 Sorting signals, N-terminal modifications and abundance of t... | MARK AS OVER ANNOTATED | Summary: The source study explicitly classifies this detected protein as non-plastid. Reason: PMID:18431481 distinguishes MS detection from its curated chloroplast assignment. The publisher supplemental abundance workbook (journal.pone.0001994.s003) and annotated-protein workbook (s005) explicitly label AT1G78370 (GSTU20) "not plastid"; the exact files and row identities are recorded in the gene notes. Thus the chloroplast annotation overinterprets detection contrary to the authors' own target-specific classification. This conclusion does not rest merely on absence of a predicted transit peptide, and it does not deny the peptide identifications. Supporting Evidence: PMID:18431481 30 proteins were considered non-plastid contaminations (Table S4). |
| GO:0009629 response to gravity | IEP PMID:23281391 A proteomics approach identifies novel proteins involved in ... | MARK AS OVER ANNOTATED | Summary: iTRAQ proteomics of gravistimulated inflorescence stems found GSTU20 among 82 proteins changing in abundance; functional follow-up was done for GSTF9 and HSP81-2, not GSTU20. Cache is abstract-only. Reason: An expression change after gravistimulation is not evidence of a role in gravitropism, and the study's functional follow-up was on other proteins. Treated like the single-dataset localisation rows in this review. Supporting Evidence: PMID:23281391 A total of 82 proteins showed significant differential quantification between treatment and controls. |
| GO:0019899 enzyme binding | IPI PMID:17220357 Glutathione S-transferase interacting with far-red insensiti... | MODIFY | Summary: GSTU20/FIP1 binding stimulates FIN219 enzymatic activity. Reason: The interaction and later complex structure are supported across the two cited papers; the measured functional consequence is stimulation of FIN219 adenylation. Refine both enzyme-binding rows consistently to enzyme activator activity rather than retaining an uninformative duplicate term under a different evidence source. Proposed replacements: enzyme activator activity Supporting Evidence: PMID:17220357 we demonstrate that FIN219-interacting protein 1 (FIP1) interacts with FIN219 in vitro and in vivo PMID:28223489 the adenylation activity per mole of FIN219–FIP1 was approximately double the adenylation activity per mole of FIN219 alone |
| GO:0019899 enzyme binding | IPI PMID:28223489 Structural basis of jasmonate-amido synthetase FIN219 in com... | MODIFY | Summary: GSTU20/FIP1 binding stimulates FIN219 enzymatic activity. Reason: The interaction and later complex structure are supported across the two cited papers; the measured functional consequence is stimulation of FIN219 adenylation. Refine both enzyme-binding rows consistently to enzyme activator activity rather than retaining an uninformative duplicate term under a different evidence source. Proposed replacements: enzyme activator activity Supporting Evidence: PMID:28223489 the adenylation activity per mole of FIN219–FIP1 was approximately double the adenylation activity per mole of FIN219 alone PMID:28223489 interaction with FIP1 triggers enhanced activity of FIN219 |
| GO:0043295 glutathione binding | IDA PMID:28223489 Structural basis of jasmonate-amido synthetase FIN219 in com... | ACCEPT | Summary: GSH co-crystallised with FIP1 shows clear density in the G-site and contacts conserved K40, E66 and S67. Reason: Direct structural evidence of GSH binding, which is intrinsic to the GST catalytic activity. Supporting Evidence: PMID:28223489 The substrate GSH shows a clear F o - F c map contour in the ligand-binding pocket of FIP1 and interacts with conserved K40, E66, and S67 |
| GO:0048046 apoplast | HDA PMID:18538804 Hydroponic isotope labelling of entire plants (HILEP) for qu... | UNDECIDED | Summary: The GSTU20-specific apoplast evidence remains inaccessible. Reason: PMID:18538804 reports isotope-based quantitation of apoplastic proteins under oxidative stress, but its abstract does not identify GSTU20 or provide the wash-fluid purity controls or target peptide data. A fresh normal fetch still yielded abstract only. Cytoplasmic/nuclear FIP1 imaging does not disprove stress-dependent extracellular detection; without the target table and controls, neither an accepted apoplast assignment nor a contamination claim can be justified. This unresolved source-access case differs from the adjudicable SUBAcon-derived organelle labels in PMID:28887381. Supporting Evidence: PMID:18538804 it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress |
| GO:2000030 regulation of response to red or far red light | IMP PMID:17220357 Glutathione S-transferase interacting with far-red insensiti... | ACCEPT | Summary: FIP1 gain- and partial loss-of-function lines have a hyposensitive hypocotyl phenotype in continuous far-red light and late flowering; the effect is linked to its interaction with the phyA-signalling component FIN219. Reason: The phenotype is supported, and the later structural/biochemical work gives a mechanism (FIP1 binds and stimulates FIN219, a phyA-pathway component) [PMID:28223489]. Both over- and under-expression give the same phenotype, consistent with a stoichiometric complex, so the regulatory role is accepted as one of the two documented functions of GSTU20. Supporting Evidence: PMID:17220357 The gain of function and partial loss of function of FIP1 resulted in a hyposensitive hypocotyl phenotype under continuous FR (cFR) light PMID:28223489 interaction with FIP1 triggers enhanced activity of FIN219 |
Loading supporting content…
Download this section (compressed HTML)Q: Does recombinant GSTU20 conjugate glutathione to the CYP83A1 product (the aci-nitro/nitrile oxide derived from aliphatic aldoximes) faster than the spontaneous reaction, i.e. is it the enzyme of the GSH-conjugation step or does it act indirectly on aliphatic glucosinolate accumulation?
Q: Should GO carry an acts_upstream_of_or_within IMP annotation to glucosinolate biosynthetic process (GO:0019761) for GSTU20 from PMID:35145536, analogous to the GSTU13 annotation to indole glucosinolate catabolic process from PMID:29122987, pending biochemical confirmation of its catalytic role?
Q: Is the FIN219/JAR1-activating role of FIP1 independent of its GST catalytic activity, and does FIN219 binding alter GSTU20 activity toward glucosinolate intermediates?
Experiment: Couple CYP83A1 microsomes with recombinant GSTU20 (and GSTF11) and GSH using methionine-derived aldoximes, and measure S-(alkylacetohydroximoyl)-GSH formation against enzyme-free controls to test catalysis of the native conjugation step.
Experiment: Profile GSH conjugate and aldoxime-derived intermediates (and core pathway transcript levels) in gstu20 and gstf11 gstu20 null lines to distinguish a catalytic block from indirect regulatory effects on aliphatic glucosinolate biosynthesis.
Experiment: Complement gstu20 with a catalytically impaired G-site mutant (e.g. at the GSH-contacting residues K40/E66/S67) to test whether GST activity is required for the glucosinolate phenotype and for the FIN219-related far-red phenotype.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)