id: Q9SAL0 gene_symbol: RGLG4 product_type: PROTEIN status: DRAFT taxon: id: NCBITaxon:3702 label: Arabidopsis thaliana description: >- RGLG4 (RING DOMAIN LIGASE 4, At1g79380) is a 401-residue soluble RING-type E3 ubiquitin-protein ligase of the plant RGLG/copine-like family, built from a central von Willebrand factor A (VWFA) domain and a C-terminal C3HC4-type RING finger. It has in vitro E3 ligase activity, forms K48-type chains like its closest paralog RGLG3, and is distributed between the cytoplasm and the nucleus. RGLG4 and RGLG3 act redundantly as positive modulators of jasmonate (JA) signalling upstream of the COI1 receptor: rglg3 rglg4 double mutants show reduced JA- and coronatine-inhibited root growth, reduced JA-responsive and wound-responsive gene expression, reduced wound-induced JA biosynthesis, resistance to coronatine-producing Pseudomonas syringae, and reduced fumonisin B1-triggered programmed cell death, the last requiring an intact RING domain. RGLG3/RGLG4 work with the E2 UBC30 and mediate ubiquitination and degradation of the monothiol glutaredoxin GRXS17; the truncated hemoglobin GLB3 binds them but appears to inhibit rather than serve as a substrate. The substrates that link RGLG4 to JA signalling are unknown. existing_annotations: - term: id: GO:0004842 label: ubiquitin-protein transferase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables supporting_entities: - AGI_LocusCode:AT1G79380 - AGI_LocusCode:AT3G01650 - AGI_LocusCode:AT5G14420 - AGI_LocusCode:AT5G63970 - PANTHER:PTN002247669 review: summary: >- PAINT IBD at node PTN002247669 (seeded by RGLG1-4 and rice Q69V56) assigns ubiquitin-protein transferase activity. RGLG4 is a descendant of that node and itself has direct experimental E3 ligase activity, so the IBA is sound and its own appearance among the seeds is expected. action: MODIFY reason: >- The claim is correct but more general than warranted. RGLG4 is a RING-type E3 whose activity is RING-dependent in vivo, so the appropriate term is the child GO:0061630 ubiquitin protein ligase activity, which the gene already carries (IEA). Same change as the IDA row for this term. propagation_review: root_cause: TERM_SCOPING_PROBLEM failure_modes: - GRANULARITY_MISMATCH source_entities: - source_id: PANTHER:PTN002247669 source_status: SUPPORTS_TRANSFER comment: Node seeded by experimental ligase/transferase evidence from RGLG1-4 (including RGLG4 itself); placement is sound and RGLG4 is inside the clade, only the term granularity is refined. proposed_replacement_terms: - id: GO:0061630 label: ubiquitin protein ligase activity supported_by: - reference_id: PMID:22898498 supporting_text: Both RGLG3 and RGLG4 possessed ubiquitin ligase activities - reference_id: file:ARATH/RGLG4/RGLG4-uniprot.txt record_section: ["DR:PANTHER"] supporting_text: DR PANTHER; PTHR45751:SF16; E3 UBIQUITIN-PROTEIN LIGASE RGLG4; 1. - term: id: GO:0004842 label: ubiquitin-protein transferase activity evidence_type: IDA original_reference_id: PMID:22898498 qualifier: enables review: summary: >- Zhang et al. 2012 showed that recombinant RGLG4 (and RGLG3) has ubiquitin ligase activity in vitro. action: MODIFY reason: >- Activity is experimentally established for RGLG4 itself. The assay is an E3 ligase assay of a RING protein, and RING-domain mutants fail to promote FB1-induced cell death in planta (PMID:25788731), so the specific child term GO:0061630 ubiquitin protein ligase activity is supported. The cached record is abstract-only; the abstract states the ligase activity directly. proposed_replacement_terms: - id: GO:0061630 label: ubiquitin protein ligase activity supported_by: - reference_id: PMID:22898498 supporting_text: Both RGLG3 and RGLG4 possessed ubiquitin ligase activities - reference_id: PMID:25788731 supporting_text: when the RING domain was mutated, RGLG3 and RGLG4 did not promote PCD - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:27497447 qualifier: enables supporting_entities: - UniProtKB:Q67XG0 review: summary: >- Interaction with the truncated hemoglobin GLB3 (Q67XG0), found by substrate trapping; GLB3 appeared to obstruct RGLG3 activity rather than being a substrate. action: REMOVE reason: >- Bare protein binding is uninformative. The interaction is not disputed, but GLB3 is neither substrate nor E2, and its proposed inhibitory effect was reported for RGLG3, so there is no more specific molecular function to assign to RGLG4. supported_by: - reference_id: PMID:27497447 supporting_text: The truncated hemoglobin GLB3 also interacted with RGLG3 and RGLG4 but appeared to obstruct RGLG3 ubiquitination activity rather than being its substrate. - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:27497447 qualifier: enables supporting_entities: - UniProtKB:Q9FKT3 review: summary: >- Interaction with the E2 UBC30 (Q9FKT3), identified in a UBC panel screen as a cognate E2 for RGLG3 and RGLG4. action: MODIFY reason: >- The partner is an E2 ubiquitin-conjugating enzyme, and the interaction reflects the E2-E3 pairing of a RING ligase, so the informative term is GO:0031624 ubiquitin conjugating enzyme binding. proposed_replacement_terms: - id: GO:0031624 label: ubiquitin conjugating enzyme binding supported_by: - reference_id: PMID:27497447 supporting_text: UBC30 as a cognate E2 UBC capable of interacting with RGLG3 and RGLG4 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:27497447 qualifier: enables supporting_entities: - UniProtKB:Q9ZPH2 review: summary: >- Interaction with the monothiol glutaredoxin GRXS17 (Q9ZPH2), trapped as a substrate with RING-dead RGLG3/RGLG4. action: REMOVE reason: >- Bare protein binding is uninformative. The biologically meaningful content (GRXS17 as an ubiquitination substrate) is captured by the ligase activity and protein ubiquitination terms; GO has no substrate-binding term that adds to them. Removal does not dispute the interaction. supported_by: - reference_id: PMID:27497447 supporting_text: we identified the monothiol glutaredoxin GRXS17 as a substrate of the Arabidopsis E3 ubiquitin ligases RING DOMAIN LIGASE 3 (RGLG3) and RGLG4 - term: id: GO:0005634 label: nucleus evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - AGI_LocusCode:AT1G79380 - AGI_LocusCode:AT3G01650 - AGI_LocusCode:AT5G14420 - AGI_LocusCode:AT5G63970 - PANTHER:PTN002247669 review: summary: >- PAINT IBD nucleus at PTN002247669, consistent with direct GFP and fractionation evidence for RGLG4. action: ACCEPT reason: >- RGLG4 descends from the node and nuclear localization is shown for RGLG4 itself. Note that PAINT did not propagate the plasma-membrane localization of the myristoylated paralogs RGLG1/RGLG2; RGLG4 lacks the N-terminal Gly2 myristoylation motif (starts MTMGNF), consistent with that omission. supported_by: - reference_id: PMID:25788731 supporting_text: Confocal images showed that GFP-RGLG3 and GFP-RGLG4 were present in both the cytoplasm and nucleus - term: id: GO:0005634 label: nucleus evidence_type: IDA original_reference_id: PMID:25788731 qualifier: located_in review: summary: >- GFP-RGLG4 was nuclear and cytoplasmic in confocal imaging, and FLAG-RGLG4 was found in the nuclear fraction (H3 marker). action: ACCEPT reason: >- Two independent methods support nuclear presence. The GFP pattern resembles free GFP, so imaging alone is weak, but cell fractionation with compartment markers confirms it. supported_by: - reference_id: PMID:25788731 supporting_text: With the subcellular compartment marker proteins H3 (nuclear) and GDPase (cytoplasmic) indicating effective separation, FLAG-RGLG3 and FLAG-RGLG4 were detected in both fractions - term: id: GO:0005634 label: nucleus evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0191 review: summary: UniProt subcellular-location mapping, derived from the curated PMID:25788731 statement. action: ACCEPT reason: Consistent with the IDA evidence for nuclear localization. supported_by: - reference_id: PMID:25788731 supporting_text: RGLG3 and RGLG4 are localized in both the cytoplasm and nucleus - term: id: GO:0005737 label: cytoplasm evidence_type: IDA original_reference_id: PMID:25788731 qualifier: located_in review: summary: GFP-RGLG4 and FLAG-RGLG4 were detected in the cytoplasm/cytoplasmic fraction. action: ACCEPT reason: Supported by imaging and fractionation (GDPase cytoplasmic marker). supported_by: - reference_id: PMID:25788731 supporting_text: With the subcellular compartment marker proteins H3 (nuclear) and GDPase (cytoplasmic) indicating effective separation, FLAG-RGLG3 and FLAG-RGLG4 were detected in both fractions - term: id: GO:0005737 label: cytoplasm evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0086 review: summary: UniProt subcellular-location mapping, derived from the curated PMID:25788731 statement. action: ACCEPT reason: Consistent with the IDA evidence for cytoplasmic localization. supported_by: - reference_id: PMID:25788731 supporting_text: RGLG3 and RGLG4 are localized in both the cytoplasm and nucleus - term: id: GO:0005739 label: mitochondrion evidence_type: HDA original_reference_id: PMID:28887381 qualifier: located_in review: summary: >- High-throughput assignment from a membrane-fraction protein correlation profiling study of Arabidopsis; RGLG4 is not discussed in the main text. action: MARK_AS_OVER_ANNOTATED reason: >- Focused GFP imaging and marker-controlled fractionation place RGLG4 in the cytoplasm and nucleus, with no mitochondrial signal reported, and RGLG4 has no mitochondrial targeting features in its UniProt record. A co-fractionation call in a proteome-wide membrane study is weak evidence of mitochondrial residence and should not be treated as a site of action. Not removed because the detection itself is a real high-throughput observation. supported_by: - reference_id: PMID:25788731 supporting_text: RGLG3 and RGLG4 are localized in both the cytoplasm and nucleus - term: id: GO:0009611 label: response to wounding evidence_type: IMP original_reference_id: PMID:22898498 qualifier: acts_upstream_of_or_within review: summary: >- rglg3 rglg4 reduced wound-stunted growth, wound-induced JA-responsive gene expression and wound-induced JA biosynthesis; RGLG4 expression responds to wounding. action: KEEP_AS_NON_CORE reason: >- The double-mutant phenotype supports a role in the JA-dependent wound response, but it is one output of RGLG4's modulation of JA signalling and rests on redundant RGLG3/RGLG4 genetics; the mechanism (substrate) is unknown. Retained as a non-core, JA-mediated process. supported_by: - reference_id: PMID:22898498 supporting_text: rglg3 rglg4 repressed wound-stunted plant growth, wound-stimulated expression of JA-responsive genes, and wound-induced JA biosynthesis, indicating their roles in JA-dependent wound response - term: id: GO:0009867 label: jasmonic acid mediated signaling pathway evidence_type: IMP original_reference_id: PMID:22898498 qualifier: acts_upstream_of_or_within review: summary: >- Altered RGLG3/RGLG4 expression changes MeJA root inhibition and JA-induced gene expression in a COI1-dependent manner; the authors call RGLG3/4 upstream modulators of JA signalling. action: MODIFY reason: >- RGLG4 is not a component of the core COI1-JAZ-MYC pathway: it does not interact with COI1, AXR1, MYC2/3/4 or CYP94B3, and its ligase activity is proposed to act on an unidentified negative regulator. Its effect is to modulate the pathway, positively (overexpression enhances, the double mutant attenuates responses). GO has no "positive regulation" child of GO:2000022, so the replacement is GO:2000022 regulation of jasmonic acid mediated signaling pathway. proposed_replacement_terms: - id: GO:2000022 label: regulation of jasmonic acid mediated signaling pathway supported_by: - reference_id: PMID:22898498 supporting_text: these results indicate that the ubiquitin ligases RGLG3 and RGLG4 are essential upstream modulators of JA signaling in response to various stimuli - reference_id: PMID:23073017 supporting_text: RGLG3 and RGLG4 didn't interact with known key factors of the core JA pathway, rather, it might target on unknown protein that negatively regulated JA signaling - term: id: GO:0016567 label: protein ubiquitination evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - AGI_LocusCode:AT3G01650 - PANTHER:PTN002247669 review: summary: >- PAINT IBD at PTN002247669 seeded by RGLG1. RGLG4 itself mediates ubiquitination of GRXS17 in planta together with RGLG3. action: ACCEPT reason: >- The general process is correct and appropriately conservative. PAINT did not propagate RGLG1's protein K63-linked ubiquitination, which is consistent with RGLG3/RGLG4 forming K48-type chains; K63 linkage should not be transferred to RGLG4. supported_by: - reference_id: PMID:27497447 supporting_text: GRXS17 is ubiquitinated and degraded in an RGLG3- and RGLG4-dependent manner in planta - reference_id: PMID:23073017 supporting_text: RGLG3 and RGLG4 likely catalyze only Lys 48-type ubiquitination - term: id: GO:0042742 label: defense response to bacterium evidence_type: IMP original_reference_id: PMID:22898498 qualifier: acts_upstream_of_or_within review: summary: >- rglg3 rglg4 double mutants were more resistant to coronatine-secreting Pst DC3000; RGLG3/4 promote JA-mediated susceptibility. action: MARK_AS_OVER_ANNOTATED reason: >- RGLG4 does not execute antibacterial defense; loss of RGLG3/RGLG4 increases resistance, because the pathogen's coronatine hijacks the JA pathway that RGLG3/4 promote and JA antagonizes SA-dependent defense. The phenotype is a downstream organismal consequence of RGLG4's modulation of JA signalling (captured by GO:2000022) and of redundant double-mutant genetics, so a defense process term over-states RGLG4's direct role. supported_by: - reference_id: PMID:22898498 supporting_text: rglg3 rglg4 was resistant to the coronatine-secreting pathogen Pseudomonas syringae pv tomato DC3000, suggesting that RGLG3 and RGLG4 acted in response to the coronatine and promoted JA-mediated pathogen susceptibility - term: id: GO:0061630 label: ubiquitin protein ligase activity evidence_type: IEA original_reference_id: GO_REF:0000003 qualifier: enables supporting_entities: - EC:2.3.2.27 review: summary: EC 2.3.2.27 (RING-type E3) mapping. action: ACCEPT reason: >- Correct and experimentally supported for RGLG4 itself (in vitro E3 activity; RING-dependent activity in planta). This is the core molecular function. supported_by: - reference_id: PMID:22898498 supporting_text: Both RGLG3 and RGLG4 possessed ubiquitin ligase activities - reference_id: file:ARATH/RGLG4/RGLG4-uniprot.txt record_section: ["DR:InterPro"] supporting_text: DR InterPro; IPR001841; Znf_RING. references: - id: GO_REF:0000003 title: Gene Ontology annotation based on Enzyme Commission mapping findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: PMID:22898498 title: Two novel RING-type ubiquitin ligases, RGLG3 and RGLG4, are essential for jasmonate-mediated responses in Arabidopsis. findings: - statement: RGLG4 and RGLG3 have in vitro ubiquitin ligase activity. supporting_text: Both RGLG3 and RGLG4 possessed ubiquitin ligase activities - statement: RGLG3/RGLG4 are upstream modulators of JA signalling acting redundantly (double-mutant genetics). supporting_text: these results indicate that the ubiquitin ligases RGLG3 and RGLG4 are essential upstream modulators of JA signaling in response to various stimuli reference_review: relevance: HIGH correctness: VERIFIED review_notes: Primary characterization of RGLG4; PMID resolves to the paper (PMC3461557). Cached copy is abstract-only. - id: PMID:23073017 title: RGLG3 and RGLG4, novel ubiquitin ligases modulating jasmonate signaling. findings: - statement: RGLG3/RGLG4 likely form K48-type chains only, unlike RGLG2, and do not bind core JA pathway components in Y2H. supporting_text: RGLG3 and RGLG4 likely catalyze only Lys 48-type ubiquitination reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Addendum by the same group; full text cached. Linkage statement is hedged ("likely") and refers back to PMID:22898498. - id: PMID:25788731 title: Hijacking of the jasmonate pathway by the mycotoxin fumonisin B1 (FB1) to initiate programmed cell death in Arabidopsis is modulated by RGLG3 and RGLG4. findings: - statement: RGLG4 is located in both the cytoplasm and the nucleus. supporting_text: Confocal images showed that GFP-RGLG3 and GFP-RGLG4 were present in both the cytoplasm and nucleus - statement: RGLG4 promotes FB1-induced programmed cell death in a RING-dependent manner. supporting_text: when the RING domain was mutated, RGLG3 and RGLG4 did not promote PCD reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached; localization by GFP imaging and marker-controlled fractionation. - id: PMID:27497447 title: The Arabidopsis Iron-Sulfur Protein GRXS17 is a Target of the Ubiquitin E3 Ligases RGLG3 and RGLG4. findings: - statement: GRXS17 is a substrate of RGLG3/RGLG4, ubiquitinated and degraded in an RGLG3/RGLG4-dependent manner in planta; UBC30 is a cognate E2. supporting_text: GRXS17 is ubiquitinated and degraded in an RGLG3- and RGLG4-dependent manner in planta reference_review: relevance: HIGH correctness: VERIFIED review_notes: Abstract-only in cache. The in vitro E2-dependent reactions in the abstract are stated for RGLG3; RGLG4-specific in vitro data, if any, are in the full text. - id: PMID:28887381 title: Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Proteome-wide HDA source of the mitochondrion annotation; RGLG4 not discussed in the main text. - id: PMID:35743207 title: Endosomally Localized RGLG-Type E3 RING-Finger Ligases Modulate Sorting of Ubiquitylation-Mimic PIN2. findings: - statement: RGLG3/RGLG4 form a subclade distinct from RGLG1/RGLG2. supporting_text: The Arabidopsis RGLG clade consists of 5 members, with RGLG3 and RGLG4 found to form K48-linked polyubiquitin chains in vitro [12], and representing a subclade different from the RGLG1 and RGLG2 clade reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Used for the phylogenetic separation of RGLG3/4 from RGLG1/2; the endosomal-localization data concern RGLG1/RGLG2, not RGLG4. - id: PMID:17586653 title: Ubiquitin lysine 63 chain forming ligases regulate apical dominance in Arabidopsis. findings: - statement: RGLG1/RGLG2 are myristoylated membrane-associated K63-chain-forming E3s (paralog contrast; not transferred to RGLG4). supporting_text: In this work, we present the characterization of RGLG1 and RGLG2, two membrane-associated ubiquitin Lys-63 chain–forming enzymes from Arabidopsis reference_review: relevance: LOW correctness: VERIFIED review_notes: Paralog paper; cited only to delimit which paralog functions should not be transferred to RGLG4. - id: file:ARATH/RGLG4/RGLG4-uniprot.txt title: UniProt record for RGLG4 (Q9SAL0) findings: [] core_functions: - description: >- RING-type E3 ubiquitin-protein ligase that, with the E2 UBC30, ubiquitinates target proteins in the cytoplasm and nucleus; GRXS17 is an identified substrate degraded in an RGLG3/RGLG4-dependent manner. Ligase activity is shown experimentally for RGLG4 itself; chain linkage is reported as K48-type for RGLG3/RGLG4, distinguishing them from the K63-forming, membrane-associated RGLG1/RGLG2. molecular_function: id: GO:0061630 label: ubiquitin protein ligase activity directly_involved_in: - id: GO:0016567 label: protein ubiquitination locations: - id: GO:0005737 label: cytoplasm - id: GO:0005634 label: nucleus supported_by: - reference_id: PMID:22898498 supporting_text: Both RGLG3 and RGLG4 possessed ubiquitin ligase activities - reference_id: PMID:27497447 supporting_text: GRXS17 is ubiquitinated and degraded in an RGLG3- and RGLG4-dependent manner in planta - reference_id: PMID:25788731 supporting_text: RGLG3 and RGLG4 are localized in both the cytoplasm and nucleus - description: >- Positive modulator, redundantly with RGLG3, of jasmonate signalling upstream of COI1, contributing to JA-dependent root growth inhibition, wound responses, coronatine responsiveness and FB1-triggered cell death; the ligase activity is required, but the JA-relevant substrates are unknown. molecular_function: id: GO:0061630 label: ubiquitin protein ligase activity directly_involved_in: - id: GO:2000022 label: regulation of jasmonic acid mediated signaling pathway supported_by: - reference_id: PMID:22898498 supporting_text: these results indicate that the ubiquitin ligases RGLG3 and RGLG4 are essential upstream modulators of JA signaling in response to various stimuli - reference_id: PMID:25788731 supporting_text: when the RING domain was mutated, RGLG3 and RGLG4 did not promote PCD suggested_questions: - question: >- Is RGLG4 itself (not only RGLG3) active with UBC30 in vitro on GRXS17, and is GRXS17 turnover proteasome-dependent? If so, should RGLG4 carry proteasome-mediated ubiquitin-dependent protein catabolic process (GO:0043161)? No RGLG paralog currently carries that term despite comparable PP2CA-degradation evidence for RGLG1/RGLG5. experts: - Arabidopsis ubiquitin/E3 ligase biologists - TAIR curators - question: >- What linkage types does RGLG4 form with its cognate E2s, given the hedged K48-only statement and the K63 activity of RGLG1/RGLG2 with UBC35/MMZ2? experts: - Ubiquitin biochemists suggested_experiments: - hypothesis: RGLG4 modulates JA signalling by ubiquitinating an as-yet unidentified negative regulator of the pathway. description: >- Substrate trapping (RING-dead RGLG4 TAP or TurboID) in MeJA- or wound-treated seedlings, followed by in vitro ubiquitination with UBC30 and stability assays of candidates in rglg3 rglg4 versus wild type. - hypothesis: RGLG4 forms K48-linked chains exclusively. description: >- In vitro ubiquitination with UBC30 and other E2s (including UBC35/MMZ2) using lysine-less and single-lysine ubiquitin mutants, or linkage-specific antibodies/MS.