Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins

Abstract

Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

Keywords: aneuploidy; oxidative stress; respiration; thiol peroxidase.

Fig. 1.

Phenotypes of ∆8 strains. (A) H₂O₂ tolerance. Tenfold serial dilution of Wt cells, Wt-M11 cells, and two independent ∆8 mutant isolates (∆8-1 and ∆8-2) onto YPD plates containing (H₂O₂-treated) or not (control) 0.4 mM H₂O₂. Pictures were taken after 3 d. (B) Dependence on respiration. Tenfold serial dilution assays of Wt, Wt-M11, ∆8-1, and ∆8-2 cells in the presence or absence of antimycin-A, rotenone, and oligomycin. (C) Increased mtDNA copy number in ∆8 mutant cells. The ratio of the normalized number of reads corresponding to mtDNA (X_mt) and the normalized number of reads corresponding to nuclear DNA (X_nuc) is shown for each strain. (D) Distribution of mitochondria. Mitochondria were stained with MitoTracker Red. Blue color corresponds to calcofluor, which visualizes cell membranes.

Fig. 2.

Chromosome XI aneuploidy of ∆8 cells. (A) Occurrence of chr-XI disomy in ∆8 strains identified by genome sequencing. Read depth was calculated in 100-bp windows. Genome coverage is shown for two independent ∆8 mutant isolates (∆8-1 and ∆8-2), a chr-XI aneuploid strain (Wt-M11), and strains (∆3, lacking three Gpxs; ∆5, lacking five Prxs) from which ∆8 cells were prepared. (B) Increased expression of chr-XI genes revealed by RNA-seq. Total numbers of normalized FPKM reads per strain were normalized by Wt reads, and the ratio was plotted. (C) Increased average expression of chr-XI genes in Wt-M11, ∆8-1, and ∆8-2 cells. Average expression of genes on chr-XI of Wt-M11, ∆8-1, and ∆8-2 strains was calculated as fold change compared with Wt cells. Center black bar indicates median and error bars (whiskers) indicate 95% confidence interval for C and E. (D) Expression levels of genes on chr-XI in ∆8 cells compared with those in the Wt-M11 strain. Log₂ ratios of FPKM read values per gene are shown for chr-XI of ∆8-1 (red) and ∆8-2 (green) cells. Three target genes that increased expression in both ∆8 strains more than twofold are labeled with asterisks [SRX1 (green), UTH1 (blue), and CCP1 (red)]. (E) Analysis of gene expression on chr-XI. The ∆8/Wt-M11 gene expression ratios were analyzed for all chr-XI genes of ∆8-1 and ∆8-2 strains. Expression of the three genes that showed increased expression in both ∆8 strains, SRX1 (green), UTH1 (blue), and CCP1 (red), is indicated with dots. (F) Comparison of the expression of the three identified genes among strains. mRNA abundance of each gene was compared with that of Wt cells, and the ratio was plotted.

Fig. 3.

Aneuploidy is linked to elevated expression of CCP1 and UTH1. Representative colonies from each expression group of the laboratory evolution experiment (Fig. S1) were analyzed at 120 generations by colony PCR. Increased expression of genes CCP1 + UTH1 or CCP1 + UTH1 + SRX1 allows cells to lose the extra copy of chr-XI (two lower panels). Individual colonies expressing these genes lost one or the other copy of this chromosome, shown by the loss of the upper or lower bands. All colonies expressing individual genes or pairs of genes including SRX1 had both bands. Left lanes in each panel show size markers.

Fig. S1.

Experimental design of the laboratory evolution experiment. (A) The marker strain used in the laboratory evolution experiment. One copy of SRX1 was replaced with the NatMX marker. Primers were designed to match 100 bp upstream and downstream of SRX1. The upper (∼1.2-kb) band represents NatMX, and the lower (∼600-bp) band represents SRX1 in ∆8 cells. (B) Design of the laboratory evolution experiment. Single or combined expression of CCP1, UTH1, and SRX1 when transformed into the marker strain. Two empty vectors were also transformed and used as the control. Parallel lines splitting from the same branch represent three independent repetitions for each group. Each group was analyzed every 25th generation by assaying selected colonies by colony PCR.

Fig. 4.

Impact of aneuploidy on protein synthesis rates. (A) Autoradiograms of 2D gel electrophoresis performed with whole extracts of 20 min [³⁵S]Met-labeled Wt and ∆8 cells after 20 min exposure to H₂O₂ (0.6 mM) or left untreated, as indicated. A blown-up portion of the autoradiograms is shown. Spots of selected proteins are indicated by arrows. A few protein spots of interest are indicated by black (induced) or white (repressed) arrows. Ahp1 is shown as a control that was deleted in ∆8 cells. (B) Quantification of Ccp1 synthesis rate before (untreated) and after (treated) H₂O₂ treatment of Wt and ∆8 cells. (C) Transcriptional (RNA-seq) and translational (Ribo-seq) changes in CCP1 and UTH1 abundance upon 0.6 mM H₂O₂ treatment of Wt cells. Error bars indicate standard error of the mean.

Fig. S2.

Master gel of Wt cells treated with H₂O₂. The gel represents an example of 2D gels analyzed in the study. The location of protein spots identified through mass spectrometry analysis is shown. The isoelectric point is shown on the horizontal axis, and molecular mass (in kDa) is on the vertical axis.

Fig. 5.

Improved growth of evolved ∆8 cell populations that lost the extra chr-XI. At the end of the laboratory evolution experiment, each population was analyzed for their cell-growth properties. The color scheme is as follows: initial ∆8 cells (red) harboring plasmid only as control (p-only), three independent populations that express CCP1 + UTH1 (black), and three independent populations expressing CCP1 + UTH1 + SRX1 (green).

Fig. S3.

Growth of disomic ∆8 cells in which UTH1 and CCP1 were expressed individually or in combination. The nonevolved strain is the strain in which the two proteins are expressed but the extra chr-XI is present. The evolved strain lacks the extra chr-XI, following long-term expression of the two proteins. (Inset) Growth of nonevolved (Upper; blue box) and evolved (Lower; green box) populations by spot assays. The strains expressing individual genes (CCP1 or UTH1) during long-term culture are shown for comparison, and exhibit slower growth compared with the evolved strain.