Coordination between aminoacylation and editing to protect against proteotoxicity

Abstract Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.


INTRODUCTION
The genetic code is established through accurate pairing between aminoacyl-tRNAs (aa-tRNAs) and mRNA codons on the ribosome (1)(2)(3)(4).Except for selenocysteine, each of the 22 natural proteinogenic amino acids is recognized by a specialized aminoacyl-tRNA synthetase (aaRS) and ligated to the corresponding tRNAs through an aminoacylation reaction ( 5 , 6 ).Efficient aminoacylation is thus critical to supply the ribosome with aa-tRNAs for translation.In addition to aminoacylation, many aaRSs also utilize an editing step to pre v ent the mispairing of amino acids and tRNAs that would otherwise result in protein mistranslation and misfolding ( 7 , 8 ).Editing can occur before or after amino acid transfer to the tRNA by the aaRS in cis or by a separate trans -editing factor ( 9 ).
Gi v en the essential role of aaRSs in protein synthesis, it is unsurprising tha t muta tions in aaRS genes are widely associated with human diseases.Autosomal dominant mutations in six aaRSs have been shown to cause a peripher al neurodegener ati v e disor der known as Charcot-Marie-Tooth (CMT) disease (10)(11)(12)(13).CMT mutations in glycyl-(GlyRS) and alanyl-(AlaRS) tRNA synthetases do not affect aminoacylation but induce aberr ant inter actions with Nrp1 to impair signal transduction in neurons ( 13 , 14 ).Recent studies in a mouse model suggest that CMT mutations in GlyRS also activate the integrated stress response (ISR) to a ttenua te protein synthesis and cause neural toxicity ( 15 , 16 ).Inhibiting ISR or ov ere xpressing tRNAs alleviates phenotypes associated with certain CMT mutations (16)(17)(18).On the other hand, biallelic recessi v e mutations have been identified in most cytoplasmic aaRSs to cause disorders in the central nervous system, such as microcephaly and seizure ( 11 , 12 , 19-21 ).These homozygous or compound heterozygous mutations frequently lead to aminoacylation defects or protein instability.( 11 ) How these a pparent loss-of-function m uta tions cause defects a t the cellular le v el remains poorly understood.
AlaRS and threonyl-tRNA synthetase (ThrRS) belong to the Class II aaRSs and share a similar editing site to hydrolyze misacylated Ser-tRNA Ala and Ser-tRNA Thr , respecti v ely (22)(23)(24)(25)(26)(27).We previously showed that compound heterozygous mutations in AlaRS impair aminoacylation and editing and cause microcephaly with se v ere neuron degeneration in children ( 21 ).Additionally, AlaRS editing-defecti v e mutations in mice cause neurodegeneration and cardioproteinopathy ( 28 , 29 ).In human patients, ThrRS editing-site muta tions are associa ted with intellectual disability and lead to destabilization of the ThrRS protein ( 30 ).Despite such evidence from human patients and animal models, whether editing defects directly contribute to human diseases remains unknown.
In addition to being used by the ribosome during canonical protein synthesis, Thr-tRNA Thr and Ala-tRNA Ala synthesized by ThrRS and AlaRS are also used in the ribosome-associated quality control (RQC) pathway (31)(32)(33).RQC senses stalled ribosomes and adds C-terminal Ala and Thr (CAT) tails to stalled polypeptides.Formation of the CAT tail does not r equir e an mRNA template and facilita tes degrada tion or sequestra tion of trunca ted polypeptides, thus protecting cells against proteotoxicity ( 31 , 34-37 ).Impairing CAT tailing results in neurological defects in mice and humans ( 38 ).
In this study, we wished to understand the cellular impact of ThrRS editing deficiency in eukaryotes.We used genome editing to introduce the ths1-C268A mutation to abolish editing of cytoplasmic ThrRS in Sacchar om y ces cer evisiae.The equivalent mutation has been shown to be essential for ThrRS editing in bacteria ( 24 , 39 ).The ths1-C268A mutation in yeast led to Ser misincorporation at Thr codons and resulted in a se v ere growth defect under heat stress.We next e volv ed suppressor muta tions tha t restored heat resistance in the ths1-C268A strain.Unexpectedly, all suppressor mutations mapped to the THS1 gene that encodes the cytoplasmic ThrRS.Further in vitro biochemical analyses revealed that the suppressor mutations increased the aminoacyla tion ef ficiency but did not rescue the editing defect of ThrRS.Similar to some aaRS mutations in human patients, we show that the ths1-C268A mutation destabilizes ThrRS and leads to a lower tRNA Thr aminoacylation le v el, which in turn impairs both global protein synthesis and the RQC pathway.We further show that decreasing ThrRS aminoacylation or editing efficacy alone is insufficient to cause proteotoxicity.Our work thus uncov ers pre viously unknown coordination between aminoacylation and editing to protect cells against proteotoxic stress.

Plasmids and strains
All Sacchar om y ces cer evisiae strains used here were derivati v es of BY4741.Esc heric hia coli DH5 ␣ grown in Luria Broth (LB) medium was used for molecular cloning.Yeast cells were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or Synthetic Defined (SD) dropout medium (0.17% yeast nitrogen base, 0.5% ammonium sulfate , 2% glucose , and 0.14% amino acid dropout mix, -His , -Leu or -Ura).Mutant yeast strains were obtained using CRISPR / Cas9 ( 40 ).Briefly, pCas9 was transformed into the WT strain and selected on SD-Leu plates.Next, ps-gRNA targeting the THS1 gene and donor DNA fragments were electro-transformed into competent cells.All transformants were selected on SD-Leu-His agar plates.Colonies were verified by PCR and Sanger sequencing.To obtain the mutant library with varying expression levels of ThrRS, we used CRISPR / Cas9 to mutate the DNA sequence 5 to the initiation codon of THS1 from CA-GA GCAA GAAAATAAAACGGA G to TA GGGCTA G-GAAGT AT AANNNNN (N r epr esents A / T / C / G).To create gcn2 , ltn1 and rqc2 mutants, the coding regions wer e r eplaced with the LEU2 or HIS3 gene and verified by PCR.The strains and plasmids are listed in Table S2, and the primers used are listed in Table S3.

Gr owth assa ys
Single colonies of yeast strains were grown in YPD at 30 • C to sa tura tion, and diluted a t 1:50 in YPD in a 96-well pla te.Continuous growth at 30 or 37 • C was monitored with a microplate reader (BioTek Synergy H1).For the spotting assay, single colonies were grown to sa tura tion in YPD at 30 • C , diluted a t 1:50 in YPD, and grown to log phase.Cell cultur es wer e then serially diluted, spotted on YPD agar pla tes, and incuba ted both a t 30 • C and 37 • C f or 2-4 da ys before imaging.

␤-Lactamase assay
To determine Ser misincorpora tion a t Thr codons, yeast cells carrying the ␤-lactamase gene b la , b la-S68T or the empty vector (pJD1212 or pRS316) were grown in SD-Ura to log phase and adjusted to the same A 600 . 1 ml of each culture was collected and washed with phospha te-buf fered saline (PBS), and the pellets wer e r esuspended in 200 l lysis buffer (PBS pH 7.0, 1 mM phen ylmethylsulf on yl fluoride (PMSF), 10 mM dithiothreitol (DTT), 1 × protein inhibitor cocktail, and 3 mg / ml zymolyase) and incubated at room temperature for 40 min with vortexing.␤-Lactamase activity was measured in a 100 l reaction mixture containing PBS pH 7.0, 100 M nitrocefin, and cell lysate (10 l for the strains carrying bla-S68T , and 0.1 l for the strains carrying WT bla ).A486 absorption was recorded with a microplate reader (BioTek Synergy H1).The WT ␤-lactamase (with Ser at position 68) shows > 10 000-fold higher activity than the S68T mutant in the WT yeast strain.To calculate the Ser misincorporation rate of each yeast strain, the ␤lactamase activity of bla-S68T was divided by that of the WT bla .

Experimental evolution of suppressor mutants
A scheme of experimental evolution is shown in Figure S3A.Briefly, three single colonies of the ths1-C268A strain were inoculated in separate tubes in YPD and grown for 2 days to sa tura tion a t 30 • C .For each round of evolution, cell cultur es wer e diluted at 1:100 in YPD and grown at 37 • C for 2 days to sa tura tion.A fraction of the culture after 10 and 15 rounds of evolution was streaked on YPD plates to isolate single colonies for growth analysis and sequencing.

Genome sequencing
Genome sequencing was performed by Azenta Life Sciences and analyzed as described ( 41 ).PE150 libraries of the yeast strains were prepared, sequenced, and mapped by Azenta Life Sciences.Reads are deposited in Sequence Read Archi v e (SRA) under accession number PRJNA972286.To identify mutations, we used FreeBayes ( https://arxiv.org/abs/1207.3907) for the detection of candidate variants.The Integrated Genome Viewer ( https://software.broadinstitute .or g / software / igv / do wnload) was used to inspect candidate variants.True mutations were differentiated from sequencing errors and preexisting SNPs by being supported by the consensus of the reads in the e volv ed isolate(s), but not by the reads from the WT or starting ths1-C268A strain.Aneuploidy was detected by generating read co verage o ver a sliding 10K window with the Bamcoverage tool ( 42 ) and inspecting the resulting bigwig files in the IGV viewer.

Aminoacylation and deacylation assays in vitro
His 6 -tagged yeast ThrRS variants wer e expr essed and purified from E. coli Rosetta (DE3) pLysS.Aminoacylation assa ys were perf ormed as described with slight modifications ( 39 ).Briefly, Thr aminoacylation activity was determined using 0.3 M ThrRS, 5 mg / ml total yeast tRNAs containing ∼3 M of tRNA Thr (Roche), 4 mM ATP, and 18 M 14 C-Thr in aminoacylation buffer (100 mM Na-HEPES pH 7.2, 30 mM KCl and 10 mM MgCl 2 ) at 37 • C. At each time point, aliquots were spotted on 3MM W ha tman paper discs pre-soaked with 5% trichloroacetic acid (TCA), washed three times with 5% TCA, dried, and their radioacti vity le v el was measured with a scintillation counter.Ser misacylation activity was similarly determined, except that 3 ThrRS and 50 M 14 C-Ser were used instead.
For deacylation, first, 14 C-Ser-tRNA Thr was pr epar ed.Briefly, 5 M ThrRS C268S, 2 mM ATP, 50 M 14 C-Ser, 5mM DTT and 10 mg / ml total yeast tRNAs (Roche) were added in a 500 l reaction containing 100 mM Na-HEPES pH 7.2, 30 mM KCl and 10 mM MgCl 2 .The reaction was stopped with the addition of 125 mM sodium acetate pH 4.5 after 10 min at 37 • C, and extracted with acidic phenol / chloroform.After ethanol precipitation and wash, the tRNA pellet was dissolved in 50 l RNase-free water.Deacylation was performed by incubating 50 nM of abovepr epar ed 14 C-Ser-tRNA Thr with 1 ThrRS in 100 mM Na-HEPES pH 7.2, 30 mM KCl and 10 mM MgCl 2 at 37 • C.

Western blot
A FLAG tag was inserted into the C-terminus of THS1 using CRISPR / Cas9.Total proteins were extracted from overnight cultures and precipitated with TCA.The same A600 of cells were collected and suspended in 1 ml of ice-cold NaOH (0.3 M) and 1% ␤-mercaptoethanol, and then incubated on ice for 15 min with occasional vortexing.TCA was then added to a final concentration of 10%, and the samples were incubated on ice for 15 min with occasional vortexing.The mixtures were centrifuged at 13 000 rpm for 20 min, and the supernatants were carefully removed.The pellets wer e r esuspended 100 l loading buffer (8 M urea, 5% SDS, 200 mM Tris pH 6.8, 1 mM EDTA, and 100 mM DTT with bromophenol blue) and heated for 15 min before separation by 12% SDS-PAGE and western blot.The antibody dilutions used were: 1:2000 for the mouse monoclonal anti-FLAG antibody (Sigma-Aldrich), 1:5000 for the mouse monoclonal anti-PGK1 primary antibody (Invitrogen), and 1:5000 for the goat-anti-mouse IgG-HRP secondary antibody (Invitr ogen).Nitr ocellulose membranes wer e tr eated with enhanced chemiluminescent substrate reagents (Bio-Rad) and visualized using a ChemiDoc Imaging System (Bio-Rad).

RQC analysis
Yeast cells carrying pTDH3-GFP-R12-RFP (34) were grown in SD-Ura.Cell pellets were resuspended in PBS and adjusted to the same A 600 .The total proteins were extracted as described above and separated on a 12% SDS-PAGE gel.A standar d western b lot protocol was applied as described above.The mouse anti-GFP first antibody (Invitrogen) was used with 1:1000 dilution.

Acidic northern blot
Total RNAs from yeast cells were extracted using a hotphenol method.Overnight cultures were treated at 37 • C for 2 h, pelleted, and resuspended in 0.3 M sodium acetate pH 4.5.One volume of acidic phenol was then added.The samples wer e vortex ed for 10 s and incubated on ice for 15 min with occasional vortexing.The aqueous phase was collected after centrifugation at 13 000 rpm for 15 min at 4 • C. Two volumes of ice-cold 100% ethanol were added, and the samples were kept at −80 • C for 1 h.Pellets were collected by centrifugation for 20 min at 13 000 rpm, washed with 70% ethanol, dried at room temperature for 5 min, and dissolved in sodium aceta te buf fer (pH 5.2).Total RNAs were separated on an acidic urea gel pr epar ed with 0.1 M sodium acetate buffer pH 5.2.Deacylated tRNA Thr control was obtained by treating the RNA samples in Tris buffer pH 9.0 for 1 h at 42 • C. For northern blot, the nylon membrane was treated with Ultrahyb ™ hybridiza tion buf fer (Invitrogen) at 42 • C for 1 h after UV crosslinking, and incubated with 100 ng / l biotin-labeled DNA probe (Biotin-TT GAACCGAT GAT CT CCACA) at 42 • C overnight.Finall y, tRN A Thr and aa-tRN A Thr were detected using the Chemiluminescent Nucleic Acid Detection Kit (Thermo Fisher Scientific).

Ov erall pr otein synthesis assa y
The protein synthesis assay was carried out as described ( 43 ) using a methionine analog (L-homopropargylglycine (HPG) containing an alkyne moiety.Yeast cells were grown to log-phase in YPD, adjusted to the same A 600 , washed twice with SD-Met medium, and incubated with 100 l SD-Met for 1 h.Fixation and fluorophore labeling were performed according to the manufacturer's protocol (Click-iT ® HPG Alexa Fluor ® 594 protein synthesis assay kit, Thermo Fisher), and the fluorescence was determined using a Synergy HT microplate reader.The fluorescence signal indicates the overall synthesis activity of the proteome within the 1-h time frame.The fluorescence signals of the mutants were normalized with that of the WT (set at 1).

Generating a 3D homology model for yeast cytoplasmic ThrRS with bound tRNA
The cytoplasmic ThrRS from Sacchar om y ces cer evisiae (P04801) is an 84.5-kDa protein comprising 734 amino acid residues, which forms a dimer that binds two molecules of tRNA ( 44 ).Because The 3D structure of Sc ThrRS is unknown, we used HHpred ( 45 ) to identify structural homologs of Sc ThrRS present in the PDB mmCIF70 10 Jan structural database.The bioinformatic analysis yielded 169 structur e hits r eported in the protein data bank ( www.rcsb.org ), w hich mostl y consisted of members of the aminoacyl-tRN A synthetase famil y.The top solution was found with Esc heric hia coli ThrRS (PDB: 1QF6; E -value: 1.7e-77; Score 697.52), whose crystal structure was determined to 2.9-Å resolution with bound tRNA ( 46 ) and which was used as input into MODELLER ( 47 ) without the tRNA coordinates.
The resulting 3D model of Sc ThrRS (residues 74-729) was inspected using COOT 0.8.9.2 ( 48 ) and superposed onto the crystal structure of Ec ThrRS (PDB: 1QF6 A), which re v ealed steric clashes between Sc ThrRS and the superposed tRNA.To remove short contacts, Sc ThrRS was split into three rigid bodies consisting of residues 74-328, 329-619 and 620-729.Each rigid body was superposed individually onto the crystal structure of the Ec ThrRS monomer.The superposed rigid bodies were visually inspected for steric clashes and then manually connected to generate a 3D model for the Sc ThrRS monomer.The Sc ThrRS dimer was generated by a ppl ying the same 2-fold crystallo gra phic symmetry matrix observed in the crystal structure of Ec ThrRS.Figures were generated using the PyMOL Molecular Graphics System, Version 2.5 Schr ödinger, LLC.

Quantitativ e pr oteomics analysis
For proteomics analyses, three biological replicates of each y east str ain wer e pr epar ed and anal yzed.Lo g phase cells pr e-cultur ed at 30 • C wer e incuba ted a t 37 • C for 2 hours, and cell pellets from 20 ml cultur es wer e fast frozen in liquid nitrogen and stored at −80 • C. Protein extraction was pr epar ed by beads beating in lysis buffer (100 mM HEPES pH 8.0, 8 M urea, 0.5% SDS, and 1 × protease inhibitor).Supernatants were collected following centrifugation at 200 g for 20 min.Total proteins were quantified using BCA protein assay kit (Thermo Fisher Scientific).Tandem Mass Tag (TMT)-based multiplexed quantitative proteomics analysis was performed at The Thermo Fisher Center for Multiplexed Proteomics at Harvard University and analyzed on an Orbitrap Lumos mass spectrometer.MS2 spectra wer e sear ched using the COMET algorithm against a Yeast Uniprot composite database containing its re v ersed complement and known contaminants.For proteome, peptide spectral matches wer e filter ed to a 1% false discovery rate (FDR) using the target-decoy strategy combined with linear discriminant analysis.The proteins wer e filter ed to a < 1% FDR and quantified only from peptides with a summed SN threshold of > 180.

ThrRS editing-defective mutation results in serine misincorporation and heat sensitivity
To understand the physiological role of ThrRS editing, we used a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system ( 40 ) to introduce a C268A mutation in the yeast THS1 gene.Cys268 is conserved among bacterial and eukaryotic ThrRSs (Figure S1), and has been shown to be essential for editing in E. coli ThrRS ( 24 , 39 ).Mutating the equivalent residue (C182) in E. coli ThrRS causes the accumulation of Ser-tRNA Thr and misincorporation of Ser at Thr codons ( 39 , 49 ).Using a ␤lactamase reporter ( 43 ), we show that Ser misincorporation increases over 15-fold in the ths1-C268A mutant compared to the wild type (WT, Figure 1 A), indicating an editing defect.Further proteomics analysis also identified an increased le v el of peptides with Thr to Ser substitutions in the ths1-C268A mutant (Figures 1 B and C).Re v erting the genomic ths1-C268A mutation with CRISPR-Cas9 r estor ed Ser misincorporation to the WT le v el (Figure 1 A).
We next tested the growth of the yeast variants in the rich medium y east-extr act-peptone-dextrose (YPD).The ths1-C268A mutant showed normal growth at 30 • C but exhibited se v ere growth defects under heat stress at 37 • C both on agar plates and in liquid media (Figures 1 D-F).Re v erting the ths1-C268A mutation fully r estor ed growth at 37 • C, ruling out any potential effect of non-specific mutations during genome editing.Previous work suggested that activation of the ISR is at least partially responsible for the peripheral neuropathy caused by Gl yRS m utations ( 15 , 16 ), and m utations in glutamyl-prol yl-tRN A synthetase in diabetic patients also activate ISR ( 50 ).In yeast, ISR depends on Gcn2 to phosphorylate eIF2 ␣ ( 51 , 52 ).We found that abolishing ISR by deleting GCN2 in the ths1-C268A mutant did not r estor e growth at 37 • C (Figur e S2), suggesting that ISR does not account for the heat sensitivity caused by the ths1-C268A mutation.

Experimental evolution of suppressor mutants from ths1-C268A under heat stress
To understand how the ThrRS editing-defecti v e mutation causes heat sensitivity, we performed three independent evolution experiments of the ths1-C268A strain at 37 • C (Figure S3A).After 10 rounds ( ∼66 generations) of evolution, cells from two replicates had partially enhanced growth at 37 • C (Figure S3B).Whole-genome sequencing results indica ted tha t they contain a duplica tion of chromosome IX, which harbors the ths1-C268A allele (Figure S3C), but identified no other muta tions tha t could explain the enhanced growth.This indicates that duplicating the partially functional ths1-C268A allele improved growth.To test whether duplication of ths1-C268A affects heat resistance, we expressed ThrRS variants from a low-copy plasmid in the ths1-C268A str ain.An extr a copy of ths1-C268A indeed partially rescued growth at 37 • C (Figures S3D).Another replicate of experimental evolution (E10B) exhibited fully r estor ed growth at 37 • C after 10 rounds.Whole-genome sequencing shows that E10B contains a ths1-L264F mutation in addition to the ths1-C268A mutation (Figures 2 D and   S3C).Engineering this double mutant ths1-L264F,C268A from the ths1-C268A strain using CRISPR-Cas9 re v ealed that the additional L264F mutation was responsible for the r estor ed growth (Figure 2 E).After 15 rounds ( ∼100 generations), all three replicates (E15A, E15B and E15C) had e volv ed to r estor e growth at 37 • C (Figur es 2 B-C and S3B).Whole-genome sequencing shows that E15B retained the ths1-L264F mutation as expected and maintained a single copy of chromosome IX.The E15A and E15C strains still contain the duplicated chromosome IX, but also each acquired an additional muta tion a t the P173 position of one copy of THS1 ( ths1-P173L and ths1-P173R , respecti v ely, Figures 2 D and S3C).To confirm that these additional ths1 mutations r estor ed heat r esistance, we introduced them either in the genome of the ths1-C268A strain (Figure 2 E) or as an extra THS1 allele on a plasmid (Figure S3D).Our results demonstra te tha t secondary muta tions of the THS1 allele ar e r esponsible for fully r escuing the heat sensitivity caused by the ths1-C268A mutation.

Suppressor mutations do not restore editing but enhance aminoacylation
The suppressor mutations are located in the editing domain of ThrRS, prompting us to test whether these mutations r estor e the editing acti vity.We e xpressed and purified His 6 -tagged yeast ThrRS variants from Esc heric hia coli and performed Ser misacylation experiments in vitro .The WT ThrRS did not accumulate mischarged Ser-tRNA Thr due to efficient editing (Figure 2 F).The C268A mutant yielded mischarged Ser-tRNA Thr over time, supporting that C268 is essential for ThrRS editing.None of the suppressor mutations lo wered mischar ged Ser-tRNA Thr formation (Figure 2 F) or promotes deacylation of Ser-tRNA Thr (Figure S4), suggesting that these secondary mutations did not r estor e the editing activity.In line with this, the suppressor strains show similar le v els of Ser misincorporation as the ths1-C268A strain (Figure 2 G).We further tested the Thr aminoacylation activity by ThrRS variants.Interestingly, we found that all three suppressor mutants exhibited increased le v els of correctly charged Thr-tRNA Thr at 37 • C (Figure 2 H).

ths1-C268A mutation destabilizes ThrRS at 37 • C
ThrRS editing-site mutations found in human patients have been shown to lower the ThrRS protein le v el in patientderi v ed cell lines ( 30 ), leading us to test the ThrRS protein le v el of our y east variants in vi vo .We added a FLAG tag to the C-terminus of THS1 and confirmed that the tag did not affect growth, and that the FLAG-tagged  ths1-C268A strain still exhibited severe growth inhibition at 37 • C (Figure S5).Western blot results re v ealed that the ths1-C268A mutation indeed decreased the ThrRS protein le v el by a pproximatel y 40% after 2 h of incubation at 37 • C (Figure 3 A).The e volv ed strains E15A and E15C ( ths1-P173L,C268A / ths1-C268A and ths1-P173R,C268A / ths1-C268A ) had a similar ThrRS protein le v el as the WT strain a t 37 • C .W hereas the ThrRS protein le v el is not fully restor ed, the incr eased ThrRS aminoacylation activity may compensate for the production of Thr-tRNA Thr (Figure 2 H).Next, we performed a time-course degradation assa y f ollowing inhibition of protein synthesis with cy clohe ximide (CHX) and found that the ThrRS C268A protein was degraded faster than the WT at 37 • C in vivo .Consistent with the Western b lot result, quantitati v e proteomics analysis also shows that the ths1-C268A mutation decreases the ThrRS protein le v el (Figure 3 E).Lowering ThrRS protein is expected to decrease aminoacylation and protein synthesis.To test this, we extracted total RNAs from the WT and ths1-C268A cells treated at 37 • C for 2 h under acidic conditions to pre v ent aa-tRN A Thr hydrol ysis and performed acidic northern b lot.As e xpected, the ths1-C268A cells had a significantly lower percentage of aa-tRNA Thr le v el (Figure 3 F and G).Additionally, the ths1-C268A strain ex-hibited a decrease in the overall protein synthesis compared to the WT (Figure 3 H).These data indicate that the ths1-C268A mutation destabilizes the ThrRS protein, which is compensated by duplication of the ths1-C268A allele and secondary mutations in THS1 in the e volv ed strains.

ThrRS editing deficiency is not sufficient to cause heat sensitivity
To dissect the contribution of aminoacylation and editing defects to heat sensitivity, we constructed additional editing-site mutants using CRISPR-Cas9.The 3D structure of yeast cytoplasmic ThrRS remains to be determined.To facilitate our mutant design, we generated a homology model of the yeast ThrRS in silico using the crystal structure of the E. coli ThrRS [PDB: 1QF6] ( 46 ) as a structural template.In our model, the side chain of C268 forms electrostatic interactions with neighboring charged residues that position a helix-hairpin-helix (Figure 4 A).Disruption of the electrostatic network may destabilize the folding of the editing site.We predicted that the electrostatic interactions are impaired by the C268A change but maintained by the C268S mutation.In addition, His272 is also involved in the electrostatic interactions, and the H272A mutation may destabilize ThrRS.We found that as predicted, the more conservati v e C268S mutation did not significantly decrease the ThrRS protein le v el at 37 • C but still increased Ser misincorporation (Figures 4 B-D), indicating that the C268S change caused an editing defect as the C268A mutation.The H272A mutation both decreased the ThrRS protein le v el and increased Ser misincorporation, but to a lesser extent as compared to the C268A m utation.Intriguingl y, the ths1-C268S mutant strain exhibited no growth defect at 30 or 37 • C (Figures 4 E-G).At 37 • C, the ths1-H272A strain exhibited a growth defect compared to the WT but grew better than the ths1-C268A strain.These results suggest that ThrRS editing deficiency alone does not lead to growth defects under heat stress.

Combined aminoacylation and editing defects cause heat sensitivity
We have shown that the ths1-C268A mutation decreases the ThrRS protein le v el a t 37 • C .To determine how varying ThrRS le v el affects heat sensitivity, we used CRISPR-Cas9 to randomize the nucleotide sequence 5 to the translation start site of THS1 .Western blot results re v ealed that se v eral mutants exhibited various levels of ThrRS (Figures 5 A and  B).For instance, ths1-mut7 had a lower le v el of ThrRS than the WT, and its overall protein synthesis level is similar to the ths1-C268A strain (Figures 5 A-C).Howe v er, the growth of ths1-mut7 was unaf fected a t 37 • C (Figures 5 D and E), suggesting that lowering the ThrRS protein le v el alone does not lead to heat sensitivity.We next combined the editingdefecti v e ths1-C268S mutation with the reduced expression of ths1-mut7 .The double mutant ( ths1-mut7,C268S ), which expr essed a decr eased ThrRS le v el as the ths1-mut7 mutant (Figure S6), exhibited slo wer gro wth than the WT or ths1-mut7 and ths1-C268S single mutant strains (Figures 5 D, E, and S7).Collecti v ely, our results support that combined aminoacylation and editing defects synergistically impair growth under heat stress.

ThrRS C268A mutation dampens CAT tailing during ribosome-associated quality control
The ths1-C268A mutation decreased the overall protein synthesis le v el (Figure 5 C).To determine the abundance of individual proteins, we performed multiplexed quantitative proteomics of the WT, ths1-C268A , ths1-C268S , and ths1-mut7 strains treated at 37 • C for 2 h (Figure S8).Using multiplexed isobaric labeling, a total of over 4400 proteins were identified for each tested strain (Table S1).We did not observe significant changes in stress-activated chaperones that assist protein refolding between the WT and ths1-C268A mutant (Figure S8C), indicating that the capacity to deal with misfolded proteins is not enhanced in the ths1-C268A strain.
In addition to translation, Thr-tRNA Thr is also used as a substrate in RQC process for adding CAT tails to stalled polypeptides ( 31 , 36 ).CAT tailing has been shown to dri v e the degradation of stalled polypeptides both on the ribosome in an Ltn1-dependent manner and off the ribosome, thereby protecting cells against proteotoxic stress ( 36 , 37 ).We have shown that the ths1-C268A mutation decreases the aminoacylation le v el of tRNA Thr (Figure 3 F and G).We next examined CAT tail formation using an established GFP reporter ( 34 ) with ribosome stalling poly-arginine sequences.In the absence of Ltn1, which ubiquitinates stalled polypeptides for proteasome degradation, CAT tailing was indeed impaired in the ths1-C268A mutant (Figure 6 C).Deleting LTN1 further decreased the growth of the ths1-C268A strain at 37 • C but had no effect on the growth of the WT strain (Figures 6 B and C).Collecti v ely, these results suggest that the ths1-C268A mutation leads to aminoacylation deficiency and impaired CAT tailing during RQC.

ThrRS aminoacylation and editing defects predispose cells to aminoglycoside toxicity
Heat stress causes global protein misfolding and proteotoxicity ( 34 , 53 ).To determine how ThrRS aminoacylation and editing defects impact cellular defense against other proteotoxic stresses, we tested the growth rates of yeast variants in the presence of an amino gl ycoside G418, w hich slows ribosomal translation and increases translational errors ( 54 , 55 ).We show that all three editing-defecti v e mutants ( ths1-C268A , ths1-C268S and ths1-H272A ), as well as ths1-mut7 , are all more sensiti v e to G418 treatment (Figure S9), further supporting our notion that robust aminoacylation and editing are critical to defending against proteotoxicity.

DISCUSSION
Aminoacylation is a fundamental process in all living organisms and provides the ribosome with aa-tRNA build-ing blocks for protein synthesis.Decades of work has provided insights into the structural basis and kinetic mechanisms of aminoacylation and editing, as well as noncanonical functions of aaRSs beyond protein synthesis ( 5 , 9 , 56 , 57 ).On the other hand, we are only beginning to understand the physiological impact of aminoacylation and editing, which is critical not only to uncover the regulatory mechanisms of gene expression and stress responses but also to elucidate the molecular basis of human diseases caused by mutations in aaRSs.Recent advances in exome and wholegenome sequencing have revealed a ra pidl y growing number of aaRS mutations that lead to human diseases ( 11 , 12 ).Compared to dominant aaRS mutations that cause peripheral neuropathy, recessi v e mutations in cytoplasmic aaRSs lead to disorders in the central nervous system.We previously reported that compound heterozygous mutations in glutaminyl-tRNA synthetase (GlnRS) cause progressi v e microcephaly and brain atrophy ( 19 ).Since this discovery, pathogenic recessi v e mutations hav e been found in 17 (out of 20) other cytoplasmic aaRSs in patients with neurological disorders ( 11 , 12 ).Most of these mutant aaRSs are reported to exhibit a lower aminoacylation efficiency, decreased protein stability or both, leading to the speculation that their disease-causing mechanisms may be due to loss of aaRS function.Exactly how aminoacylation defects lead to cellular toxicity is unclear.In se v eral cases, protein misfolding has been implicated to worsen the disease onset caused by recessi v e aaRS mutations ( 21 ).For instance, GlnRS mutations found in microcephaly patients cause increased ubiquitina tion and aggrega tion of the GlnRS protein ( 19 ), and a pathogenic mutation in AlaRS has been shown to cause an editing defect ( 21 ).Neurons are particularly sensiti v e to proteotoxic stress ( 58 ).It is therefore tempting to specula te tha t aminoacyla tion deficiency may cause se v ere damage to cells in combination with proteotoxic stress.In this stud y, we demonstra te tha t combined aminoacyla tion and editing defects lead to se v ere proteotoxicity.
ThrRS and AlaRS share homologous editing domains to hydrolyze misacylated Ser-tRNAs ( 22 , 24 ).Editingdefecti v e AlaRS mutations have been shown to cause neurodegeneration in mice and heat sensitivity in E. coli and yeast ( 29 , 43 , 59 ).Interestingly, deleting one AlaRS allele further enhances proteotoxicity in editing-defecti v e mice ( 28 ), suggesting that as in our ThrRS study, combining editing defects with reduced protein le v els enhances phenotypic consequences.In humans, the K276E and L227P mutations in the ThrRS editing site are associated with de v elopmental disorders and intellectual disability ( 30 ).These mutations appear to destabilize ThrRS in cells, and the equivalent mutations in yeast cause lethality ( 30 ).These studies are consistent with our findings that aminoacylation and editing defects synergistically sensitize cells to proteotoxic stress.
Ef ficient aminoacyla tion by ThrRS and AlaRS is required to provide Thr-tRN A Thr and Ala-tRN A Ala substra tes to RQC , which results in CAT tailing of stalled polypeptides.A recent study suggests that Rqc2 binding to aa-tRNAs is a rate-limiting step in CAT tailing, which is slower than canonical translation ( 60 ).RQC and CAT tailing are critical for cellular defense against proteotoxicity ( 31 , 34-37 ).Mutations in NEMF (the mammalian homolog of Rqc2), which catalyzes the mRNA-independent addition of Ala and Thr to the stalled polypeptides, result in neurological defects and a shortened life span in mice ( 38 ).Such mutations are also associated with juvenile neuromuscular diseases in humans ( 38 ).CAT tails have been proposed to extend the peptide from the ribosomal exit tunnel and expose Lys residues for ubiquitination by Ltn1 ( 37 ), and also directly serve as a degron to target polypeptides for degradation ( 36 ).In addition, some CATylated polypeptides were shown to form aggregates, possibly by sequestering toxic pr ematur e polypeptides ( 35 ).Here, we show tha t the ths1-C268A muta tion decreases the aminoacylation le v el of tRNA Thr and impairs CAT tailing (Figure 6 ).This may further increase the proteotoxicity in addition to the protein misfolding stress caused by ThrRS editing deficiency and heat (Figure 7 ).

DA T A A V AILABILITY
The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE ( 61 ) partner repository under the dataset identifier PXD042145.Genome sequencing reads are deposited in Sequence Read Archi v e (SRA) under accession number PR-JNA972286.Any additional information r equir ed to reanalyze the data reported in this paper is available from the lead contact upon request.

SUPPLEMENT ARY DA T A
Supplementary Data are available at NAR Online.

Figure 1 .
Figure 1.Editing-defecti v e mutation in yeast ThrRS leads to heat sensitivity.( A ) Ser misincorporation percentage in the WT, C719A and re v ertant yeast strains was determined with the ␤-lactamase reporter.Cells were grown in SD-Ura at 30 • C. The percentage shows the activity ratio of the S68T ␤-lactamase variant over the WT ␤-lactamase.Misincorporation of Ser at Thr codons r estor es acti v e ␤-lactamase for the S68T variant.( B ) Tandem mass spectrometry of a peptide containing Ser misincorporation (highlighted in red) at a Thr codon.( C ) Relati v e abundance of the peptide in (B).In TMT-based multiplexed quantitati v e pr oteomics analyses, pr oteins fr om differ ent samples ar e labeled with differ ent isobaric tags and mixed for tandem MS.The peptide shown here (from WT and ths1-C268A samples) was detected sim ultaneousl y as a single precursor ion peak, and the relati v e abundance was determined by the signals of the isobaric tags.( D ) Yeast cells were grown in YPD to log phase and spotted on YPD agar plates with 10-fold dilutions.The plates were incubated for 2 da ys bef ore ima ging.The ima ges shown her e ar e r epr esentati v es of at least three biological r eplicates.( E ) Repr esentati v e growth curv es of y east str ains in YPD liquid medium out of at least three biological replica tes.( F ) Quantita tion of growth ra tes in YPD.Statistical analysis compares the mutants with the WT at the same temperature.Error bars represent one standar d de viation (SD) from the mean in at least three biological replicates.The P values are determined using the unpaired t -test.** P < 0.01.

Figure 2 .
Figure 2. Intragenic mutations in ThrRS rescue heat sensitivity caused by the ths1-C268A mutation.( A-C ) The growth of yeast variants was tested as in Figur e 1 .E15A-C ar e independently e volv ed strains from the ths1-C268A mutant after 15 cycles.The figur es shown her e ar e r epr esentati v es of at least three biological replicates.( D ) Sanger sequencing results of the THS1 gene in the e volv ed str ains.E15A and E15C str ains carry two THS1 alleles due to duplication of Chromosome IX. ( E ) Growth curves of yeast strains in YPD with means and standar d de via tions of a t least three biological replicates.The mutations identified in the e volv ed strains are introduced chromosomally to the ths1-C268A strain using CRISPR-Cas9.( F ) Ser misacylation by purified ThrRS (3 M) in vitro with means and standard deviations of triplicates.Efficient editing in the WT pre v ents the accumulation of Ser-tRNA Thr through hydrolysis.( G ) Ser misincorporation percentage was determined with the ␤-lactamase reporter as in Figure 1 A. ( H ) Thr aminoacylation b y ThrRS v ariants (0.3 M) with means and standar d de viations of triplicates.Mutations in the e volv ed strains increase the aminoacylation efficiency.Error bars r epr esent one SD from the mean.The P values are determined using the unpaired t -test.** P < 0.01.

Figure 3 .
Figure 3. C268A destabilizes ThrRS and decreases overall protein synthesis at 37˚C. ( A, B ) Western blot against FLAG-tagged ThrRS.Yeast cells were grown in YPD at 30 • C to log phase and then incubated at 37 • C for 2 hours before preparation of total proteins.The relati v e ThrRS le v el was normalized with the WT from Western b lot e xperiments.Statistical anal ysis compares the m utants with the WT at the same tempera ture.( C , D ) Degrada tion of ThrRS following the addition of 500 ng / l of CHX to inhibit protein synthesis.The ths1-C268A mutation caused r apid degr adation of ThrRS.( E ) Relati v e ThrRS protein le v el re v ealed by TMT-based multiple xed quantitati v e proteomics.The Y-axis indicates the ThrRS percentage from each sample relati v e to total ThrRS in the protein mixture of all samples (also see Table S1).( F, G ) Acidic northern blot against tRNA Thr .WT and ths1-C268A cells were grown at 30 • C to mid-log phase and then incubated at 37 • C for 2 hours.Alkaline (OH − ) treatment of the total RNA causes deacylation.The ths1-C268A mutant shows a lower le v el of aminoacylation.( H ) Ov erall protein synthesis le v el determined by the incorporation of a Met analo g L-homopropargylgl ycine (HPG) in the pr oteome.Err or bars r epr esent one SD from the mean in at least three biological r eplicates.The P values ar e determined using the unpaired t -test.** P < 0.01.The figures shown in (A, C, F) are representati v es of at least three biological replicates.
Figure 3. C268A destabilizes ThrRS and decreases overall protein synthesis at 37˚C. ( A, B ) Western blot against FLAG-tagged ThrRS.Yeast cells were grown in YPD at 30 • C to log phase and then incubated at 37 • C for 2 hours before preparation of total proteins.The relati v e ThrRS le v el was normalized with the WT from Western b lot e xperiments.Statistical anal ysis compares the m utants with the WT at the same tempera ture.( C , D ) Degrada tion of ThrRS following the addition of 500 ng / l of CHX to inhibit protein synthesis.The ths1-C268A mutation caused r apid degr adation of ThrRS.( E ) Relati v e ThrRS protein le v el re v ealed by TMT-based multiple xed quantitati v e proteomics.The Y-axis indicates the ThrRS percentage from each sample relati v e to total ThrRS in the protein mixture of all samples (also see Table S1).( F, G ) Acidic northern blot against tRNA Thr .WT and ths1-C268A cells were grown at 30 • C to mid-log phase and then incubated at 37 • C for 2 hours.Alkaline (OH − ) treatment of the total RNA causes deacylation.The ths1-C268A mutant shows a lower le v el of aminoacylation.( H ) Ov erall protein synthesis le v el determined by the incorporation of a Met analo g L-homopropargylgl ycine (HPG) in the pr oteome.Err or bars r epr esent one SD from the mean in at least three biological r eplicates.The P values ar e determined using the unpaired t -test.** P < 0.01.The figures shown in (A, C, F) are representati v es of at least three biological replicates.

Figure 4 .
Figure 4. Effects of ThrRS editing-defecti v e mutations on heat resistance.( A ) Homology model of S. cerevisiae cytoplasmic ThrRS with bound tRNA and adenosine monophosphate (AMP).Ribbon diagram of the 3D model for Sc ThrRS using the crystal structure of E. coli ThrRS (PDB: 1QF6)(46) as the template.The two Sc ThrRS monomers in the two-fold symmetric dimer are shown in gold and lavender, with the bound tRNA in red and blue, respecti v ely.Each Sc ThrRS monomer is bound to one molecule of AMP that is shown as a CPK model.The inset shows an enlarged view of Cys268 together with His151, His155, Asp266, and His272.The aforementioned residues are conserved from bacteria to humans.( B, C ) ThrRS protein le v els at 37 • C re v ealed by Western blot as in Figure 3 A. The relati v e ThrRS le v el was normalized with the WT. ( D ) Ser misincorporation le v els determined with the ␤-lactamase reporter as in Figure 1 A. ( E-G ) Growth of yeast variants as described in Figure 1 .Statistical analysis compares the mutants with the WT at the same temperature.Error bars represent one SD from the mean in at least three biological replicates.The P values are determined using the unpaired t -test.** P < 0.01.The figures shown in (B, E) are representati v es of at least three biological replicates.

Figure 5 .
Figure 5. Aminoacylation and editing defects of ThrRS synergistically cause heat sensitivity.( A, B ) Western blot against FLAG-ThrRS.Mut 5-7 show decreased ThrRS protein le v els.The relati v e ThrRS le v el was normalized with the WT.The figure shown in (A) is r epr esentati v e of thr ee biological r eplicates.( C ) Protein synthesis le v el determined with HPG incorpora tion.Sta tistical anal ysis compares the m utants with the WT at the same temperature.( D, E ) Growth of yeast variants.A combination of C268S and Mut 7 changes lead to a growth defect at 37 • C. Error bars r epr esent one SD from the mean in at least three biological replicates.The P values are determined using the unpaired t -test.** P < 0.01.

Figure 6 .
Figure 6.ThrRS C268A mutation dampens CAT tailing during RQC.( A ) Detection of CAT tails with the pTDH3-GFR-R12-RFP r eporter.Cells wer e grown at 30 • C to mid-log phase and then incubated at 37 • C for 5 hours prior to Western blot using an anti-GFP antibody.Ribosome stalling at the R12 motif leads to CAT tailing of GFP.Deleting LTN1 pre v ents ubiquitination and proteasome degradation of CAT-tailed proteins, and deleting RQC2 abolishes CAT tailing.The ths1-C268A mutant shows a lower le v el of CAT tailing, consistent with a decreased supply of Thr-tRNA Thr .Total proteins are detected with Ponceau staining of the same membrane following Western b lot.Representati v e images of three biological replicates are shown.( B, C ) Deleting LTN1 further decreases growth in the ths1-C268A mutant at 37 • C. Error bars r epr esent one SD from the mean in at least three biological replicates.The P values are determined using the unpaired t-test.

Figure 7 .
Figure 7. Model of proteotoxicity resulting from concerted aminoacylation and editing defects of ThrRS.Robust aminoacylation and editing of ThrRS lead to an efficiently and accurately translated proteome, which is functional and protected against proteotoxic stresses, such as heat.An aminoacylation defect in ThrRS lowers the supply of Thr-tRNA Thr , which leads to both decreased protein synthesis and impaired RQC.An editing defect further increases Ser misincorporation and protein misfolding, thereby contributing to the overall proteotoxicity.Created with BioRender.com.