GLO2

Summary: Identified in the vegetative vacuole proteome of rosette-leaf protoplasts.

Reason: Glycolate oxidase is a soluble peroxisomal matrix enzyme with a C-terminal PTS1 and is one of the most abundant proteins of photosynthetic leaves; it was identified in purified leaf peroxisomes (PMID:17951448) and acts there in the C2 cycle (PMID:22303256). Detection in a bulk organelle or fraction proteome of this kind is most simply explained by carry-over of peroxisomes or peroxisomal matrix protein (peroxisomes are physically appressed to chloroplasts in leaf cells), and no targeted study shows a functional pool of GOX in this compartment. The high-throughput detection is not contradicted, but it does not describe where the enzyme acts. Action follows the ARATH convention for single high-throughput proteomics location hits (projects/ARATH/organelle-audit.md, rule 3). The vacuole proteome itself recognized a substantial fraction of organellar contaminants, and GLO2 has no vacuolar sorting signal.

Summary: Leaf RNA interactome capture (ptRIC) recovered (S)-2-hydroxy-acid oxidase among metabolic enzymes crosslinked to poly(A) RNA.

Reason: The cited proteome-wide RNA interactome capture lists (S)-2-hydroxy-acid oxidase as a putative moonlighting RNA-binding enzyme, without validation of specific mRNA targets, binding site or any consequence for RNA fate. GLO2 is a highly abundant peroxisomal matrix enzyme; capture of abundant metabolic enzymes in RIC does not by itself establish mRNA binding as a function of the protein. The observation is not contradicted, but the MF annotation over-states what was shown.

Summary: Recombinant At-GOX2 (At3g14415) oxidized glycolate with O2, producing H2O2, and was about 10-fold more active with glycolate than with L-lactate; it complemented a Synechocystis glycolate dehydrogenase double mutant.

Reason: Core function, directly demonstrated on purified recombinant GLO2 with the correct locus (At3g14415) stated in the paper. GO:0003973 is the correct term; obsolete GO:0008891 glycolate oxidase activity is replaced by it (verified in OLS).

Summary: Kinetic and isotope-effect analysis of purified recombinant GOX2 (At3g14415) with glycolate (Km about 0.3 mM, kcat about 30 s-1).

Reason: Core function; direct biochemical evidence on recombinant GLO2, with loci stated explicitly in the paper.

Summary: Phylogenetic inference of (S)-2-hydroxy-acid oxidase activity.

Reason: GLO2 itself (AGI At3g14415) is among the experimental sources of this PAINT node, as expected for a gene with direct biochemical evidence (PMID:21828292, PMID:25416784); the IBA adds that the activity is inherited across the plant glycolate oxidase clade.

Summary: Combined automated annotation (InterPro/EC/Rhea) to (S)-2-hydroxy-acid oxidase activity.

Reason: Agrees with the experimental evidence for GLO2.

Summary: Detected in a 2-D gel/MALDI-TOF survey of the Arabidopsis nuclear proteome.

Reason: Glycolate oxidase is a soluble peroxisomal matrix enzyme with a C-terminal PTS1 and is one of the most abundant proteins of photosynthetic leaves; it was identified in purified leaf peroxisomes (PMID:17951448) and acts there in the C2 cycle (PMID:22303256). Detection in a bulk organelle or fraction proteome of this kind is most simply explained by carry-over of peroxisomes or peroxisomal matrix protein (peroxisomes are physically appressed to chloroplasts in leaf cells), and no targeted study shows a functional pool of GOX in this compartment. The high-throughput detection is not contradicted, but it does not describe where the enzyme acts. Action follows the ARATH convention for single high-throughput proteomics location hits (projects/ARATH/organelle-audit.md, rule 3). There is no nuclear-localization signal or nuclear role reported for glycolate oxidase.

Summary: Identified by mass spectrometry in purified Arabidopsis leaf peroxisomes, distinguished from its paralog by isoform-specific peptides.

Reason: Core location. The leaf peroxisome proteome specifically identified At3g14415 (named GOX1 in this paper, which uses the crossed GOX1/GOX2 naming) with PTS1 PRL>.

Summary: Phylogenetic inference that GLO2 is active in the peroxisome.

Reason: Consistent with direct proteomic identification in leaf peroxisomes (PMID:17951448) and with the conserved peroxisomal location of plant glycolate oxidases.

Summary: UniProt subcellular location (Peroxisome) mapped to GO.

Reason: Supported by the leaf peroxisome proteome (PMID:17951448).

Summary: Detected in a size-exclusion chromatography/proteomic survey of soluble protein complexes.

Reason: Glycolate oxidase is a soluble peroxisomal matrix enzyme with a C-terminal PTS1 and is one of the most abundant proteins of photosynthetic leaves; it was identified in purified leaf peroxisomes (PMID:17951448) and acts there in the C2 cycle (PMID:22303256). Detection in a bulk organelle or fraction proteome of this kind is most simply explained by carry-over of peroxisomes or peroxisomal matrix protein (peroxisomes are physically appressed to chloroplasts in leaf cells), and no targeted study shows a functional pool of GOX in this compartment. The high-throughput detection is not contradicted, but it does not describe where the enzyme acts. Action follows the ARATH convention for single high-throughput proteomics location hits (projects/ARATH/organelle-audit.md, rule 3). Soluble extracts used in complex-profiling studies include peroxisomal matrix protein released during extraction. GLO2 is translated in the cytosol before peroxisomal import, but no functional cytosolic pool has been reported. Note that the paralog GLO1 carries a curated NOT cytosol annotation from the Arabidopsis cytosolic proteome (PMID:21166475).

Summary: Detected in a large-scale chloroplast proteome.

Reason: Glycolate oxidase is a soluble peroxisomal matrix enzyme with a C-terminal PTS1 and is one of the most abundant proteins of photosynthetic leaves; it was identified in purified leaf peroxisomes (PMID:17951448) and acts there in the C2 cycle (PMID:22303256). Detection in a bulk organelle or fraction proteome of this kind is most simply explained by carry-over of peroxisomes or peroxisomal matrix protein (peroxisomes are physically appressed to chloroplasts in leaf cells), and no targeted study shows a functional pool of GOX in this compartment. The high-throughput detection is not contradicted, but it does not describe where the enzyme acts. Action follows the ARATH convention for single high-throughput proteomics location hits (projects/ARATH/organelle-audit.md, rule 3). GLO2 has no chloroplast transit peptide.

Summary: Detected in chloroplast/stroma proteomics of a cpSRP54 mutant.

Reason: Glycolate oxidase is a soluble peroxisomal matrix enzyme with a C-terminal PTS1 and is one of the most abundant proteins of photosynthetic leaves; it was identified in purified leaf peroxisomes (PMID:17951448) and acts there in the C2 cycle (PMID:22303256). Detection in a bulk organelle or fraction proteome of this kind is most simply explained by carry-over of peroxisomes or peroxisomal matrix protein (peroxisomes are physically appressed to chloroplasts in leaf cells), and no targeted study shows a functional pool of GOX in this compartment. The high-throughput detection is not contradicted, but it does not describe where the enzyme acts. Action follows the ARATH convention for single high-throughput proteomics location hits (projects/ARATH/organelle-audit.md, rule 3).

Summary: UniPathway mapping to the oxidative photosynthetic carbon pathway (C2 cycle), a child of photorespiration.

Reason: Core process. GLO2 catalyzes the peroxisomal glycolate-to-glyoxylate step of the C2 cycle; it is co-regulated with photorespiratory genes (PMID:22303256), shares a redundant photorespiratory role with GLO1 (PMID:26896850), and gox2-1 mutants are impaired under high light (PMID:27225899). GO:0009854 is part_of GO:0009853 photorespiration and is the most precise available term. The core function nonetheless uses the broader GO:0009853 photorespiration, so that every C2-cycle step and ammonia refixation share one process term (see modules/photorespiration_c2_cycle.yaml).

Summary: InterPro mapping to FMN binding.

Reason: Glycolate oxidase is an FMN-dependent flavoprotein; FMN-binding residues are conserved with spinach GOX (UniProt, by similarity to P05414). Cofactor binding is intrinsic to the catalytic activity.

Summary: Identified in affinity-purified stress granule isolates (Rbp47b-GFP) from Arabidopsis seedlings.

Reason: Cached text is abstract-only, so the supporting data for GLO2 cannot be seen; per projects/ARATH/organelle-audit.md (rule 4) an experimental row whose supporting figures are not available is left UNDECIDED. The study reports that only a quarter of the proteins identified in stress granule isolates were known or predicted SG components, the remainder being enriched in metabolic enzymes and regulators. GLO2 is an abundant peroxisomal matrix enzyme (PMID:17951448) without any reported cytosolic or RNA-granule function, so the co-purification may reflect association during isolation rather than a functional stress granule pool; whether a cytosolic GLO2 pool joins stress granules needs the full data.

Summary: InterPro mapping to the generic oxidoreductase activity term.

Reason: Correct but uninformative; GLO2 has experimentally demonstrated (S)-2-hydroxy-acid oxidase activity (GO:0003973), a descendant of this term already carried by the gene.

Proposed replacements: (S)-2-hydroxy-acid oxidase activity

Summary: ARBA machine-learning annotation to defense response to bacterium.

Reason: Agrees with the experimental IMP row from Rojas et al. 2012 (PMID:22286136). A secondary role of the H2O2 product, not the core metabolic function.

Summary: Arabidopsis gox T-DNA mutants are compromised for nonhost resistance and show reduced H2O2, callose deposition and electrolyte leakage after nonhost bacterial inoculation.

Reason: Cached text is abstract-only; Kerchev et al. 2016 (PMID:27225899) state in their full text that the gox2-1 allele was described in Rojas et al. 2012, consistent with this IMP annotation to GLO2. Deferring to the curator; defence is a secondary role of the H2O2 product.

Summary: Rhea mapping (RHEA:25311 glycolate + O2 = glyoxylate + H2O2) to alcohol oxidase activity.

Reason: GO:0047639 alcohol oxidase activity (primary alcohol + O2 = aldehyde + H2O2) is a sibling, not a parent, of GO:0003973 in GO. The GLO2 reaction is the oxidation of a (S)-2-hydroxy acid to a 2-oxo acid, which is what GO:0003973 describes (the term that replaced obsolete glycolate oxidase activity). Replace with GO:0003973.

Proposed replacements: (S)-2-hydroxy-acid oxidase activity

Summary: Detected in an apoplast-focused quantitative proteomics study (HILEP) under oxidative stress.

Reason: Glycolate oxidase is a soluble peroxisomal matrix enzyme with a C-terminal PTS1 and is one of the most abundant proteins of photosynthetic leaves; it was identified in purified leaf peroxisomes (PMID:17951448) and acts there in the C2 cycle (PMID:22303256). Detection in a bulk organelle or fraction proteome of this kind is most simply explained by carry-over of peroxisomes or peroxisomal matrix protein (peroxisomes are physically appressed to chloroplasts in leaf cells), and no targeted study shows a functional pool of GOX in this compartment. The high-throughput detection is not contradicted, but it does not describe where the enzyme acts. Action follows the ARATH convention for single high-throughput proteomics location hits (projects/ARATH/organelle-audit.md, rule 3). GLO2 has no signal peptide.

Summary: ARBA machine-learning annotation to hydrogen peroxide biosynthetic process.

Reason: H2O2 is a direct product of the GLO2-catalyzed reaction, demonstrated for the recombinant enzyme (PMID:21828292); a true but secondary process view of the core activity.

Summary: gox mutants accumulate less H2O2 after nonhost pathogen inoculation.

Reason: GLO2 produces H2O2 stoichiometrically (PMID:21828292) and gox mutants have lower H2O2 during defence (abstract of PMID:22286136; full text not cached, curator deferred to). Non-core: H2O2 is a by-product of the photorespiratory step.

Summary: Recombinant At-GOX2 oxidizes L-lactate to pyruvate with O2 in vitro, with a much lower Vmax than for glycolate.

Reason: The activity is real in vitro (UniProt kinetics: Km 0.356 mM, Vmax 0.742 umol/min/mg for L-lactate versus 35.64 umol/min/mg for glycolate), but GLO2 is a glycolate oxidase: it is about 10-fold more active, and has a 40-fold higher Vmax, with glycolate. The paper uses At-GOX2 as the plant glycolate oxidase comparator for cyanobacterial lactate oxidase. No physiological role of GLO2 in L-lactate oxidation is known. The term is a descendant of GO:0003973 and is precise for the measured reaction, so it is kept but as non-core.

Summary: Rhea mapping (RHEA:55868 (S)-lactate + O2 = pyruvate + H2O2) to L-lactate oxidase activity.

Reason: Derived from the UniProt catalytic-activity line based on the in vitro data of PMID:21828292; a minor side activity relative to glycolate oxidation. Kept as non-core, consistent with the EXP row.