Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover

Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome-mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature

The fungus Candida albicans tolerates ambiguity at multiple codons

The ascomycete Candida albicans is a normal resident of the gastrointestinal tract of humans and other warm-blooded animals. It occurs in a broad range of body sites and has high capacity to survive and proliferate in adverse environments with drastic changes in oxygen, carbon dioxide, pH, osmolarity, nutrients, and temperature. Its biology is unique due to flexible reassignment of the leucine CUG codon to serine and synthesis of statistical proteins. Under standard growth conditions, CUG sites incorporate leucine (3% of the times) and serine (97% of the times) on a proteome wide scale, but leucine incorporation fluctuates in response to environmental stressors and can be artificially increased up to 98%. In order to determine whether such flexibility also exists at other codons, we have constructed several serine tRNAs that decode various non-cognate codons. Expression of these tRNAs had minor effects on fitness, but growth of the mistranslating strains at different temperatures, in medium with different pH and nutrients composition was often enhanced relatively to the wild type (WT) strain, supporting our previous data on adaptive roles of CUG ambiguity in variable growth conditions. Parallel evolution of the recombinant strains (100 generations) followed by full genome resequencing identified various strain specific single nucleotide polymorphisms (SNP) and one SNP in the deneddylase (JAB1) gene in all strains. Since JAB1 is a subunit of the COP9 signalosome complex, which interacts with cullin (Cdc53p) to mediate degradation of a variety of cellular proteins, our data suggest that neddylation plays a key role in tolerance and adaptation to codon ambiguity in C. albicans.publishe

Glucose uptake in the ancestor and the evolved lines.

<p>After 15 h of growth, optical densities and medium glucose content were measured in parallel in the ancestor and the evolved lines. Glucose uptake rates were estimated by the drop of glucose content per cell. Glucose uptake rate per cell was normalized to that of the ancestor value. The bars indicate mean ± 95% confidence interval. Two-sample <i>t</i> test was used to assess difference in glucose uptake between the ancestor and the evolved lines. */**/*** indicates <i>p</i>-value < 0.05/0.01/0.001, respectively. To ensure that changes in glucose uptake reflect the impact of the accumulated mutations in the evolved lines (rather than the direct effects of mistranslation), tRNA_CAG^Ser was swapped for the corresponding empty vector in the ancestor and the evolved lines.</p

Side effects of adaptation to mistranslation.

<p>(A) Survival of the ancestor and the evolved lines upon prolonged starvation. Colony forming units were used as a proxy for live cell numbers. The colony forming units were counted every 3 d, and the ratio of live cells were calculated and normalized to the ancestor (upper left panel). The bars indicate mean ± 95% confidence interval. To ensure that changes in cell survival reflect the impact of the accumulated mutations (rather than the direct effects of mistranslation), tRNA_CAG^Ser was swapped for the corresponding empty vector in the ancestor and in the evolved lines. (B) Distribution of Rpl25p-GFP in ancestor and evolved lines carrying tRNA_CAG^Ser. (C) Rpl25p-GFP relocalization upon starvation in the ancestor and the evolved lines carrying tRNA_CAG^Ser. Cells were grown to mid-log phase in starvation conditions for 24 h and were analyzed by epifluorescence microscopy. Cells with vacuolar fluorescence were manually counted. At least eight randomly chosen microscopic fields were counted, and approximately 100 cells per line were counted. The bars indicate mean ± 95% confidence interval. Mann-Whitney U test was used to assess the difference in Rpl25p-GFP relocalization between ancestor and evolved lines. *** indicates <i>p</i>-value < 0.001.</p

Fitness changes during laboratory evolution.

<p>(A) Fitness of the ancestor and the evolved lines under high mistranslation rate. The ancestor and evolved lines carry the mistranslation-causing tRNA_CAG^Ser construct. The ancestor is isogenic to the wild type, with the only exception being that the latter carries an empty vector instead of tRNA_CAG^Ser. Growth rate (calculated by monitoring optical density) was used as a proxy for fitness. Fitness values were normalized to the wild-type control. The bars indicate mean ± 95% confidence interval. Mann-Whitney U test was used to assess difference in fitness between ancestor and evolved lines. *** indicates <i>p</i> < 0.001. (B) Fitness of the ancestor and evolved lines under low mistranslation rate. tRNA_CAG^Ser was swapped for the corresponding empty vector in the ancestor and the evolved lines. Growth rate (calculated by monitoring optical density) was used as a proxy for fitness. Fitness values were normalized to the wild-type control carrying no tRNA_CAG^Ser construct. The bars indicate mean ± 95% confidence interval. Mann-Whitney U test was used to assess difference in fitness between ancestor and evolved lines. */**/*** indicates <i>p</i> < 0.05/0.01/0.001, respectively.</p

Evolution of protein synthesis rate.

<p>Amino acid incorporation rate was measured by protein pulse labeling with [¹⁴C(U)]-L-Amino Acid mixture. The bars indicate mean ± 95% confidence interval. Two-sample <i>t</i> test was used to assess difference in protein synthesis rate between ancestor and evolved lines. */**/*** indicates <i>p</i>-value < 0.05/0.01/0.001.</p

Evolution of protein aggregation rate.

<p>(A) Distribution of VHL-mCherry in ancestor and evolved cells. (B) Changes of protein aggregation rate during evolution. The ancestor and evolved lines carry the mistranslation-causing tRNA_CAG^Ser construct. The ancestor is isogenic to the wild type, with the only exception being that the latter carries an empty vector instead of tRNA_CAG^Ser. The number of cells with and without aggregated foci was counted, and the ratio of cells with aggregated foci was calculated. This ratio calculated in ancestor and evolved lines was normalized to the ratio calculated in the wild type. The ratio of the cells with aggregated foci can be used as a proxy for protein aggregation rate. The bars indicate mean ± 95% confidence interval. Mann-Whitney U was used to assess difference in fitness between ancestor and evolved lines. * indicates <i>p</i> < 0.05.</p

Evolution of mistranslation rate.

<p>(A) Mutations in the variable arm of the tS(AGA)D3 serine tRNAs. The variable arm mutated in three lines independently (lines 6, 9, and 10). The identity elements for the SerRS are indicated in red (discriminator base G73 and the GC base pairs in the variable arm). Structure of the molecule was predicted by tRNAscan-SE analysis. (B) Evolution of mistranslation rate. The figure shows β-galactosidase enzyme activities in the ancestor and evolved lines, all of which carry the mistranslation causing tRNA_CAG^Ser construct. The ancestor is isogenic to the wild type, with the only exception being that the latter carries an empty vector instead of tRNA_CAG^Ser. Enzyme activities were normalized to the enzyme activity measured in wild-type control carrying no tRNA_CAG^Ser by normalization to the total amount of β-galactosidase protein (quantified by western blot). The bars indicate mean ± 95% confidence interval. Mann-Whitney U test was used to assess difference in fitness between ancestor and evolved lines. **/*** indicates <i>p</i> < 0.01/0.001, respectively. (C) β-galactosidase enzyme activities in the ancestor and the evolved lines carrying no tRNA_CAG^Ser. Enzyme activities were normalized to the enzyme activity measured in the wild type after normalization to the total amount of β-galactosidase protein quantified using western blot. The bars indicate mean ± 95% confidence interval. Mann-Whitney U test was used to assess difference in fitness between ancestor and evolved lines */** indicates <i>p</i> < 0.05/0.01, respectively.</p

Changes in the ubiquitin-proteasome system during evolution.

<p>(A) Inhibition of the ancestor and the evolved lines by cycloheximide. To test the sensitivity of the populations to protein synthesis inhibitor, the growth medium was supplemented with sublethal dosage of cycloheximide (0.06 μg/ml). Inhibition rates were calculated by comparing growth rates in drug-containing and drug-free media, and were normalized to that of the wild-type control. The bars indicate mean ± 95% confidence interval. Mann-Whitney U test was used to assess difference in fitness between ancestor and evolved lines. */**/*** indicates <i>p</i>-value < 0.05/0.01/0.001, respectively. To ensure that changes in inhibition rates do not simply reflect the direct effects of mistranslation, tRNA_CAG^Ser was swapped for the corresponding empty vector in the ancestor and the evolved lines. (B) Changes in proteasome activity during evolution. Proteasome activities were normalized to the activity measured in wild-type control carrying no tRNA_CAG^Ser. The bars indicate mean ± standard error. Two-sample <i>t</i> test was used to assess the difference in proteasome activity between the ancestor and the evolved lines. */**/*** indicates <i>p</i>-value < 0.05/0.01/0.001, respectively.</p