The YBR056W-A and Its Ortholog YDR034W-B of S. cerevisiae Belonging to CYSTM Family Participate in Manganese Stress Overcoming

The CYSTM (cysteine-rich transmembrane module) protein family comprises small molecular cysteine-rich tail-anchored membrane proteins found in many eukaryotes. The Saccharomyces cerevisiae strains carrying the CYSTM genes YDRO34W-B and YBR056W-A (MNC1) fused with GFP were used to test the expression of these genes under different stresses. The YBR056W-A (MNC1) and YDR034W-B genes are expressed under stress conditions caused by the toxic concentrations of heavy metal ions, such as manganese, cobalt, nickel, zinc, cuprum, and 2.4-dinitrophenol uncoupler. The expression level of YDR034W-B was higher than that of YBR056W-A under alkali and cadmium stresses. The Ydr034w-b-GFP and Ybr056w-a-GFP proteins differ in the cellular localization: Ydr034w-b-GFP was mainly observed in the plasma membrane and vacuolar membrane, while Ybr056w-a-GFP was observed in the cytoplasm, probably in intracellular membranes. The null-mutants in both genes demonstrated decreased cell concentration and lytic phenotype when cultivated in the presence of excess manganese. This allows for speculations about the involvement of Mnc1 and Ydr034w-b proteins in manganese stress overcoming.


Introduction
Yeast cells acquire a variety of stress-tolerant mechanisms, including the induction of stress proteins via stress-triggered signal-transduction pathways [1][2][3][4][5]. The proteins of the CYSTM superfamily were proposed to be a part of cellular protective mechanisms against stresses in yeast [6]. Multiple sequence alignment revealed a cysteine-rich transmembrane module, CYSTM, in a wide range of small molecular tail-anchored membrane proteins in eukaryotes, including humans [6]. To date, the proteins of the family have not been fully annotated. The CYSTM1 gene has been identified in mammals; however, its function is still unclear (https://www.ebi.ac.uk/interpro/entry/InterPro/IPR043240/, accessed on 24 April 2023). The protein encoded by the CYSTM1 gene has been shown to be a biomarker of Huntington's disease [7].
The functions of proteins belonging to the CYSTM superfamily are best studied in plants. The significance of many CYSTM proteins in stress overcoming in plant cells has been demonstrated [8][9][10][11][12][13]. Some of the plant CYSTM proteins confer tolerance to cadmium and copper in Digitaria ciliaris and Oryza sativa [8]. Heterologous expression in yeast of plant CDT1 belonging to this group conferred metal resistance by preventing the uptake of metal ions into the yeast cell [8]. The Arabidopsis representative of the superfamily, agents mitomycin C, the replication inhibitor methotrexate, the oxidizing agent hydrogen peroxide, and the membrane destabilizing agent 1,8-nonadiene [6]. YDL012C and YDR210W were shown to overlap in the chemicals against which they provide resistance, suggesting that these proteins might function together as a complex [6]. YDL012C and YBR016W target GFP in the plasma membrane [15]. Both YDL012C and YBR016W encoded proteins are localized mostly in regions of new membrane synthesis, toward the emergent buds of dividing cells [15].
YDR034W-B has a paralog, YBR056W-A. DNA microarray analysis made it possible to reveal a global set of yeast genes induced and repressed in response to various stresses, including variations in temperature, oxidation, nutrients, pH, and osmolarity, which indicates that nearly half of the genome is involved in the responses to environmental changes [5]. Among these genes, the so-called common environmental response (CER) genes were identified; their expression changed under a variety of stresses, including temperature shift, peroxide, osmotic and pH stresses [5]. One of the CER genes with its expression increasing under the above stress conditions was YBR056W-A [5]. The enhanced expression of YBR056W-A (MNC1) in S. cerevisiae cells adapted to growth in the presence of 2 mM Mn 2+ and the lytic phenotype of the ∆ybr056w-a strain in the presence of excess manganese have been shown, suggesting the possible role of this protein in manganese stress overcoming [16]. As for YDR034W-B encoded protein, there is no information about its role in stress conditions. Under normal growth conditions, the expression of this protein is negligible similar to YBR056W-A encoded protein [16]. The yeast co-expression network analysis suggests that the proteins YDL012C, YDR210W, and YBR016W are together involved in resistance against the DNA-damaging agents mitomycin C, the replication inhibitor methotrexate, the oxidizing agent hydrogen peroxide, and the membrane destabilizing agent 1,8-nonadiene [6].
YDL012C and YDR210W were shown to overlap in the chemicals against which they provide resistance, suggesting that these proteins might function together as a complex [6]. YDL012C and YBR016W target GFP in the plasma membrane [15]. Both YDL012C and YBR016W encoded proteins are localized mostly in regions of new membrane synthesis, toward the emergent buds of dividing cells [15].
YDR034W-B has a paralog, YBR056W-A. DNA microarray analysis made it possible to reveal a global set of yeast genes induced and repressed in response to various stresses, including variations in temperature, oxidation, nutrients, pH, and osmolarity, which indicates that nearly half of the genome is involved in the responses to environmental changes [5]. Among these genes, the so-called common environmental response (CER) genes were identified; their expression changed under a variety of stresses, including temperature shift, peroxide, osmotic and pH stresses [5]. One of the CER genes with its expression increasing under the above stress conditions was YBR056W-A [5]. The enhanced expression of YBR056W-A (MNC1) in S. cerevisiae cells adapted to growth in the presence of 2 mM Mn 2+ and the lytic phenotype of the ∆ybr056w-a strain in the presence of excess manganese have been shown, suggesting the possible role of this protein in manganese stress overcoming [16]. As for YDR034W-B encoded protein, there is no information about its role in stress conditions. Under normal growth conditions, the expression of this protein is negligible similar to YBR056W-A encoded protein [16].
This study aimed to compare the stress conditions in which the expression of YDR034W-B and YBR056W-A is manifested and to assess the localization of GFP-fused proteins in the cells of S. cerevisiae. The effect of knockout mutations in YDR034W-B and YBR056W-A on resistance to stress caused by heavy metal ions was also assessed.

Fluorescence Microscopy
To test the effect stress factors on the expression of GFP fusion proteins, the cells of GFP fusion strains were cultivated in the control YPD medium and YPD media supplemented with one of the following components: 40 mM KOH, 0.2 mM Cd(CH 3 The cultivation was performed in Erlenmeyer flasks in 100 mL of YPD at 28 • C and 145 rpm for 24 h. To test the effect of 2,4-dinitrophenol, manganese, and peroxide on GFP fusion proteins expression during a short incubation time, the cells of the YDRO34W-B-GFP and YBR056W-A-GFP strains were cultivated in 2 mL of YPD with stirring for 24 h to a culture absorption of 20 (measured in a 1-cm cuvette at 600 nm). After cultivation, the medium was supplemented with one of the following components: 0.2 mM 2,4-dinitrophenol (Sigma, St. Louis, MO, USA), or 5 mM MnSO 4 ·4H 2 O, or 2 mM H 2 O 2 , and the cultivation was continued for 0.5, 1 and 1.5 h. The control cultivation was performed for both strains under the same conditions in YPD.
After cultivation, the cells were examined in an AXIO Imager A1 fluorescent microscope (Zeiss, Jena, Germany) with a 56HE filter set (Zeiss) at a wavelength of 480 nm (maximum excitation) and 512-630 (emission). Images were obtained with Axiocam 506 (Zeiss).

Staining with Nile Red
The cells of YDRO34W-B-GFP and YBR056W-A-GFP strains were cultivated in YPD supplemented with 4 mM Mn 2+ for 24 h. For visualization of lipids, the living cells were stained with Nile red (N1142, Thermo Scientific, Waltham, MA, USA) [17]. The cells were washed once in 0.025 M Hepes-KOH, pH 7.0, then incubated for 10 min at room temperature in the same buffer supplemented with the Nile red (the stock solution containing 1 mg/mL of Nile red in ethanol was diluted l:100 with Hepes-KOH, pH 7.0).
The cells were examined by phase-contrast and fluorescent microscopy in an AXIO Imager A1 (Zeiss, Jena, Germany) with a filter set of 56HE (Zeiss, Jena, Germany) at a wavelength of 450-500 nm for excitation and 512 + 630 nm for emission. An Axiocam 506 camera (Zeiss, Jena, Germany) was used to obtain images.

Determination of Effects of Different Concentrations of Heavy Metal Ions
The GFP strains YDRO34W-B-GFP and YBR056W-A-GFP were used to assess the effects of different concentrations of cadmium and manganese ions on GFP protein fluorescence intensity and fluorescence cell count.
The S. cerevisiae wild-type (WT) strain BY4742 and BY4742-derived mutant strains (∆ydro34w-b and ∆ybro56w-a) were used to assess the effects of different concentrations of cadmium and manganese ions on cell growth.
The yeast strains were cultivated in a liquid YPD medium in sterile multi-well plates at 28 • C and 400 rpm for 24 h in a thermoshaker. Yeast samples were added to the normalized initial cell concentration (0.1 × 10 8 cell/ mL) to the wells containing 0.2 mL of YPD medium supplemented with different concentrations of Cd(CH 3 COO) 2 ·2H 2 O or MnSO 4 ·4H 2 O. After cultivation, the cell concentration in culture samples was measured with a NovoCyte Flow cytometer. The samples were also examined with an AXIO Imager A1 ZEISS microscope (Oberkochen, Germany).

Flow Cytometry
The cell concentration in the cultivation medium was determined by flow cytometry. After a series of dilutions of the cell suspension with water, the number of cells in 25 µL in each sample was counted on a NovoCyte Flow cytometer (Agilent, Santa Clara, CA, USA). Expression of GFP-tagged proteins was determined on a NovoCyte flow cytometer using 488 nm for excitation and 585 nm for emission. A total of 100,000 cells were counted at each experimental point. All assays were repeated 4-5 times, and the mean results are presented.

Statistics
The experiments were performed in triplicate, and the results are presented as the mean with standard deviation (Excel). Statistical analysis was performed by Excel using Student's t-test. The typical and most representative micrographs selected from 10-20 images obtained in independent experiments are presented.

Results
The S. cerevisiae strains YDRO34W-B-GFP and YBR056W-A-GFP carrying the YDRO34W-B and YBR056W-A (MNC1) genes fused with GFP were used to test the expression of the above genes in the presence of heavy metal ions, alkali, and H 2 O 2 by fluorescence microscopy. The cells were cultivated for 24 h. Stress factor concentrations were chosen based on our previous work with related strain BY4741 [18]. The cells grown in the control YPD medium demonstrated negligible expression of both proteins ( Figure 2). No expression of these proteins was observed upon the addition of 2 mM H 2 O 2 to the YPD medium, the picture was the same as control (Figure 2). Green fluorescence of the cells indicating the enhanced expression of both MNC1 and YDRO34W-B was observed in the cells grown in YPD supplemented with Mn 2+ or Co 2+ or Ni 2+ or Zn 2+ or Cu 2+ (Figure 2). In the medium with KOH or Cd 2+ , the fluorescence of YDRO34W-B-GFP cells was much more pronounced compared with the fluorescence of YBR056W-A-GFP cells (Figure 2).
For quantification, fluorescence levels at a wavelength of 585 nm were compared using flow cytometry of cells grown in the presence of manganese and cadmium ions as an example. The data obtained (Supplementary Figure S1) confirmed that the fluorescence of the cells of both strains grown in the presence of 4 mM Mn 2+ was at a similar level. When the cells were grown in the presence of 0.2 mM Cd 2+ , the fluorescence level of cells of the YDRO34W-B-GFP strain was significantly higher than that of the YBR056W-A-GFP strain (Supplementary Figure S1).
We also assessed the dependence of the proportion of fluorescing cells on concentrations of cadmium and manganese by flow cytometry. In the case of Cd 2+ , the proportion of fluorescing cells was higher for the YDRO34W-B-GFP strain, and in the case of Mn 2+ ,  Figure S1).
We also assessed the dependence of the proportion of fluorescing cells on concentrations of cadmium and manganese by flow cytometry. In the case of Cd 2+ , the proportion of fluorescing cells was higher for the YDRO34W-B-GFP strain, and in the case of Mn 2+ , this proportion was higher for the cells of the YBR056W-A-GFP strain (Figure 3). This was observed over the entire range of used concentrations of metal ions. Thus, we have observed differences in the expression of Ydro34w-b and Mnc1 proteins depending on the type of stress.
The localization of CYSTM proteins in membranes, including the cytoplasmic membrane, was predicted due to the analysis of their structure [6] and was experimentally confirmed for some yeast proteins belonging to this family [14,15]. Our microscopic data do not contradict this view (Figure 2). Thus, we have observed differences in the expression of Ydro34w-b and Mnc1 proteins depending on the type of stress.
The localization of CYSTM proteins in membranes, including the cytoplasmic membrane, was predicted due to the analysis of their structure [6] and was experimentally confirmed for some yeast proteins belonging to this family [14,15]. Our microscopic data do not contradict this view (Figure 2).
To improve the visualization of membrane structures, we used Nile red to stain the cells grown in the presence of 4 mM Mn 2+ (Figure 4). Because excitation of the Nile red starts at a wavelength of 410 nm, we can contemporaneously observe both lipids and GFP proteins in yeast cells using a Zeiss 56HE filter kit (a wavelength of 450-500 nm for excitation and 512 + 630 nm for emission). The fluorochrome stained vacuoles, but the plasma membrane was not visualized because of a high level of GFP fluorescence. Nevertheless, the obtained microphotographs show that in the cells of the YDRO34W-B-GFP strain, the GFP-fused protein is localized in the periphery of the cell and the vacuolar membrane, while in the cells of the YBR056W-A-GFP strain, the GFP-fused protein is localized in the cytoplasm, without concentrating in the cell periphery or vacuole. This method did not allow for precise determination of the To improve the visualization of membrane structures, we used Nile red to stain the cells grown in the presence of 4 mM Mn 2+ (Figure 4). Thus, we have observed differences in the expression of Ydro34w-b and Mnc1 proteins depending on the type of stress.
The localization of CYSTM proteins in membranes, including the cytoplasmic membrane, was predicted due to the analysis of their structure [6] and was experimentally confirmed for some yeast proteins belonging to this family [14,15]. Our microscopic data do not contradict this view (Figure 2).
To improve the visualization of membrane structures, we used Nile red to stain the cells grown in the presence of 4 mM Mn 2+ (Figure 4). Because excitation of the Nile red starts at a wavelength of 410 nm, we can contemporaneously observe both lipids and GFP proteins in yeast cells using a Zeiss 56HE filter kit (a wavelength of 450-500 nm for excitation and 512 + 630 nm for emission). The fluorochrome stained vacuoles, but the plasma membrane was not visualized because of a high level of GFP fluorescence. Nevertheless, the obtained microphotographs show that in the cells of the YDRO34W-B-GFP strain, the GFP-fused protein is localized in the periphery of the cell and the vacuolar membrane, while in the cells of the YBR056W-A-GFP strain, the GFP-fused protein is localized in the cytoplasm, without concentrating in the cell periphery or vacuole. This method did not allow for precise determination of the Because excitation of the Nile red starts at a wavelength of 410 nm, we can contemporaneously observe both lipids and GFP proteins in yeast cells using a Zeiss 56HE filter kit (a wavelength of 450-500 nm for excitation and 512 + 630 nm for emission). The fluorochrome stained vacuoles, but the plasma membrane was not visualized because of a high level of GFP fluorescence. Nevertheless, the obtained microphotographs show that in the cells of the YDRO34W-B-GFP strain, the GFP-fused protein is localized in the periphery of the cell and the vacuolar membrane, while in the cells of the YBR056W-A-GFP strain, the GFP-fused protein is localized in the cytoplasm, without concentrating in the cell periphery or vacuole. This method did not allow for precise determination of the Mnc1protein localization. Taking into account the presence of a membrane domain, we speculate that Mnc1 can be localized in the membranes of the endoplasmic reticulum. The data obtained confirm the idea of membrane localization of these members of the CYSTM family and indicate differences in their localization.
Many types of stress lead to a decrease in the electrochemical gradient on the yeast plasma membrane, and this disturbance may be the primary step of the stress response [19,20].
In this regard, we have checked how the known uncoupler 2,4-dinitrophenol [21] affects the expression of MNC1 and YDRO34W-B. The short-term incubation of the YDRO34W-B-GFP and YBR056W-A-GFP strains with 2,4-dinitrophenol, Mn 2+, and H 2 O 2 was used for this purpose. No green fluorescence of the cells of both strains was observed in the control cells ( Figure 5).
Mnc1protein localization . Taking into account the presence of a membrane domain, we speculate that Mnc1 can be localized in the membranes of the endoplasmic reticulum. The data obtained confirm the idea of membrane localization of these members of the CYSTM family and indicate differences in their localization.
Many types of stress lead to a decrease in the electrochemical gradient on the yeast plasma membrane, and this disturbance may be the primary step of the stress response [19,20]. In this regard, we have checked how the known uncoupler 2,4-dinitrophenol [21] affects the expression of MNC1 and YDRO34W-B. The short-term incubation of the YDRO34W-B-GFP and YBR056W-A-GFP strains with 2,4-dinitrophenol, Mn 2+, and H2O2 was used for this purpose. No green fluorescence of the cells of both strains was observed in the control cells ( Figure 5). The fluorescence was not detected by fluorescence microscopy after 30 and 60 min of incubation with 2,4-dinitrophenol or Mn 2+ . Green fluorescence of the cells of both strains appeared in the case of cultivation for 1.5 h in the presence of 2,4-dinitrophenol and Mn 2+ ( Figure 5), but not H2O2. The cell demonstrated no fluorescence in the case of H2O2 (the picture was the same as control, Figure 5). Hence, 2,4-dinitrophenol stimulated the expression of both genes similarly to Mn 2+ , and we suggest that the disturbance of the electrochemical gradient on the plasma membrane may serve as a signal of this stimulation.
We have also checked the effects of the knockout mutations in MNC1 and YDRO34W-B on the growth and cell morphology under Cd 2+ -and Mn 2+ -induced stresses. The changes in cell concentrations ( Figure 6a) and cellular morphology (Figure 6c) in the WT-type  Figure 5). Hence, 2,4-dinitrophenol stimulated the expression of both genes similarly to Mn 2+ , and we suggest that the disturbance of the electrochemical gradient on the plasma membrane may serve as a signal of this stimulation.
We have also checked the effects of the knockout mutations in MNC1 and YDRO34W-B on the growth and cell morphology under Cd 2+ -and Mn 2+ -induced stresses. The changes in cell concentrations ( Figure 6a) and cellular morphology (Figure 6c) in the WT-type strain and ∆mnc1 and ∆ydro34w-b strains under Cd 2+ stress were similar. In the presence of Mn 2+ (2-4 mM), cell concentration decreased for the ∆mnc1 and ∆ydro34w-b strains (Figure 6b). Cell lysis was also observed by light microscopy (Figure 6c). The effect of knockout mutations was not significant but it suggested the possibility of the participation of the studied proteins in overcoming stress caused by a toxic Mn 2+ concentration. strain and ∆mnc1 and ∆ydro34w-b strains under Cd 2+ stress were similar. In the presence of Mn 2+ (2-4 mM), cell concentration decreased for the ∆mnc1 and ∆ydro34w-b strains (Figure 6b). Cell lysis was also observed by light microscopy (Figure 6c). The effect of knockout mutations was not significant but it suggested the possibility of the participation of the studied proteins in overcoming stress caused by a toxic Mn 2+ concentration.

Discussion
The role of CYSTM proteins in metal homeostasis was proposed long ago: the arrangement of sulfhydryl groups of the protein within the membrane could alter the redox potential of the membrane or directly chelate metal ions [6]. The enhanced expression of MNC1 but not YDR034W-B was observed in S. cerevisiae cells adapted to the growth at toxic manganese concentrations [16]. In this article, the transcriptome of cells of the stationary growth stage has been analyzed (120-h cultivation in the presence of 2.5 mM manganese salt); in this study, we have analyzed the cells of the early active-growth stage (24-h cultivation in the presence of 2.5 mM manganese salt). Possibly, the observed difference in the YDR034W-B expression is associated with the growth stage. Previously we suggested the plasma membrane localization of Mnc1 [16] based on data on the structure and localization of other CYSTM proteins [6,15]. The data obtained for the strains containing the GFP-fusion gene in the present work indicate that Mnc1 is localized mainly in the cytoplasm, probably in intracellular membranes.
The expression of YDR034W-B and MNC1 in the cells grown in the control YPD medium was very low [16]. In this work, we have first identified the stress conditions in which YDR034W-B gene expression increased. This expression increased in yeast cells cultivated in the presence of toxic concentrations of Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , or KOH. The MNC1 gene is expressed in the presence of toxic concentrations of Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , or Zn 2+ , while its expression shows a lower level in the presence of Cd 2+ or KOH.
To our surprise, we did not observe the expression of these proteins under peroxide stress, although an increase in the expression of YBR056W-A under this type of stress has been previously reported [6]. Perhaps, this is due to differences in cultivation conditions and stress exposure. We did not observe any effect on resistance to cadmium ions of knockout mutations in both genes studied. At the same time, we noted a decrease in the resistance of knockout mutant cells to manganese ions. Ions of cadmium [22,23] and manganese [24][25][26] significantly differ in the mechanism of toxic action, the range of toxic concentrations, and the biological role. Cadmium does not participate in metabolic processes in yeast, while manganese is an essential trace element for yeast, being a cofactor for numerous metalloenzymes [25] and a necessary component of the antioxidant system [26]. In this regard, the system of manganese homeostasis in yeast includes a number of membrane proteins that differ in the mechanism of action and cellular localization. We speculate that Mnc1 and Ydro34w-b proteins may be components of the manganese homeostasis system in yeast cells. We propose MNC2 (manganese-chelating protein 2) as a possible gene name for YDRO34W-B.
The significance of studying the yeast manganese homeostasis system is summarized in the review, where the authors indicate the functional conservation of proteins involved in manganese homeostasis from yeast to humans [25]. This fact makes yeast a relevant model to find out new aspects of human neurodegenerative diseases and to propose new therapeutic approaches to their treatment [25]. It is noteworthy that one of the markers of Hadington's disease is a protein of the CYSTM family [7]. Thus, the study of CYSTM proteins is of interest for further research on stress adaptation in eukaryotes.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14050987/s1, Figure S1: The cell count and mean X (characterizing the fluorescence intensity) for the cells of YDRO34W-B-GFP and YBR056W-A-GFP strains of S. cerevisiae cultivated for 24 h in control YPD (C), and in YPD supplemented with 0.2 mM Cd 2+ (Cd) or 4 mM Mn 2+ (Mn).