hal3 (SPAC15E1.04) encodes a 625-residue fission-yeast fusion protein whose N-terminal half is a Hal3/HFCD-family flavoprotein domain and whose C-terminal half is thymidylate synthase. The protein carries two essential biosynthetic activities: it is the sole (monogenic) phosphopantothenoylcysteine decarboxylase (PPCDC) of S. pombe, converting 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in coenzyme A biosynthesis, and its C-terminal domain is a thymidylate synthase that methylates dUMP to dTMP (it rescues a budding-yeast cdc21 mutant). Unlike budding yeast, where PPCDC is a Cab3-Sis2/Vhs3 heterotrimer and thymidylate synthase is the separate Cdc21 protein, both activities reside in one S. pombe polypeptide, which is not proteolytically processed. Like budding-yeast Hal3, the N-terminal domain also binds and modestly inhibits the Ppz-family Ser/Thr phosphatases (S. pombe Pzh1) in vitro. The gene is essential and the protein is cytoplasmic/cytosolic.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO 'catalytic activity' from the flavoprotein/HFCD signatures IPR003382 and IPR036551 of the N-terminal Hal3-like domain. True but uninformative; this domain is the PPCDC, shown biochemically for Sp Hal3. Reason: Root MF term; the specific activity of the HFCD domain of this protein is phosphopantothenoylcysteine decarboxylase activity (IDA, Molero et al. 2013). Proposed replacements: phosphopantothenoylcysteine decarboxylase activity Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt In the N-terminal section; belongs to the HFCD (homo- PMID:23962284 also exhibit PPCDC activity in vitro and provide PPCDC function in vivo, indicating that Sp Hal3 is a monogenic PPCDC in fission yeast |
| GO:0004633 phosphopantothenoylcysteine decarboxylase activity | IDA PMID:23962284 The Schizosaccharomyces pombe fusion gene hal3 encodes three... | ACCEPT | Summary: IDA (Molero et al. 2013): Sp Hal3 and its N-terminal domain show PPCDC activity in vitro and supply PPCDC function in vivo, making it a monogenic (homo-oligomeric-type) PPCDC, in contrast to the budding-yeast Cab3-Sis2/Vhs3 heterotrimer. Core MF. Supporting Evidence: PMID:23962284 also exhibit PPCDC activity in vitro and provide PPCDC function in vivo, indicating that Sp Hal3 is a monogenic PPCDC in fission yeast |
| GO:0004799 thymidylate synthase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the thymidylate synthase node PTN000167391; hal3 itself (PomBase:SPAC15E1.04) is among the experimental sources, reflecting its own IGI evidence. The C-terminal half is a canonical TS domain. Core MF. Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt In the C-terminal section; belongs to the thymidylate PMID:23962284 the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function |
| GO:0004799 thymidylate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: IEA from InterPro IPR000398/IPR020940 (thymidylate synthase, active-site signature) and RHEA:12104 / EC 2.1.1.45. Correct for the C-terminal TS domain. Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8- |
| GO:0004799 thymidylate synthase activity | IGI PMID:23962284 The Schizosaccharomyces pombe fusion gene hal3 encodes three... | ACCEPT | Summary: IGI (Molero et al. 2013) with S. cerevisiae CDC21: full-length Sp Hal3 and its C-terminal domain rescue the cdc21 TS mutant. Direct functional evidence for TS activity of this protein. Core MF. Supporting Evidence: PMID:23962284 the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function |
| GO:0004799 thymidylate synthase activity | ISS GO_REF:0000024 | ACCEPT | Summary: ISS from S. cerevisiae Cdc21 (P06785), the source of UniProt's by-similarity TS reaction. Consistent with the IGI complementation evidence. Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8- PMID:23962284 the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function |
| GO:0004864 protein phosphatase inhibitor activity | IDA PMID:23962284 The Schizosaccharomyces pombe fusion gene hal3 encodes three... | KEEP AS NON CORE | Summary: IDA (Molero et al. 2013): Sp Hal3 and its N-terminal domain bind and modestly inhibit S. cerevisiae Ppz1 and S. pombe Pzh1 in vitro. This is the conserved moonlighting activity of the Hal3 family; its physiological importance in S. pombe is not established (the abstract describes only partial mimicry of Sc Hal3 functions). Pzh1 is a Ser/Thr phosphatase, so GO:0004865 would be the more specific term. Reason: Experimentally supported in vitro, but modest and of unclear in vivo significance relative to the two essential biosynthetic activities. Supporting Evidence: PMID:23962284 retain the ability to bind to and modestly inhibit in vitro S. cerevisiae Ppz1 as well as its S. pombe homolog Pzh1 |
| GO:0005737 cytoplasm | EXP PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: EXP from the Matsuyama et al. 2006 ORFeome YFP localization screen (UniProt subcellular location Cytoplasm). Consistent with cytosolic CoA and dTMP synthesis. Supporting Evidence: PMID:16823372 we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome file:SCHPO/hal3/hal3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:16823372}. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: IEA from UniProt subcellular location SL-0086 (Cytoplasm), itself derived from the ORFeome localization data. Correct. Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:16823372}. |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from PTN002610699, seeded by human TYMS (P04818), Arabidopsis DHFR-TS (AT4G34570) and a trypanosome DHFR-TS, which have mitochondrial pools. No mitochondrial localization or targeting signal has been reported for Sp Hal3; the ORFeome data place it in the cytoplasm/cytosol. As for budding-yeast Cdc21, the requirement for TS in mtDNA synthesis does not imply mitochondrial residence. Reason: Lineage-specific mitochondrial pools of animal/plant TS do not transfer to a cytosolic fungal fusion protein without supporting data. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH LINEAGE OR TAXON MISMATCH Sources checked: UniProtKB:P04818 · human TYMS (mitochondrial pool reported) SUPPORTS SOURCE BUT NOT TARGET AGI_LocusCode:AT4G34570 · Arabidopsis bifunctional DHFR-TS SUPPORTS SOURCE BUT NOT TARGET Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:16823372}. |
| GO:0005829 cytosol | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: HDA from the Matsuyama et al. 2006 ORFeome localization screen (cytosol). Consistent with both biosynthetic activities. Supporting Evidence: PMID:16823372 we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: IBA (PTN000167391, seeded by E. coli ThyA) for cytosolic activity. For hal3 this is independently supported by the HDA cytosol annotation. Supporting Evidence: PMID:16823372 we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome |
| GO:0006231 dTMP biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA (PTN000167391; hal3 is among the sources). TS catalyses the de novo formation of dTMP. Core BP for the C-terminal domain. Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8- PMID:23962284 the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function |
| GO:0006231 dTMP biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: IEA from InterPro IPR000398/IPR020940 (thymidylate synthase). Correct. Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt In the C-terminal section; belongs to the thymidylate |
| GO:0006231 dTMP biosynthetic process | IGI PMID:23962284 The Schizosaccharomyces pombe fusion gene hal3 encodes three... | ACCEPT | Summary: IGI (Molero et al. 2013) with CDC21: rescue of the budding-yeast TS mutant demonstrates that the C-terminal domain makes dTMP in vivo. Supporting Evidence: PMID:23962284 the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function |
| GO:0006235 dTTP biosynthetic process | IEA GO_REF:0000041 | ACCEPT | Summary: IEA from UniPathway UPA00575 (dTTP biosynthesis). dTMP made by the TS domain is phosphorylated onward to dTTP; TS performs a step of this pathway. Acceptable but broader than dTMP biosynthetic process. Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt PATHWAY: Pyrimidine metabolism; dTTP biosynthesis. |
| GO:0010181 FMN binding | IDA PMID:23962284 The Schizosaccharomyces pombe fusion gene hal3 encodes three... | KEEP AS NON CORE | Summary: IDA (Molero et al. 2013) for FMN binding. HFCD-family PPCDCs are FMN-dependent flavoproteins; the abstract does not describe the flavin assay, so defer to the curator who read the full text. FMN is the cofactor of the core PPCDC activity. Reason: Cofactor binding that is implied by and subordinate to the PPCDC activity. Supporting Evidence: file:SCHPO/hal3/hal3-uniprot.txt In the N-terminal section; belongs to the HFCD (homo- PMID:23962284 also exhibit PPCDC activity in vitro and provide PPCDC function in vivo, indicating that Sp Hal3 is a monogenic PPCDC in fission yeast |
| GO:0015937 coenzyme A biosynthetic process | IC PMID:23962284 The Schizosaccharomyces pombe fusion gene hal3 encodes three... | ACCEPT | Summary: IC from GO:0004633 (Molero et al. 2013): as the monogenic PPCDC, hal3 performs the decarboxylation step of CoA biosynthesis. Core BP for the N-terminal domain. Supporting Evidence: PMID:23962284 also exhibit PPCDC activity in vitro and provide PPCDC function in vivo, indicating that Sp Hal3 is a monogenic PPCDC in fission yeast |
| GO:0016741 transferase activity, transferring one-carbon groups | IEA GO_REF:0000002 | MODIFY | Summary: IEA from InterPro IPR045097 (thymidylate synthase/dCMP hydroxymethylase) to the generic one-carbon transferase parent. Correct but uninformative. Reason: Thymidylate synthase activity is the specific, experimentally supported activity. Proposed replacements: thymidylate synthase activity Supporting Evidence: PMID:23962284 the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function |
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Download this section (compressed HTML)Q: Does the Hal3-like domain of Sp Hal3 inhibit Pzh1 in vivo enough to affect cation tolerance, or is the in vitro inhibition a vestigial property of the fused protein?
Q: Does fusing PPCDC and thymidylate synthase in one polypeptide allow co-regulation of CoA and dTMP supply (e.g. during S phase), or is the fusion functionally neutral?
Experiment: Express the N-terminal and C-terminal domains as separate genes in a hal3 deletion background to test whether the split proteins support viability, growth rate and cation tolerance like the fusion.
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