MSD1 (YPL104W) encodes the mitochondrial aspartyl-tRNA synthetase (AspRS) of Saccharomyces cerevisiae, a nuclear-encoded class II aminoacyl-tRNA synthetase of the bacterial-type (type 1) AspRS lineage, distinct from the cytoplasmic AspRS encoded by DPS1. The 658-residue protein has an N-terminal OB-fold anticodon-binding domain, a class II catalytic core with the bacterial-type GAD insertion domain between motifs 2 and 3, and conserved aspartate- and ATP-binding residues. It catalyzes the ATP-dependent attachment of L-aspartate to mitochondrial tRNA(Asp), forming aspartyl-tRNA(Asp) for translation on mitochondrial ribosomes. Disruption of MSD1 abolishes aspartylation of mitochondrial tRNA(Asp) in isolated mitochondria and produces a respiratory-deficient phenotype typical of loss of mitochondrial protein synthesis, with secondary instability of the mitochondrial genome. A missense allele (S388N, in the GAD insertion domain) suppresses an acceptor-stem mutation in the mitochondrially encoded tRNA(Asp), consistent with direct recognition of that tRNA by the enzyme. The protein is found in purified mitochondria and, by analogy with other mitochondrial synthetases, acts in the matrix. MSD1 is the yeast ortholog of human DARS2 and is used as a complementation model for DARS2 disease variants.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro-based nucleotide binding; true but entailed by the more specific ATP binding retained in this review. Reason: Class II aminoacyl-tRNA synthetases bind ATP to form the aminoacyl-adenylate. The specific nucleotide is known (ATP; UniProt binding sites 248-250, 553, 604-607 by similarity), and GO:0005524 ATP binding is accepted below, so this broader term adds nothing. Supporting Evidence: file:yeast/MSD1/MSD1-uniprot.txt CC -!- SIMILARITY: Belongs to the class-II aminoacyl-tRNA synthetase family. |
| GO:0003676 nucleic acid binding | IEA GO_REF:0000002 | MODIFY | Summary: Mapped from the OB-fold anticodon-binding domain (IPR004365); the bound nucleic acid is tRNA, so the specific term is tRNA binding. Reason: IPR004365 is the N-terminal OB-fold anticodon-binding domain of AspRS/LysRS/AsnRS, which binds the tRNA anticodon. In yeast, the S388N MSD1 allele suppresses an acceptor-stem mutation in mitochondrial tRNA(Asp), consistent with direct enzyme-tRNA contact. The correct specific term is GO:0000049 tRNA binding. This is a substrate-binding activity intrinsic to the ligase reaction, not a separate core function. Proposed replacements: tRNA binding Supporting Evidence: PMID:10710420 We conclude that the base substitution causes the respiratory rescue and discuss the possible mechanism as one which enhances interaction between the mutated tRNA Asp and the suppressor version of AspRS. |
| GO:0004812 aminoacyl-tRNA ligase activity | IEA GO_REF:0000120 | TOO GENERAL | Summary: True parent of the established aspartate-tRNA ligase activity; uninformative here. Reason: GO:0004815 aspartate-tRNA ligase activity is experimentally supported in yeast (loss of mitochondrial tRNA(Asp) acylation in msd1 disruptant mitochondria) and accepted below; it is an is_a descendant of this term. Supporting Evidence: PMID:2668951 Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. |
| GO:0004815 aspartate-tRNA ligase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference at PTN000502597, a node uniting bacterial AspS and mitochondrial AspRS; MSD1 is itself one of the experimental donors. Reason: MSD1 sits in PANTHER PTHR22594:SF5 (ASPARTATE--TRNA LIGASE, MITOCHONDRIAL) together with human DARS2 and E. coli AspS, the donors listed here. MSD1 appearing in its own WITH/FROM is expected - its own experimental annotation was one of the descendant evidences used to place the IBD. The IBA additionally asserts that Asp-tRNA ligase activity is ancestral to the bacterial/mitochondrial AspRS clade rather than a yeast-specific property. The experimental basis in yeast itself is the disruptant acylation assay. Supporting Evidence: PMID:2668951 Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. GO_REF:0000033 |
| GO:0004815 aspartate-tRNA ligase activity | IEA GO_REF:0000120 | ACCEPT | Summary: EC/Rhea-mapped electronic annotation of the same core activity; consistent with the experimental evidence. Reason: The EC 6.1.1.12 assignment in UniProt is curator-inferred from the yeast genetic and biochemical data (ECO:0000305, PMID:2668951) and matches the experimentally established function. Supporting Evidence: file:yeast/MSD1/MSD1-uniprot.txt CC -!- FUNCTION: Catalyzes the attachment of aspartate to tRNA(Asp) in the |
| GO:0004815 aspartate-tRNA ligase activity | IMP PMID:2668951 Homology of aspartyl- and lysyl-tRNA synthetases. | ACCEPT | Summary: Core experimental evidence in yeast; mitochondria from an msd1 disruptant cannot acylate mitochondrial tRNA(Asp). Reason: Gampel and Tzagoloff (1989) cloned MSD1, showed it encodes a protein similar to the cytoplasmic AspRS, and showed that mitochondria from an MSD1-disrupted strain cannot acylate mitochondrial aspartyl-tRNA. This is a loss-of-activity (IMP) result in mitochondrial extracts rather than an assay of purified recombinant Msd1, but combined with the class II AspRS domain architecture and conserved Asp/ATP binding residues it establishes the activity. The cached record is abstract-only; the curator saw the full text. Supporting Evidence: PMID:2668951 Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. |
| GO:0004815 aspartate-tRNA ligase activity | ISA PMID:2668951 Homology of aspartyl- and lysyl-tRNA synthetases. | ACCEPT | Summary: Sequence-alignment inference from the cytoplasmic AspRS DPS1; the claim is correct and independently established experimentally. Reason: The ISA donor is DPS1 (SGD:S000003941), the yeast cytoplasmic AspRS. DPS1 is a paralog in a different PANTHER family (PTHR43450:SF1, eukaryotic cytoplasmic AspRS; MSD1 is PTHR22594:SF5), so similarity to DPS1 alone would only support the amino-acid specificity class. Because the specificity (aspartate) is shared and the activity is also shown directly by the IMP acylation assay in the same paper, the claim is accepted; this row should not be read as an orthology-based transfer. Supporting Evidence: PMID:2668951 The primary structure of the protein derived from the gene sequence is similar to that of the yeast cytoplasmic aspartyl-tRNA synthetase. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: ATP is a substrate of the aspartylation reaction; ATP-binding residues are conserved. Reason: Class II AspRS activates aspartate with ATP to form aspartyl-adenylate. UniProt annotates conserved ATP-binding positions by similarity to characterized AspRS structures. Retained as the specific nucleotide-binding statement; it is part of the catalytic mechanism rather than an independent function. Supporting Evidence: file:yeast/MSD1/MSD1-uniprot.txt CC Reaction=tRNA(Asp) + L-aspartate + ATP = L-aspartyl-tRNA(Asp) + AMP + |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | TOO GENERAL | Summary: Electronic cytoplasm call, likely from bacterial AspS homology; true only in the GO sense that mitochondria are part of the cytoplasm. Reason: MSD1 is the mitochondrial isoform; the cytosolic AspRS in yeast is DPS1. GO mitochondrion is part_of cytoplasm, so the statement is not false, but it is entailed by the accepted mitochondrion and mitochondrial matrix annotations and would mislead if read as cytosolic localization. There is no evidence for a cytosolic pool of Msd1. Supporting Evidence: PMID:35820270 The yeast homologue of human DARS2, MSD1, encodes the mitochondrial isoform of aspartyl-tRNA synthetase. |
| GO:0005739 mitochondrion | HDA PMID:14576278 The proteome of Saccharomyces cerevisiae mitochondria. | ACCEPT | Summary: Detected in the high-purity yeast mitochondrial proteome. Reason: Large-scale MS of highly purified mitochondria; consistent with the genetic evidence that MSD1 is required for mitochondrial tRNA(Asp) charging. Supporting Evidence: PMID:14576278 The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. |
| GO:0005739 mitochondrion | HDA PMID:16823961 Toward the complete yeast mitochondrial proteome: multidimen... | ACCEPT | Summary: Detected in the PROMITO mitochondrial proteome dataset. Reason: Independent proteomic confirmation of mitochondrial localization. Supporting Evidence: PMID:16823961 A total of 851 different proteins (PROMITO dataset) were identified by use of multidimensional LC-MS/MS |
| GO:0005739 mitochondrion | HDA PMID:24769239 Quantitative variations of the mitochondrial proteome and ph... | ACCEPT | Summary: Detected in quantitative proteomics of isolated mitochondria. Reason: Third independent proteomic detection in isolated mitochondria. Supporting Evidence: PMID:24769239 we performed an overall quantitative proteomic and phosphoproteomic study of isolated mitochondria extracted from yeast grown on fermentative (glucose or galactose) and respiratory (lactate) media |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference at PTN000502598, the eukaryotic mitochondrial AspRS node, with MSD1 and DARS2 as donors. Reason: The mitochondrial location is placed at the node uniting the eukaryotic mitochondrial AspRS orthologs (yeast MSD1, human DARS2), not at the deeper node shared with bacterial AspS, which is the correct placement. MSD1 being its own donor is expected. Supporting Evidence: GO_REF:0000033 PMID:2668951 The yeast nuclear gene MSD1 coding for mitochondrial aspartyl-tRNA synthetase has been cloned and sequenced. |
| GO:0005739 mitochondrion | IMP PMID:2668951 Homology of aspartyl- and lysyl-tRNA synthetases. | ACCEPT | Summary: Location inferred from loss of mitochondrial tRNA(Asp) acylation in disruptant mitochondria. Reason: The disruptant phenotype (loss of aspartyl-tRNA acylation activity in isolated mitochondria) implies the enzyme resides in mitochondria; this is consistent with three independent proteomic detections. IMP is an unusual code for localization but the claim is correct. Supporting Evidence: PMID:2668951 Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000044 | ACCEPT | Summary: Matrix location from UniProt subcellular-location curation; the expected site of mitochondrial tRNA charging. Reason: UniProt assigns Mitochondrion matrix by curator inference (ECO:0000305) from PMID:2668951. Mitochondrial tRNAs and ribosomes are in the matrix, so this is where the enzyme must act. Note that no sub-mitochondrial fractionation of Msd1 has been reported in yeast; the matrix placement is inferred, not directly measured. Supporting Evidence: file:yeast/MSD1/MSD1-uniprot.txt CC -!- SUBCELLULAR LOCATION: Mitochondrion matrix |
| GO:0006418 tRNA aminoacylation for protein translation | IEA GO_REF:0000120 | TOO GENERAL | Summary: True parent of the mitochondrial-specific aminoacylation process. Reason: GO:0070127 tRNA aminoacylation for mitochondrial protein translation, proposed below as the replacement for the obsolete process terms, is an is_a descendant of this term and states the compartment correctly. Supporting Evidence: PMID:2668951 In situ disruption of MSD1 in a respiratory-competent haploid strain of yeast induces a pleiotropic phenotype consistent with a lesion in mitochondrial protein synthesis. |
| GO:0006422 obsolete aspartyl-tRNA aminoacylation | IBA GO_REF:0000033 | MODIFY | Summary: Sound PAINT inference whose term has been obsoleted because it restates the aspartate-tRNA ligase molecular function. Reason: GO:0006422 was obsoleted as a restatement of GO:0004815, with the process to be captured by tRNA aminoacylation for protein translation. The IBD at PTN000502597 (with E. coli AspS and MSD1 as donors) is valid. For MSD1 the compartment-specific process term GO:0070127 applies, since the enzyme charges mitochondrial tRNA(Asp) for mitochondrial translation. Original id preserved; no fault in the donor evidence is implied. Propagation Review Root cause: TERM SCOPING PROBLEM Sources checked: PANTHER:PTN000502597 SUPPORTS TRANSFER The IBD and its Asp-charging biology are valid for MSD1, which is itself an experimental donor; only the process term has since been obsoleted. Proposed replacements: tRNA aminoacylation for mitochondrial protein translation Supporting Evidence: GO_REF:0000033 PMID:2668951 Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. |
| GO:0016874 ligase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: Very general parent of aspartate-tRNA ligase activity. Reason: Entailed by the accepted GO:0004815 aspartate-tRNA ligase activity. Supporting Evidence: PMID:2668951 Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. |
| GO:0070146 obsolete mitochondrial aspartyl-tRNA aminoacylation | IGI PMID:10710420 A missense mutation in the nuclear gene coding for the mitoc... | MODIFY | Summary: Genetic interaction between an MSD1 suppressor allele (NSM3, S388N) and the mitochondrial tRNA(Asp) gene; the obsolete term is replaced by the mitochondrial tRNA aminoacylation process. Reason: Chiang and Liaw (2000) identified the nuclear suppressor NSM3 as an MSD1 allele carrying S388N, which restores respiration in a strain whose mitochondrial tRNA(Asp) gene has an acceptor-stem C72U mutation; allele-swap experiments showed the substitution is causal. This allele-specific suppression places Msd1 in direct functional contact with mitochondrial tRNA(Asp) and supports its role in charging that tRNA. GO:0070146 is obsolete (it restates GO:0004815); its stated process replacement is GO:0070127. Proposed replacements: tRNA aminoacylation for mitochondrial protein translation Supporting Evidence: PMID:10710420 The comparison of NSM3 DNA sequences between the suppressor and the wild-type version, cloned from the parental strain FF1210-6C/170, revealed a G to A transition that causes the replacement of amino acid serine (AGU) by an asparagine (AAU) at position 388. PMID:10710420 In experiments switching restriction fragments between the wild type and suppressor versions of the NSM3 gene, the rescue of respiratory deficiency was demonstrated only when the substitution was present in the construct. |
| GO:0070146 obsolete mitochondrial aspartyl-tRNA aminoacylation | IMP PMID:2668951 Homology of aspartyl- and lysyl-tRNA synthetases. | MODIFY | Summary: Disruptant mitochondria cannot acylate tRNA(Asp) and the strain shows a mitochondrial protein-synthesis defect; obsolete term replaced by the mitochondrial tRNA aminoacylation process. Reason: Msd1 directly performs the aminoacylation step that supplies Asp-tRNA(Asp) to mitochondrial translation, so process participation is warranted (it is the catalyst, not a substrate). GO:0070146 is obsolete; replace with GO:0070127. Downstream phenotypes (respiratory deficiency, loss of mtDNA) are consequences of lost mitochondrial translation and are not annotated as separate processes. Proposed replacements: tRNA aminoacylation for mitochondrial protein translation Supporting Evidence: PMID:2668951 In situ disruption of MSD1 in a respiratory-competent haploid strain of yeast induces a pleiotropic phenotype consistent with a lesion in mitochondrial protein synthesis. PMID:2668951 Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. |
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Download this section (compressed HTML)Q: Does purified recombinant Msd1 aminoacylate yeast mitochondrial tRNA(Asp) in vitro, and with what kinetics relative to human DARS2?
Q: Does the S388N (NSM3) enzyme aminoacylate the C72U mutant mitochondrial tRNA(Asp) more efficiently than wild-type Msd1, as proposed by Chiang and Liaw?
Q: Does Msd1 misacylate mitochondrial tRNA(Asn), as reported for human DARS2, given that the yeast mitochondrial Asn-tRNA(Asn) formation route differs from bacteria lacking AsnRS?
Experiment: Express and purify Msd1 (without its predicted presequence) and measure aspartylation of in vitro transcribed mitochondrial tRNA(Asp), wild type and C72U, with wild-type and S388N enzyme.
Type: in vitro aminoacylation assay
Experiment: Sub-mitochondrial fractionation (carbonate extraction, protease protection) of tagged Msd1 to confirm matrix localization and test for any membrane association as reported for DARS2.
Type: subcellular fractionation
Mitochondrial aspartyl-tRNA synthetase; required for acylation of aspartyl-tRNA; yeast and bacterial aspartyl-, asparaginyl-, and lysyl-tRNA synthetases contain regions with high sequence similarity, suggesting a common ancestral gene
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