Old yellow enzymes protect against acrolein toxicity in the yeast Saccharomyces cerevisiae

Abstract

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce alpha,beta-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of alpha,beta-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small alpha,beta-unsaturated carbonyl compounds, such as acrolein, in vivo.

FIG. 1.

Functional grouping of deletion mutant sensitivity data. (A) Localization was assigned based on the “Component” term of the Saccharomyces Genome Database GO term mapper. Cyt, cytoplasm; Nuc, nucleus; Mit, mitochondrion; Golgi, Golgi apparatus; ER, endoplasmic reticulum; Vac, vacuole; Plasma/bud, plasma membrane and bud; O/U, other/unknown. (B) Gene products were grouped into functional categories according to the MIPS functional database and the Saccharomyces Genome Database, combined with visual inspection. More-detailed functional data are available in Table 1. PS, protein synthesis; Cell, cell cycle and differentiation; Signal, cellular communication/signal transduction; Metab, metabolism; Transport, cellular transport; Transcript, transcription; Prot fate, protein fate; O/U, other/unknown.

FIG. 2.

Deletion of OYE2 causes sensitivity to acrolein. Sensitivity was determined by spotting strains on YEPD plates containing various concentrations of acrolein, H₂O₂, or crotonaldehyde. Cultures of wild-type (wt), oye2, oye3, and oye2 oye3 cells were grown to stationary phase and the A₆₀₀ was adjusted to 1, 0.5, 0.1, 0.05, or 0.01 before the cells were spotted onto plates. Growth was monitored after 3 days of incubation at 30°C. Results are shown for plates containing no oxidant (YEPD), 3 mM acrolein, 4 mM H₂O₂, and 2 mM crotonaldehyde.

FIG. 3.

Overexpression of OYE2 or OYE3 increases resistance to acrolein. (A) Wild-type strains containing empty vector pYES/CT (v) or vector-containing OYE2 (mcOYE2) or OYE3 (mcOYE3) were grown to exponential phase in minimal SD media. Cells were washed and resuspended in minimal media containing glucose or galactose (Gal) to induce the expression of OYE2 and OYE3. Cultures were adjusted to A₆₀₀ values of 1, 0.1, and 0.01 before being spotted onto plates. Results are shown for plates containing no oxidant (YEPD), 4 mM acrolein, 4 mM H₂O₂, and 2.5 mM crotonaldehyde (Crot). Chemical structures are shown for acrolein (Acr) and crotonaldehyde. (B) Overexpression of OYE2 and OYE3 was confirmed by means of Western blot analysis.

FIG. 4.

Old yellow enzymes promote resistance to methyl vinyl ketone but not to 3-penten-2-one. (A) Cultures of wild-type (wt), oye2, oye3, and oye2 oye3 cells were grown to stationary phase and the A₆₀₀ was adjusted to 1, 0.1, or 0.01 before the cells were spotted onto plates containing MVK or 3-penten-2-one. Growth was monitored for 3 days, and results are shown for 1 mM MVK and 1.5 mM 3-penten-2-one. (B) Overexpression of OYE2 or OYE3 increases resistance to MVK but not to 3-penten-2-one. Wild-type strains containing vector (v), mcOYE2, or mcOYE3 were tested for sensitivity to MVK or 3-penten-2-one as described for Fig. 3. Chemical structures are shown for MVK and 3-penten-2-one.

FIG. 5.

Acrolein and crotonaldehyde cause similar levels of cellular damage. (A) Protein carbonylation is induced by exposure to acrolein and crotonaldehyde. Wild-type cells were grown in minimal media to exponential phase (A₆₀₀ = 0.6) and treated with 1 mM or 2 mM acrolein (A) or crotonaldehyde (C) for 1 h. Protein extracts were treated with the carbonyl-specific probe, DNPH, and analyzed by Western blot analysis using an antibody against DNPH. (B) Acrolein and crotonaldehyde (crot) deplete cellular glutathione. Wild-type cells were grown in minimal media to exponential phase (A₆₀₀ = 0.6) and treated with 0.2 mM acrolein or 0.5 mM crotonaldehyde for 1 h. The GSH concentrations shown are the means for three determinations and are given as nmol/ml/A₆₀₀.

FIG. 6.

Propionaldehyde and butyraldehyde are not toxic to yeast cells. Wild-type cells were grown in minimal media to exponential phase (A₆₀₀ = 0.6) and treated with a range of concentrations of acrolein or propionaldehyde (A) or crotonaldehyde and butyraldehyde (B). Sensitivity was determined by measuring A₆₀₀ readings following 24 h of growth. The values shown are the means for three independent readings.

FIG. 7.

Regulation of OYE2 and OYE3 expression in response to acrolein. Gene expression of OYE2 (A) and OYE3 (B) was determined by RT-PCR. Wild-type (wt), yap1, skn7, and msn2 msn4 mutant cells were grown to exponential phase and treated with 0.05 mM or 0.1 mM acrolein for 1 h. Induction (n-fold) is expressed relative to ACT1 and relative to the untreated sample for each strain. The values shown are the means for three determinations.