Introduction

Cold atmospheric pressure plasma (CAP) mainly consists of ions and electrons and emits reactive species and UV photons (Gaunt et al. 2006). CAP has been used in various medical applications including wound healing and non-inflammatory anti-cancer therapy (Kong et al. 2009; Weltmann and von Woedtke 2016; Tanaka and Hori 2017). Especially, argon (Ar)-based CAP is widely used for coagulation therapy (Manner 2008) and known to enhance wettability and osseointegration on dental implant metals (Duske et al. 2012; Giro et al. 2013; Canullo et al. 2016). Additionally, exposure to CAP is projected as a new sterilization method in the fields of food processing, sanitation, and medicine (Heinlin et al. 2010; Hoffmann et al. 2013; Shaw et al. 2015; Mir et al. 2016). The CAP system as a sterilization technique is advantageous because of the low-associated costs and minimal generation of residual toxicity and thermal damage (Hoffmann et al. 2013; Lackmann and Bandow 2014). Previous studies reported the sterilization effects of CAP on bacterial cells such as Escherichia coli, Salmonella enterica, and Staphylococcus aureus (Klämfl et al. 2012; Maisch et al. 2012a, b; Fernández et al. 2013; Homma et al. 2013; Niemira et al. 2014; Sun et al. 2014; Ziuzina et al. 2014; Maeda et al. 2015). Bacterial cells are mainly inactivated by reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced by CAP exposure (Gaunt et al. 2006; van Gils et al. 2013; Lackmann and Bandow 2014).

Inactivation of spoilage yeasts including Saccharomyces cerevisiae is an important issue to be solved in food industries (Loureiro and Malfeito-Ferreira 2003; Krisch et al. 2016), and CAP exposure is expected as an effective way to prevent the spoilage. However, the fungicidal effects of CAP have not yet been investigated as extensively as its bactericidal effects. Previous studies reported that yeast cells were also inactivated by a CAP treatment via the accumulation of ROS and RNS (Feng et al. 2010; Koban et al. 2010; Maisch et al. 2012a, b; Ryu et al. 2013). However, the fungicidal mechanisms of CAP and the cellular responses of eukaryotic microorganisms to CAP stress have not yet been elucidated in detail.

We recently demonstrated via a fluorescence microscopic analysis that CAP promoted the formation of Hsp104 aggregates (Itooka et al. 2016). Hsp104 is a stress-responsive chaperone that plays a role in the segregation of denatured proteins (Glover and Lindquist 1998; Bösl et al. 2006). Since Hsp104 forms complexes with denatured proteins, it is possible to visualize protein aggregates using Hsp104-GFP (Liu et al. 2010; Zhou et al. 2011; Escusa-Toret et al. 2013; Wallace et al. 2015). Therefore, our finding of the formation of Hsp104-GFP aggregates following a CAP treatment strongly suggests that CAP promotes the denaturation of yeast proteins. Additionally, we found that CAP induced changes in the intracellular localization of Ire1, a key sensor of ER stress and trigger of the unfolded protein response (UPR) (Kimata et al. 2003, 2004, 2007; Mori 2009). Yeast UPR is induced via the activation of the Ire1-Hac1 pathway (Mori et al. 1996; Mori 2009; Brodsky and Skach 2011). The accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to the activation of Ire1 via the self-association and oligomerization of Ire1 in yeast cells (Kimata et al. 2007). Activated Ire1 subsequently splices HAC1 mRNA, leading to the synthesis of the Hac1 protein, which functions as a transcription factor and induces the transcriptional activation of UPR-related genes (Chapman and Walter 1997; Kawahara et al. 1997). Although the non-activated form of Ire1-GFP diffuses throughout the ER, highly self-oligomerized Ire1-GFP exhibits a punctate intracellular localization (Kimata et al. 2007; Promlek et al. 2011; Mathuranyanon et al. 2015; Kawazoe et al. 2017). Since Ire1-GFP in CAP-treated cells shows a punctate localization (Itooka et al. 2016), CAP has been proposed to cause ER stress in yeast cells.

Although the findings of our previous fluorescence microscopy study suggested that CAP causes protein denaturation and ER stress in yeast (Itooka et al. 2016), it was almost impossible to perform more detailed analyses because of the limited processing capacity of the CAP system used in that study. In order to conduct biochemical and molecular biological analyses, we developed a new CAP system using a glass funnel with improved processing capacity. We herein demonstrated that CAP elevated the levels of insoluble aggregated proteins and ubiquitinated proteins and also impaired protein folding in the ER. Additionally, we found that the pretreatment of yeast cells at 37 °C alleviated damage and cell death caused by CAP exposure. Our results strongly indicate that the fungicidal effects of CAP are partially derived from protein damage.

Materials and methods

Strains and medium

S. cerevisiae YPH250 (MATα trp1-1 his3-200 lys2-801 leu2-1 ade2-101 ura3-52) and its isogenic tsa1∆ null mutant were used in the present study. The tsa1∆ null mutant was constructed by introducing the DNA fragment encoding tsa1∆::kanMX, which was amplified from the genomic DNA of the tsa1∆::kanMX strain in the Yeast Knockout Collection (Open Biosystems Inc., Huntsville, AL). In order to monitor the localization of Ire1-GFP, KMY1015 (MATα leu2-3112 ura3-52 his3-Δ200 trp1-Δ901 lys2-801 ire1Δ::TRP1) expressing Ire1-GFP was used (Mathuranyanon et al. 2015). The methods of cell cultivation and medium were described in our previous study (Itooka et al. 2016). Exponentially growing cells were harvested at an optical density at 600 nm (OD600) of 0.5 and treated with CAP.

The CAP system and CAP treatment

CAP was produced in dielectric barrier discharges on a glass funnel. Copper tape (5.0 mm in width) was attached to the inner and outer surfaces of the funnel (Fig. 1). A high voltage of 20.0 kV was applied at 11 kHz between the outer and inner tapes. The inner tape was grounded for an electrical circuit. Ar gas was introduced into the funnel at a flow rate of 0.8 standard liters per minute (SLM), and the distance between the bottom of the glass funnel and ground was adjusted to 3.0 mm. Copper tape was cooled by a blower (YMS-A107, Yamazen, Osaka, Japan) to prevent overheating. In the CAP treatment of yeast cells, 25 ml of the liquid culture (OD600 = 0.5) was centrifuged, and precipitated cells were resuspended in 2 ml of fresh SD medium. The cell solution was added to a petri dish (diameter, 53 mm; Corning Falcon® 351007) and exposed to CAP. The CAP treatment was carried out in a room at 25 °C, and the sample temperature was measured using a radiation thermometer (AD-5611A, A&D Company, Limited, Tokyo, Japan).

Fig. 1
figure 1

Schematic of the experimental setup for CAP exposure. a CAP was produced in dielectric barrier discharges on a glass funnel. Copper tape (5.0 mm in width) was attached to the inner and outer surfaces of the funnel. A high voltage of 20.0 kV at 11 kHz between the inside and outside copper tape caused a barrier discharge using a dielectric of the glass. Ar gas was fed to the funnel at a flow rate of 0.8 SLM. Samples in a petri dish were exposed to CAP produced inside the funnel. b, c Yeast cells in SD medium were treated with CAP (closed circles and black bars) or Ar gas alone (open squares and white bars). b The relative survival rate was calculated as colony-forming units (CFU). c Intracellular oxidation levels were assayed using an oxidant-sensitive probe H2DCFDA. Data are shown as the mean ± SE (n = 3)

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Plasmid construction

YIp-SSA1-GFP

A part of the open reading frame (ORF) of SSA1 was amplified by a polymerase chain reaction (PCR) using the primers 5′-GGACCCAGTTGAAAAGGTCTAGAGAGATGCTAAATTGGACAAATCTC-3′ and 5′-CGTTATTATTCAATTGCCGCACCAATCTCGAGAATCACTTCTTCAAC-3′. The amplicon was cloned into the XbaI/XhoI sites of YIp-SSA4-GFP (Itooka et al. 2016) after digestion with XbaI/XhoI to construct YIp-SSA1-GFP. Regarding the integration of the SSA1-GFP gene at the chromosomal SSA1 locus, YIp-SSA1-GFP was linearized by SalI and introduced into cells.

YIp-TSA1-GFP

A part of the ORF of TSA1 was amplified by PCR using the primers 5′-CAACCGAGCTCATTGCTTTCTCAGAAGCTGCTAAG-3′ and 5′-GCAAGCTCGAGATTTGTTGGCAGCTTCGAAGTATT-3′. The amplicon was digested with SacI/XhoI and cloned into the SacI/XhoI sites of YIp-SSA4-GFP to construct YIp-TSA1-GFP. YIp-TSA1-GFP was linearized by BstXI and introduced into yeast cells.

The construction of the YIp-HSP104-GFP plasmid was previously described (Itooka et al. 2016).

Western blotting analysis

A BiP aggregation analysis was performed as described by Promlek et al. (2011). Briefly, total cell lysates treated with CAP were fractionated by centrifugation at 19,300×g for 10 min. Pellet samples were applied to a 8.0% polyacrylamide gel for an SDS-PAGE analysis. BiP protein levels were monitored using an anti-BiP antibody and quantified using ImageJ software (http://imagej.nih.gov/ij/).

Protein ubiquitination was analyzed as described by Collins et al. (2010). Extracted proteins were resolved on an 8.0% polyacrylamide gel for the SDS-PAGE analysis. Ubiquitinated proteins were detected using an anti-ubiquitin antibody (P4D1, Santa Cruz Biotechnology, Dallas, TX). Ponceau S staining was used for the verification and normalization of protein loading abundance.

Measurement of insoluble aggregated proteins

Insoluble aggregated proteins were analyzed as described by Koplin et al. (2010) with modifications. In order to prepare cell lysates, cells were frozen rapidly in liquid nitrogen and resuspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.0, 1.0 mM EDTA, and 5% glycerol). After an incubation with Zymolyase 20 T (2.5 mg/ml) and protease inhibitors at 25 °C for 20 min, cells were disrupted by vortexing with glass beads. In order to remove intact cells, samples were centrifuged at 200×g for 20 min. The total protein concentration of each sample was measured using the Protein Assay CBB Solution kit (Nacalai Tesque, Kyoto, Japan) and normalized. Insoluble aggregated proteins were obtained by centrifugation at 16,000×g for 20 min. These proteins were washed twice with lysis buffer containing 2% Nonidet® P-40 (NP-40), and centrifuged at 16,000×g at 4 °C for 20 min to remove membrane proteins. The final sediment proteins were solubilized in 50 μl of urea buffer (50 mM Tris-HCl, pH 7.5, 6.0 M urea, and 5% SDS), separated by a 10% polyacrylamide gel, and visualized by silver staining using Sil-Best Stain One (Nacalai Tesque, Kyoto, Japan). The quantities of insoluble aggregated proteins were measured by the BCA assay using the Protein Assay Bicinchoninate kit (Nacalai Tesque, Kyoto, Japan).

Microscopic analysis and survival assay

A fluorescence microscope system (IX83, Olympus, Tokyo, Japan) was used for the microscopic analysis. Cells treated with CAP were immediately observed without fixation. The relative survival rate was calculated as colony-forming units (CFU), as previously described (Itooka et al. 2016). Levels of intracellular oxidation were measured using an oxidant-sensitive probe 2′,7′-dichlorofluorescein diacetate (H2DCFDA; Molecular Probes, OR, USA) as described previously (Allen et al. 2010; Itooka et al. 2016).

Results

Fungicidal activity of the new CAP system

In our previous study, we used a typical laboratory-scale CAP system that processes only 100 μl of culture medium each time (Itooka et al. 2016). In order to increase this capacity, we developed a new CAP system using a glass funnel that functions as a non-conductor as well as a cover. This allows the efficient exposure of CAP to the target via the retention of Ar plasma inside the funnel (Fig. 1a). We initially examined the effects of the new CAP system on the survival of yeast cells (Fig. 1b). The CAP exposure efficiently killed yeast cells, while exposure of Ar gas alone did not kill yeast cells. Furthermore, the CAP treatment for 20 min elevated the level of intracellular oxidation (Fig. 1c). On the other hand, exposure of Ar gas alone did not elevate the intracellular oxidation level, as reported previously (Itooka et al. 2016). The sample temperature hardly changed before and after CAP exposure for 25 min (from 23.6 ± 0.3 to 23.5 ± 0.4 °C). These results were consistent with previous findings (Koban et al. 2010; Maisch et al. 2012a, b; Itooka et al. 2016), and clearly indicate an improvement of the processing capacity of our CAP systems; the new CAP system processed 2 ml of culture medium each time and efficiently killed yeast cells without thermal stress.

CAP increased insoluble aggregated protein levels in yeast cells

Since we succeeded to improve the processing capacity of our CAP system (from 100 μl to 2 ml), it became possible to conduct biochemical and molecular biological analyses to find out more about the effects of CAP on yeast cells. Previous studies reported that CAP causes the inactivation and denaturation of bacterial proteins (Yasuda et al. 2008; Lackmann et al. 2013; Lackmann and Bandow 2014). We also demonstrated that Hsp104-GFP, a marker of insoluble protein aggregation (Liu et al. 2010; Zhou et al. 2011; Escusa-Toret et al. 2013; Wallace et al. 2015), formed foci in CAP-treated yeast cells (Itooka et al. 2016). This finding suggested that CAP causes the accumulation of denatured proteins in yeast cells. In order to verify this possibility, we used the new system to examine whether CAP promotes the formation of insoluble protein aggregates. Consistent with our previous study, the new CAP system also induced the formation of Hsp104-GFP foci (Fig. 2a). We reconfirmed that the formation of Hsp104-GFP foci was not induced by exposure of Ar gas alone (data not shown), as reported in our previous report (Itooka et al. 2016). We assayed the levels of insoluble aggregated proteins in CAP-treated cells. As shown in Fig. 2b, c, the CAP treatment for more than 20 min significantly elevated the levels of insoluble aggregated proteins to a similar extent as those in cells exposed to heat shock at 42 °C. These results clearly indicate that CAP induces the denaturation of yeast proteins.

Fig. 2
figure 2

CAP enhanced insoluble aggregated protein levels in yeast cells. a Cells carrying a GFP-tagged chromosomal copy of HSP104 were treated with CAP for 20 min. GFP signals were immediately observed after the treatment without cell fixation. BF bright field. The white bar indicates 3 μm. b, c Cell lysates were prepared after the treatment with CAP or heat shocked at 42 °C for the indicated time. Insoluble aggregated proteins were isolated by centrifugation and washed with NP-40 containing buffer, and then solubilized in buffer containing urea and SDS. b Samples were separated using 10% polyacrylamide gel electrophoresis and visualized by silver staining. c The quantities of insoluble aggregated proteins were measured by the BCA assay. The levels of insoluble aggregated proteins in non-stressed cells were considered to be onefold. Data are represented as the mean ± SE (n = 3)

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CAP promoted protein ubiquitination

In the ubiquitin-proteasome system, ubiquitin functions as a signal for protein degradation. Denatured proteins conjugated with ubiquitin are degraded via the proteasome (Finley et al. 2012; Amm et al. 2014). Since CAP induced the denaturation of yeast proteins (Fig. 2), we performed western blotting using an anti-ubiquitin antibody to examine whether protein ubiquitination is enhanced in cells treated with CAP. The CAP treatment, as well as heat shock at 42 °C, significantly increased the levels of ubiquitinated proteins (Fig. 3). This result supports the finding that CAP induces protein denaturation in yeast cells.

Fig. 3
figure 3

CAP promoted protein ubiquitination. Cells were treated with CAP for the indicated time or heat shocked at 42 °C for 30 min and then lysed using glass beads in EZ buffer. Extracted proteins were resolved on an 8% polyacrylamide gel, and ubiquitinated proteins were detected by western blotting using an anti-ubiquitin antibody. The protein loading quantity was verified by Ponceau S staining

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H2O2-dependent protein aggregation was induced in CAP-treated cells

Tsa1, a major thioredoxin peroxidase in yeast (Garrido and Grant 2002), is a molecular chaperone and protects against the accumulation of H2O2-induced denatured protein aggregates (Jang et al. 2004; Rand and Grant 2006; Hanzén et al. 2016). Since sulfenylated Tsa1 recruits Hsp70 to denatured proteins in a H2O2-dependent manner and forms aggregates, Tsa1-GFP and Ssa1-GFP form foci in the cytoplasm under H2O2 stress, but not under heat shock conditions (Hanzén et al. 2016; Hill et al. 2017). We verified that the formation of the Tsa1-GFP focus was induced by H2O2, but not by heat shock, and the formation of Tsa1-GFP focus was also induced by the treatment with CAP (Fig. 4a). Additionally, the formation of the Ssa1-GFP focus was not observed in tsa1∆ cells treated with CAP (Fig. 4b). These results suggest that the formation of protein aggregates in CAP-treated cells was induced, at least partially, in a H2O2-dependent manner.

Fig. 4
figure 4

CAP caused the formation of the Tsa1-GFP focus in the cytoplasm. a Yeast cells carrying the GFP-tagged chromosomal copy of TSA1 were treated with CAP or the indicated stress conditions. Tsa1-GFP was immediately observed after the CAP treatment without fixation. b Wild-type and tsa1∆ cells carrying a GFP-tagged chromosomal copy of SSA1 were treated with CAP for 20 min. BF bright field. The white bar indicates 3 μm

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The CAP treatment promoted the accumulation of unfolded proteins in the ER

We previously reported that CAP but not Ar gas alone induced changes in the localization of Ire1, a key sensor of ER stress (Kimata et al. 2003, 2004, 2007; Mori 2009), which suggests that CAP causes the oligomerization of Ire1 and ER stress in yeast cells (Itooka et al. 2016). In the present study, we investigated whether CAP enhances the levels of unfolded proteins in the ER. Previous studies reported that BiP, a major ER chaperone (Rose et al. 1989; Mori et al. 1996), binds unfolded proteins to form aggregates under ER stress conditions (Kimata et al. 2003). Therefore, it is possible to detect ER stress by measuring the levels of sedimentable BiP (Promlek et al. 2011; Kawazoe et al. 2017). A treatment with dithiothreitol (DTT), an ER stress inducer that disrupts disulfide bond formation (Kimata et al. 2003, 2007), elevated the levels of sedimentable BiP (Fig. 5a). The treatment with CAP also increased the levels of sedimentable BiP over time in the pellet fraction. The levels of sedimentable BiP in cells treated with CAP for 25 min were similar to those in cells treated with DTT. These results clearly indicate that the CAP treatment promotes the formation of unfolded protein aggregates in the yeast ER.

Fig. 5
figure 5

CAP increased BiP aggregate levels in the ER. a After the treatment with CAP or 10 mM DTT for the indicated time, total cell lysates were prepared using glass beads in Triton X-100-containing buffer. Pellet fractions were prepared by the centrifugation of total cell lysates and subjected to a western blotting analysis with an anti-BiP antibody. The protein levels of sedimentable BiP were quantified using ImageJ, and the intensity of the BiP band in cells treated without stress was considered to be onefold. Data are shown as the mean ± SE (n = 3). b Cells were treated with CAP for the indicated time or with 10 mM DTT for 60 min. Ire1-GFP was immediately observed after the CAP treatment

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Since BiP levels in the pellet fraction were increased by CAP, the oligomerization of Ire1 was re-examined using the new CAP system. We observed that Ire1-GFP formed punctate foci following the CAP treatment (Fig. 5b), confirming that CAP induced the oligomerization of Ire1 and ER stress in yeast cells.

A pretreatment with mild heat shock improved yeast resistance to CAP

Since we found that, as described above, CAP as well as heat shock induced protein denaturation, we next examined whether the pretreatment of cells with mild heat shock improves cell survival under CAP stress. Cells pretreated with sublethal mild heat shock at 37 °C exhibit increased resistance to subsequent severe stress because the pretreatment causes adaptive responses including the induced expression of molecular chaperones and intracellular accumulation of trehalose via the activation of the stress-responsive transcription factors Hsf1 (heat shock factor 1) and Msn2/Msn4 (general stress responsive transcription factors) (Sanchez and Lindquist 1990; Izawa et al. 2004; Morano et al. 2012; Verghese et al. 2012; Gibney et al. 2013). In order to examine the effects of the pretreatment on yeast resistance to CAP, cells in the exponential phase were pretreated at 37 °C for 1 h and then exposed to CAP. We found that the survival rate under CAP stress was significantly increased by the pretreatment at 37 °C (Fig. 6a), indicating that the pretreatment with mild heat shock improved resistance to the subsequent CAP treatment.

Fig. 6
figure 6

A pretreatment with mild heat shock alleviated cell death caused by CAP. Cells were pretreated with or without mild heat shock at 37 °C for 1 h prior to the CAP treatment. a The relative survival rate was calculated as colony-forming units (CFU). Data are shown as the mean ± SE (n = 3). b Cells were treated with CAP for 20 min after or without the pretreatment at 37 °C for 1 h. The intracellular localization of Hsp104-GFP and Ire1-GFP was immediately observed after the stress treatment without fixation. The white bar indicates 3 μm

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The pretreatment also affected the formation of Hsp104-GFP foci upon CAP exposure. The formation of Hsp104-GFP foci was significantly less in cells treated with CAP after the treatment at 37 °C for 1 h than in those directly challenged with the CAP treatment (Fig. 6b). Additionally, the oligomerization of Ire1-GFP was hardly induced by CAP in pretreated cells (Fig. 6b). These results suggest that the pretreatment at 37 °C alleviated the accumulation of denatured proteins in CAP-treated yeast cells.

Discussion

In the present study, we improved the processing capacity of our CAP system by preventing the rapid diffusion of Ar plasma using a glass funnel as a cover. We increased the amount of culture medium that may be processed by CAP each time, with efficient fungicidal effects. Therefore, this system became available to perform molecular biological analyses in order to confirm that CAP causes protein denaturation in yeast cells. CAP led to increased levels of insoluble protein aggregates and ubiquitinated proteins, clearly indicating that CAP causes severe damage to yeast proteins. Protein denaturation appears to have strongly contributed to the inactivation of yeast cells by CAP. Previous studies reported that CAP causes protein denaturation in bacterial cells (Yasuda et al. 2008; Lackmann et al. 2013; Lackmann and Bandow 2014). Thus, the induction of protein denaturation may be a common cytotoxic activity utilized by CAP against bacteria and eukaryotic microorganisms.

However, ER stress is a phenomenon specific to eukaryotic cells. CAP enhanced the levels of unfolded proteins in the ER as well as Ire1 oligomerization, indicating that CAP induces ER stress. The induction of ER stress by CAP indicates that CAP damages not only preexisting proteins but also newly synthesized secretory and transmembrane proteins in yeast cells. The accumulation of these inadequacies may lead to the quick inactivation of yeast cells by CAP.

This idea may be supported by our novel result showing that the pretreatment at 37 °C alleviated cell death caused by CAP (Fig. 6). A treatment with mild heat shock induces the expression of molecular chaperones via the activation of Hsf1 and Msn2/Msn4, and they counteract protein denaturation and prevent intracellular disorder (Vabulas et al. 2010; Kim et al. 2013; Mackenzie et al. 2016). Therefore, pretreated cells appear to have an enhanced ability to cope with protein damage caused by CAP and improved tolerance to CAP stress.

CAP emits ROS and causes the endogenous production of ROS in yeast cells (Ryu et al. 2013). Indeed, we observed elevated levels of intracellular oxidation in cells treated with CAP (Fig. 1c) (Itooka et al. 2016). ROS are known to induce protein denaturation and aggregation in yeast cells (Costa and Moradas-Ferreira 2001; Hanzén et al. 2016; Weids et al. 2016). Additionally, the formation of the Tsa1-GFP focus in the cytoplasm, which is induced in a H2O2-dependent manner and forms aggregates with denatured proteins (Hanzén et al. 2016; Hill et al. 2017), was also induced in CAP-treated yeast cells. Collectively, these results indicate that ROS, including H2O2, are the main cause of protein denaturation in the cytoplasm of CAP-treated cells. Another possible cause of protein denaturation is UV photons derived from CAP (Park et al. 2003; Heise et al. 2004; Gaunt et al. 2006). It is widely known that UV radiation causes modifications to proteins in cells via ROS production (Ichihashi et al. 2003) and a direct photochemical reaction (Pattison and Davies 2006; Pattison et al. 2012). Lackmann et al. (2013) demonstrated the synergistic effects of UV photons and particles, such as ROS, on enzyme activities and protein structures; thus, ROS and UV photons may synergistically contribute to protein denaturation in yeast cells treated with CAP.

Although CAP is projected as an alternative method of sterilization in various fields, its fungicidal effects have not fully been examined. Only a small amount of samples may be processed each time in the typical laboratory-scale CAP system; therefore, we improved the processing capacity of CAP in order to acquire sufficient amounts of CAP-treated cells for biochemical and molecular biological analyses. In the present study, we demonstrated that CAP caused the denaturation of yeast proteins, at least partially, in a H2O2-dependent manner. We also showed that CAP-induced ER stress in yeast was specific to eukaryotic cells. Furthermore, a pretreatment at 37 °C significantly alleviated cell death caused by CAP. Our results strongly suggest that the induction of protein denaturation is a common cytotoxic function of CAP against prokaryotic and eukaryotic microorganisms.