ufsp2 encodes a UFM1-specific cysteine protease (peptidase C78 family) that functions as the primary deUFMylase in the UFM1 conjugation pathway. The enzyme cleaves UFM1 from conjugated substrates (deconjugation) and processes UFM1 precursors to expose the C-terminal glycine required for conjugation (maturation). Its principal substrate is UFMylated RPL26 on ER-bound 60S ribosomal subunits, where deUFMylation by ufsp2 is required for ribosome recycling during ER-associated ribosome quality control. All annotations for zebrafish ufsp2 are inferred from mammalian orthologs (human UFSP2 and mouse Ufsp2) by sequence similarity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005783 endoplasmic reticulum | IBA GO_REF:0000033 | ACCEPT | Summary: Correct. UFSP2 is recruited to the ER via its N-terminal domain interaction with DDRGK1, and acts on UFMylated RPL26 on ER-bound ribosomes. This is a functionally important localization site. Reason: The ER localization is well supported by structural studies showing the N-terminal domain recruits UFSP2 to the ER via DDRGK1, and functional studies demonstrating its role in deUFMylation of ER-associated ribosomal subunits. Supporting Evidence: PMID:21228277 plays a role in the recognition of its cellular substrate C20orf116 and thus in the recruitment of UfSP2 to the endoplasmic reticulum PMID:30626644 RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER file:DANRE/ufsp2/ufsp2-deep-research-falcon.md In the current pathway-centric view, this ER tethering spatially matches UFSP2 to major UFMylation events on **ER-associated ribosomes**, and thus to ER proteostasis/ribosome quality control processes. |
| GO:0071567 deUFMylase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Correct. deUFMylase activity is the core molecular function of UFSP2. The enzyme cleaves UFM1 from conjugated substrates with strict specificity, using a catalytic cysteine-dependent mechanism. Reason: This is the defining molecular function of UFSP2, supported by direct biochemical evidence from the mammalian orthologs showing specific cleavage of UFM1 conjugates, loss of activity upon Cys-to-Ser mutation, and accumulation of UFMylated RPL26 when UFSP2 is disrupted. Supporting Evidence: PMID:17182609 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin PMID:17182609 replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities PMID:30626644 RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. file:DANRE/ufsp2/ufsp2-deep-research-bioreason-sft.md A cytoplasmic cysteine protease that specializes in processing the ubiquitin-like modifier UFM1 file:DANRE/ufsp2/ufsp2-deep-research-falcon.md UFSP2 is described as a UFM1-specific **cysteine protease/isopeptidase**, with activity dependent on a catalytic cysteine and a conserved **Cys–Asp–His** catalytic triad (often discussed alongside an additional conserved Tyr typical of cysteine-based Ub/UBL proteases). UFSP activity can be inhibited by thiol-reactive reagents such as **N-ethylmaleimide (NEM)** or by mutation of the catalytic cysteine. file:DANRE/ufsp2/ufsp2-deep-research-falcon.md - **UFSP2**: a major **de-UFMylase** for ER-associated substrates, particularly efficiently removing UFM1 from the ribosomal protein **RPL26 (uL24)**; UFSP2 knockout causes a **drastic accumulation of UFMylated proteins** in cell systems summarized in reviews. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Likely correct but non-core. Nuclear localization of UFSP2 has been reported for the mammalian ortholog, and UFSP2 has been observed to be recruited to DNA double-strand breaks. However, the primary functional site is the ER. Reason: Nuclear localization is reported for the mammalian ortholog and there is evidence for UFSP2 function at DNA damage sites, but the core function of UFSP2 is at the ER where it deUFMylates RPL26 on ribosomal subunits. Falcon deep research notes that reviews list nucleus/cytoplasm/ER localization, but ER tethering is the strongest mechanistic conclusion, consistent with retaining nucleus as non-core. Supporting Evidence: file:DANRE/ufsp2/ufsp2-deep-research-falcon.md Some reviews also list nucleus/cytoplasm/ER localization, but ER tethering is the strongest mechanistic conclusion. |
| GO:0006508 proteolysis | IBA GO_REF:0000033 | MODIFY | Summary: Correct but too general. UFSP2 performs a highly specific form of proteolysis -- it cleaves the isopeptide bond between UFM1 and its conjugated substrates. The term proteolysis does not capture this specificity. Reason: While proteolysis is technically accurate, it is overly broad for this specialized isopeptidase. The specific process is removal of the ubiquitin-like modifier UFM1 from substrate proteins (deUFMylation). No dedicated GO term for protein deUFMylation exists, so the closest correct existing BP term is GO:0070646 protein modification by small protein removal, whose definition explicitly covers removal of ubiquitin-like proteins (a sibling of protein deneddylation and protein desumoylation). A dedicated protein deUFMylation term is requested in proposed_new_terms. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN008489070 · UFSP UFM1-specific protease ancestral node SUPPORTS TRANSFER The node correctly transfers proteolytic activity to UFSP2, so the propagation is sound. The problem is granularity: GO:0006508 (proteolysis) is far broader than the specific UFM1 deconjugation (deUFMylation) that UFSP2 catalyzes. UniProtKB:Q9NUQ7 · Human UFSP2 SUPPORTS TRANSFER Experimentally characterized ortholog donor establishing UFM1-specific isopeptidase activity. Proposed replacements: protein modification by small protein removal Supporting Evidence: file:DANRE/ufsp2/ufsp2-deep-research-falcon.md Cross-species evidence supports that UFSP2 catalyzes: - **Proteolytic maturation of pro-UFM1** to mature UFM1 required for UFMylation, and - **Hydrolysis of isopeptide bonds** linking UFM1 to substrate proteins (de-UFMylation). |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Redundant with the IBA annotation for nucleus. Consistent with subcellular localization data from the mammalian ortholog. Reason: Redundant with the IBA-supported nucleus annotation. Retained as non-core since the primary functional site is the ER. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Correct. UFSP2 is a soluble enzyme that resides in the cytoplasm and is recruited to the ER membrane surface where it acts on UFMylated substrates. Reason: Cytoplasm is listed in UniProt subcellular location data and is consistent with UFSP2 being a soluble protease. However, the mechanistic picture points to constitutive ER tethering via the N-terminal domain interaction with DDRGK1/ODR4 rather than dynamic recruitment, so cytoplasm is retained as a non-core localization. Supporting Evidence: file:DANRE/ufsp2/ufsp2-deep-research-falcon.md Some reviews also list nucleus/cytoplasm/ER localization, but ER tethering is the strongest mechanistic conclusion. |
| GO:0071567 deUFMylase activity | IEA GO_REF:0000117 | ACCEPT | Summary: Redundant with the IBA annotation for deUFMylase activity. Correct annotation from ARBA machine learning models. Reason: Redundant with the IBA-supported annotation but independently correct. deUFMylase activity is the core molecular function. Supporting Evidence: PMID:17182609 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin file:DANRE/ufsp2/ufsp2-deep-research-falcon.md - **UFSP2**: a major **de-UFMylase** for ER-associated substrates, particularly efficiently removing UFM1 from the ribosomal protein **RPL26 (uL24)**; UFSP2 knockout causes a **drastic accumulation of UFMylated proteins** in cell systems summarized in reviews. |
| GO:0032790 ribosome disassembly | ISS GO_REF:0000024 | ACCEPT | Summary: Correct. UFSP2-mediated deUFMylation of RPL26 is a required step in the recycling of 60S ribosomal subunits from the ER. UFMylation marks stalled ribosomes for quality control, and UFSP2 removes UFM1 to enable ribosome recycling. Reason: Supported by studies showing that RPL26 UFMylation at the ER triggers ribosome quality control and that UFSP2-mediated deUFMylation is needed for 60S subunit recycling. Supporting Evidence: PMID:30626644 RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. PMID:37036982 UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs PMID:38383785 UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are file:DANRE/ufsp2/ufsp2-deep-research-falcon.md A 2024 Nature study (“UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER”) provides primary, high-authority support for a model in which UFMylation machinery promotes **recycling of 60S ribosomal subunits** from ER-associated states, and it explicitly frames UFSP2 as an **ER-tethered UFM1-specific hydrolase** in this functional context. |
| GO:0006508 proteolysis | ISS GO_REF:0000024 | MODIFY | Summary: Correct but too general. Same issue as the IBA proteolysis annotation. UFSP2 performs specific isopeptide bond cleavage of UFM1 conjugates, not general proteolysis. Reason: Same granularity problem as the IBA row: GO:0006508 is the top-level proteolysis term, whereas UFSP2 cleaves one specific isopeptide bond, UFM1 from its conjugate (UniProt: thiol-dependent isopeptidase that specifically cleaves UFM1). GO:0070646 protein modification by small protein removal is the closest existing BP term and is the parent of the analogous protein desumoylation and protein deneddylation terms; a dedicated protein deUFMylation term would be better still. The ISS donor is human UFSP2 (Q9NUQ7), whose UFM1 specificity was demonstrated directly (PMID:17182609). Proposed replacements: protein modification by small protein removal Supporting Evidence: file:DANRE/ufsp2/ufsp2-uniprot.txt Thiol-dependent isopeptidase that specifically cleaves UFM1, file:DANRE/ufsp2/ufsp2-uniprot.txt deUFMylase that mediates deconjugation of UFM1 from target proteins. PMID:17182609 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin |
| GO:0005634 nucleus | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Nucleus: UniProt records a nuclear pool of Ufsp2 by similarity to mouse Ufsp2 (Q99K23); no zebrafish imaging exists and no nuclear substrate is established for this protein. Reason: Kept because the mammalian localisation record is consistent across IBA, IEA and ISS routes, but graded non-core with the matching IBA/IEA rows: the characterised catalytic work of UFSP2 is on ER-associated UFMylated RPL26, and no nuclear deUFMylation substrate has been demonstrated. Supporting Evidence: file:DANRE/ufsp2/ufsp2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum {ECO:0000250|UniProtKB:Q99K23}. Cytoplasm {ECO:0000250|UniProtKB:Q99K23}. Nucleus {ECO:0000250|UniProtKB:Q99K23}. PMID:30626644 RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Cytoplasm: UFSP2 is a soluble protease with a cytosolic pool, recruited from there onto the cytoplasmic face of the ER membrane. Reason: Correct as a compartment but not where the enzyme does its characterised work: UFSP2 acts on the cytoplasmic surface of the ER on translocon-bound ribosomes, so the cytoplasm row is kept non-core alongside the matching IEA row, with the ER rows carrying the core location. Supporting Evidence: file:DANRE/ufsp2/ufsp2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum {ECO:0000250|UniProtKB:Q99K23}. Cytoplasm {ECO:0000250|UniProtKB:Q99K23}. Nucleus {ECO:0000250|UniProtKB:Q99K23}. PMID:30626644 RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. |
| GO:0005783 endoplasmic reticulum | IEA GO_REF:0000120 | ACCEPT | Summary: Endoplasmic reticulum: UFSP2 is tethered to the ER surface through its N-terminal domain binding DDRGK1/C20orf116, where it deUFMylates RPL26 on translocon-bound 60S subunits. Reason: This is the functionally decisive location and is asserted identically by the IBA and by UniProt's location record inherited from mouse Ufsp2 (Q99K23). The structural work on UFSP2 shows the N-terminal domain mediates recruitment to its ER-membrane partner (PMID:21228277), and the UFMylated substrate pool is enriched on ER-bound ribosomes (PMID:30626644). Supporting Evidence: file:DANRE/ufsp2/ufsp2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum {ECO:0000250|UniProtKB:Q99K23}. Cytoplasm {ECO:0000250|UniProtKB:Q99K23}. Nucleus {ECO:0000250|UniProtKB:Q99K23}. PMID:21228277 plays a role in the recognition of its cellular substrate C20orf116 and thus in the recrui PMID:30626644 RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. |
| GO:0005783 endoplasmic reticulum | ISS GO_REF:0000024 | ACCEPT | Summary: Endoplasmic reticulum: UFSP2 is tethered to the ER surface through its N-terminal domain binding DDRGK1/C20orf116, where it deUFMylates RPL26 on translocon-bound 60S subunits. Reason: This is the functionally decisive location and is asserted identically by the IBA and by UniProt's location record inherited from mouse Ufsp2 (Q99K23). The structural work on UFSP2 shows the N-terminal domain mediates recruitment to its ER-membrane partner (PMID:21228277), and the UFMylated substrate pool is enriched on ER-bound ribosomes (PMID:30626644). Supporting Evidence: file:DANRE/ufsp2/ufsp2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum {ECO:0000250|UniProtKB:Q99K23}. Cytoplasm {ECO:0000250|UniProtKB:Q99K23}. Nucleus {ECO:0000250|UniProtKB:Q99K23}. PMID:21228277 plays a role in the recognition of its cellular substrate C20orf116 and thus in the recrui PMID:30626644 RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. |
| GO:0071567 deUFMylase activity | ISS GO_REF:0000024 | ACCEPT | Summary: deUFMylase activity by similarity to human UFSP2 (Q9NUQ7): thiol-dependent isopeptidase cleaving UFM1 from conjugated substrates. Reason: Same claim as the IBA and ARBA rows and graded identically. The activity is defined by the C78 peptidase fold and the catalytic cysteine; the human/mouse orthologs cleave the UFM1 C-terminal extension but not ubiquitin, and Cys-to-Ser abolishes the activity (PMID:17182609). Zebrafish Ufsp2 keeps the C78 domain, so the ISS transfer is sound. Supporting Evidence: file:DANRE/ufsp2/ufsp2-uniprot.txt Thiol-dependent isopeptidase that specifically cleaves UFM1, file:DANRE/ufsp2/ufsp2-uniprot.txt Belongs to the peptidase C78 family. PMID:17182609 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin PMID:17182609 replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 a |
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Download this section (compressed HTML)Q: Does zebrafish ufsp2 act on the same substrate as its mammalian orthologs, i.e. is UFMylated Rpl26 detectable and does it accumulate when ufsp2 is disrupted?
Q: Does zebrafish retain a separate ufsp1, and if so which protease performs UFM1 precursor maturation versus deconjugation in vivo?
Experiment: Generate ufsp2 loss-of-function zebrafish and blot for UFM1 conjugates (particularly Rpl26) in ER-enriched fractions of embryos.
Hypothesis: Loss of ufsp2 causes accumulation of UFMylated Rpl26 on ER-bound ribosomes, as in mammalian cells.
Experiment: Express tagged zebrafish Ufsp2 and a catalytic-cysteine mutant and follow localisation relative to an ER marker and to Ddrgk1.
Hypothesis: Zebrafish Ufsp2 is recruited to the ER membrane by Ddrgk1 through its N-terminal domain, with cytoplasmic and nuclear pools that are not catalytically engaged.
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