EARS2

Summary: tRNA recognition is an integral substrate-binding activity of EARS2.

Reason: The IPR008925 anticodon-binding domain maps to tRNA binding. Human EARS2 charges native mitochondrial tRNA-Glu and tRNA-Gln in PMID:19805282, independently supporting physical substrate recognition as part of its catalytic mechanism. This is not a generic protein-binding claim and does not require assigning an additional RNA-processing activity.

Summary: ATP is the specified nucleotide substrate.

Reason: The three literal InterPro sources support a class-I aminoacyl-tRNA synthetase nucleotide-binding site. The demonstrated human reaction uses ATP to activate glutamate before transfer to tRNA. ATP binding is therefore a supported descendant of nucleotide binding; this refinement does not assert an independent ATPase.

Proposed replacements: ATP binding

Summary: Both demonstrated glutamylation activities refine the generic aminoacyl-tRNA ligase term.

Reason: Human EARS2 transfers glutamate to mitochondrial tRNA-Glu and also to mitochondrial tRNA-Gln. GO:0004818 and GO:0050561 are distinct current children of aminoacyl-tRNA ligase activity and preserve both substrate reactions. The latter forms Glu-tRNA-Gln, not Gln-tRNA-Gln: downstream amidation belongs to GatCAB. Rhea:23540 and Rhea:64612 distinguish the two substrates; Rhea:18397 represents their broader Glx reaction.

Proposed replacements: glutamate-tRNA ligase activity glutamate-tRNA(Gln) ligase activity

Summary: The PAINT glutamate-tRNA ligase inference is directly corroborated in human EARS2.

Reason: Current PTHR43311 places the exact 523-residue Q5JPH6 sequence beneath PTN000012469, and the literal IBD for GO:0004818 is recovered at that node. Current E. coli and Plasmodium donor records include experimental grounding, while the human target is itself a legitimate experimentally supported descendant. Its occurrence among the donors is not circularity. Human tRNA-Glu aminoacylation independently supports the inherited activity.

Summary: Recombinant human EARS2 glutamylates mitochondrial tRNA-Glu.

Reason: PMID:19805282 tested a transit-sequence-deleted His-tagged human mtGluRS expressed in E. coli with native bovine and human placental mitochondrial tRNAs. The observed glutamate charging supports GO:0004818. The named NP_001077083 reference parent matches current Q5JPH6-1; complete sequencing of the experimental insert and equivalent activity of the alternative natural product are not established.

Summary: The electronic chemistry mapping specifies the correct tRNA-Glu reaction.

Reason: InterPro:IPR004527 identifies the bacterial/mitochondrial glutamate-tRNA ligase family; EC:6.1.1.17 and Rhea:23540 specify ATP-dependent glutamate transfer to tRNA-Glu. This mapping agrees with direct human aminoacylation in PMID:19805282 and does not imply that EARS2 discriminates against tRNA-Gln.

Summary: ATP binding participates directly in the aminoacylation reaction.

Reason: The literal class-I synthetase domain mappings and current sequence features identify the nucleotide-binding machinery. ATP is consumed during glutamyl-adenylate formation before glutamate is transferred to tRNA. Retain ATP binding as a mechanistic component of EARS2 catalysis, without interpreting it as an unrelated ATP hydrolysis activity.

Summary: MitoCoP directly detects the human EARS2 protein group in mitochondria; matrix is the finer curated target location.

Reason: PMID:34800366 Table S1(A), row 333, identifies group 6714 containing Q5JPH6, H3BTB7 and Q5JPH6-2. Subtractive proteomics is Class 1 (mean log2 purified/crude mitochondrial enrichment 1.116; 85.925% sequence coverage), spatial profiling is Cluster 1 and importomics is Class 1. Table S1(B), row 301, includes EARS2 in MitoCoP. These are gene/protein-group measurements, not isoform-resolved matrix assays. Carbonate/sonication classifies the group as peripheral; this does not identify a membrane face or refute matrix-side association. The matrix refinement uses the independently curated experimental matrix assertion and UniProt SL-0170 assignment citing PMID:19805282. The inspected main article establishes mitochondrial targeting but does not independently resolve the matrix subcompartment; its supplementary Methods were unavailable. The curated positive matrix assertion is retained with this access limit, rather than rejected from incomplete text.

Proposed replacements: mitochondrial matrix

Summary: The inherited mitochondrial localization can be refined at target level to matrix.

Reason: The exact current target sequence descends from PTN008450384, where the literal mitochondrion IBD is recovered. The named Arabidopsis AT5G64050 donor maps to Q9FEA2/OVA3 and the yeast SGD:S000005393 donor to P48525/MSE1; their current records contain experimental mitochondrial annotations. The target self-donor is legitimate. The matrix refinement uses the independently curated experimental matrix assertion and UniProt SL-0170 assignment citing PMID:19805282. The inspected main article establishes mitochondrial targeting but does not independently resolve the matrix subcompartment; its supplementary Methods were unavailable. The curated positive matrix assertion is retained with this access limit, rather than rejected from incomplete text.

Proposed replacements: mitochondrial matrix

Summary: Human EARS2 mitochondrial targeting is supported, with a separately curated matrix assertion.

Reason: Full-length human EARS2 fused to C-terminal EGFP localizes to HeLa mitochondria in PMID:19805282. This differs from the transit-sequence-deleted recombinant enzyme used for aminoacylation. The matrix refinement uses the independently curated experimental matrix assertion and UniProt SL-0170 assignment citing PMID:19805282. The inspected main article establishes mitochondrial targeting but does not independently resolve the matrix subcompartment; its supplementary Methods were unavailable. The curated positive matrix assertion is retained with this access limit, rather than rejected from incomplete text.

Proposed replacements: mitochondrial matrix

Summary: The ARBA mitochondrial prediction agrees with independent target evidence and can be refined to matrix.

Reason: The current ARBA00026962 record still outputs GO:0005739. Its 380 alternative condition sets include FunFam assignments; the available target InterPro/PANTHER cross-references do not reconstruct a complete condition match, and a shared structural superfamily is not proof of FunFam membership. That rule-trace limit does not negate independently observed mitochondrial localization. The matrix refinement uses the independently curated experimental matrix assertion and UniProt SL-0170 assignment citing PMID:19805282. The inspected main article establishes mitochondrial targeting but does not independently resolve the matrix subcompartment; its supplementary Methods were unavailable. The curated positive matrix assertion is retained with this access limit, rather than rejected from incomplete text.

Proposed replacements: mitochondrial matrix

Summary: Retain the positive experimental mitochondrial matrix assertion.

Reason: GOA assigns EXP and UniProt assigns ECO:0000269 matrix localization to PMID:19805282. Its inspected main text supports human mitochondrial targeting; the detailed supplementary Methods could not be recovered, so the specific matrix assay has not been independently reconstructed. This is not evidence that the curator lacked such support. Retain the positive matrix annotation, explicitly distinguishing curator-supported subcompartment assignment from the main-text imaging scope.

Summary: SL-0170 maps specifically to mitochondrial matrix.

Reason: The current official subcellular-location record SL-0170 maps to GO:0005759 and is part of mitochondrion. EARS2 carries that location with experimental literature provenance in UniProt. This is a transparent mapping of a positive curated target assertion; it is not an additional independent localization experiment. The primary source access limit is preserved in the reference assessment.

Summary: The translation-linked charging process is specifically mitochondrial.

Reason: The conserved class-I synthetase site supports aminoacylation for translation, and direct human EARS2 biochemistry identifies mitochondrial tRNA-Glu and tRNA-Gln substrates. GO:0070127 is a current descendant of GO:0006418 and specifies this demonstrated process. The low in-vitro activity reported on cytoplasmic tRNA-Gln does not establish a physiological cytoplasmic translation role.

Proposed replacements: tRNA aminoacylation for mitochondrial protein translation

Summary: Preserve the sound ancestral glutamylation inference while replacing its obsolete process term.

Reason: PTN000012469 is on the current target lineage and carries the literal GO:0006424 IBD, with direct experimental donor records including the target itself. No loss or wrong-clade placement is inferred. Current official GO marks GO:0006424 obsolete; the imported source ID is preserved. The obsolete process label is not evidence against glutamylation. GO:0070127 captures the demonstrated mitochondrial translation-linked charging process, while existing GO:0004818 and GO:0050561 preserve the two chemical activities.

Proposed replacements: tRNA aminoacylation for mitochondrial protein translation

Summary: Human mitochondrial glutamylation remains valid despite process-term obsoletion.

Reason: Current official GO marks GO:0006424 obsolete; the imported source ID is preserved. The obsolete process label is not evidence against glutamylation. GO:0070127 captures the demonstrated mitochondrial translation-linked charging process, while existing GO:0004818 and GO:0050561 preserve the two chemical activities. PMID:19805282 directly establishes the EARS2 charging step; GatCAB, not EARS2, performs the subsequent tRNA-bound glutamate amidation. The article’s GatCAB knockdowns must not be attributed to EARS2.

Proposed replacements: tRNA aminoacylation for mitochondrial protein translation

Summary: The InterPro glutamylation mapping needs a current mitochondrial process term.

Reason: Both literal InterPro entries still list the imported glutamylation process mapping, and the target matches the family/domain. Current official GO marks GO:0006424 obsolete; the imported source ID is preserved. The obsolete process label is not evidence against glutamylation. GO:0070127 captures the demonstrated mitochondrial translation-linked charging process, while existing GO:0004818 and GO:0050561 preserve the two chemical activities.

Proposed replacements: tRNA aminoacylation for mitochondrial protein translation

Summary: The zinc-binding domain inference is plausible but not independently resolved for human EARS2.

Reason: The source is an IPR033910 match over residues 36–361, not a mistaken protein or missing domain. A current family alignment, with both full sequences authenticated, maps the four curated E. coli P04805 Zn ligands C98/C100/C125/H127 to human C140/C142/C167/N169. Three cysteines are retained; the histidine correspondence differs. This does not prove loss of zinc binding, and absence of a human UniProt metal-site feature is not negative experimental evidence. No human occupancy or metal-dependence assay was recovered, so retain uncertainty rather than automatically accepting the family-level metal mapping or rejecting it.

Summary: The broad charging annotation can be made specific to mitochondrial translation.

Reason: IPR000924 and IPR020058 support the aminoacyl-tRNA synthetase mechanism. Human EARS2 performs the glutamate-loading step on two mitochondrial tRNAs, a direct contribution to mitochondrial translation. GO:0070127 is a current descendant of GO:0043039 and captures that supported biological context without assigning downstream amidotransferase chemistry.

Proposed replacements: tRNA aminoacylation for mitochondrial protein translation

Summary: Human EARS2 glutamylates mitochondrial tRNA-Gln.

Reason: PMID:19805282 demonstrates a nondiscriminating human mtGluRS that charges native mitochondrial tRNA-Gln with glutamate. GO:0050561 names this activity; its current definition uses the broader Glx wording, while Rhea:64612 explicitly describes the tRNA-Gln reaction. The product is Glu-tRNA-Gln, which GatCAB subsequently converts to Gln-tRNA-Gln. This does not assign glutaminyl-tRNA synthetase or amidotransferase activity to EARS2.

Summary: The electronic mappings preserve the tRNA-Gln glutamylation reaction.

Reason: Rhea:64612 directly specifies ATP-dependent glutamate transfer to tRNA-Gln; Rhea:18397 is its broader Glx parent reaction and EC:6.1.1.24 maps to GO:0050561. These are consistent with direct human biochemical evidence and must not be mistaken for direct glutamine charging. The broader Rhea record does not replace the separately supported tRNA-Glu activity.

Summary: EARS2 directly supplies charged mitochondrial tRNAs for translation.

Reason: The human enzyme catalyses the glutamate-loading step on mitochondrial tRNA-Glu and tRNA-Gln in PMID:19805282. This is direct biochemical participation in tRNA aminoacylation for mitochondrial translation, not an inference from disease necessity or from GatCAB knockdown. Natural alternative-product equivalence and detailed matrix imaging remain separate evidence questions.