Functional Substitution of a Eukaryotic Glycyl-tRNA Synthetase with an Evolutionarily Unrelated Bacterial Cognate Enzyme

Abstract

Two oligomeric types of glycyl-tRNA synthetase (GlyRS) are found in nature: a α2 type and a α2β2 type. The former has been identified in all three kingdoms of life and often pairs with tRNAGly that carries an A73 discriminator base, while the latter is found only in bacteria and chloroplasts and is almost always coupled with tRNAGly that contains U73. In the yeast Saccharomyces cerevisiae, a single GlyRS gene, GRS1, provides both the cytoplasmic and mitochondrial functions, and tRNAGly isoacceptors in both compartments possess A73. We showed herein that Homo sapiens and Arabidopsis thaliana cytoplasmic GlyRSs (both α2-type enzymes) can rescue both the cytoplasmic and mitochondrial defects of a yeast grs1 - strain, while Escherichia coli GlyRS (a α2β2-type enzyme) and A. thaliana organellar GlyRS (a (αβ)2-type enzyme) failed to rescue either defect of the yeast mull allele. However, a head-to-tail αβ fusion of E. coli GlyRS effectively supported the mitochondrial function. Our study suggests that a α2-type eukaryotic GlyRS may be functionally substituted with a α2β2-type bacterial cognate enzyme despite their remote evolutionary relationships.

GARS‐related disease in infantile spinal muscular atrophy: Implications for diagnosis and treatment

“…The empty plasmid did not support yeast growth, consistent with GRS1 being an essential gene (Figure c). The wild‐type GARS ΔMTSΔWHEP expression construct supported robust yeast growth, which indicates that human GARS can complement the loss of the endogenous GRS1 locus, consistent with previous findings (Chien et al, ; Oprescu et al, ). The p.Ile334Asn GARS ΔMTSΔWHEP expression construct was unable to support yeast growth (Figure c), suggesting that p.Ile334Asn GARS represents a loss‐of‐function allele.…”

“…Yeast complementation assays have revealed loss‐of‐function characteristics for the majority of CMT‐associated GARS variants (Antonellis et al, ; Griffin et al, ) indicating that this model can be employed to predict pathogenicity (Oprescu et al, ). GARS with both the mitochondrial targeting sequence (MTS) and WHEP domain deleted (ΔMTSΔWHEP) has been shown to allow robust cell growth (Chien et al, ; Oprescu et al, ). We therefore compared the ability of wild‐type and p.Ile334Asn human GARS ΔMTSΔWHEP to complement the loss of endogenous yeast GRS1 (the yeast ortholog of GARS ).…”

“…Yeast complementation assays were performed as previously described (Oprescu et al, ). The p.Ile334Asn GARS variant was modeled in the human wild‐type GARS ΔMTSΔWHEP open‐reading frame (Chien et al, ; Oprescu et al, ). Mutagenesis was performed using the Quickchange II XL Site‐Directed Mutagenesis Kit, a sequence‐validated wild‐type GARS ΔMTSΔWHEP pDONR221 construct, and mutation‐specific primers (available upon request).…”

“…Reactions were transformed into Escherichia coli , and plasmid DNA was purified from individual colonies and sequenced to confirm the presence of the mutation and the absence of PCR‐induced errors. Sequence‐validated constructs for wild‐type and p.Ile334Asn GARS ΔMTSΔWHEP were cloned into pYY1 (Chien et al, ) using Gateway cloning technology (Invitrogen). The resulting reactions were transformed into E. coli , and plasmid DNA was purified from individual colonies and digested with Bsr GI to confirm proper recombination.…”

A recurrent GARS mutation causes distal hereditary motor neuropathy

“…The p.Gly327Arg GARS variant was introduced into the ΔMTSΔWHEP GARS open‐reading frame using site‐directed mutagenesis (primers available upon request). The open‐reading frame was fully sequenced to rule out cycle‐induced errors and recombined into the pYY1 expression construct (adapted from Reference using Gateway cloning technology (Invitrogen). The resulting GARS expression construct was transformed into a haploid yeast strain in which the endogenous yeast ortholog ( GRS1 ) was deleted; cell viability was maintained with an exogenous copy of GRS1 on pRS316, which is a URA3 ‐bearing plasmid.…”