Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast

Abstract

Evolving lineages face a constant intracellular threat: most new coding sequence mutations destabilize the folding of the encoded protein. Misfolded proteins form insoluble aggregates and are hypothesized to be intrinsically cytotoxic. Here, we experimentally isolate a fitness cost caused by toxicity of misfolded proteins. We exclude other costs of protein misfolding, such as loss of functional protein or attenuation of growth-limiting protein synthesis resources, by comparing growth rates of budding yeast expressing folded or misfolded variants of a gratuitous protein, YFP, at equal levels. We quantify a fitness cost that increases with misfolded protein abundance, up to as much as a 3.2% growth rate reduction when misfolded YFP represents less than 0.1% of total cellular protein. Comparable experiments on variants of the yeast gene orotidine-5'-phosphate decarboxylase (URA3) produce similar results. Quantitative proteomic measurements reveal that, within the cell, misfolded YFP induces coordinated synthesis of interacting cytosolic chaperone proteins in the absence of a wider stress response, providing evidence for an evolved modular response to misfolded proteins in the cytosol. These results underscore the distinct and evolutionarily relevant molecular threat of protein misfolding, independent of protein function. Assuming that most misfolded proteins impose similar costs, yeast cells express almost all proteins at steady-state levels sufficient to expose their encoding genes to selection against misfolding, lending credibility to the recent suggestion that such selection imposes a global constraint on molecular evolution.

Fig. 1.

Protein variants display characteristic misfolding phenotypes. (A) Western blotting reveals partitioning of YFP variants into the insoluble fraction of a two-phase lysis. Blots of total (n = 2), soluble (n = 5), and insoluble (n = 4) cell fractions were probed from strains expressing 3×FLAG-YFP variants for 2 h, with a representative of each shown. Strains carried an rpn4 deletion (a regulator of the proteasome) and were treated with 100 μM of the proteasome inhibitor bortezomib during induction (43) to reduce misfolded protein degradation. A reference protein (P_GAL1-3×Myc-YFPref) present in each strain was used to normalize galactose induction and loading between strains. Error bars show 95% confidence intervals about the mean reference-normalized fold change over YFPwt. (B) YFP variants fluoresce at different levels. Densities show single-cell variability across >10,000 cells, with arbitrary heights set to visually separate distributions. (C) YFP-Ura3wt dispersed throughout the cytosol, whereas YFP-Ura3m1 often localized in a single bright focus. Scale bar is estimated.

Fig. 2.

YFP variants display different competitive growth rates. YFP (test strains) vs. RFP (reference strain) cell count ratios, assessed by flow cytometry and normalized to an initial mean YFP/RFP ratio of 1, changed log-linearly with the number of reference strain generations. Lines show least-squares regression fits.

Fig. 3.

Misfolded proteins impose a fitness disadvantage. (A) Fitness disadvantage of YFP-variant–expressing strains relative to a strain expressing YFPwt as a percentage of RFP-expressing reference strain growth rate, when fully induced with 27.5 mM galactose (induced) and 0 mM galactose (uninduced). Error bars show 95% confidence intervals about the mean. (B) Fitness disadvantage relative to strains expressing YFPwt correlates with the relative amount of insoluble YFP (Fig. 1A). (C) The fitness cost of YFPm3 increases linearly with protein level monitored by average YFPm3 fluorescence across all four time points. (D) Misfolded YFP-Ura3m1 confers an induction-dependent fitness cost relative to folded YFP-Ura3wt.

Fig. 4.

Chronic low-level expression of a misfolded protein induces a cytosolic unfolded protein response. (A) Mean protein ratios for 3,031 proteins, plotted against the number of measurements used to generate each mean ratio, between strains KG079 (expressing misfolded YFPm3) and KG071 (expressing folded YFPwt). Gray envelope shows expected variation for the most variable proteins as a function of number of measurements (in the text). Error bars show 95% confidence interval on the mean assuming log ratios are drawn from a normal distribution. Other protein quality control proteins are highlighted for comparison (open squares), such as cytosolic proteins SSB1/SSB2/ZUO1/SSZ1, which associate with nascent polypeptide chains at the ribosome, and JEM1/LHS1/SCJ1/ERJ5, ER chaperones up-regulated in the UPR. (B) Without galactose induction of YFP variants, no protein levels differ significantly.