pos5 (SPAC323.01c) encodes the mitochondrial NAD(H) kinase of fission yeast, an NAD kinase family enzyme that phosphorylates NADH (and NAD+) with ATP to make NADPH (NADP+) inside mitochondria. Because NADP(H) cannot be imported into yeast mitochondria, this kinase is the main source of the mitochondrial NADP(H) pool, although a substantial residual pool remains in its absence. The protein carries an N-terminal mitochondrial targeting sequence and localises to mitochondria, most likely to the matrix as shown for its budding-yeast ortholog Pos5. Deletion lowers mitochondrial NADP+ and total NADP(H) and reduces cellular coenzyme Q10 to about 20% of wild type. The mutant cannot grow on non-fermentable carbon sources, is hypersensitive to hydrogen peroxide, overproduces sulfide, is partially arginine-requiring and has rounded cells. These defects are rescued by budding-yeast POS5, or by the cytosolic NAD kinase Utr1 only when it is targeted to mitochondria. The CoQ deficiency points to an unidentified NADPH-dependent step upstream of the quinone ring modifications rather than at the Coq6 hydroxylase.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005739 mitochondrion | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: Genome-wide ORFeome YFP-fusion localisation places Pos5 in mitochondria. A plasmid-expressed Pos5-GFP co-localising with MitoTracker confirms this independently (PMID:41926382). Reason: Direct whole-organelle localisation evidence, which cannot resolve the sub-compartment. Under the root organelle policy (projects/ORGANELLES.md) this is ACCEPT. The location is central to the function, since the gene's role is to supply the mitochondrial NADP(H) pool, and mitochondrial targeting is required for function (PMID:41926382). The matrix sub-compartment is carried by the separate IBA row. The one caveat is that an endogenous C-terminal GFP fusion mislocalised and was non-functional in Nishihara et al. The plasmid GFP result would support an additional IDA row from PMID:41926382; it is not added as NEW because the term is already in GOA. Supporting Evidence: PMID:16823372 we determined the localization of 4,431 proteins PMID:41926382 The Pos5-GFP fluorescence overlapped with the MitoTracker Red FM signal (Fig 5), indicating that Pos5 localizes to mitochondria as a mitochondrial NAD(H) kinase |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping. The UniProt location line rests on the ORFeome study (ECO:0000269, PubMed:16823372). Reason: I checked the source. The SubCell keyword is grounded in the S. pombe experimental localisation (the HDA row above), not in a prediction. It agrees with the predicted transit peptide and with the independent GFP co-localisation in PMID:41926382. So the organelle claim holds, and the row is consistent with the experimental rows. Supporting Evidence: file:SCHPO/pos5/pos5-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:16823372}. PMID:41926382 The Pos5-GFP fluorescence overlapped with the MitoTracker Red FM signal (Fig 5), indicating that Pos5 localizes to mitochondria as a mitochondrial NAD(H) kinase |
| GO:0005759 mitochondrial matrix | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference from the S. cerevisiae Pos5 node. ScPos5 was shown to be in the mitochondrial matrix. Reason: The PAINT node is seeded by ScPOS5, which was localised to the matrix experimentally (PMID:12727869). S. pombe pos5 is the 1:1 ortholog (PANTHER subfamily PTHR20275:SF26). Several independent lines support the matrix in S. pombe. First, there is an N-terminal mitochondrial transit peptide, and deleting the first 83 residues abolishes function. Second, the protein's product, mitochondrial NADP(H), is a matrix cofactor pool, and that pool is depleted in ฮpos5. Third, an unrelated NAD kinase rescues only when given a matrix-targeting presequence (the SpCoq3 MTS). No sub-mitochondrial fractionation has been done in S. pombe. Even so, the matrix is where the activity is expected to act, so this is the core location. Supporting Evidence: PMID:12727869 is localized to the mitochondrial matrix of yeast and appears to be important PMID:41926382 CoQ quantification showed that only MTS36UTR1 restored CoQ production, whereas neither ฮMTS83pos5 nor UTR1 could recover CoQ levels in the ฮpos5 strain PMID:41926382 Mitochondrial NADP+ level and total NADP(H) level in ฮpos5 were decreased to 0.6- and 0.8-fold, respectively, of those in the wild-type strain |
| GO:0006741 NADP+ biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the NAD kinase family (IPR002504). Pos5 catalyses the phosphorylation step that makes NADP(H) from NAD(H). Reason: The term is defined as formation of NADP+ from NAD+, which is exactly the reaction class the NAD kinase family catalyses. Pos5 therefore performs the step itself. S. pombe ฮpos5 mitochondria have less NADP+ and less total NADP(H) (PMID:41926382). This is the core biological process. The S. pombe deletion data would justify an additional experimental (IMP) row from PMID:41926382, which is noted for PomBase rather than added here because the term is already in GOA. Supporting Evidence: file:SCHPO/pos5/pos5-uniprot.txt Belongs to the NAD kinase family. PMID:41926382 Mitochondrial NADP+ level and total NADP(H) level in ฮpos5 were decreased to 0.6- and 0.8-fold, respectively, of those in the wild-type strain |
| GO:0016301 kinase activity | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning assignment of generic kinase activity. It is correct but uninformative. Reason: The term is true but too general. The specific, supported activity is NADH kinase activity (GO:0042736), an is_a descendant that the gene already carries (IBA and IEA rows). Support for it comes from NAD kinase family membership, the ScPos5 biochemistry (PMID:12727869), and rescue of S. pombe ฮpos5 by ScPOS5 or by mitochondrially targeted Utr1 (PMID:41926382). Proposed replacements: NADH kinase activity Supporting Evidence: file:SCHPO/pos5/pos5-uniprot.txt Belongs to the NAD kinase family. PMID:41926382 CoQ levels in the NSP7 strain were recovered to 0.9-fold of that observed in the ฮpos5 + pos5 strain (NSP11) |
| GO:0016773 phosphotransferase activity, alcohol group as acceptor | IEA GO_REF:0000117 | MODIFY | Summary: ARBA assignment of a generic phosphotransferase parent term. It is correct but uninformative. Reason: NADH kinase activity (GO:0042736) is an is_a descendant of this term. It is the activity supported for this gene and is already annotated by the IBA and IEA rows, so the general parent should be refined to it. Proposed replacements: NADH kinase activity Supporting Evidence: file:SCHPO/pos5/pos5-uniprot.txt Belongs to the NAD kinase family. |
| GO:0019674 NAD+ metabolic process | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping (IPR017437, ATP-NAD kinase C-terminal domain). Pos5 consumes NAD(H) as the substrate for NADP(H) formation. Reason: The term is true but too general. The specific process Pos5 carries out is NADP+ biosynthesis from NAD(H) (GO:0006741). That term is an is_a child of NAD+ metabolic process and is already on the gene (IEA row). Proposed replacements: NADP+ biosynthetic process Supporting Evidence: PMID:41926382 Mitochondrial NADP+ level and total NADP(H) level in ฮpos5 were decreased to 0.6- and 0.8-fold, respectively, of those in the wild-type strain |
| GO:0034599 cellular response to oxidative stress | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference from ScPos5, the "peroxide-sensitive" mutant that is needed for resistance to oxidative stress. S. pombe ฮpos5 is also hypersensitive to H2O2. Reason: The IBA placement is sound. The ScPos5 seed is required for oxidative stress resistance (PMID:12727869), and the S. pombe mutant grows slowly on H2O2 (PMID:41926382). The role is indirect, though. Pos5 supplies the mitochondrial NADPH that antioxidant reductases use. CoQ deficiency alone also makes S. pombe H2O2-sensitive, and ฮpos5 is CoQ-deficient. The term is kept because the gene contributes to the response through its reductant supply, but it is peripheral to the core enzymatic function. Supporting Evidence: PMID:12727869 to a broad range of oxidative stress conditions, arginine biosynthesis and PMID:41926382 The ฮpos5 strain failed to grow on YEGES medium containing glycerol and ethanol as non-fermentable carbon sources and showed retarded growth on YES containing hydrogen peroxide as well as on minimal medium |
| GO:0042736 NADH kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference from ScPos5, whose recombinant protein has NADH kinase activity. S. pombe pos5 is its ortholog, and ScPOS5 complements S. pombe ฮpos5. Reason: This is the core molecular function. The node is seeded by ScPos5 NADH kinase activity measured on the recombinant protein (PMID:12727869). S. pombe Pos5 belongs to the same PANTHER subfamily, keeps the conserved NAD kinase motifs, and is functionally replaced by ScPOS5. The S. pombe defect is also rescued by an unrelated NAD kinase (Utr1) once it is targeted to mitochondria (PMID:41926382). The S. pombe enzyme has not been assayed directly. Supporting Evidence: PMID:12727869 recombinant Pos5p has NADH kinase activity PMID:41926382 CoQ levels in the NSP7 strain were recovered to 0.9-fold of that observed in the ฮpos5 + pos5 strain (NSP11) PMID:41926382 CoQ quantification showed that only MTS36UTR1 restored CoQ production, whereas neither ฮMTS83pos5 nor UTR1 could recover CoQ levels in the ฮpos5 strain |
| GO:0042736 NADH kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated mapping from the UniProt catalytic-activity line (RHEA:12260, EC 2.7.1.86). It is consistent with the IBA row. Reason: This row makes the same core claim as the IBA row, from the UniProt catalytic activity curated by similarity to ScPos5. It is consistent with family membership and with the complementation data from S. pombe. Supporting Evidence: file:SCHPO/pos5/pos5-uniprot.txt Phosphorylates both NADH and NAD(+), with a preference for NADH. PMID:12727869 recombinant Pos5p has NADH kinase activity |
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Download this section (compressed HTML)Q: Should Pos5 carry GO:0006744 ubiquinone biosynthetic process? This review does not add it. Pos5 supplies mitochondrial NADPH, but no CoQ biosynthetic step is assigned to it. A comparator query (QuickGO, GO:0006744 and descendants, S. pombe, S. cerevisiae and human) finds the Coq enzymes and the ferredoxin/ferredoxin reductase electron donors to Coq6 (arh1, etp1, ARH1, YAH1, FDXR, FDX2), but no NADPH-generating enzyme (ScPOS5, UTR1, ZWF1, NADK, NADK2, G6PD, zwf1). The CoQ phenotype would be better captured as a FYPO phenotype (decreased ubiquinone level). If the consuming step is identified, a has_input/causal link in a GO-CAM model would express the dependency better than a process annotation.
Q: Does S. pombe Pos5 prefer NADH over NAD+ as its ScPos5 ortholog does? In S. pombe ฮpos5 mitochondria NADP+ falls more than NADPH, whereas in S. cerevisiae ฮpos5 NADPH falls most. Could this reflect different substrate preference, or compensating NADP+-reducing activities?
Q: Where is the processing site of the Pos5 presequence? UniProt predicts a 42-residue transit peptide, whereas the authors align residues 1-83 to the 62-residue ScPos5 presequence.
Q: Why does the endogenous C-terminal GFP fusion mislocalise and lose function, while the plasmid-expressed fusion localises to mitochondria? Is the C-terminus needed for oligomerisation or import?
Experiment: Purify recombinant mature S. pombe Pos5 and measure its kinetics with NADH and NAD+ (ATP and polyphosphate as donors) to confirm the inferred NADH kinase activity and substrate preference.
Hypothesis: S. pombe Pos5 is an NADH-preferring NAD kinase.
Type: in vitro enzyme assay
Experiment: Use sub-mitochondrial fractionation and protease protection assays (mitoplasts, carbonate extraction) of functional tagged Pos5 to place it in the matrix.
Hypothesis: Pos5 acts in the mitochondrial matrix.
Type: biochemical fractionation
Experiment: Use LC-MS profiling of CoQ intermediates and of PHB/4-hydroxybenzaldehyde in wild type, ฮpos5 and ฮpos5 with PHB supplementation, with mitochondrial Fe-S enzyme activity measurements to separate a direct cofactor role from indirect Fe-S effects.
Hypothesis: A specific NADPH-dependent step upstream of ring modification limits CoQ synthesis in ฮpos5.
Type: metabolomics
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