RRT2 (also DPH7, ERE1; YBR246W) encodes a WD40-repeat beta-propeller protein, ortholog of human DPH7/WDR85, that catalyses the third step of eukaryotic diphthamide biosynthesis on translation elongation factor 2 (eEF2): hydrolysis of the diphthine methyl ester made by Dph5 to diphthine and methanol (EC 3.1.1.97), which allows Dph6 to amidate diphthine to diphthamide. rrt2/dph7 null cells accumulate (methylated) diphthine on eEF2, show enhanced Dph5-eEF2 association, increased -1 frameshifting and resistance to sordarin. It is the first WD40 protein shown to have enzymatic activity; the catalytic residues are not established. Rrt2 is mainly cytoplasmic; independently, as Ere1 it is transiently recruited to endosomes with Ere2 and is required for retromer-dependent recycling of the arginine permease Can1 to the plasma membrane. It lacks an ATP-binding domain and is not the diphthamide synthetase. The name RRT2 (regulator of rDNA transcription) comes from a transposon screen in which its disruption altered an rDNA-linked Pol I reporter; no direct role in rDNA transcription has been described.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: PANTHER IBA (PTN000522690) cytoplasm; consistent with direct observation that Ere1/Rrt2-GFP is mostly cytoplasmic. Supporting Evidence: PMID:21880895 In wild-type cells, Ere1-GFP was mostly cytoplasmic |
| GO:0005737 cytoplasm | IDA PMID:21880895 Two novel WD40 domain-containing proteins, Ere1 and Ere2, fu... | ACCEPT | Summary: Shi et al. (2011) observed functional Ere1-GFP to be mostly cytoplasmic in wild-type cells. Supporting Evidence: PMID:21880895 In wild-type cells, Ere1-GFP was mostly cytoplasmic file:yeast/RRT2/RRT2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:21880895}. Endosome |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SL-0086 mapping of the experimentally observed cytoplasmic localization. Supporting Evidence: file:yeast/RRT2/RRT2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:21880895}. Endosome |
| GO:0005768 endosome | IDA PMID:21880895 Two novel WD40 domain-containing proteins, Ere1 and Ere2, fu... | KEEP AS NON CORE | Summary: Ere1-GFP colocalized with the endosomal PI3P marker in about 10% of wild-type cells and more prominently in ESCRT mutants, indicating transient recruitment. Reason: Genuine observation tied to the retromer/Can1 recycling role, which is separate from the diphthamide enzyme function; non-core location. Supporting Evidence: PMID:21880895 suggesting that Ere1-GFP may be transiently recruited to endosomes |
| GO:0005768 endosome | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt SL-0101 mapping of the endosomal localization from Shi et al. 2011. Reason: Same evidence as the IDA row; transient, non-core. Supporting Evidence: file:yeast/RRT2/RRT2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:21880895}. Endosome |
| GO:0005829 cytosol | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways default cytosol compartment. Appropriate here: Rrt2 is a soluble cytoplasmic protein acting on cytosolic eEF2. Supporting Evidence: PMID:21880895 In wild-type cells, Ere1-GFP was mostly cytoplasmic Reactome:R-SCE-5367021 Yeast cytosolic diphthamide biosynthesis protein 7 (RRT2) catalyzes the removal |
| GO:0005829 cytosol | TAS Reactome:R-SCE-5367021 | ACCEPT | Summary: Reactome R-SCE-5367021 describes cytosolic Dph7 (RRT2) removing the methyl group from Me-diphthine eEF2 (EFT1); consistent with the cytoplasmic GFP data. Supporting Evidence: Reactome:R-SCE-5367021 Yeast cytosolic diphthamide biosynthesis protein 7 (RRT2) catalyzes the removal |
| GO:0017178 diphthine-ammonia ligase activity | RCA GO_REF:0000123 | REMOVE | Summary: YeastPathways (PWY-6482-1) assigns RRT2/YBR246W to DIPHTINE--AMMONIA-LIGASE-RXN, citing Su et al. 2012, and the GO-CAM conversion turns that into diphthine-ammonia ligase activity. Reason: Incorrect enzyme assignment. Su et al. 2012 only showed that ybr246w cells accumulate diphthine. Rrt2/Dph7 lacks the ATP-binding (adenine nucleotide alpha hydrolase) domain needed for the ATP-dependent amidation; the amidating ligase is Dph6/YLR143W, and Dph7 is the diphthine methylesterase acting one step earlier. Supporting Evidence: PMID:24739148 However, Dph7 is not the diphthamide synthetase, as it lacks the ATP-binding domain required. PMID:23169644 we thought it was a scaffold or adaptor protein instead of the actual enzyme that catalyzes the amidation reaction because it lacks an ATP-dependent catalytic domain PMID:24739148 Dph7 is a methylesterase that hydrolyzes methylated diphthine to produce diphthine |
| GO:0017183 protein histidyl modification to diphthamide | IBA GO_REF:0000033 | ACCEPT | Summary: PANTHER IBA for diphthamide formation from the DPH7 family node (human WDR85 and yeast Rrt2 experimental data); correct. Supporting Evidence: PMID:24739148 Dph7 is a methylesterase that hydrolyzes methylated diphthine to produce diphthine PMID:23468660 dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2 |
| GO:0017183 protein histidyl modification to diphthamide | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway UPA00559 (peptidyl-diphthamide biosynthesis) mapping; correct for this pathway enzyme. Supporting Evidence: file:yeast/RRT2/RRT2-uniprot.txt FUNCTION: Catalyzes the demethylation of diphthine methyl ester to form |
| GO:0017183 protein histidyl modification to diphthamide | IMP PMID:22188241 YBR246W is required for the third step of diphthamide biosyn... | ACCEPT | Summary: Deletion of YBR246W blocks diphthamide completion and causes diphthine-modified eEF2 to accumulate (Su et al. 2012). Supporting Evidence: PMID:22188241 the deletion of YBR246W leads to the accumulation of diphthine |
| GO:0017183 protein histidyl modification to diphthamide | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways RCA placing RRT2 in diphthamide biosynthesis. The pathway membership is correct even though the specific reaction (amidation) is wrong. Supporting Evidence: PMID:24739148 Dph7 is a methylesterase that hydrolyzes methylated diphthine to produce diphthine PMID:23468660 dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2 |
| GO:0032456 endocytic recycling | IMP PMID:21880895 Two novel WD40 domain-containing proteins, Ere1 and Ere2, fu... | KEEP AS NON CORE | Summary: Shi et al. (2011) identified YBR246W as Ere1 in a canavanine chemical-genetic screen; ere1 vps23 cells fail to recycle Can1 to the plasma membrane via the retromer pathway. Reason: Supported by direct mutant phenotype, but this cargo-specific endosomal recycling role is a second, apparently independent function of the protein, not its diphthamide enzymatic role. Supporting Evidence: PMID:21880895 Ere1 is required for Can1 recycling via the retromer-mediated pathway |
| GO:0051723 protein methylesterase activity | EXP PMID:24739148 Dph7 catalyzes a previously unknown demethylation step in di... | ACCEPT | Summary: Lin et al. (2014) showed that Dph7 hydrolyses the methyl ester on eEF2-bound methylated diphthine, i.e. removes a methyl ester from a protein residue. Reason: Accurate description of the activity on a protein substrate; complementary to the more specific diphthine methylesterase term (the two are not linked by is_a in GO). Supporting Evidence: PMID:24739148 Dph7 is a methylesterase that hydrolyzes methylated diphthine to produce diphthine PMID:24739148 Diphthamide was formed on Ξdph7 eEF-2 only in the presence of both Dph6 and Dph7 |
| GO:0061685 diphthine methylesterase activity | IDA PMID:24739148 Dph7 catalyzes a previously unknown demethylation step in di... | ACCEPT | Summary: In vitro, purified Dph7 decreased methylated diphthine and increased diphthine on dph7 eEF2 (LC-MS), and Dph7 pre-treatment made dph7 eEF2 a Dph6 substrate. Supporting Evidence: PMID:24739148 Dph7 is a methylesterase that hydrolyzes methylated diphthine to produce diphthine PMID:24739148 Diphthamide was formed on Ξdph7 eEF-2 only in the presence of both Dph6 and Dph7 |
| GO:0061685 diphthine methylesterase activity | IEA GO_REF:0000120 | ACCEPT | Summary: RHEA:42656 / EC 3.1.1.97 mapping from UniProt catalytic activity; correct. Supporting Evidence: file:yeast/RRT2/RRT2-uniprot.txt Reaction=diphthine methyl ester-[translation elongation factor 2] + H2O |
| GO:0061685 diphthine methylesterase activity | IMP PMID:24739148 Dph7 catalyzes a previously unknown demethylation step in di... | ACCEPT | Summary: dph7 deletion accumulates methylated diphthine that Dph6 cannot amidate (Lin et al. 2014). Supporting Evidence: PMID:24739148 Dph7 is a methylesterase that hydrolyzes methylated diphthine to produce diphthine PMID:24739148 Diphthamide was formed on Ξdph7 eEF-2 only in the presence of both Dph6 and Dph7 |
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Download this section (compressed HTML)Q: Is the Ere1 endosomal recycling role mechanistically linked to the methylesterase activity (e.g. a methyl-esterified cargo or regulator), or is it an independent moonlighting function of the WD40 scaffold?
Q: Should GO:0061685 diphthine methylesterase activity be classified under GO:0051723 protein methylesterase activity, since its substrate is a protein residue?
Experiment: Identify catalytic residues (conserved Ser/His/Asp candidates) by mutagenesis and in vitro esterase assays on dph7 eEF2, then test the catalytically dead alleles for Can1-GFP recycling in vps23 background.
Hypothesis: The Rrt2 esterase active site is dispensable for Can1 recycling
Type: mutagenesis / separation-of-function
Methylesterase performing penultimate step of diphthamide biosynthesis; hydrolyzes methylated diphthine to produce diphthine and allows Dph6-catalyzed amidation reaction to occur; deletion leads to resistance to sordarin and accumulation of methylatediphthine; WD40 domain-containing protein; involved in endosomal recycling; forms complex with Rtt10p that functions in retromer-mediated pathway for recycling internalized cell-surface proteins
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