NSP1 (nitrile-specifier protein 1; At3g16400; also called JAL28, and AtNSP3 in the nomenclature of Kissen and Bones) is a cytosolic specifier protein of the glucosinolate-myrosinase defence system. It has two parts: an N-terminal jacalin-related lectin (JAL) domain with a beta-prism fold, and a C-terminal Kelch-repeat six-bladed beta-propeller that carries the active site, including a predicted EXXXDXXXH-type iron-binding triad. The protein is a monomer. NSP1 has no activity on intact glucosinolates. After tissue damage it acts on the unstable aglucone released by myrosinase and converts it to a simple nitrile (with release of sulfur and sulfate; EC 4.8.1.5) instead of the default isothiocyanate. It does this with aliphatic, aromatic (benzyl) and indolic glucosinolates, but it does not form epithionitriles or thiocyanates. In Columbia-0, where basal rosette-leaf ESP activity is low, NSP1 accounts for most constitutive simple-nitrile formation in rosette leaves and for the rise in nitrile formation after feeding by Pieris rapae larvae; its transcript is induced by that herbivory. It is also the main nitrile-forming activity in seedlings and, together with NSP3, in roots. nsp1 mutants show altered glucosinolate content in seeds and during germination, which suggests an additional role in glucosinolate turnover. No function of the lectin domain, such as carbohydrate or myrosinase binding, has been demonstrated experimentally.
Definition: Catalysis of the reaction: a (Z)-N-(sulfonatooxy)alkanimidothioate (a glucosinolate aglucone) = a nitrile + sulfur + sulfate (EC 4.8.1.5; RHEA:59956). Converts myrosinase-released glucosinolate aglucones to simple nitriles instead of isothiocyanates.
Justification: Nitrile-specifier proteins are characterized catalysts with a curated EC number and Rhea reaction. GO has no term below carbon-sulfur lyase activity (GO:0016846) for this reaction. It is the sister activity of the epithionitrile-forming (ESP) and thiocyanate-forming (TFP) sulfolyases proposed in those reviews.
Parent term: carbon-sulfur lyase activity
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003729 mRNA binding | HDA PMID:27729395 In Planta Determination of the mRNA-Binding Proteome of Arab... | UNDECIDED | Summary: The NSP1-specific mRNA-capture result requires the unavailable target data. Reason: PMID:27729395 uses in-planta mRNA interactome capture and explicitly includes metabolic enzymes among candidate RNA-binding proteins; NSP1 enzyme function is therefore not grounds for dismissing the HDA annotation. A fresh normal fetch remained abstract-only and the PMC full-text endpoint returned a browser challenge. The target-specific enrichment, confidence-set membership and controls could not be inspected. Keep this genuinely unresolved claim pending those data, rather than calling it promiscuous binding or applying a generic organelle-fraction argument. Supporting Evidence: PMID:27729395 Other At-RBP set proteins include major signaling proteins, cytoskeleton-associated proteins, membrane transporters, and enzymes, suggesting the scope and function of RNA-protein interactions within a plant cell is much broader than previously appreciated. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from the specifier-protein node PTN002355837. The sole experimental donor is ESP (AT1G54040), whose nuclear pool depends on interaction with WRKY53. Reason: The only grounding for this node is ESP's WRKY53-dependent nuclear import. In wrky53 knockout cells ESP is exclusively cytoplasmic. That is a conditional, interaction-specific localization of one paralog, not an ancestral property of the family. No WRKY53 interaction or nuclear localization has been reported for NSP1. Proteomics places NSP1 in the cytosol, where it meets myrosinase-released aglucones. The nucleus is therefore an over-propagation to NSP1. The action is MARK_AS_OVER_ANNOTATED rather than REMOVE because a minor nuclear pool of a soluble ~50 kDa protein cannot be excluded. Propagation Review Root cause: PROPAGATION BAD Failure modes: CONTEXT OR TISSUE MISMATCH Sources checked: PANTHER:PTN002355837 SUPPORTS SOURCE BUT NOT TARGET Specifier-protein node. Its nuclear 'is_active_in' is seeded only by ESP's WRKY53-dependent nuclear import, which is not shown to be ancestral and has no counterpart reported for the NSPs. AGI_LocusCode:AT1G54040 SUPPORTS SOURCE BUT NOT TARGET ESP nuclear localization (IDA, PMID:17369373) holds for ESP only in the presence of WRKY53. Supporting Evidence: PMID:17369373 ESR is exclusively in the cytoplasm in W53-KO cells, indicating that ESR is brought to the nucleus by the interaction PMID:25293756 we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes |
| GO:0005737 cytoplasm | ISM GO_REF:0000122 | MODIFY | Summary: The supported cytoplasmic compartment is the cytosol. Reason: Refine the true general cytoplasm location to cytosol, already supported elsewhere in this review. This refinement does not exclude distinct secondary compartments reported under other terms. Proposed replacements: cytosol Supporting Evidence: PMID:25293756 we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes |
| GO:0005829 cytosol | HDA PMID:25293756 A proteomic strategy for global analysis of plant protein co... | ACCEPT | Summary: NSP1 was detected by label-free proteomics in the crude cytosolic fraction of Arabidopsis leaves (size-exclusion profiling). Reason: High-throughput but direct. It is consistent with NSP1 being a soluble specifier protein acting on aglucones in the cytosol. This is the core location. Supporting Evidence: PMID:25293756 Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins |
| GO:0005829 cytosol | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | ACCEPT | Summary: Detected in the soluble/cytosolic fraction in a proteome-wide fractionation-SEC study of Arabidopsis leaves. Reason: Independent proteomic support for cytosolic localization. Supporting Evidence: PMID:28887381 Over 150 proteins had a complicated localization pattern, and were clearly partitioned between cytosolic and membrane-associated pools. |
| GO:0009845 seed germination | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA machine-learning annotation, most likely learned from NSP-family germination-stage data (PMID:31850033). Reason: The underlying observation is that NSP1 affects the time course of glucosinolate content during germination, a metabolic phenotype already captured by glucosinolate catabolic process. The full text of PMID:31850033 (now cached) measures only glucosinolate content; it reports no germination assay (germination rate, radicle emergence) for nsp1-1, so NSP1 is not shown to do any of the work of seed germination. Marked as over-annotated, consistently with the IEP and IMP seed germination rows from the same paper. Supporting Evidence: PMID:31850033 The time course of total glucosinolate content during ten days of germination depended on functional NSP1. PMID:31850033 In contrast to Col-0 and tgg1 tgg2, germinating seeds of nsp1-1 did not contain higher total glucosinolate levels on day 4 relative to days 0 and 10 (Figure 7C). |
| GO:0009845 seed germination | IEP PMID:31850033 Glucosinolate Content in Dormant and Germinating Arabidopsis... | MARK AS OVER ANNOTATED | Summary: Expression-pattern annotation. NSP1 protein accumulates progressively after germination and in young seedlings (UniProt DEVELOPMENTAL STAGE). The full text is now cached; its expression data are public ATH1 microarray series extracted from Genevestigator. Reason: An expression pattern during germination supports expression in germinating seeds, not participation in germination. The full text confirms that the expression evidence is re-analysed microarray data for seed maturation and germination, and that the only nsp1 phenotypes are glucosinolate-content changes; no germination assay was performed. The 'involved_in seed germination' claim therefore overstates the evidence. Marked as over-annotated (not removed) because NSP1 is genuinely expressed in this developmental window. Supporting Evidence: PMID:31850033 Expression of NSP genes during maturation and germination of A. thaliana Col-0 seeds. PMID:31850033 mutants deficient in either seed-expressed NSP2 or seedling-expressed NSP1 were affected in glucosinolate content in seeds and during stratification or germination when compared to Col-0 indicating a direct role in turnover |
| GO:0009845 seed germination | IMP PMID:31850033 Glucosinolate Content in Dormant and Germinating Arabidopsis... | MARK AS OVER ANNOTATED | Summary: IMP from nsp1 T-DNA mutants analysed during germination. The abstract reports altered glucosinolate content, not altered germination. The full text is now cached. Reason: Having read the full text, it contains no germination-rate or radicle-emergence data for nsp1-1; the mutant phenotype is purely metabolic (time course of seed glucosinolate content during germination), and the authors themselves state that further experiments are needed to establish a direct role of NSPs in turnover. That phenotype is already captured by the glucosinolate catabolic process IMP from the same paper. The acts_upstream_of_or_within seed germination row is therefore an over-interpretation of a metabolic phenotype; it is marked as over-annotated rather than removed because the observation itself is sound. Supporting Evidence: PMID:31850033 Although our data confirm glucosinolate turnover during seed-seedling transition and provide some indication for involvement of NSPs, more experiments are required to establish a direct participation of NSPs in turnover pathways. PMID:31850033 The time course of total glucosinolate content during ten days of germination depended on functional NSP1. |
| GO:0010162 seed dormancy process | IMP PMID:31850033 Glucosinolate Content in Dormant and Germinating Arabidopsis... | MARK AS OVER ANNOTATED | Summary: IMP from nsp1 mutant seeds (dry/dormant seeds and stratification). The abstract reports higher glucosinolate levels in dry nsp1-1 seeds but no dormancy phenotype. The full text is now cached. Reason: The full text reports no dormancy assay (dormancy depth, after-ripening or germination of fresh versus stored seed) for nsp1-1; seeds were stored for months before analysis and the measured trait is glucosinolate content in dry and stratified seed. Glucosinolate content of dormant seeds is a metabolic trait, not the seed dormancy process. Marked as over-annotated rather than removed because the underlying metabolic observation is sound. Supporting Evidence: PMID:31850033 Mutant lines deficient in NSP1 or NSP2 had significantly higher glucosinolate contents in seeds than Col-0 (Figures 2B, C), but did not differ from Col-0 seeds in weight (Figure S1). PMID:31850033 The mutant lines nsp1-1, nsp2-1 and nsp2-2 had significantly higher glucosinolate levels in dry seeds than Col-0. |
| GO:0019762 glucosinolate catabolic process | IDA PMID:18987211 The genetic basis of constitutive and herbivore-induced ESP-... | ACCEPT | Summary: Purified recombinant NSP1 converts allyl-, 4-methylsulfinylbutyl-, 4-methylthiobutyl- and benzylglucosinolate to their simple nitriles in the presence of myrosinase. Reason: Core process. NSP1 carries out the post-myrosinase conversion of the glucosinolate aglucone. Supporting Evidence: PMID:18987211 The protein purified from bacterial extracts promoted the formation of the simple nitrile, but not the epithionitrile, upon myrosinase-catalyzed hydrolysis of allylglucosinolate PMID:18987211 AtNSP1 also converts 4-methylsulfinylbutylglucosinolate, 4-methylthiobutylglucosinolate, and benzylglucosinolate to their corresponding simple nitriles in the presence of myrosinase |
| GO:0019762 glucosinolate catabolic process | IEA GO_REF:0000120 | ACCEPT | Summary: Combined electronic annotation from ARBA and the InterPro NSP1/2/3 family signature. Reason: Correct and consistent with experimental data for NSP1. Supporting Evidence: PMID:18987211 The protein purified from bacterial extracts promoted the formation of the simple nitrile, but not the epithionitrile, upon myrosinase-catalyzed hydrolysis of allylglucosinolate |
| GO:0019762 glucosinolate catabolic process | IMP PMID:31850033 Glucosinolate Content in Dormant and Germinating Arabidopsis... | ACCEPT | Summary: nsp1 mutants have altered glucosinolate content in seeds and during germination. The authors interpret this as a direct role of NSPs in glucosinolate turnover. Reason: Consistent with NSP1's role in glucosinolate breakdown. The abstract explicitly links NSP1 to glucosinolate content during germination. Supporting Evidence: PMID:31850033 mutants deficient in either seed-expressed NSP2 or seedling-expressed NSP1 were affected in glucosinolate content in seeds and during stratification or germination when compared to Col-0 indicating a direct role in turnover |
| GO:0030234 enzyme regulator activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA reflecting the historical view of specifier proteins as myrosinase cofactors that regulate myrosinase product outcome. Reason: NSP1 has its own active site in the Kelch propeller and converts the aglucone released by myrosinase to a simple nitrile. UniProt curates this as EC 4.8.1.5 (thiohydroximate-O-sulfate sulfur/sulfate-lyase, nitrile-forming). Specifier proteins are now regarded as Fe(II)-dependent lyases, not regulators of myrosinase. The node placement is sound, but the activity should be captured as carbon-sulfur lyase activity (GO:0016846), consistent with the ESP and TFP reviews. No more specific GO term exists; see proposed_new_terms. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: ROLE CONFLATION Sources checked: PANTHER:PTN001509219 SUPPORTS TRANSFER Specifier-protein ancestral node. Placing the specifier activity at this node is sound, but it carries the historical myrosinase "regulator/cofactor" MF term instead of the lyase activity. The fix is a term swap for all descendants. Proposed replacements: carbon-sulfur lyase activity Supporting Evidence: PMID:30900313 Based on these insights, we propose that specifier proteins are catalysts that might be classified as Fe2+ -dependent lyases. PMID:28479247 the R292 residue in AtNSP1 displayed a conformation different from those of the corresponding residues in ESP and TFP proteins, which might account for the product specificity and catalytic mechanism of AtNSP1 |
| GO:0030246 carbohydrate binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the plant jacalin-like lectin domain (IPR033734) in the N-terminal ~145 residues of NSP1 (JAL28). This is separate from the specifier activity, which lies in the Kelch propeller. ESP lacks the JAL domain yet has the same class of activity. Reason: Structurally plausible: NSP1 has a jacalin beta-prism domain, and jacalin domains typically bind mannose or galactose. Sugar binding by NSP1 has not been tested, and its authors state the domain's role is unknown. The domain is not required for nitrile-specifier activity, since ESP and NSP5 lack it. The domain-based inference is kept as non-core, pending experimental glycan binding data. Supporting Evidence: PMID:30395611 In addition, AtNSP1 possesses an N-terminal JAL domain PMID:19224919 Although Kelch repeats are involved in protein-protein interactions ( 24 ) and lectins are known to bind carbohydrates ( 40 ), the role of these domains in AtESP and homologous proteins is not yet known. |
| GO:0080027 response to herbivore | IMP PMID:18987211 The genetic basis of constitutive and herbivore-induced ESP-... | KEEP AS NON CORE | Summary: Pieris rapae feeding induces NSP1 transcript and increases simple-nitrile formation in wild-type Col-0 leaves. The increase is absent in the nsp1 T-DNA mutant (SALK_072600). Reason: Experimental and full text available. NSP1 is required for the herbivore-induced shift toward nitrile formation, so it acts within a chemical response to herbivory. Whether this protects the plant has not been shown. Simple nitriles are proposed to act as volatile signals in direct and indirect defence against P. rapae, and in the paralog ESP, nitrile formation increased generalist feeding. The response is real but downstream of the core biochemical activity, so it is kept as non-core. Supporting Evidence: PMID:18987211 While nitrile formation in Col-0 wild-type plants was herbivore inducible, herbivory by P. rapae larvae did not increase the proportion of nitriles formed upon leaf homogenization in SALK_072600 plants PMID:18987211 While feeding by P. rapae larvae on wild-type plants led to increased At3g16400 transcript levels in the damaged leaves |
| GO:0080028 nitrile biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA at the specifier-protein node. NSP1's own experimental annotation (AT3G16400) is among the donors, as expected. Reason: Correct and core. Simple-nitrile formation is the defining activity of the NSPs, the evolutionarily oldest specifier proteins. Supporting Evidence: PMID:18987211 AtNSP1 is one of five Arabidopsis ESP homologues that promote simple nitrile, but not epithionitrile or thiocyanate, formation. |
| GO:0080028 nitrile biosynthetic process | IDA PMID:18987211 The genetic basis of constitutive and herbivore-induced ESP-... | ACCEPT | Summary: Purified recombinant NSP1 forms simple nitriles from several glucosinolates with myrosinase, and nsp1 leaves make only trace nitriles. Reason: Direct biochemical evidence for the core activity. Supporting Evidence: PMID:18987211 a line with a T-DNA insertion in the second exon of At3g16400 (SALK_072600) produced no or only trace amounts of simple nitriles of the endogenous glucosinolates in rosette leaf homogenates PMID:18987211 AtNSP1 did not promote thiocyanate or epithionitrile formation from any of the tested substrates. |
| GO:0080028 nitrile biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Combined electronic annotation (ARBA and InterPro NSP1/2/3 family). Reason: Correct and consistent with experimental data. Supporting Evidence: PMID:19224919 3-butenylnitrile was also detected upon incubation with AtNSP1 ( Fig. 5 A ) and AtNSP3 ( Fig. 5 C ), showing that these proteins also possess nitrile-specifier activity. |
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Download this section (compressed HTML)Q: Does the N-terminal jacalin-related lectin domain of NSP1 bind carbohydrates (or glycosylated myrosinase), and does it contribute to capturing the aglucone by bringing NSP1 close to myrosinase?
Q: Is NSP1 truly Fe(II)-dependent in vivo? Recombinant NSP1 was insensitive to EDTA in one study, while paralogs bind iron through a conserved EXXXDXXXH triad.
Q: Is the seed and germination glucosinolate phenotype of nsp1 a direct turnover role of NSP1 in intact tissue, or an indirect result of feedback on hydrolytic enzymes?
Experiment: Glycan-array or affinity binding assays with the isolated NSP1 JAL domain, and pull-down/proximity labelling with TGG1/TGG2 and PYK10-type myrosinases, to test lectin function and myrosinase association.
Experiment: ICP-MS/Ferene S iron quantification and E386Q/D390N/H394A mutagenesis of purified NSP1, to confirm the iron cofactor and its requirement for nitrile formation.
Experiment: Germination-rate and dormancy assays (after-ripening, stratification response) on nsp1 alleles and complemented lines, to test whether any developmental phenotype exists beyond altered glucosinolate content.
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