LONRF3 is a poorly characterized LONRF-family protein predicted to function as a RING-type ubiquitin E3 ligase on the basis of phylogenetic annotation and two conserved RING-type zinc-finger regions. It also contains four TPR repeats and a C-terminal region homologous to the substrate-binding N-terminal domain of Lon proteases. LONRF3 lacks the AAA+ ATPase and proteolytic domains of complete Lon peptidases; its isolated Lon-domain homology therefore does not confer the complete architecture of a Lon peptidase. Direct ubiquitin-transfer activity, E2 partners, physiological substrates, biological process, and native subcellular location have not been established. Seven candidate partners have been recovered by systematic binary- interaction screens, but none is validated as a substrate, cofactor, or stable-complex component. Two alternative isoforms alter the TPR or C-terminal Lon-domain architecture, but their functions have not been compared.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0061630 ubiquitin protein ligase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference supports LONRF3 as a RING-type ubiquitin E3 ligase. Reason: The IBA is the only informative catalytic annotation for LONRF3 and is consistent with its conserved LONRF-family placement and two annotated RING-type zinc-finger regions. No direct LONRF3 ubiquitin-transfer assay or substrate is available, so the broad ligase term is retained without transferring the protein-quality-control substrates or neuronal biology established for the paralog LONRF2. Propagation Review Root cause: NO FAILURE CORE Sources checked: MGI:MGI:1920209 · mouse Lonrf1 SUPPORTS TRANSFER A mammalian LONRF-family donor supports conservation of broad RING E3 activity, not LONRF3-specific substrates or processes. PANTHER:PTN009070514 · LONRF-family RING E3 node SUPPORTS TRANSFER The family node is appropriately scoped to the broad ubiquitin ligase activity. PomBase:SPAC6B12.07c · fission-yeast LONRF-family protein SUPPORTS TRANSFER The distant fungal donor supports only the conserved broad RING E3 activity. |
| GO:0046872 metal ion binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro maps the RING-finger signature to broad metal ion binding. Reason: The source signature is a C3HC4 RING-type zinc-finger signature, so structural zinc coordination is the informative activity implied by the mapping. Generic metal ion binding is not false, but it discards the relevant ion specificity and should be replaced by the current term zinc ion binding. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: InterPro:IPR018957 · RING-type zinc-finger signature SUPPORTS TRANSFER The signature supports structural zinc binding, but its mapping to unspecified metal binding is unnecessarily broad. Proposed replacements: zinc ion binding |
| GO:0005515 protein binding | IPI PMID:20195357 A comprehensive resource of interacting protein regions for ... | MARK AS OVER ANNOTATED | Summary: A large-scale mRNA-display interaction-region screen reported an interaction between LONRF3 and prohibitin-1. Reason: The source is a high-throughput in vitro interaction-resource study rather than a focused functional analysis of LONRF3. The edge is valid source evidence, but generic protein binding is uninformative and does not establish prohibitin-1 as a physiological substrate, cofactor, or stable-complex partner. Supporting Evidence: PMID:20195357 Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs), including 12 transcription-related proteins. |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | MARK AS OVER ANNOTATED | Summary: A proteome-scale binary-interactome screen reported LONRF3 interactions with NOTCH2NLA and APPBP2. Reason: These systematic screen edges are useful candidate relationships but do not define a specific molecular function or establish either partner as an endogenous LONRF3 substrate or cofactor. The generic protein-binding term therefore overstates the functional information available from the assay. Supporting Evidence: PMID:25416956 Here, we describe a systematic map of ?14,000 high-quality human binary protein-protein interactions. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: The HuRI reference map reported four LONRF3 binary partners: CYSRT1, desmin, CTAG1B, and TRAF2. Reason: HuRI is a systematic proteome-wide binary-interaction resource. Its LONRF3 edges are hypothesis-generating physical associations, but generic protein binding does not identify a specific activity and cannot make the partners substrates, cofactors, or members of one physiological complex without targeted validation. Supporting Evidence: PMID:32296183 Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. |
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Download this section (compressed HTML)Q: Does full-length LONRF3 directly catalyze ubiquitin transfer, which E2 enzymes support it, and do both RING regions contribute to the reaction?
Q: Which endogenous proteins are direct LONRF3 substrates, and does their modification alter degradation, localization, interaction, or activity?
Q: Where does endogenous LONRF3 function, and which of the seven screen-derived partners, if any, are reproducible cofactors or substrates rather than assay-context contacts?
Q: Do the isoform-2 TPR4 deletion and isoform-3 Lon-domain truncation alter substrate recognition or regulation relative to the canonical product?
Experiment: Purify sequence-verified full-length LONRF3 and reconstitute ubiquitination with an E1, ubiquitin, and an E2 panel. Measure E2~ubiquitin discharge, autoubiquitination, and free chain formation, comparing wild type with separate and combined zinc-coordinating- residue mutants of the two RING regions.
Hypothesis: LONRF3 is an active RING-type E3 ligase whose activity requires one or both intact RING regions and a restricted subset of human E2 enzymes.
Type: reconstituted ubiquitination assay
Experiment: Engineer an acute degron at the endogenous LONRF3 locus, then perform matched diGly ubiquitin-remnant proteomics, total proteomics, and pulse-chase turnover analysis. Rescue candidate changes with wild-type versus RING-disrupted LONRF3 and test direct modification in a purified reaction.
Hypothesis: Acute loss of LONRF3 changes ubiquitination and abundance of a limited set of direct physiological substrates.
Type: acute perturbation and ubiquitin proteomics
Experiment: Introduce a minimally disruptive endogenous fluorescent or proximity-labeling tag, map localization and proximal proteins under basal and proteotoxic-stress conditions, and validate high-confidence partners by reciprocal endogenous co-immunoprecipitation.
Hypothesis: LONRF3 occupies a regulated cellular compartment and engages a limited local network not resolved by systematic binary screens.
Type: endogenous localization and proximity proteomics
Experiment: Express sequence-verified isoforms from the same genomic landing site at matched levels and compare localization, E2 binding, ubiquitination activity, proximity interactomes, and modification of substrates identified in endogenous loss-of-function studies.
Hypothesis: Isoforms 2 and 3 retain potential RING ligase activity but differ from the canonical product in substrate or partner selection because of their altered TPR or Lon-domain architecture.
Type: isoform-comparative functional analysis
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Intrinsic ubiquitin protein ligase activity by LONRF3 has not been demonstrated, and its E2 requirements, active RING region or regions, and ubiquitin-chain products are unknown.
OPEN BIOLOGY MF_DARK
What is known: An IBA supports broad ubiquitin ligase activity, and UniProt predicts two RING-type zinc fingers; neither source is a direct LONRF3 ubiquitin-transfer experiment.
Significance: Direct catalysis is required to distinguish an active E3 ligase from a RING-containing interaction scaffold and to determine whether one or both RING regions contribute.
What would resolve it: Reconstitute sequence-verified full-length LONRF3 with an E1, a panel of human E2 enzymes, and ubiquitin; measure E2~ubiquitin discharge, autoubiquitination, and chain formation while comparing wild type with structure-guided mutants in each RING region.
Provenance (the field's own admissions):
Gap: No physiological LONRF3 substrate or biological process has been established.
OPEN BIOLOGY BP_DARK
What is known: Seven proteins occur as candidate partners in three systematic interaction resources, but those binary edges do not demonstrate LONRF3-dependent ubiquitination, degradation, or another substrate outcome; LONRF2 substrates and protein-quality- control biology are paralog-specific evidence.
Significance: Substrate identity is necessary to connect the inferred ligase activity to a cellular pathway and to determine whether LONRF3 resembles or diverges from other LONRF paralogs.
What would resolve it: Combine acute endogenous LONRF3 depletion with diGly ubiquitin-remnant proteomics and quantitative protein-turnover measurements, then require rescue by wild-type but not RING-disrupted LONRF3 and validate prioritized substrates in a purified reaction.
Provenance (the field's own admissions):
Gap: The native subcellular location of active LONRF3 and any regulated cofactor assembly are unknown.
OPEN BIOLOGY CC_DARK
What is known: UniProt carries only electronic cytoplasmic localization, while the interaction resources do not establish endogenous co-localization or a physiological stable complex.
Significance: Compartment and transient cofactors constrain which E2 enzymes and substrates can encounter LONRF3 and prevent screen-derived partners from being interpreted as one complex.
What would resolve it: Endogenously tag LONRF3 and combine quantitative imaging, biochemical fractionation, and proximity labeling under basal and proteotoxic-stress conditions, validating candidate cofactors by reciprocal endogenous interaction assays.
Provenance (the field's own admissions):
Gap: Whether the three annotated LONRF3 isoforms differ in ligase activity, substrate selection, localization, or stability is unknown, and the isoform used for each interaction-screen edge is not established in the cached main texts.
OPEN BIOLOGY MF_DARK
What is known: Isoform 2 removes most of TPR4 while retaining both RING regions and the C-terminal Lon-domain homolog; isoform 3 retains both RING regions but truncates most of the C-terminal Lon-domain homolog. These architectural differences have not been tested functionally.
Significance: The altered TPR and putative substrate-binding architectures could change partner or substrate selection without eliminating the retained RING regions.
What would resolve it: Compare sequence-verified isoforms expressed at matched endogenous levels for E2 engagement, ubiquitination activity, partner selection, stability, localization, and rescue of any substrate or phenotype discovered after LONRF3 loss.
Provenance (the field's own admissions):
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