aspartyl-tRNA Synthetase Research Articles

The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake systems could also be exploited by a Trojan horse strategy to facilitate the transport of antibiotics into bacterial cells. Several natural antibiotics mimic substrates of peptide uptake routes. In this study, we analyzed an ABC transporter involved in the uptake of nucleoside peptidyl antibiotics. Our data might help to design drug conjugates that may hijack this uptake system to gain access to cells.

Dual Routes for B. subtilis Asparaginyl‐tRNA Formation

Two distinct routes for attaching asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis, are known. The one-step, direct pathway uses an asparaginyl-tRNA synthetase (AsnRS) to aminoacylate Asn to tRNAAsn. In organisms lacking AsnRS, a two-step pathway is used. First a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches aspartate (Asp) to tRNAAsn. The Asp is then amidated to Asn by GatCAB. Despite Bacillus subtilis using AsnRS for Asn-tRNAAsn production and GatCAB for Gln-tRNAGln formation, we predicted the lone B. subtilis AspRS is non-discriminating to enable the organism to also use the two-step pathway for Asn-tRNAAsn formation. Our in vitro and in vivo results are consistent with the AspRS using tRNAAsn as a substrate as the first step in tRNA-dependent Asn biosynthesis. B. subtilis encoding a tRNA-dependent route for Asn production would explain why the organism is able to grow in the absence Asn when all three of its glutamine-dependent Asn synthetases are knocked out. This work was supported by Skidmore College and the National Science Foundation (MCB-1244326)

Two Distinct Routes for Asparaginyl‐tRNA Formation in B. halodurans

In nature, two distinct routes have evolved for attaching the amino acid asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis. In the direct pathway Asn is ligated to tRNAAsn by an asparaginyl-tRNA synthetase (AsnRS). In a second, two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) first misacylates Asp to tRNAAsn. This tRNA-bound Asp is then amidated to Asn by the amidotransferase GatCAB. Bioinformatic analysis shows that the Gram-positive Bacillus halodurans can use the direct route. However, whether it uses the indirect route depends on whether its AspRS is non-discriminating. Using both in vivo and in vitro assays, we demonstrate the B. halodurans AspRS can aspartylate tRNAAsn and can be used with GatCAB to form Asn-tRNAAsn. Aminoacylation of tRNAAsn by AsnRS is five-fold more efficient than with AspRS. Retention of the two-step pathway for Asn-tRNA formation in B. halodurans may be vestigial though is might be advantageous unde...

Structure based approach for understanding organism specific recognition of protein-RNA complexes.

BackgroundProtein-RNA interactions perform diverse functions within the cell. Understanding the recognition mechanism of protein-RNA complexes has been a challenging task in molecular and computational biology. In earlier works, the recognition mechanisms have been studied for a specific complex or using a set of non–redundant complexes. In this work, we have constructed 18 sets of same protein-RNA complexes belonging to different organisms from Protein Data Bank (PDB). The similarities and differences in each set of complexes have been revealed in terms of various sequence and structure based features such as root mean square deviation, sequence homology, propensity of binding site residues, variance, conservation at binding sites, binding segments, binding motifs of amino acid residues and nucleotides, preferred amino acid-nucleotide pairs and influence of neighboring residues for binding.ResultsWe found that the proteins of mesophilic organisms have more number of binding sites than thermophiles and the binding propensities of amino acid residues are distinct in E. coli, H. sapiens, S. cerevisiae, thermophiles and archaea. Proteins prefer to bind with RNA using a single residue segment in all the organisms while RNA prefers to use a stretch of up to six nucleotides for binding with proteins. We have developed amino acid residue-nucleotide pair potentials for different organisms, which could be used for predicting the binding specificity. Further, molecular dynamics simulation studies on aspartyl tRNA synthetase complexed with aspartyl tRNA showed specific modes of recognition in E. coli, T. thermophilus and S. cerevisiae.ConclusionBased on structural analysis and molecular dynamics simulations we suggest that the mode of recognition depends on the type of the organism in a protein-RNA complex.ReviewersThis article was reviewed by Sandor Pongor, Gajendra Raghava and Narayanaswamy Srinivasan.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0039-8) contains supplementary material, which is available to authorized users.

Neuroprotection induced by NMDA preconditioning as a strategy to understand brain tolerance mechanism.

Excitotoxicity refers to toxicity caused by abnormal concentrations of glutamate in the synaptic cleft that may lead to neuronal death. Since its description, the phenomenon of glutamatergic excitotoxicity has been implicated in the physiopathology of a wide range of neurological and psychiatric disorders, from acute brain damage such as traumatic brain injury, ischemia as well as chronic conditions like epilepsy, depression and neurodegenerative pathologies such as Huntington's, Parkinson's and Alzheimer's diseases. Excessive stimulation of glutamatergic receptors, mainly N-methyl-D-aspartate (NMDA) receptors (NMDAR), can have numerous adverse effects on the cell viability, including increased nitric oxide release (NO), activation of proteases, increased production of reactive oxygen (ROS) and nitrogen (RNS) species and massive influx of calcium ions (Ca2+), resulting in cell death. Thus, the use of strategies that modulate the excitotoxic cell damage represents a perspective for the treatment of diseases such as Parkinson's and Alzheimer's diseases, ischemia, traumatic brain injury (TBI) and seizures.

Differentially expressed proteins in fluconazole-susceptible and fluconazole-resistant isolates of Candida glabrata.

The current study aimed to identify the differences presented in the proteome of fluconazole-susceptible isolates of Candida glabrata compared to those with fluconazole-resistant ones. Two-dimensional differential gel electrophoresis was applied to identify proteins that were differentially expressed in fluconazole-susceptible and fluconazole-resistant isolates of C. glabrata. Eight proteins including aspartyl-tRNA synthetase, translation elongation factor 3, 3-phosphoglycerate kinase, ribosomal protein L5, coproporphyrinogen III oxidase, pyruvate kinase, G-beta like protein, and F1F0-ATPase alpha subunit were found to be more abundantly represented, while four proteins including vitamin B12-(cobalamin)-independent isozyme of methionine synthase, microtubule-associated protein, adenylosuccinate synthetase, and aldose reductase were found to be less abundantly represented in fluconazole-resistant strains versus those with fluconazole-susceptible ones. These differentially expressed proteins were primarily associated with energy metabolism, stress response, and macromolecule synthesis. Proteins associated with energy metabolism, stress response, and macromolecule synthesis may play a role in the development of fluconazole resistance in the clinical isolates of C. glabrata. Multiple different mechanisms are involved in the development of fluconazole resistance in C. glabrata. These findings provide a scientific basis for discovering new genes and mechanisms associated with fluconazole resistance in C. glabrata.

Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA(Asn), and instead synthesize asparagine from tRNA(Asn)-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA(Asn), and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA(Asn) to form Asn-tRNA(Asn) for use in protein synthesis. ND-AspRS, GatCAB, and tRNA(Asn) may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNA(Asn). We report the 3.7-Å resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNA(Asn) molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNA(Asn) formation evolutionarily preceded the direct attachment of Asn to tRNA(Asn), our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.

DARS-associated leukoencephalopathy can mimic a steroid-responsive neuroinflammatory disorder.

To describe the expanding clinical spectrum of a recently described hereditary leukoencephalopathy, hypomyelination with brainstem and spinal cord involvement and leg spasticity, which is caused by mutations in the aspartyl tRNA-synthetase encoding gene DARS, including patients with an adolescent onset. Three patients with mutations in DARS were identified by combining MRI pattern recognition and genetic analysis. One patient had the typical infantile presentation, but 2 patients with onset in late adolescence had a disease mimicking an acquired inflammatory CNS disorder. Adolescent-onset patients presented with subacute spastic paraplegia and had positive response to steroids. They had only minor focal supratentorial white matter abnormalities, but identical spinal cord changes involving dorsal columns and corticospinal tracts. Clinical presentation included subacute spastic paraplegia with partial improvement on steroids. Focal T2 hyperintense white matter changes on brain MRI in combination with spinal cord signal abnormalities usually suggest acquired inflammatory conditions such as multiple sclerosis, especially in the context of relapsing course and a positive response to steroid treatment. Adolescents with mutations in DARS can present with a comparable clinical picture, broadening the clinical spectrum of hypomyelination with brainstem and spinal cord involvement and leg spasticity.

Leukoencephalopathy

Just 11 years ago, a new genetic white matter disease, leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL), was described in 7 children and 1 teenager.1 Clinical features were dominated by progressive motor deterioration without sphincter dysfunction; half of the patients had loss of position and vibratory sensation and learning problems, but no vision or hearing loss. CSF was acellular without oligoclonal bands. Somatosensory evoked potentials, nerve conduction velocities, and a single sural nerve biopsy were normal. MRI showed a symmetrical leukoencephalopathy (figure, top) with a predilection for the brainstem and spinal cord. Magnetic resonance spectroscopy revealed decreased N -acetylaspartate and elevated lactate levels in brain white matter. Disease was due to a deficiency in mitochondrial aspartyl-tRNA synthetase.2

Biochemical and Structural Characterization of Mycobacterial Aspartyl-tRNA Synthetase AspS, a Promising TB Drug Target

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.

The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

Crystal structure of human cytosolic aspartyl-tRNA synthetase

Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at 2.25 Å resolution. DRS is a homodimer with a dimer interface 3,750.5 Å2which comprises of 16.6% of the monomeric surface area. Our structure reveals the C-terminal end of the N-helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNAAsp to elongation factor 1α. From our analyses of the crystal structure and post-translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.

The RimL transacetylase provides resistance to translation inhibitor microcin C.

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.

Translational Approach to Predictive Biomarker Discovery in Liver Transplant Rejection.

Objectives: Monitoring of immune status after liver transplantation is important for prediction of acute cellular rejection or operational tolerance. However, there is no unified standard to know the degree of rejection and the timing when immunosuppressive therapy should be increased, reduced, or terminated. Therefore, the complete understanding of rejection and graft acceptance is important matter for improving the quality of life after transplantation. Methods: By using a proteomic approach, rejection-specific serum biomarkers in experimental orthotopic liver transplantation (OLT) models were identified. Selected serum biomarkers were verified both in experimental and clinical liver transplantation. Results: Many serum proteins specifically lower molecular weight proteins (under 24 kDa) were significantly upregulated or downregulated in the course of rejection (7 to 28 days after OLT). Several upregulated proteins were gradually decreased after overcoming rejection, and the expression levels in the tolerogenic phase (>60 days after OLT) were similar to the naive rats, suggesting that those proteins are potential biomarkers for prediction of acute cellular rejection after OLT. By using an UltraFlex MALDI-ToF/ToF Mass Spectrometer, we have identified 22 proteins (4 proteins: downregulated and 18 proteins: upregulated). Our pilot data demonstrated that at least five biomarkers including hemopexin, vitamin D-binding protein, threonyl-tRNA synthetase, aspartyl-tRNA synthetase, and Ig κ light chain were highly expressed not only in experimental OLT models but also in OLT patients with rejection episodes. Conclusions: We have successfully identified rejection-specific biomarkers from experimental OLT models, and our translational approach (animal findings to the clinical application) may narrow down the target biomarkers for future clinical verification and validation.

The discovery of the BABA receptor: scientific implications and application potential.

The discovery of the BABA receptor: scientific implications and application potential.

Enzymatic synthesis of bioinformatically predicted microcin C-like compounds encoded by diverse bacteria.

ABSTRACTThe Trojan horse Escherichia coli antibiotic microcin C (McC) consists of a heptapeptide attached to adenosine through a phosphoramidate linkage. McC is synthesized by the MccB enzyme, which terminally adenylates the ribosomally synthesized heptapeptide precursor MccA. The peptide part is responsible for McC uptake; it is degraded inside the cell to release a toxic nonhydrolyzable aspartyl-adenylate. Bionformatic analysis reveals that diverse bacterial genomes encoding mccB homologues also contain adjacent short open reading frames that may encode MccA-like adenylation substrates. Using chemically synthesized predicted peptide substrates and recombinant cognate MccB protein homologs, adenylated products were obtained in vitro for predicted MccA peptide-MccB enzyme pairs from Helicobacter pylori, Streptococcus thermophilus, Lactococcus johnsonii, Bartonella washoensis, Yersinia pseudotuberculosis, and Synechococcus sp. Some adenylated products were shown to inhibit the growth of E. coli by targeting aspartyl-tRNA synthetase, the target of McC.

Plant perception of β-aminobutyric acid is mediated by an aspartyl-tRNA synthetase.

Specific chemicals can prime the plant immune system for augmented defence. β-aminobutyric acid (BABA) is a priming agent that provides broad-spectrum disease protection. However, BABA also suppresses plant growth when applied in high doses, which has hampered its application as a crop defence activator. Here we describe a mutant of Arabidopsis thaliana that is impaired in BABA-induced disease immunity (ibi1) but hypersensitive to BABA-induced growth repression. IBI encodes an aspartyl-tRNA synthetase. Enantiomer-specific binding of R-BABA to IBI1 primed the protein for non-canonical defence signalling in the cytoplasm after pathogen attack. This priming was associated with aspartic acid accumulation and tRNA-induced phosphorylation of translation initiation factor eIF2α. However, mutation of eIF2α-phosphorylating GCN2 kinase did not affect BABA-induced immunity, but relieved BABA-induced growth repression. Hence, BABA-activated IBI1 controls plant immunity and growth via separate pathways. Our results open new opportunities to separate broad-spectrum disease resistance from the associated costs on plant growth.

Discovery of an asymmetric ATPase with two catalytic centers that are separately activated by two different misacylated‐tRNAs (751.2)

A majority of microorganisms lack a complete set of aminoacyl‐tRNA synthetases but still utilize the twenty standard amino acids in their proteomes. The pathogenic bacterium Helicobacter pylori is missing both the glutaminyl‐ and asparaginyl‐tRNA synthetases and instead uses indirect tRNA aminoacylation to generate Gln‐tRNAGln and Asn‐tRNAAsn. This process proceeds via misacylation of these tRNAs to produce Glu‐tRNAGln and Asp‐tRNAAsn, followed by transamidative conversion of the two amino acid side chains to produce Gln‐tRNAGln and Asn‐tRNAAsn. Both misacylated tRNA conversions rely on an amidotransferase called GatCAB. We have identified a new enzyme from H. pylori that induces the formation of a macromolecular complex that also contains this organism’s misacylating aspartyl‐tRNA synthetase (ND‐AspRS) and GatCAB. This enzyme contains two different ATPase active sites, one based on a classic P‐loop ATPase domain and the second based on an [[Unsupported Character ‐ Symbol Font □]]‐adenosine nucleotide hydrolase (AANH) domain. Remarkably, the P‐Loop ATPase is activated by binding of Asp‐tRNAAsn, whereas the AANH ATPase is activated by Glu‐tRNAGln. These ATPase activities modulate GatCAB activity to drive conversion of both misacylated tRNAs into their cognate products. The asymmetric nature of this enzyme and its activation by different misacylated tRNAs make it an unusual new participant in tRNA aminoacylation and protein translation.

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl‐tRNA synthetase enables RNA‐dependent asparagine biosynthesis

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA(Asn). The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA(Asn). S. aureus thus encodes both the direct and indirect routes for Asn-tRNA(Asn) formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.

Tissue-Specific Loss of DARS2 Activates Stress Responses Independently of Respiratory Chain Deficiency in the Heart

Adaptive stress responses activated upon mitochondrial dysfunction are assumed to arise in order to counteract respiratory chain deficiency. Here, we demonstrate that loss of DARS2 (mitochondrial aspartyl-tRNA synthetase) leads to the activation of various stress responses in a tissue-specific manner independently of respiratory chain deficiency. DARS2 depletion in heart and skeletal muscle leads to the severe deregulation of mitochondrial protein synthesis followed by a strong respiratory chain deficit in both tissues, yet the activation of adaptive responses is observed predominantly in cardiomyocytes. We show that the impairment of mitochondrial proteostasis in the heart activates the expression of mitokine FGF21, which acts as a signal for cell-autonomous and systemic metabolic changes. Conversely, skeletal muscle has an intrinsic mechanism relying on the slow turnover of mitochondrial transcripts and higher proteostatic buffering capacity. Our results show that mitochondrial dysfunction is sensed independently of respiratory chain deficiency, questioning the current view on the role of stress responses in mitochondrial diseases.