The strains and primers used in our study are listed in Tables 1, 2. Wild-type (WT) strain BWP17 was used as the background strain to construct the cnb1Δ/Δ, yvc1Δ/Δ, and cnb1Δ/Δyvc1Δ/Δ mutant strains by the PCR-mediated homologous recombination method. The ARG4 cassettes were amplified and transformed into the WT. The heterozygous mutants (cnb1:ARG4/CNB1) were identified by PCR. After that, the URA3 fragment was transformed into the heterozygous mutant above to construct the cnb1Δ/Δ mutant strains (cnb1:ARG4/cnb1:URA3). The yvc1Δ/Δ and double mutant cnb1Δ/Δyvc1Δ/Δ were constructed with a similar method.

Besides, the pP_CCH1-GFP plasmid was digested with StuI for 1 h and transferred into the WT and other mutant strains to measure the CCH1 expression level. In general, the strains were cultured in YPD (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose) medium. The SC (2% glucose, 0.67% yeast nitrogen base, 0.2% amino acid mixture) medium without uracil was used to separate and select the URA3-tagged strains.

The YPD plates containing different concentrations of TN, β-mercaptoethanol, dithiothreitol (DTT), or calcofluor white (CFW) were used to measure the sensitivity to ER stress or cell wall stress (Su et al., 2021). Besides, 5 mM reductive agent ascorbic acid (VC) was added into these stress-related plates to investigate the relationship between stress susceptibility and oxidative stress response (OSR).

The content of cellular calcium and the calcium flux were measured with a Fluo-4 (C₅₁H₅₀F₂N₂O₂₃, Beyotime, Shanghai, China, dissolved in DMSO) probe (Bartoli et al., 2019). Log-phase cells were treated with 2 μg/ml TN for 2 h. The cells were collected, washed with phosphate-buffered saline (PBS, 8 g/l NaCl, 0.2 g/l KCl, 1.42 g/l Na₂HPO₄, 0.27 g/l KH₂PO₄, pH 7.4), and resuspended with PBS buffer. The 2-mM Fluo-4 probe was added and incubated with 70 r/min at 30°C for 1 h. The fluorescence intensity (excitation wavelength at 488 nm, emission wavelength at 525 nm) was detected using a fluorescent microplate reader (Bode et al., 2020). The scanning time was sustained for 7 min with a scan gap for 1 s.

5-(6)-Carboxy-2′,7′-dichlorofluorescein diacetate (C-DCFDA) or 7-amino-4 chloromethyl coumarin (CMAC) was used to measure the vacuolar membrane permeability (VMP) (Andrei-Selmer et al., 2001). In the normal cells, C-DCFDA with green fluorescence or CMAC with blue fluorescence was concentrated in the vacuolar cavity, while it was spread to the whole cell in VMP-positive cells. The TN-treated strains were resuspended in PBS buffer and added with 1 mg/ml C-DCFDA (C₂₅H₁₄Cl₂O₉, Heowns, Tianjin, China, dissolved in DMSO) or 1 mg/ml CMAC (C₁₀H₈ClNO₂, Beyotime, China, dissolved in DMSO). The cells were incubated for 10 min and observed by fluorescence microscopy. At least 1,000 cells for each group were photographed to count the percent of VMP-positive cells.

The log-phase cells treated with 2 μg/ml TN were collected, washed, and resuspended in PBS buffer. Two micrograms per milliliter of JC-1 (2 mg/ml, dissolved in DMSO, Sigma) was added into the suspension. The cells were incubated for 1 h, and the mitochondrial membrane potential (MMP) was recorded with the flow cytometer (FACSCalibur, BD, San Jose, CA, United States). The red fluorescence (excitation wavelength at 525 nm, emission wavelength at 590 nm) with J-aggregates could be detected in the cells with normal MMP. The green fluorescence (excitation wavelength at 490 nm, emission wavelength at 530 nm) with J-monomer was detected in the cells with decreased MMP (Yu et al., 2021).

The strains treated with TN were cultured to log-phage. Total RNA was extracted with Eastep^TM Total RNA Extraction Kit (Promega, Madison, WI, United States) and transcribed reversely to cDNA. The HAC1 splicing assay was detected by the PCR method with primers HAC1-5RT and HAC1-3RT. The PCR product was separated in agarose gel electrophoresis for 3 h (Li J. et al., 2018).

The cellular viability was detected by MTT reagent [3-(4,5)-dimethylthiazo(-z-y1)-3,5-di-phenytetrazoliumromide, 4 mg/ml, Beyotime, China] (Rong et al., 2020). The log-phage cells were collected and resuspended in PBS buffer. The 0.5-mg/ml MTT reagent was added and incubated with 70 r/min at 37°C for 1 h. The cells were centrifuged, and the supernatant was removed. Dimethyl sulfoxide (DMSO, Beyotime, China) was added into the precipitated cells, and the cells were centrifuged. The dissolved supernatant was collected to measure the absorption wavelength at 570 nm.

Propidium iodide (PI) was used to measure the cell death rate for the PI-positive cells which were stained with red fluorescence completely (Priante et al., 2018). The strains treated with TN were cultured to log-phase. Five micrograms per milliliter of PI (1 mg/ml, Sigma) was added into cells and incubated for 5 min. Mortality rate was represented by the PI-positive cells with a flow cytometer.

Real-time PCR assay was used to measure the expression level of the calcium channel-related gene CCH1 and UPR response-related genes PMT4 and PRB1. Cells were collected, and the total RNA was extracted with Eastep^TM Total RNA Extraction Kit (Promega, United States) and transcribed reversely to cDNA (Meng et al., 2018). The RealMasterMix (SYBR Green) kit (TransGen, China) was used for real-time PCR analysis (Gomes-Neto et al., 2017). The following primers used are listed in Table 2: ACT1-5RT, ACT1-3RT, CCH1-5RT, CCH1-3RT, PRB1-5RT, PRB1-3RT, PMT4-5RT, and PMT4-3RT. The 2^–ΔΔ CT method was used to calculate the expression level of different genes, and ACT1 was used as the internal control.

Each experiment mentioned above was repeated at least three times under the tested conditions. The standard deviations and means were calculated by the separate three replicates. The one-tailed Student’s t test was used to calculate p values. The p values less than 0.05 were considered as statistically significant difference.

Firstly, we constructed the cnb1Δ/Δ mutant and measured its sensitivity to ER stress reagent TN. The results showed that on the YPD plate, cnb1Δ/Δ grew normally as a wild-type (WT) strain, while cnb1Δ/Δ could hardly grow on the 2-μg/ml TN plate (Figure 1A). Besides, the growth of liquid medium indicated that the WT strain grew rapidly during the 24-h culture period, whose value increased from 0.8 to 24. However, the OD₆₀₀ of cnb1Δ/Δ was always maintained at 0.8 and the maximum value at 24 h was just 3 (Figure 1B). Since calcineurin regulated the cellular calcium concentration through inhibition of plasma membrane channel CCH1 (Xu et al., 2019), we assumed that the deletion of CNB1 might have an impact on the CCH1 expression. It was interesting that under TN treatment, the expression level of the CCH1 promoter in the cnb1Δ/Δ strain was near as 1.5 times as WT (Figure 1C). Moreover, the qPCR analysis indicated that CCH1 was upregulated in the transcription level of cnb1Δ/Δ in response to ER stress (Figure 1D). In general, the deletion of CNB1 leads to the sensitivity to ER stress and overexpression of CCH1.

Now that the sensitivity to ER stress of the cnb1Δ/Δ strain was related with the overexpression of CCH1, we speculated that the inhibition of the plasma membrane calcium channel might improve its growth status. Verapamil or nifedipine, as the universal calcium channel blocker, was usually applied to cure the hypertension or angina. A different concentration of verapamil or nifedipine was added into the YPD plates containing 2 μg/ml TN. The spot assay result showed that the colony of cnb1Δ/Δ could grow in both the plates treated with verapamil and nifedipine (Figure 2A). Meanwhile, the liquid growth measurement indicated that during the 24-h culture, 40 or 80 μg/ml verapamil could improve the growth of cnb1Δ/Δ under ER stress (Figure 2B). These results implied that the inhibition of the plasma membrane calcium channel to decrease the cytosolic calcium concentration could recover the cnb1Δ/Δ growth under ER stress.

Since the inhibition of the cytosolic calcium concentration recovered the growth of cnb1Δ/Δ and Yvc1 was the unique vacuolar membrane calcium channel in yeast, we doubted whether the deletion of YVC1 could improve the growth status of cnb1Δ/Δ under ER stress. We constructed yvc1Δ/Δ and cnb1Δ/Δyvc1Δ/Δ strains and measured their susceptibility to environmental stress. CFW and caspofungin could cause the cell wall stress. Besides, both β-mercaptoethanol and DTT could interfere the formation of disulfide bonds, and TN could inhibit glycosylation. These three reagents could lead to ER stress. We found that similar with the WT strain, cnb1Δ/Δ, yvc1Δ/Δ, and cnb1Δ/Δyvc1Δ/Δ strains were resistant to CFW, β-mercaptoethanol, and DTT (Figure 3A, panels 2–4). However, the cnb1Δ/Δ and cnb1Δ/Δyvc1Δ/Δ strains were sensitive to caspofungin and TN. Since Yvc1 regulated the OSR in yeast (Yu et al., 2014b), the antioxidant vitamin C (VC) was added into the plates to verify whether the sensitivity was related with abnormal OSR. It was interesting that the addition of VC recovered the strains on the caspofungin-treated plate, indicating that the sensitivity to cell wall stress was related with impaired OSR reaction (Figure 3A, panels 5–6). Nevertheless, the addition of VC could not change the strains’ susceptibility to TN (Figure 3A, panels 7–8), which revealed the novel interaction between Yvc1 and Cnb1 with ER stress independently of OSR.

Under TN treatment, we measured the strain growth in the liquid medium during the 24-h cultivation. WT and yvc1Δ/Δ strains grew rapidly to the log phase and eventually maintained with the maximum quantity. Although cnb1Δ/Δ and cnb1Δ/Δyvc1Δ/Δ mutants grew slowly, cnb1Δ/Δyvc1Δ/Δ grew marginally faster than the cnb1Δ/Δ strain did (Figure 3B). Besides, the cell death rates under ER stress of these strains were calculated by flow cytometry with PI dye. All of the strains grew well in the control group, and their dead rates were low (Figures 3C,D, control). Under the TN treatment, WT and yvc1Δ/Δ strains were still with the small dead cells, and the death rate was 26.8% in the cnb1Δ/Δyvc1Δ/Δ strains, which was increased to 39.72% in the cnb1Δ/Δ strain (Figures 3C,D). These results indicated that even if cnb1Δ/Δyvc1Δ/Δ showed sensitivity to TN, the cell death rate was lower than that of the cnb1Δ/Δ strain, indicating that the deletion of YVC1 recovered the cell vitality of cnb1Δ/Δ.

Normally, the vacuole cavity could be dyed by CMAC, while the damaged cells were stained in the whole cell or failed to be stained (Andrei-Selmer et al., 2001). The vacuolar membrane permeability of the mutants showed that deletion of CNB1 caused the damaged vacuolar membrane under TN treatment, with the whole cells stained by CMAC or failing to be stained (Figure 4A). Besides, the calculation of the VMP-positive rate showed that cultured in the TN-treated medium, the WT and yvc1Δ/Δ strains maintained the integrity of vacuoles, with a low percentage of the VMP-positive rate. However, the positive rate was up to 70% in the vacuolar severely impaired cnb1Δ/Δ strain, although for the cnb1Δ/Δyvc1Δ/Δ strain with impaired vacuolar membrane, the VMP-positive rate was lower than cnb1Δ/Δ, just about 45% (Figure 4B).

The mitochondrial activities under ER stress were measured as well. In the control group, the cnb1Δ/Δyvc1Δ/Δ, cnb1Δ/Δ, and yvc1Δ/Δ strain, as the WT strain, maintained the normal mitochondrial function, with the low percentage of damaged rate (Figures 4C,D, control). However, compared with the other strains, the mitochondrial function was interfered by TN in the cnb1Δ/Δ strain, and the rate of decreased MMP was up to 41.72%. Nevertheless, the rate of impaired mitochondria of cnb1Δ/Δyvc1Δ/Δ was just 16.71% (Figures 4C,D). In conclusion, the deletion of YVC1 recovered the function of vacuole and mitochondria of the cnb1Δ/Δ strain.

The UPR pathway is the classical response process in the ER stress of yeast strains (Zhang et al., 2019). Since the deletion of YVC1 recovered the cell vitality of cnb1Δ/Δ under the treatment of TN, we speculated whether the UPR pathway was overactivated in this mutant. To verify the possibility, the total RNA of these three mutants and WT strains was extracted and transcribed reversely to cDNA (Gomes-Neto et al., 2017). The Hac1 splicing level and the expression level of PRB1 and PMT4 were measured. However, much unexpectedly, similar with the WT or yvc1Δ/Δ mutants, the UPR pathway was activated in cnb1Δ/Δ and cnb1Δ/Δyvc1Δ/Δ effectively. The unspliced HAC1 was 581 bp in the WT and mutant strains of the control group (Figure 5A). Moreover, under TN treatment for 2 h, HAC1 was spliced normally in the cnb1Δ/Δ and cnb1Δ/Δyvc1Δ/Δ strains and other strains with the size at 562 bp (Figure 5A).

Besides, qPCR analysis indicated that the expression levels of PMT4 and PRB1 in the cnb1Δ/Δ and cnb1Δ/Δyvc1Δ/Δ strains were similar with WT, which were all upregulated in the transcriptional level under ER stress (Figures 5B,C). In summary, these figures showed that the URP pathway in all of the mutants did not fail to evoke, implying a novel regulation mechanism within susceptibility.

Yvc1 and Cnb1 were related with the cellular calcium regulation (Cyert and Philpott, 2013), and the inhibition of CCH1 to decrease the cytosolic calcium content could improve the growth status in the cnb1Δ/Δ strain. Therefore, we speculated whether the disruption of YVC1 reduces the vacuolar calcium release to improve the cell vitality. Moreover, the results showed that calcium fluctuation was enhanced in the cnb1Δ/Δ strain under ER stress. In the detected period, the calcium fluctuation was increased within 3 min, and the maximum concentration was higher than other strains. Nevertheless, the calcium flux of the cnb1Δ/Δyvc1Δ/Δ strain was steady with a low peak value like the WT strain (Figure 6A).

Next, we measured the specific concentration of cellular calcium, which showed a similar tendency with the calcium flux. We found that the calcium concentration was increased significantly with TN treatment for 2 h of cnb1Δ/Δ, in which the concentration was much higher than those of other strains. The calcium content of the cnb1Δ/Δyvc1Δ/Δ strain was close to that of yvc1Δ/Δ, lower than that of cnb1Δ/Δ (Figure 6B). In conclusion, the calcium concentration was decreased significantly and its fluctuation was reduced obviously under ER stress in the cnb1Δ/Δyvc1Δ/Δ strain, indicating that the disruption of YVC1 could reduce the cellular calcium concentration, thereby improving the vitality.

To test and verify our hypothesis that the deletion of YVC1 could decrease the cytosolic calcium concentration and recover the cell growth, CaCl₂ or its chelating agent EGTA was added into the culture medium in the presence of TN. Although TN treatment in cnb1Δ/Δ caused a decreased MTT level, the supplement of EGTA recovered the cellular vitality, in which the MTT level was increased. Moreover, the supplement of CaCl₂ led to significantly decreased vitality level in cnb1Δ/Δ (Figure 7A). Moreover, the PI death rate in the EGTA group was 4.08%, which increased to 12.28% in the CaCl₂ group. It indicated that the addition of EGTA improved the cell growth as well (Figure 7B). The VMP assay showed that the permeability was improved in the addition of EGTA, while CaCl₂ caused severely damaged VMP in the cnb1Δ/Δ strain (Figure 7C). Furthermore, the VMP-positive rate calculation displayed that under TN treatment, the addition of CaCl₂ led to a 60% positive rate of the mutant, while under EGTA treatment, the positive rate was down to 48% (Figure 7D). In conclusion, we determine that it is the overloaded calcium ions that cause the susceptibility to ER stress in the cnb1Δ/Δ strain, and the disruption of YVC1 reduces the cytosolic calcium and improves the cell vitality (Figure 8).