THI3 (YDL080C, KID1) encodes a nuclear thiamine diphosphate (TPP)-binding protein of the TPP-dependent 2-oxo-acid decarboxylase family. It is a paralog of the pyruvate decarboxylases Pdc1/5/6p and of Aro10p, but it is a regulatory protein. Thi3p senses intracellular TPP. When TPP is low (thiamine starvation), TPP-free Thi3p associates with the transcription factors Thi2p and Pdc2p and is required for their activation of the THI regulon, the genes for thiamine biosynthesis and uptake. It also autoregulates its own promoter. TPP binding to Thi3p disrupts these complexes, and Thi3p mutants unable to bind TPP constitutively derepress THI genes. Early multiple-mutant studies suggested a role in leucine and isoleucine decarboxylation in the Ehrlich pathway, but individually expressed Thi3p shows no decarboxylase activity with pyruvate, branched-chain, aromatic or linear 2-oxo acids. Those phenotypes are therefore probably indirect consequences of its regulation of thiamine/TPP supply.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000287 magnesium ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO Mg2+ binding from the TPP-enzyme domain (IPR012000). In TPP enzymes Mg2+ anchors the diphosphate of the cofactor. Thi3p binds thiamine diphosphate, which plausibly needs this Mg2+ site, but Mg2+ binding by Thi3p has not been tested. Supporting Evidence: PMID:19013460 Thi3p is a TPP-binding protein and upregulates THI genes expression when TPP is not bound. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO root 'catalytic activity' (IPR011766), transferred because Thi3p has the TPP-decarboxylase fold. When Thi3p was expressed alone in a pdc1 pdc5 pdc6 aro10 thi3 strain, no decarboxylation of any 2-oxo acid tested was detected. Thi3p appears to be a non-catalytic, TPP-sensing regulatory paralog. Reason: Catalytic activity not detected in direct assays; Thi3p is a regulatory TPP sensor, not a decarboxylase. Supporting Evidence: PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. file:yeast/THI3/THI3-uniprot.txt no decarboxylase enzymatic activity could be detected in cell extracts of |
| GO:0004737 pyruvate decarboxylase activity | IEA GO_REF:0000116 | REMOVE | Summary: RHEA:54360 (4-methyl-2-oxopentanoate decarboxylation) mapped to pyruvate decarboxylase activity. Thi3p does not decarboxylate pyruvate, and UniProt itself lists pyruvate as a non-substrate. Romagnoli et al. found no decarboxylase activity with any 2-oxo acid, including pyruvate. Reason: Contradicted by direct assay; Thi3p has no pyruvate (or other 2-oxo acid) decarboxylase activity. Supporting Evidence: PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. |
| GO:0005515 protein binding | IPI PMID:19013460 Thiamin-dependent transactivation activity of PDC2 in Saccha... | MODIFY | Summary: Nosaka et al. 2008 showed Thi3p interacts with Pdc2p, the DNA-binding transcription factor for THI and PDC genes, and that this interaction increases under thiamine starvation. The informative term is already present as RNA polymerase II-specific DNA-binding transcription factor binding from the same paper. Reason: 'protein binding' is uninformative; the partner Pdc2p is a DNA-binding transcription factor. Proposed replacements: RNA polymerase II-specific DNA-binding transcription factor binding Supporting Evidence: PMID:19013460 We found here that Pdc2p could transactivate gene expression and interact with Thi3p, both of which were enhanced by thiamin starvation. |
| GO:0005634 nucleus | HDA PMID:14562095 Global analysis of protein localization in budding yeast. | ACCEPT | Summary: Global GFP-fusion screen localised Thi3p to the nucleus, consistent with its role as a transcriptional coregulator. Supporting Evidence: PMID:14562095 Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. file:yeast/THI3/THI3-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000269|PubMed:14562095}. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA nucleus at PTN001056938 (donors THI3, PDC1, PDC5). For Thi3p the nuclear localisation is supported by its own data and its regulatory function; correct. Supporting Evidence: file:yeast/THI3/THI3-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000269|PubMed:14562095}. |
| GO:0005634 nucleus | IDA PMID:16194233 Genetic regulation mediated by thiamin pyrophosphate-binding... | ACCEPT | Summary: Nosaka et al. 2005 studied Thi3p as a transcriptional regulator of THI genes; the nuclear localisation is consistent with that role and with the GFP data. Supporting Evidence: PMID:16194233 suggesting that Thi2p and Thi3p participate simultaneously in the induction file:yeast/THI3/THI3-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000269|PubMed:14562095}. |
| GO:0005634 nucleus | IDA PMID:16850348 Pdc2 coordinates expression of the THI regulon in the yeast ... | ACCEPT | Summary: Mojzita and Hohmann studied Thi3/Thi2/Pdc2 control of the THI regulon. The cached abstract reports nuclear enrichment of Pdc1/Pdc5; the curator's Thi3p nucleus IDA presumably comes from the full text. It is consistent with the GFP localisation, so the curator is followed. Supporting Evidence: PMID:16850348 While the Thi2, in conjunction with Thi3, seems to control expression of THI genes with respect to thiamine availability file:yeast/THI3/THI3-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000269|PubMed:14562095}. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SubCell (SL-0191) mapping of the nuclear localisation; correct. Supporting Evidence: file:yeast/THI3/THI3-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000269|PubMed:14562095}. |
| GO:0005829 cytosol | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT IBA cytosol inherited from the pyruvate decarboxylase clade node (donor PDC1). For the catalytic Pdc enzymes cytosol is right, but Thi3p is a nuclear regulatory paralog that has lost decarboxylase activity, and GFP-Thi3p was scored as nuclear. A cytosolic pool cannot be excluded but has no support. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE COMPARTMENT OR COMPLEX MISMATCH Sources checked: SGD:S000004034 · PDC1, cytosolic pyruvate decarboxylase isozyme 1 SUPPORTS SOURCE BUT NOT TARGET Cytosol is correct for the catalytic Pdc1p donor, but THI3 diverged into a non-catalytic nuclear TPP-sensing coregulator; GFP-Thi3p is nuclear. Supporting Evidence: file:yeast/THI3/THI3-uniprot.txt SUBCELLULAR LOCATION: Nucleus {ECO:0000269|PubMed:14562095}. PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. |
| GO:0005829 cytosol | RCA GO_REF:0000123 | REMOVE | Summary: YeastPathways conversion from PWY3O-4109 (isoleucine degradation) places a decarboxylation that Thi3p does not catalyse in the cytosol. The location claim rests on a non-existent activity. Reason: Derived from an RCA reaction assignment that is contradicted by direct assay. Supporting Evidence: PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. |
| GO:0006550 L-isoleucine catabolic process | RCA GO_REF:0000123 | REMOVE | Summary: YeastPathways conversion from PWY3O-4109 (isoleucine degradation). Thi3p performs no step of isoleucine catabolism because it has no detectable 2-oxo acid decarboxylase activity. Any effect of thi3 deletion on fusel alcohols is probably indirect, through its regulation of thiamine (TPP) supply for the active decarboxylases. Reason: Thi3p does not catalyse the decarboxylation step; at most an indirect regulatory effect. Supporting Evidence: PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. PMID:15933030 In addition, Thi3p has been assumed to be a positive regulator of the thiamine biosynthetic pathway. |
| GO:0016831 carboxy-lyase activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO carboxy-lyase activity (IPR012110, pyruvate decarboxylase family), transferred because Thi3p has the TPP-decarboxylase fold. When Thi3p was expressed alone in a pdc1 pdc5 pdc6 aro10 thi3 strain, no decarboxylation of any 2-oxo acid tested was detected. Thi3p appears to be a non-catalytic, TPP-sensing regulatory paralog. Reason: Catalytic activity not detected in direct assays; Thi3p is a regulatory TPP sensor, not a decarboxylase. Supporting Evidence: PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. file:yeast/THI3/THI3-uniprot.txt no decarboxylase enzymatic activity could be detected in cell extracts of |
| GO:0030976 thiamine pyrophosphate binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO thiamine pyrophosphate binding from the TPP-enzyme domains. For Thi3p it is experimentally supported: Thi3p binds TPP, and mutants lacking TPP-binding activity derepress THI genes in thiamine-replete conditions. TPP binding is how Thi3p senses thiamine status. Supporting Evidence: PMID:19013460 Thi3p is a TPP-binding protein and upregulates THI genes expression when TPP is not bound. PMID:16194233 When mutant Thi3p proteins lacking TDP-binding activity were produced in the thi3Delta strain, THI genes were expressed even under thiamin-replete conditions. |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IMP PMID:16194233 Genetic regulation mediated by thiamin pyrophosphate-binding... | ACCEPT | Summary: Nosaka et al. 2005: THI gene induction under thiamine starvation requires Thi3p together with Thi2p, and THI3 autoregulates its own promoter. Thi3p therefore positively regulates RNA polymerase II transcription of THI genes as a non-DNA-binding coregulator. Supporting Evidence: PMID:16194233 suggesting that Thi2p and Thi3p participate simultaneously in the induction PMID:16194233 When mutant Thi3p proteins lacking TDP-binding activity were produced in the thi3Delta strain, THI genes were expressed even under thiamin-replete conditions. |
| GO:0047433 branched-chain-2-oxoacid decarboxylase activity | EXP PMID:10753893 An investigation of the metabolism of isoleucine to active A... | REMOVE | Summary: Dickinson et al. 2000 inferred from multiple-mutant metabolite studies that any member of the PDC1/PDC5/PDC6/ARO10/THI3 family allows isoleucine catabolism, and earlier work attributed leucine-derived 2-oxo-acid decarboxylation to YDL080C (THI3). Later work showed the broad-specificity decarboxylase is Aro10p. When each family member was expressed alone in a quintuple-null strain, Thi3p showed no decarboxylation of any branched-chain 2-oxo acid. The earlier genetic effects are better explained by Thi3p's regulatory role. The UniProt entry carries a CAUTION recording this conflict. Reason: Direct enzyme assays (Romagnoli et al. 2012) found no branched-chain 2-oxo acid decarboxylase activity for Thi3p; the earlier genetic inference is superseded. Supporting Evidence: PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. file:yeast/THI3/THI3-uniprot.txt no decarboxylase enzymatic activity could be detected in cell extracts of PMID:10753893 Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. |
| GO:0047433 branched-chain-2-oxoacid decarboxylase activity | IEA GO_REF:0000120 | REMOVE | Summary: ARBA/RHEA:21108/EC 4.1.1.72 IEA propagated from the UniProt catalytic-activity line, which rests on genetic inference. Contradicted by direct assay of individually expressed Thi3p. Reason: No decarboxylase activity detected for Thi3p in direct assays. Supporting Evidence: PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. |
| GO:0047433 branched-chain-2-oxoacid decarboxylase activity | RCA GO_REF:0000123 | REMOVE | Summary: YeastPathways conversion from PWY3O-4109 (isoleucine degradation) attaches the isoleucine-derived 2-oxo-acid decarboxylation (EC 4.1.1.72) to Thi3p. YeastCyc lists every family member; direct assays found no activity for Thi3p. Reason: No decarboxylase activity detected for Thi3p in direct assays. Supporting Evidence: PMID:22904058 No decarboxylation activity was found for Thi3 with any of the substrates tested. |
| GO:0061629 RNA polymerase II-specific DNA-binding transcription factor binding | IPI PMID:19013460 Thiamin-dependent transactivation activity of PDC2 in Saccha... | ACCEPT | Summary: Nosaka et al. 2008: Thi3p physically interacts with Thi2p, a DNA-binding transcriptional activator of THI genes. The interactions are enhanced by thiamine starvation and are needed for full transactivation. This is Thi3p's mechanistic MF as a TPP-sensing coregulator. Supporting Evidence: PMID:19013460 Thi3p is a TPP-binding protein and upregulates THI genes expression when TPP is not bound. PMID:19013460 We found here that Pdc2p could transactivate gene expression and interact with Thi3p, both of which were enhanced by thiamin starvation. PMID:19013460 This enhancement of the transactivation activity was not observed in a thi3 strain. |
| GO:0061629 RNA polymerase II-specific DNA-binding transcription factor binding | IPI PMID:19013460 Thiamin-dependent transactivation activity of PDC2 in Saccha... | ACCEPT | Summary: Nosaka et al. 2008: Thi3p physically interacts with Pdc2p, a DNA-binding transcriptional activator of THI genes. The interactions are enhanced by thiamine starvation and are needed for full transactivation. This is Thi3p's mechanistic MF as a TPP-sensing coregulator. Supporting Evidence: PMID:19013460 Thi3p is a TPP-binding protein and upregulates THI genes expression when TPP is not bound. PMID:19013460 We found here that Pdc2p could transactivate gene expression and interact with Thi3p, both of which were enhanced by thiamin starvation. PMID:19013460 This enhancement of the transactivation activity was not observed in a thi3 strain. |
| GO:0090180 positive regulation of thiamine biosynthetic process | IMP PMID:16194233 Genetic regulation mediated by thiamin pyrophosphate-binding... | ACCEPT | Summary: Genetic evidence from Nosaka et al. 2005: Thi3p and Thi2p act together, as a complex disrupted by TPP binding to Thi3p, to induce THI (thiamine biosynthesis) genes when thiamine is scarce. Core process. Supporting Evidence: PMID:16194233 Furthermore, Thi2p and Thi3p were demonstrated to bind each other PMID:16194233 suggesting that Thi2p and Thi3p participate simultaneously in the induction PMID:16194233 When mutant Thi3p proteins lacking TDP-binding activity were produced in the thi3Delta strain, THI genes were expressed even under thiamin-replete conditions. |
| GO:0090180 positive regulation of thiamine biosynthetic process | IMP PMID:19013460 Thiamin-dependent transactivation activity of PDC2 in Saccha... | ACCEPT | Summary: Genetic evidence from Nosaka et al. 2008: Thi3p binds Pdc2p and is required for the thiamine-starvation-enhanced transactivation by Pdc2p of THI genes. Core process. Supporting Evidence: PMID:19013460 Thi3p is a TPP-binding protein and upregulates THI genes expression when TPP is not bound. PMID:19013460 We found here that Pdc2p could transactivate gene expression and interact with Thi3p, both of which were enhanced by thiamin starvation. PMID:19013460 This enhancement of the transactivation activity was not observed in a thi3 strain. |
| GO:0090180 positive regulation of thiamine biosynthetic process | IPI PMID:16194233 Genetic regulation mediated by thiamin pyrophosphate-binding... | ACCEPT | Summary: Physical interaction with Thi2p evidence from Nosaka et al. 2005: Thi3p and Thi2p act together, as a complex disrupted by TPP binding to Thi3p, to induce THI (thiamine biosynthesis) genes when thiamine is scarce. Core process. Supporting Evidence: PMID:16194233 Furthermore, Thi2p and Thi3p were demonstrated to bind each other PMID:16194233 suggesting that Thi2p and Thi3p participate simultaneously in the induction PMID:16194233 When mutant Thi3p proteins lacking TDP-binding activity were produced in the thi3Delta strain, THI genes were expressed even under thiamin-replete conditions. |
| GO:0090180 positive regulation of thiamine biosynthetic process | IPI PMID:19013460 Thiamin-dependent transactivation activity of PDC2 in Saccha... | ACCEPT | Summary: Physical interaction with Pdc2p evidence from Nosaka et al. 2008: Thi3p binds Pdc2p and is required for the thiamine-starvation-enhanced transactivation by Pdc2p of THI genes. Core process. Supporting Evidence: PMID:19013460 Thi3p is a TPP-binding protein and upregulates THI genes expression when TPP is not bound. PMID:19013460 We found here that Pdc2p could transactivate gene expression and interact with Thi3p, both of which were enhanced by thiamin starvation. PMID:19013460 This enhancement of the transactivation activity was not observed in a thi3 strain. |
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Download this section (compressed HTML)Q: Are the reported thi3 effects on leucine/isoleucine-derived fusel alcohols explained entirely by reduced TPP supply to Aro10 and the Pdc isozymes, or does Thi3p modulate decarboxylase activity post-translationally (e.g. by hetero-oligomerising with Aro10 or Pdc proteins)?
Q: Has Thi3p lost the catalytic residues (e.g. the catalytic glutamate/histidines of PDC) that would explain its lack of decarboxylase activity?
Experiment: Supplement thi3 mutants with excess thiamine, or express THI genes constitutively, and measure isoamyl alcohol and active amyl alcohol formation to test whether the Ehrlich phenotype is indirect.
Experiment: Assay purified Thi3p with TPP for binding affinity and for residual 2-oxo-acid decarboxylase or carboligase activity.
Regulatory protein that binds Pdc2p and Thi2p transcription factors; activates thiamine biosynthesis transcription factors Pdc2p and Thi2p by binding to them, but releases and de-activates them upon binding to thiamine pyrophosphate (TPP), the end product of the pathway; has similarity to decarboxylases but enzymatic activity is not detected
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