NCU06005 encodes the glycerol kinase of Neurospora crassa, an FGGY-family carbohydrate kinase that phosphorylates glycerol with ATP to form sn-glycerol 3-phosphate. It belongs to the glycerol kinase subfamily (PANTHER PTHR10196:SF69) together with the experimentally characterized enzymes of budding yeast (GUT1), Arabidopsis (GLPK) and Escherichia coli (GlpK), and it is the only glycerol kinase gene in the N. crassa reference genome. Glycerol phosphorylation is the first step of the inducible glycerol dissimilation pathway, in which sn-glycerol 3-phosphate is then oxidized by mitochondrial glycerol-3-phosphate dehydrogenase to dihydroxyacetone phosphate. Classical genetics and enzymology identified a cytosolic, glycerol-, cold- and deoxyribose-inducible glycerokinase encoded by the glp-4 locus on linkage group VI, where NCU06005 also lies; no molecular complementation of glp-4 by NCU06005 has been published.
Summary: Glycerol kinase activity is inherited from the PTN000023392 node and NCU06005 sits in the glycerol kinase subfamily SF69 with the characterized yeast, plant and E. coli enzymes. It carries the glycerol-kinase-specific IPR005999/TIGR01311 signatures and is the only glycerol kinase gene in the N. crassa proteome, so the classical N. crassa cytosolic glycerokinase activity (glp-4, linkage group VI) is best explained as the product of this locus. Core function.
Reason: Sound phylogenetic transfer within subfamily SF69, consistent with organism-level enzymology.
Propagation Review
Root cause:NO FAILURE CORE
Sources checked:
PANTHER:PTN000023392SUPPORTS TRANSFER
Node seeded by experimentally characterized glycerol kinases including yeast GUT1 and E. coli GlpK, both in the same PANTHER subfamily (SF69) as NCU06005; no evidence of loss in the target.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Summary: Same claim as the IBA row; the glycerol-kinase-specific InterPro and NCBIfam signatures and subfamily SF69 membership support it. Core function.
Reason: Consistent with the IBA row and the organism-level enzymology.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Summary: The mitochondrion IBD at PTN000023394 is seeded by mammalian GK (porin-bound outer-membrane pool, SF56) and by yeast GUT1, the SF69 sister of NCU06005, whose in vitro mitochondrial import shows an additional pool of a mainly cytosolic enzyme. For N. crassa the only localization evidence is classical enzymology placing glycerokinase in the cytosol, distinct from mitochondrial glycerol-3-phosphate dehydrogenase; that assay reports the main pool and cannot exclude a minor mitochondrial one. Its N-terminal extension is net acidic and lacks presequence-like composition, which argues against a matrix-targeted form but not against outer-membrane association or a minor imported pool. With no target-specific evidence of loss, the inherited location is retained as a plausible minor, non-core location, consistent with the treatment of the same IBA on yeast GUT1 and with the family-level judgment that the eukaryotic glycerol kinase clade inherits both cytosol and mitochondrion.
Reason: Phylogenetically sound inheritance of a secondary location from an SF69 sister seed (Gut1p); the main Neurospora activity is cytosolic, so the mitochondrial pool is at most a minor, non-core location. Changed from MARK_AS_OVER_ANNOTATED because the cytosolic fractionation does not contradict a minor pool.
Propagation Review
Root cause:NO FAILURE NON CORE
Sources checked:
PANTHER:PTN000023394SUPPORTS TRANSFER
Seeds have real but additional mitochondrial pools (mammalian outer-membrane GK, yeast imported Gut1p, the latter in the same SF69 subfamily as NCU06005); transfer of a minor location is sound. The sibling cytosol IRD at this node is contested in interpro/panther/PTHR10196/PTHR10196-review.yaml (LOSS_CONTRADICTED).
we performed mitochondrial import experiments and confirmed import of both Gut1p and Gpd2p, implying a novel dual cytosolic and mitochondrial localization
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Summary: Generic FGGY-family mapping. True but uninformative; glycerol catabolic process (a descendant of carbohydrate metabolic process in GO, and already carried by the gene) states the established role of the enzyme in the inducible glycerol dissimilation pathway.
Reason: Correct but too general; refine to the specific descendant the gene already carries.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Summary: Inheritance of a glycerol-metabolism role from the eukaryotic glycerol kinase node is sound. In N. crassa the enzyme is part of the inducible glycerol dissimilatory system, so glycerol catabolic process is the more precise descendant.
Reason: Correct but general; glycerol catabolic process is the specific supported descendant.
Propagation Review
Root cause:TERM SCOPING PROBLEM
Failure modes:GRANULARITY MISMATCH
Sources checked:
PANTHER:PTN000023394SUPPORTS TRANSFER
Node transfer is sound; only the granularity is refined.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Summary: The kinase directly forms sn-glycerol 3-phosphate, so the specific descendant glycerol-3-phosphate biosynthetic process (already carried by the gene via IBA) is the precise term.
Reason: Correct but general; refine to the biosynthetic descendant.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Summary: The IBD is seeded only by mammalian GK (subfamily SF56), where glycerol phosphorylation feeds triglyceride synthesis and recycling in liver and adipose tissue. In N. crassa the enzyme is part of an inducible glycerol dissimilatory (catabolic) system. Supplying sn-glycerol 3-phosphate that could be acylated is substrate supply, not a step of triglyceride metabolism, and there is no fungal evidence for such a role. The previous KEEP_AS_NON_CORE is changed for consistency with the same judgment on the SF69 sister enzyme yeast GUT1.
Reason: Mammal-specific physiological role propagated to a fungal glycerol-catabolic enzyme; precursor supply is not participation.
Propagation Review
Root cause:PROPAGATION BAD
Failure modes:CONTEXT OR TISSUE MISMATCHROLE CONFLATION
Sources checked:
PANTHER:PTN000023394SUPPORTS SOURCE BUT NOT TARGET
Seeds are mammalian SF56 glycerol kinases whose triglyceride roles reflect tissue lipid physiology; not transferable to the Neurospora glycerol-catabolic enzyme.
Summary: Correct but general; the glycerol-kinase-specific signatures and subfamily resolve the activity to glycerol kinase activity, which the gene already carries.
Reason: Refine to the specific descendant glycerol kinase activity.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
GO:0016773 phosphotransferase activity, alcohol group as acceptor
IEA GO_REF:0000002
MODIFY
Summary: Correct but general; glycerol kinase activity is the specific supported descendant.
Reason: Refine to the specific descendant glycerol kinase activity.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Summary: ATP-dependent phosphorylation of glycerol is the first step of the N. crassa inducible glycerol dissimilatory pathway (glycerokinase followed by mitochondrial glycerol-3-phosphate dehydrogenase). The enzyme performs this step, so it participates in glycerol catabolism. Core process.
Reason: The gene product catalyses the entry step of the pathway.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
GO:0046167 glycerol-3-phosphate biosynthetic process
IBA GO_REF:0000033
ACCEPT
Summary: The glycerol kinase reaction itself forms sn-glycerol 3-phosphate, so the enzyme catalyses this biosynthetic step directly. Sound inheritance from the eukaryotic glycerol kinase node.
Reason: Direct consequence of the core catalytic activity.
Propagation Review
Root cause:NO FAILURE CORE
Sources checked:
PANTHER:PTN000023394SUPPORTS TRANSFER
Product formation follows from the conserved catalytic activity.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
IC PMID:6284716 Genetic and enzymatic characterization of the inducible glyc...
NEW
Summary: Classical N. crassa enzymology placed the inducible glycerokinase in the cytosol, distinct from the mitochondrial glycerol-3-phosphate dehydrogenase. NCU06005 is the only glycerol kinase gene in the genome and lies on the same linkage group (VI) as glp-4, so the cytosolic activity is attributable to this locus. The sister SF69 enzyme yeast Gut1p is likewise cytosolic. PAINT placed a cytosol IRD at PTN000023394; that call is contested at family level (PTHR10196 review, LOSS_CONTRADICTED) because it negates the localization of its own seeds, and organism-level evidence for N. crassa supports cytosol.
Reason: Organism-specific evidence for the cytosolic location of the activity. IC rather than IDA because the 1982 enzymology assayed the glycerokinase activity, and its attribution to NCU06005 rests on curator inference (sole glycerol kinase gene, same linkage group as glp-4), not on a molecular identification in the paper.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively.
Q: Does an NCU06005 deletion strain (FGSC knockout collection) phenocopy glp-4 mutants (no growth on glycerol, no inducible glycerokinase activity), and does NCU06005 complement a glp-4 allele?
Q: Is there a minor mitochondrion-associated pool of Neurospora glycerol kinase, as reported for yeast Gut1p?
Suggested Experiments
Experiment: Assay glycerokinase activity and growth on glycerol in the NCU06005 deletion strain, and complement a classical glp-4 mutant with NCU06005, to confirm the locus identity.
Experiment: Subcellular fractionation or fluorescent-protein tagging of NCU06005 to test for a mitochondrial pool alongside the reported cytosolic activity.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Review rationale: The glycerol-kinase-specific family assignment and classical N. crassa glycerol-dissimilation enzymology ground transfer of glycerol phosphorylation. This directly produces glycerol-3-phosphate, supporting the existing biosynthetic-process annotation. The predicted metabolic process is biologically correct but less precise than biosynthesis.
PMID:6284716: "Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively."