LONRF1 is a poorly characterized RING-family protein containing two conserved RING-type zinc-finger regions and a C-terminal region homologous to the substrate-binding N-terminal domain of Lon proteases. Conserved family relationships, preservation of the RING architectures, and a yeast two-hybrid interaction with the ubiquitin-conjugating enzyme UBE2L6 support ubiquitin protein ligase activity, although ubiquitin transfer by LONRF1 itself has not been directly demonstrated and no physiological substrate is established. The protein lacks the AAA+ ATPase and proteolytic domains required for Lon peptidase activity. Its native site of action is unresolved, and direct localization evidence is lacking. An alternative isoform lacks residues 452-462 upstream of the second RING region, but no isoform-specific functional difference has been tested.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0061630 ubiquitin protein ligase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference supports LONRF1 as a RING-type ubiquitin E3 ligase. Reason: LONRF1 contains two annotated RING-type zinc fingers, and a human E2/E3-RING interaction screen detected interaction with UBE2L6 (PMID:19549727). Together with the IBA donors, these features support ubiquitin protein ligase activity as the principal conserved molecular function. Propagation Review Root cause: NO FAILURE CORE Sources checked: MGI:MGI:1920209 · mouse Lonrf1 SUPPORTS TRANSFER Orthologous mammalian LONRF1 source supporting conservation of RING E3 activity. PANTHER:PTN009070514 · PANTHER LONRF1-family node SUPPORTS TRANSFER Phylogenetic node used by the IBA inference and consistent with the target's two RING domains. PomBase:SPAC6B12.07c · fission-yeast LONRF-family protein SUPPORTS TRANSFER Distant fungal family donor; supportive at the broad RING E3 activity level. Supporting Evidence: PMID:19549727 In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. |
| GO:0046872 metal ion binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro maps the RING-finger signature to the broad term metal ion binding. Reason: The inference is structurally sound but unnecessarily broad. LONRF1 has two C3HC4 RING-type zinc fingers, so zinc ion binding is the informative activity implied by the source signature rather than unspecified metal 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 zinc coordination, but its mapping to generic metal ion binding loses the biologically relevant specificity. Proposed replacements: zinc ion binding |
| GO:0005515 protein binding | IPI PMID:19549727 Analysis of the human E2 ubiquitin conjugating enzyme protei... | MODIFY | Summary: A focused human E2/E3-RING screen detected interaction between LONRF1 and UBE2L6. Reason: The IPI result supports the specific activity ubiquitin-conjugating enzyme binding rather than the uninformative generic protein-binding term. The Y2H edge is consistent with an E3-ligase model but is not a biochemical ligase assay. Proposed replacements: ubiquitin conjugating enzyme binding Supporting Evidence: PMID:19549727 In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. |
| 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 27 LONRF1 interaction partners. Reason: These high-throughput IPI observations may identify candidate partners, but pooling heterogeneous screen hits under generic protein binding neither defines a specific molecular activity nor establishes that the partners are physiological substrates or cofactors of LONRF1. |
| GO:0005515 protein binding | IPI PMID:29892012 An interactome perturbation framework prioritizes damaging m... | MARK AS OVER ANNOTATED | Summary: An interactome-perturbation study retained a binary interaction between LONRF1 and GORASP2. Reason: The experimental interaction can be retained as candidate relationship evidence, but GO:0005515 is too generic to describe LONRF1 function, and this screen does not by itself show that GORASP2 is a ubiquitination substrate or physiological regulator. |
| GO:0005515 protein binding | IPI PMID:31515488 Extensive disruption of protein interactions by genetic vari... | MARK AS OVER ANNOTATED | Summary: A variant-centered interaction study reported binary interactions of LONRF1 with XIAP and NTAQ1. Reason: These IPI results are useful candidate-interactor data but cannot be promoted to a defined LONRF1 molecular function. Generic protein binding is non-specific, and the assay does not establish either protein as a physiological substrate or cofactor. |
| 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 screen reported a union of 73 binary interaction partners for LONRF1. Reason: A large high-throughput binary-interaction union is hypothesis-generating and includes many biologically heterogeneous partners. It does not define a coherent molecular function or establish physiological E3 substrates, so the generic protein binding annotation is over-annotated. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-983140 | MARK AS OVER ANNOTATED | Summary: Reactome places LONRF1 in the cytosolic step transferring ubiquitin from an E2 to an E3-bound substrate. Reason: LONRF1 inherits cytosol by membership in Reactome's generic candidate E3 set for this ubiquitination-cycle reaction, not from a LONRF1 localization experiment. The broad location is therefore plausible but insufficiently supported. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-983147 | MARK AS OVER ANNOTATED | Summary: Reactome places LONRF1 in the cytosolic release of an E3 from its polyubiquitinated substrate. Reason: LONRF1 inherits cytosol by membership in Reactome's generic candidate E3 set for this ubiquitination-cycle reaction. No LONRF1 localization experiment supports the asserted compartment, so the pathway-derived location is over-annotated. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-983156 | MARK AS OVER ANNOTATED | Summary: Reactome places LONRF1 in a cytosolic substrate-polyubiquitination step. Reason: Reactome propagates cytosol to LONRF1 through membership in a generic E3-ligase candidate set for substrate polyubiquitination, rather than through direct localization evidence. Compatibility with the pathway model is not positive compartment evidence. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-983157 | MARK AS OVER ANNOTATED | Summary: Reactome places LONRF1 in the cytosolic interaction of an E3 with substrate and an E2-ubiquitin complex. Reason: The cytosol assertion is inherited from Reactome's generic candidate E3 set for this ubiquitination-cycle reaction. Because no LONRF1 localization experiment underlies it, the broad pathway compartment is over-annotated. |
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Download this section (compressed HTML)Q: Does full-length human LONRF1 directly catalyze ubiquitin transfer, and which E2 enzyme or enzymes support that reaction under physiological conditions?
Q: Which endogenous proteins are direct LONRF1 substrates, and does their modification alter stability, localization, interaction, or activity rather than necessarily targeting them for proteasomal degradation?
Q: Where does endogenous LONRF1 act, and are the numerous binary-interaction hits reproducible partners, transient substrates, or screening-context artifacts?
Q: Does the deletion of residues 452-462 in isoform 2 alter the nearby RING module or any other property relative to the canonical product?
Experiment: Reconstitute purified full-length LONRF1 with an E1, ubiquitin, and a panel of candidate E2 enzymes including UBE2L6. Measure E2~ubiquitin discharge, autoubiquitination, and free or substrate-linked ubiquitin chains, comparing wild type with structure-guided zinc-coordinating-residue mutants in each RING region.
Hypothesis: LONRF1 is an active RING-type E3 whose ubiquitin-transfer activity requires an intact RING region and is supported by a restricted subset of human E2 enzymes.
Type: in vitro ubiquitination assay
Experiment: Engineer an acute degron at the endogenous LONRF1 locus, then perform matched diGly ubiquitin-remnant proteomics, quantitative total proteomics, and pulse- chase turnover analysis before and after depletion. Rescue prioritized changes with wild-type versus RING-disrupted LONRF1 and test direct ubiquitination of validated candidates in a purified system.
Hypothesis: Acute loss of LONRF1 changes ubiquitination and abundance of a small set of endogenous direct 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 and size-exclusion or native-complex analysis.
Hypothesis: LONRF1 occupies a regulated cellular compartment and engages a limited local interaction network that is obscured by proteome-scale binary screens.
Type: endogenous localization and proximity proteomics
Experiment: Compare sequence-verified canonical and isoform-2 products expressed from the same genomic landing site at matched levels for stability, localization, E2 binding, in vitro ubiquitination, and validated substrate modification, with equivalent RING-disrupting variants as mechanistic controls.
Hypothesis: The sequence deletion in isoform 2 modulates the nearby RING module without necessarily creating an isoform-exclusive function.
Type: isoform-comparative functional analysis
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Direct ubiquitin ligase activity by LONRF1 and its E2 requirements, chain products, and physiological substrates have not been established.
OPEN BIOLOGY MF_DARK
What is known: An IBA annotation, two conserved RING regions, and a yeast two-hybrid edge to UBE2L6 support a RING-E3 model, but none is a LONRF1-specific ubiquitin-transfer assay and interaction-screen partners cannot be assumed to be substrates.
Significance: Direct catalysis and substrate identification are required to distinguish an active ubiquitin ligase from a RING-containing interaction scaffold and to refine the molecular activity beyond the broad parent term.
What would resolve it: Reconstitute full-length LONRF1 with candidate human E2 enzymes and ubiquitin, compare wild type with RING-disrupting variants, and measure E2~ubiquitin discharge, autoubiquitination, substrate ubiquitination, and chain linkage.
Gap: The biological process in which LONRF1-mediated ubiquitination acts is unknown.
OPEN BIOLOGY BP_DARK
What is known: Reactome supplies only generic ubiquitination-cycle reactions, proteome-scale interaction screens do not identify a validated endogenous substrate, and transcriptomic associations in mouse cells do not demonstrate LONRF1-dependent protein quality control, proteasomal degradation, oxidative-stress defense, or tissue remodeling.
Significance: Without a causal substrate and phenotype, no substrate-specific ubiquitination or downstream protein-quality-control process can be assigned to human LONRF1.
What would resolve it: Combine acute endogenous LONRF1 loss with diGly ubiquitin-remnant proteomics and quantitative protein-turnover measurements, then rescue candidate changes with wild-type but not RING-disrupted LONRF1 and validate direct substrates biochemically.
Gap: The native subcellular location and any stable macromolecular complex of LONRF1 are unknown.
OPEN BIOLOGYCURATION CC_DARK
What is known: Reactome models LONRF1 in the cytosol, but those annotations derive from generic pathway reactions rather than direct localization experiments, and the many binary interaction-screen partners do not define a stable complex.
Significance: Compartment and complex context constrain which E2 enzymes and candidate substrates can encounter LONRF1 physiologically.
What would resolve it: Endogenously tag LONRF1 and combine quantitative imaging, biochemical fractionation, proximity labeling, and orthogonal co-complex validation under basal and proteotoxic-stress conditions.
Gap: Whether the canonical product and isoform 2 differ in activity, stability, localization, or partner selection is unknown.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: Isoform 2 lacks residues 452-462 upstream of the second RING region, but no GO annotation targets this isoform and no isoform-comparative experiment is available; proximity to the RING region alone does not establish an isoform-specific effect.
Significance: This local deletion could affect regulation of the inferred E3 module, but treating the two products as functionally distinct without direct evidence would overstate current knowledge.
What would resolve it: Express matched, sequence-verified isoforms at endogenous levels and compare E2 engagement, ubiquitination activity, stability, localization, and validated substrate recognition, including RING-disrupting controls.
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