Figure 2. Environmental screens under protein burden.

(A) Impact of translation inhibition on protein burden in wild type yeast. The bar plot shows the cost of yEVenus in wild type strain as a function of increasing cycloheximide concentration. Cycloheximide is a widely used chemical agent to inhibit translation. Treatment of cells with sub-inhibitory concentration (0.18 µg/ml) of this chemical agent leads to a 3.7-fold increase in protein burden (t-test, p<0.001). For the calculation of fitness cost of yEVenus, see Materials and methods. The bars indicate mean ±95% confidence interval, based on four technical measurements of 17 biological replicates for each concentration. Source file is available as Supplementary file 5. (B) Impact of transcription inhibition on protein burden in wild type yeast. The bar plot shows the cost of yEVenus in wild type strain in response to mycophenolic acid (MPA) stress. MPA is a well-known transcription elongation inhibitor. Treatment of cells with sub-inhibitory concentration (30 µg/ml) of this chemical agent leads to a two-fold increase in protein burden (Mann Whitney U-test, p<0.001). For the calculation of fitness cost of yEVenus, see Materials and methods. The bars indicate mean ±95% confidence interval, based on at least 12 technical measurements of 15 biological replicates for each concentration. Source file is available as Supplementary file 5. (C) Impact of amino acid availability on protein burden. The bar plot shows the cost of yEVenus in wild type strain as a function of amino acid concentration. Auxotrophic amino acids were supplied at normal concentration to the medium, while non-auxotrophic amino acids were serially diluted from the regular one. Arbitrary units are relative concentrations normalized to the regular amino acid level. Total depletion of non-essential amino acids (0 arbitrary unit) from the growth medium resulted in a 2.5-fold increase in protein burden, compared to the regular one (t-test, p<0.001). For the calculation of fitness cost of yEVenus, see Materials and methods. The bars indicate mean ±95% confidence interval, based on at least five technical measurements of 12 biological replicates for each condition. Source file is available as Supplementary file 5. (D) The impact of protein burden across different carbon sources. The left panel shows the cost of yEVenus in wild type strain on different carbon sources. The right panel shows the absolute fitness (arbitrary units estimated by measuring colony size on solid agar media) of the yEVenus overexpressing wild type strain on different carbon sources. Growth media with alternative carbon sources (respirato-fermentative galactose, respirative raffinose) led to a reduction of absolute fitness by 27–32% (right panel, t-test, p<0.001), compared to that on the standard carbon source (fermentative glucose). However, the relative fitness cost of yEVenus overexpression (left panel) was not affected by the change of carbon source. Specifically, the cost of yEVenus on glucose is comparable to that on galactose (t-test, p=0.14) or raffinose (t-test, p=0.07). For the calculation of absolute fitness and fitness cost of yEVenus, see Materials and methods. The bars indicate mean ±95% confidence interval, based on at least 12 technical measurements of 15 biological replicates for each of the genotype. Source file is available as Supplementary file 5.