Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response in Saccharomyces cerevisiae

Abstract

Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae, it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER) and unfolded protein response (UPR) has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v). Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1Δ and hac1Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid) and mild ethanol stress (5% ethanol) induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH.

Keywords: BiP; ER stress; Hac1p; Ire1p; Saccharomyces cerevisiae; acetic acid; lactic acid; unfolded protein response.

FIGURE 1

BiP aggregation upon acetic acid stress. Exponentially growing cells were exposed to stress with acetic acid or 10 mM DTT for 3 h and lysed using glass beads in Triton X-100-containing buffer. (A) Total cell lysates were fractionated into the supernatant and pellet fractions by centrifugation and analyzed using an anti-BiP antibody. Protein loading abundance was verified and normalized by Ponceaus S staining. (B) Protein levels of BiP in the pellet fraction were quantified using ImageJ, and the intensity of the BiP band in cells treated without stress was considered to be 1-fold. Data are shown as the mean ± standard error (n = 3). Lane 1, w/o stress; lane 2, 0.2% acetic acid; lane 3, 0.3% acetic acid; lane 4, 10 mM DTT. ^∗∗P-value < 0.01.

FIGURE 2

Changes in the localization of Ire1p-GFP upon acetic acid stress. Exponentially growing cells were exposed to the indicated conditions. Ire1p-GFP was immediately observed after the treatment. Representative pictures are shown. (Upper), Ire1p-GFP; (Bottom), bright field. The white bar indicates 3 μm. Quantitative data are shown as means ± standard error. One hundred cells under each condition were examined and experiments were repeated three times (300 cells in total were examined). Cells were treated with acetic acid for 30 min (gray bars) or 60 min (black bars). ^∗∗P-value < 0.01. N.S., no statistically significant difference.

FIGURE 3

Acetic acid induced the splicing of HAC1 mRNA and transcriptional activation of KAR2, a target gene of Hac1p. Exponentially growing cells were exposed to the indicated stress conditions. (A) Total RNA samples from cells treated with acetic acid or DTT were subjected to RT-PCR in order to amplify the HAC1 products. HAC1^u and HAC1ⁱ were fractionated using 2.0% agarose gel electrophoresis. Lane 1, w/o stress; lane 2, 0.3% acetic acid for 15 min, lane 3, 0.3% acetic acid for 30 min; lane 4, 0.3% acetic acid for 60 min; lane 5, 10 mM DTT for 60 min. (B) KAR2 mRNA levels were analyzed by qRT-PCR. In order to compare expression levels, the mRNA level of KAR2 was normalized to that of ACT1 under each condition. The mRNA level in cells without a stress treatment was considered to be 100%. Data are shown as the mean ± standard error (n = 3). Lane 1, w/o stress; lane 2, 0.1% acetic acid for 60 min, lane 3, 0.2% acetic acid for 60 min; lane 4, 0.3% acetic acid for 60 min; lane 5, 10 mM DTT for 60 min. ^∗∗P-value < 0.01. N.S., no statistically significant difference.

FIGURE 4

ire1Δ and hac1Δ cells showed higher susceptibility to acetic acid stress than wild-type cells. Exponentially growing cells (OD₆₀₀ = 0.5) were harvested and resuspended in fresh SD medium to obtain an initial OD₆₀₀ value of 0.1. To examine their susceptibility to acetic acid, the cells were treated with 0.3% acetic acid (AA) for 5 h. (A) Samples were diluted in 10^-1 steps with SD medium, dripped (10 μl) onto SD agar plates, and incubated at 28°C for 2 days. (B) Samples were diluted 500-fold and aliquots (100 μl) were plated onto YPD agar plates. Relative survival rates were calculated as colony-forming units (CFUs). The CFUs of cells before the stress treatment was considered to be 100%. Data are shown as the mean ± standard error (n = 3). ^∗P-value < 0.05.

FIGURE 5

Effects of various carboxylic acids on the induction of UPR. Exponentially growing cells were treated with various carboxylic acids for 30 min. (A) Ire1p-GFP was immediately observed after the treatment without fixation. Representative pictures are shown. BF, bright field. The white bar indicates 3 μm. (B) The mRNA levels of KAR2 were analyzed by qRT-PCR. In order to compare expression levels, the mRNA level of KAR2 was normalized to that of ACT1 under each condition. The mRNA level in cells without a stress treatment was considered to be 100%. Data are shown as the mean ± standard error (n = 3). Lane 1, w/o stress; lane 2, 3.0% lactic acid, lane 3, 0.3% propionic acid; lane 4, 0.6% sorbic acid. ^∗∗P-value < 0.01. N.S., no statistically significant difference.

FIGURE 6

Effects of the combination of mild ethanol stress and mild acetic acid stress on the induction of the UPR. Exponentially growing cells were treated with ethanol (EtOH) and acetic acid (AA). (A) Ire1p-GFP was immediately observed after the treatment without fixation. Representative pictures are shown. BF, bright field. The white bar indicates 3 μm. (B) The mRNA levels of KAR2 were analyzed by qRT-PCR. In order to compare expression levels, the mRNA level of KAR2 was normalized to that of ACT1 under each condition. The mRNA level in cells without a stress treatment was considered to be 100%. Data are shown as the mean ± standard error (n = 3). Lane 1, w/o stress; lane 2, 0.1% AA for 60 min, lane 3, 5% EtOH for 60 min; lane 4, 0.1% AA and 5% EtOH for 60 min. ^∗∗P-value < 0.01. N.S., no statistically significant difference.

FIGURE 7

Effects of extracellular dissociated acetic acid on the UPR. Exponentially growing cells were exposed to the indicated conditions for 60 min using the SD-PPB medium whose pH was adjusted to 6.80 by potassium phosphate buffer. Final pH values after addition of acetic acid, ethanol, and DTT into the SD-PPB medium are indicated. Ire1p-GFP was immediately observed after the treatment. Representative pictures are shown. BF, bright field. The white bar indicates 3 μm.