Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast

BBA - General Subjects 1864 (2020) 129516

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Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast Yun-ying Zhaoa, Chun-lei Caoa, Ying-li Liub, Jing Wangb, Shi-yun Lic, Jie Lid, Yu Denga,c,

T

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a National Engineering Laboratory for Cereal Fermentation Technology (NELCF), School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China b China-Canada Joint Lab of Food Nutrition and Health (Beijing), Beijing Technology & Business University, Beijing 100048, China c School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China d Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Yeast Cobalt Reactive oxygen species (ROS) Endoplasmic reticulum stress

Background: Cobalt is an important metal cofactor of many living cells. However, excessive cobalt is toxic and can cause cell death and even several diseases in humans. Saccharomyces cerevisiae is a useful tool for studying metal homeostasis and many of the genes and pathways are highly conserved in higher eukaryotes including humans. Methods: The intracellular cobalt and reactive oxygen species (ROS) levels were measured by an atomic absorption spectrometer and DHE staining method, respectively. The expression of genes involved in scavenging oxidative stress was tested by qPCR method, while the expression of UPRE-lacZ report gene was analyzed via βgalactosidase activity assay. Results: Using a genome-scale genetic screen, 153 cobalt-sensitive and 37 cobalt-tolerant gene deletion mutants were identified from Saccharomyces cerevisiae. We showed that 101 of the cobalt-sensitive mutants accumulated higher intracellular cobalt compared to wild-type. The intracellular ROS levels in 112 of the mutants were induced by cobalt, which might be caused by the decreased expression of genes involved in scavenging oxidative stress in response to cobalt. Moreover, more than one-third of the cobalt-sensitive mutants were also sensitive to tunicamycin, and cobalt stress might induce the unfolded protein response (UPR) through serine/threonine kinase and endoribonuclease Ire1. Conclusions: This study reinforced the fact that cobalt toxicity might be due to the high intracellular cobalt and ROS levels, and the endoplasmic reticulum stress responses induced by cobalt. General significance: Elucidating the toxicity mechanisms of cobalt stress response will help reveal new routes for the treatment of the diseases induced by cobalt.

1. Introduction Cobalt is an essential metal cofactor of many enzymes and vitamin 12 in living cells [1]. However, when the extracellular cobalt concentration is high, it can result in the accumulation of reactive oxygen species (ROS) inside cells, culminating in diseases such as lung cancer, pneumonia, asthma, and contact dermatitis in humans [2]. Additionally, cobalt can replace other ions such as magnesium and calcium that are essential for living cells [3]. In the budding yeast,

Saccharomyces cerevisiae, cytosolic cobalt homeostasis is maintained and regulated mainly by the manganese transporter, Smf2, the phosphate transporter, Pho84, and the low-affinity iron transporter, Fet4 [4–6]. Yap1, a basic leucine zipper (bZIP) regulator that regulates gene expression through recognition by its Yap1 response element (YRE; 5′TT/GAC/GTC/AA-3′), can control cobalt uptake by regulating PHO84 [7]. The budding yeast Saccharomyces cerevisiae can defend against cobalt-induced toxicity by activating the iron responsive transcription

Abbreviations: ROS, Reactive oxygen species; ER, Endoplasmic reticulum; VPS, vacuolar protein sorting; VMA, vacuolar membrane ATPase; TM, tunicamycin; DTT, dithiothreitol; ORF, open reading frame; RQC, ribosome quality control complex; SRP, signal recognition particle; RQC, ribosome quality control complex; UPRE, unfolded protein response element; RFU, Relative fluorescence units ⁎ Corresponding author at: National Engineering Laboratory for Cereal Fermentation Technology (NELCF), School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. E-mail address: [email protected] (Y. Deng). https://doi.org/10.1016/j.bbagen.2020.129516 Received 23 August 2019; Received in revised form 7 December 2019; Accepted 31 December 2019 Available online 03 January 2020 0304-4165/ © 2020 Published by Elsevier B.V.

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factor Aft1, and genes involved in reactive oxygen species (ROS)regulated by the transcription factor, Yap1 [8,9]. Cobalt stress induces the expression of genes involved in iron uptake by regulating the nuclear accumulation of the transcriptional factor Aft1 involved in iron homeostasis [8]. Yap1 is required for oxidative stress tolerance and DNA damage repair pathways induced by cobalt stress and can control the iron homeostasis by regulating the expression of genes involved in iron uptake including FIT2, FIT3, FET4, MRS4 and ISU2 [7,10]. Overexpression of the vacuolar protein Cot1, a target of Aft1 and involved in zinc and cobalt transport into the vacuole, increases tolerance to cobalt [11]. While overexpression of the YLR162W gene, an un-characterized open reading frame (ORF), inhibits cell proliferation via cell cycle progression, leads to cell death and decreased mitochondrial membrane potential, and renders cells susceptible to the hypoxic conditions induced by cobalt in Saccharomyces cerevisiae [12]. Cobalt toxicity not only activates homologous recombination (HR) by inhibiting DNA synthesis and causing G1-S arrest in Saccharomyces cerevisiae [13], but also induces cell death, which is attenuated by MgSO4 through inhibition of HIF-1α and activation of ERK1/2/MAPK via mitochondrial apoptotic signaling suppression in a neuronal cell line [14]. Screening of a Schizosaccharomyces pombe nonessential gene knockout library identified 54 cobalt-sensitive deletion gene mutants and 56 cobalt-resistant deletion strains [15]. Meanwhile, 226 gene deletion strains were found to be sensitive to 2.5 mM CoCl2 based on a colony area ratio < 0.5 using high-density arrays in S. cerevisiae [16]. However, the toxicity mechanisms of cobalt remain poorly understood in eukaryotic cells. The present work aims to elucidate the toxicity mechanisms induced by cobalt stress in S. cerevisiae. To obtain a global view of cobalt stress in eukaryotic cells, we herein screened cobalt-sensitive and -tolerant mutations in a yeast nonessential gene deletion library and identified 153 cobalt-sensitive and 37 cobalt-tolerant mutants. Next, we examined the cellular responses of cultured yeast cells to CoCl2 in terms of intracellular cobalt concentration, ROS levels, and the expression of genes associated with antioxidant defenses was also analyzed. Specifically, the cell growth of the cobalt-sensitive mutants under endoplasmic reticulum (ER) stress induced by tunicamycin (TM) was tested and the activities of UPRE-lacZ induced by cobalt in these mutants were also investigated.

relative to wild-type cells in CoCl2-free medium were considered cobaltsensitive or cobalt-resistant. Sensitive and resistant mutants were subjected to a secondary screen using the serial dilution assay method on solid YPD plates with 2 mΜ CoCl2 as described previously [17]. The number of viable cells of the identified cobalt-sensitive mutants was determined by the CFU method described previously after treatment with 2 mΜ CoCl2 [18].

To probe cellular oxidative stress in cobalt-sensitive mutants, we measured the intracellular ROS levels using dihydroethidium (DHE) as previously described [19]. Briefly, overnight cell cultures were inoculated in YPD to an OD600nm of 0.1, grown to the middle log phase, split into two aliquots, and grown in the absence or presence of 1 mM CoCl2 for an additional 2 h or 12 h. Next, about 5 × 106 cells were harvested by centrifugation and resuspended in 250 μL phosphatebuffered saline (PBS) with 2.5 μg/mL DHE and incubated in the dark for 30 min. Relative fluorescence units (RFU) were counted using a Synergy H4 fluorescence reader (BioTek).

2. Material and methods

2.6. Total RNA extraction and quantitative real-time PCR (qPCR) assay

2.1. Strains and media

To extract the total RNA, the indicted mutant cells were fist collected from the middle log phase cultures which were treated with or without 1 mM CoCl2 for 1 h. Next, the total RNA was extracted by the hot phenol method. The first-strand cDNA was synthesized from 1 μg total RNA of each simple via the Primer Script RT reagent kit (Cwbiotech, China) according to the manufacturer's instructions. qPCR reactions were performed in a Thermo Scientific CFX96 instrument, using SYBR Premix Ex Taq (Cwbiotech, China). The PGK1 gene was used as an internal control. Each reaction was carried out in triplicate, and the expression levels of each were calculated using the –ΔΔCt method [20]. The primers used in the experiment are listed in Table S1.

2.3. Genotype assay of cobalt-sensitive mutants using tunicamycin To investigate whether cobalt-sensitive genes are involved in the endoplasmic reticulum response, the effects of tunicamycin on the phenotypes of cobalt-sensitive mutants were assessed by adding an appropriate amount of tunicamycin to YPD plates. The growth of cobalt-sensitive mutants was assayed on plates containing YPD alone and YPD with 1 μg/mL tunicamycin by the serial dilution method as described above. 2.4. Measurement of the intracellular cobalt concentration An atomic absorption spectrometer in flame emission mode was used for measuring the intracellular cobalt concentration in yeast cells treated with 1 mM CoCl2 at 30 °C for 2 h or 12 h in YPD medium as described previously for calcium measurement [17]. Three individual colonies for each mutant were measured, and wild-type BY4743 served as a control. 2.5. Oxidative stress assay for cobalt-sensitive mutants

All S. cerevisiae strains were derived from the S288C background. Yeast cells were grown at 30 °C in YPD medium (1% yeast extract, 2% peptone, 2% glucose). CoCl2 was purchased from Sangon Biotech (Shanghai, China), and dihydroethidium was purchased from Sigma (Beijing, China). 2.2. Genome-wide screening of cobalt-sensitive mutations S. cerevisiae deletion mutants of 4757 non-essential genes in a BY4743 background were purchased from Thermo Fisher SCIENTIFIC (http://clones.thermofisher.com/cloneinfo.php?clone=yeast, catalog number: 95400.BY4743) and frozen at −80 °C in 96-well microtitre plates in liquid YPD medium containing 15% glycerol. For a primary screen of cobalt-sensitive mutations in S. cerevisiae, the deletion mutant library was first transferred to fresh liquid YPD medium (pH ~5.5) and cultured at 30 °C in new 96-well microtitre plates. A 20 μL sample of each mutant was then transferred to 180 μL fresh liquid YPD medium with or without 1 mΜ CoCl2 and cultured at 30 °C for 6 to 12 h. Growth rates of each mutant in YPD medium with and without 1 mΜ CoCl2 were measured from the absorbance at 600 nm (OD600nm) to identify cobalt-sensitive and -resistant mutants. Mutants with a relative OD600nm reduced or increased by > 30% in liquid YPD medium containing CoCl2

2.7. Construction and cellular localization of N-terminally green fluorescent protein (GFP)-tagged Ire1 A construct for expressing N-terminally GFP-tagged GFP-Ire1 under the control of the ADH1 promoter was generated by integrating the His3MX6-pADH1-GFP cassette, which was amplified with primers ScIRE1-N-GFP-F and ScIRE1-N-GFP-R from plasmid pEASY-T1His3MX6-pADH1-GFP (this study), to the N-terminus of Ire1 in the wild-type BY4741 cells. Correct integration was confirmed by PCR with primers GFP-Check-F and ScIRE1-Check-R. The primers used in the construction are listed in Table S1. Subcellular localization of GFP-Ire1 was visualized using a Nikon Eclipse 80i epifluorescence microscope 2

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(Wuxi, Jiangsu Province, China) equipped with a Plan fluor 100/1.3 oil objective lens and FITC (EX 465–495 and EM515–555) cube filters. A mercury lamp served as the light source.

genes) was significantly increased compared with the levels in wildtype cells (Fig. S1A) when they were treated with 1 mM CoCl2 for 2 h. However, when the treated time was lengthened to 12 h, 101 mutants (66% of the total 153 genes) accumulated higher intracellular cobalt levels than that of the wild type cells (Fig. 2A and B), indicating their crucial role in regulating the intracellular cobalt homeostasis. Meanwhile, the rest of the 52 cobalt-sensitive mutants accumulated fewer or similar intracellular cobalt ions compared with the wild-type strain (Fig. S1B). Based on these results, we speculate that cobalt toxicity of these mutants has a lot to do with the high levels of the intracellular cobalt after long processing hours. Cobalt can generate reactive oxygen species (ROS) when it accumulates to toxic levels in cells [23]. Interestingly, it was reported that 77 mutants were sensitive to oxidative stress previously (Table S3). Therefore, we measured intracellular ROS levels evoked by cobalt stress and showed that the intracellular ROS levels of 77 or 112 mutants were increased after 2 h' or 12 h' cobalt treatment (Fig. S2 and Fig. 3), including 57 gene deletion mutants that were also sensitive to oxidative stress, as previously reported (Table S3). We listed some genes as the representative genes of their categories as below.

2.8. Protein extraction and galactosidase activity assay To measure unfolded protein response element (UPRE)-driven βgalactosidase activity in yeast cells, we introduced into test strains pMCZ-Y plasmid DNA harboring a UPRE in the CYC1 promoter in front of the lacZ gene in a 2 μm-based plasmid [21]. Total proteins were extracted from yeast cells and the protein concentration was quantified prior to β-galactosidase assays as described previously [17]. Data are presented as the mean ± standard deviation (SD) from six independent experiments. 2.9. Meta-analysis for the identified genes Enrichment and protein-protein interaction (PPI) network analyses of cobalt-sensitive genes were performed using the powerful web-based Metascape tool (http://metascape.org/gp/index.html#/main/step1), and the results were modified by Cytoscape v3.6.0 [22].

3.3. Increased intracellular cobalt contents and ROS levels in mutation of genes involved in cell cycle and DNA processing

3. Results 3.1. Genome-wide screening of mutated genes involved in cobalt tolerance

Twenty mutants encoding proteins involved in cell cycle or DNA processing were sensitive to 2 mM CoCl2, and ten (RAD57, RAD9, XRS2, IES6, SLX8, RAD6, CNM67, POL32, PHO80 and PHO85) were also sensitive to 1 mM CoCl2 (Table S2). Interestingly, 14 mutants of these mutants accumulated higher intracellular cobalt in response to cobalt stress (Fig. 2A), including nine mutants (RAD57, UBC13, XRS2, CLG1, RAD6, SGS1, DSK2, UME1 and HDA3) that also accumulated higher intracellular ROS levels than that of wide type strain. Moreover, the intracellular cobalt levels of five mutants (PHO80, PHO85, RRD2, CSM4 and CLN2), and the intracellular ROS levels of six mutants (RAD9, IES6, SLX8, POL32, CNM67 and HDA3) were induced by cobalt stress, respectively (Fig. 2A and Fig. 3A). All the results indicate that these genes play essential roles in maintaining intracellular cobalt and/or ROS homeostasis, and the high associated manner between ROS signaling and DNA processing. Many studies have reported that exposure to high cobalt concentrations can be toxic to living cells due to ROS production, but cobalt can exert toxicity by affecting DNA replication, damaging DNA, and inhibiting the SOS repair pathway via an oxidative stressindependent pathway [24]. Therefore, it is unsurprising that intracellular ROS levels in some of the mutants involved in cell cycle and DNA processing were not induced by cobalt stress, as demonstrated for PHO80, PHO85, RRD2, CSM4 and CLN2.

To study the effects of cobalt ions on the growth of S. cerevisiae cells, the yeast diploid non-essential gene deletion library was screened to identify genes whose deletion caused yeast cells to become sensitive to 2 mM CoCl2, and 153 gene deletion mutants were identified (Table S2). Among these mutants, 76 were sensitive to an even lower concentration of 1 mM CoCl2 (Table S2, underlined). It has been reported previously that 23 of the mutants (http://www.yeastgenome.org), namely AFT1, CDC50, COT1, DBF2, NHX1, POP2, RCY1, RPL14A, SAM37, SWI3, UBC7, YAP1, VMA13, VMA5, VPH1, VPS16, VPS3, VPS4, VPS9, YFR049W, YIR016W, YLR065C and YOR331C are sensitivity to cobalt [16], while cobalt sensitivity of the other 130 mutants is reported for the first time. These genes are categorized into the following groups based on functional analysis of the Munich Information Center for Protein Sequences database (MIPS) functional catalog (http://mips. helmholtz-muenchen.de/proj/funcatDB/): metabolism, cell cycle and DNA processing, transcription, protein synthesis, folding, modification and destination, protein with binding function or cofactor requirement (structural or catalytic), cellular transport, other and unknown functions (Table S2). Gene Ontology (GO) enrichment analysis of cobalt-sensitive genes was performed based on the Metascape database with p-value < .01, min overlap genes = 3, and min enrichment factor > 1.5 as the cutoff criteria. The most significant results include the regulation of biological quality, endosome, cytosolic transport, late endosome to vacuole transport, positive regulation of macroautophagy, and protein retention in the Golgi apparatus among the top 20 GO terms in cluster groups (Fig. 1A). Furthermore, the above enriched cluster groups were all closely connected with each other and clustered into intact networks (Fig. 1B). We found that all enriched terms of the top six cluster groups were mainly associated with the regulation of cellular pH, endosome functions, cellular transport and regulation of macroautophagy.

3.4. Mutation of genes involved in cellular transport affects cobalt sensitivity At least 63 nonessential vacuolar protein sorting (VPS) genes in the genome of S. cerevisiae have been reported in previous research [25], and these VPS genes have been classed into six groups according to their characteristics, such as the vacuolar morphology of their mutants [26]. In the present study, 17 vps mutants were identified sensitive to 2 mΜ CoCl2, and 12 mutants (VPS3, VPS60, VPS45, VPS24, VPS1, VPS51, VPS37, VPS18, VPS65, VPS16, VPS31 and VPS4) were also sensitive to 1 mΜ CoCl2 (Table S2). Nine cobalt-sensitive mutants (VPS45, VPS24, VPS37, VPS18, VPS65, VPS5, VPS28, VPS60 and VPS31) accumulated higher intracellular cobalt and ROS levels than the wild-type strain in response to cobalt stress (Fig. 2B and Fig. 3C). Also, the intracellular cobalt levels in two vps mutants for VPS13 and VPS9, and intracellular ROS levels in three vps mutants for VPS1, VPS51 and VPS16, were increased compared with the wide type strain, respectively. The observed correlation between cobalt-sensitive phenotypes and intracellular cobalt of these VPS mutants suggests that the VPS pathway also plays a

3.2. High intracellular cobalt contents and oxidative stress involved in cobalt tolerance Since cobalt is toxic to cell growth when present in excess, to investigate correlations between cobalt sensitivity and intracellular cobalt content for all cobalt-sensitive mutants, we measured the intracellular cobalt concentrations in yeast cells stressed with 1 mM CoCl2. The intracellular cobalt content of only 30 mutants (20% of the total 153 3

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Fig. 1. Meta-enrichment analysis summary of cobalt-sensitive genes. (A) Heatmap of the top 20 enriched GO terms. For GO terms, each band represents one enriched term coloured according to its -log 10 p-value. The dominant term within each group is used as a group heading. (B) Network of enriched terms coloured based on cluster ID. Nodes represent enriched terms, and node size indicates the number of genes involved. Nodes sharing the same cluster are typically close to each other, and the thicker the edge, the higher the similarity. Heatmaps (A) and networks (B) were produced using Metascape (http://metascape.org/gp/index.html#/main/ step1).

H+-ATPase genes can be inhibited by various toxic metals including cobalt [30]. It is therefore unsurprising that the deletion of VMA2, VMA3, VMA5, VMA10, VMA16, VMA13, VPH1 and VMA21, encoding seven of the fourteen subunits of the V-ATPase and one of the three assembly factors, rendered yeast cells hypersensitive to 2 mM CoCl2. Three mutants (VMA16, VMA5 and VPH1) and six mutants (VMA2, VMA3, VMA5, VMA10, VMA13, VPH1 and VMA21) accumulated higher

significant role in maintaining intracellular cobalt ion homeostasis, which could help to maintain calcium and lithium ion homeostasis in yeast cells, too [27,28]. The H+-ATPase localized in the vacuole membrane (V-ATPase) is composed of the catalytic V1 subcomplex and the proton-translocating membrane V0 subcomplex [29]. In S. cerevisiae, cell growth inhibition by metal salts is generally pH dependent and the growth of mutants for

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Fig. 2. Increased intracellular cobalt content of 101 cobalt-sensitive gene mutants in response to cobalt stress. Log-phase cells were cultured with 1 mM CoCl2 for 12 h before harvesting and measurement of intracellular cobalt content. The relative cobalt content of these cobalt-sensitive mutants is normalized against wild-type BY4743 cells (given an arbitrary value of 1.0). Results are the average of three independent assays for each strain. Bars indicate SD, P < .01 for the comparison of the intracellular cobalt content between each mutants and the wild type strain.

3.5. Regulation of the redox homeostasis genes by cobalt stress

intracellular cobalt and ROS levels in response to cobalt stress (Fig. 2B and Fig. 3C). Consistent with a previous study [16], we found that deletion of COT1, encoding a protein transporting cobalt into vacuoles, rendered yeast cells sensitive to both 2 mΜ CoCl2 and 1 mΜ CoCl2, and decreased the intracellular cobalt content of yeast cells in response to cobalt stress (Fig. S1B). Deletion of SPF1, encoding P5-ATPase-Spf1/ Cod1 involved in endoplasmic reticulum function as well as calcium and manganese homeostasis [31,32], also caused yeast cells to be sensitive to cobalt, and increased the intracellular cobalt content and ROS levels in response to cobalt stress (Fig. 2B and Fig. 3C). Deletion of ARV1, SEC22 and TLG2 involved in protein translocation, ARC18 involved in cytoskeleton/actin patch [33] and VRP1 involved in cytoskeletal organization and cytokinesis [34], respectively, resulted in mutants sensitive to cobalt stress that accumulated higher intracellular ROS levels in response to cobalt stress compared with wild-type cells (Fig. 3C). In addition, mutation of GCS1 involved in ER-Golgi transport [35], and mutation of MRS4 and FRE8 involved in iron homeostasis [36,37], also caused yeast cells to be sensitive to cobalt stress, and increased intracellular ROS levels in response to cobalt stress (Fig. 3C). Interestingly, we found that 26 of these cobalt-sensitive mutants harboring altered genes related to cellular transport were sensitive to oxidative stress (Table S3), indicating their critical roles for S. cerevisiae cells in responding to high levels of cobalt in the environment and orchestrating oxidative stress responses.

As previously reported that cobalt stress could up-regulate genes involved in oxidative stress scavenging to alleviate the oxidative damage caused by this metal through activating the transcription factor Yap1, including GSH1, TRX2, SOD1, GPX2, etc. [7]. The induction rates of ROS levels in 11 mutants for UBP1, PGD1, VPS5, VPH1, VPS28, VMA13, HDA3, UBC13, NRK1, EPS1, and SLX8 were all higher than that of the other mutants and wild-type cells when treatment with cobalt for 2 h (Fig. S2). Therefore, we next tested the expression of CTT1 (cytosolic catalase T), TRX2 (thioredoxin 2), TRR1 (thioredoxin reductase), GSH1 (glutamylcysteine synthetase), SOD1 (copper/zinc superoxide dismutase) and GPX2 (2-Cys peroxiredoxin) in these mutants with cobalt treatment. The results showed that all the above genes were significantly induced by cobalt stress in the wild-type cells (Fig. 4). Notably, the expression of GSH1 was all decreased in the 11 mutants compared with wild type cells (Fig. 4A). Interestingly, the expression levels of CTT1, GPX2 and SOD1 were all reduced in ten of these mutants except the mutant for SLX8, EPS1 or UBP1, respectively (Fig. 4B-4D). It has been reported that GPX2 is not only regulated by oxidative stress through two transcription factors Yap1 and Skn7, but also by the calcium signal response through the calcineurin-dependent transcription factor Crz1 [38]. We found that the deletion of EPS1, encoding a member of the protein disulfide isomerase family and involved in 5

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Fig. 3. Increased intracellular ROS levels of cobalt-sensitive gene mutants in response to cobalt stress. Log-phase cells were grown with or without 1 mM CoCl2 for 12 h before harvesting and measurement of intracellular ROS levels using dihydroethidium. Results are averages of three independent assays for each strain. Bars indicate SD, P < .01 for the comparison the intracellular ROS level of each mutant grown with or without 1 mM CoCl2 for 12 h.

another ubiquitin ligase Slx8 (encoded by SLX8), and these three genes play a crucial role in controlling the DNA repair pathway as well as the genome stability and sumoylation via ubiquitination [41–43]. Vph1 is a subunit of vacuolar-ATPase V0 domain and is relative distribution to the decreases of vacuolar membrane under DNA replication stress [43], while Hda3 might be also involved in DNA repair pathway by activating the yeast histone deacetylase HDA1 [44]. In addition to the effects of oxidative stress, the expression of SOD1 in ubp1/ubp1 mutant, CTT1 in slx8/slx8 mutant, TRX2 in slx8/slx8 and ubc13/ubc13 mutants, as well as TRR1 in vph1/vph1 and hda3/hda3 mutants, might be related to DNA

retention of resident ER proteins [39], did not influence the expression of GPX2, suggesting that it might be regulated by another way except the oxidative stress. The expression of TRX2, a gene encoding thioredoxin, and TRR1, a gene encoding thioredoxin reductase, are both regulated by two transcription factors Yap1 and Skn7 [40]. In the present study, we found that the induction of TRX2 was all inhibited in these mutants except the mutant for UBC13 and SLX8 (Fig. 4E), while the expression of TRR1 was all decreased in these mutants except the mutant for VPH1 and HDA3, respectively (Fig. 4F). UBC1 and UBC13, both encoding E2 ubiquitin-conjugating enzyme, could be stimulated 6

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Fig. 4. Relative expression levels of genes involved in oxidative stress response. Gene expression is quantified using RT-qPCR and comparative critical threshold (2) method. The PGK1 gene was used as internal control and the ratio of the fold-change without treatment was standardized to 1.0. These values represent the average of three independent experiments. The asterisks of “*”and “**” show statistically significant differences of P < .05 and P < .01, respectively.

ΔΔCt

functions of the ER, such as dithiothreitol (DTT), tunicamycin (TM), cadmium, platinum or cobalt, may lead to ER stress [47–51]. In the present study, to investigate whether the phenotypes of the 153 cobaltsensitive mutants are related to ER stress, we next analyzed their sensitivity to TM, and 58 mutants were sensitive to 1 μg/mL TM (Table S2, Fig. 5A). Interestingly, 46 of the 58 TM-sensitive mutants (nearly 80%) were also sensitive to 1 mM CoCl2, most notably genes involved in protein sorting and cellular transport (Table S2). In S. cerevisiae, the transmembrane kinase/nuclease Ire1 is the only ER membrane sensory protein, and this protein cleaves precursor RNA encoding the transcription factor Hac1 to form mature mRNA [52]. In addition to Hac1, transcription factor Gcn4 binds the promoters of target genes to stimulate the transcription of UPR-responsive genes [53]. In order to determine whether cobalt stress induces the UPR response through Ire1,

repair pathway and DNA replication stress in some way. Based on these analyses, we concluded that the decreased expression of genes involved in scavenging oxidative stress was regulated by different mechanisms, and the high intracellular ROS levels evoked by cobalt stress might be responsible for some of these regulations. 3.6. Analysis of endoplasmic reticulum stress in cobalt-sensitive mutants In eukaryotic cells, the ER is critical for synthesizing new secretory proteins, lipid- and membrane-associated proteins, protein targeting and secretion, and protein modification and degradation. ER stress can be triggered by various factors, including accumulation of unfolded or misfolded proteins, perturbations in ionic homeostasis, and ER lipid and glycolipid imbalance [45,46]. Thus, any factors that disturb the normal 7

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Fig. 5. Phenotypes of tunicamycin-sensitive deletion mutants and activation of the UPR pathway by cobalt stress. (A) Phenotypes of tunicamycin-sensitive deletion mutants. Wild-type BY4743 cells and cobalt-sensitive gene deletion mutants identified from the genome-scale screen were grown at 30 °C in liquid YPD overnight, serially diluted 10-fold, spotted on YPD plates with or without 1 μg/mL tunicamycin, and incubated for 2 days at 30 °C. (B) Subcellular localization of GFP-Ire1 fusion protein. Wild-type BY4741 cells carrying a GFP-Ire1 allele cultured to log-phase in YPD, YPD + 1 μg/mL TM, or YPD + 1 mM CoCl2 media. Cells were visualized using a Nikon Eclipse 80i epifluorescence microscope. TM, tunicamycin.

foci at the nuclear envelope and cortical ER in response to TM stress, consistent with a previous study [54,55]. As expected, the GFP-Ire1 fusion protein also exhibited a punctate distribution in cells exposed to cobalt stress (Fig. 5B), indicating that cobalt stress might activate the UPR pathway by altering the localization of Ire1. In addition, the activities of the UPRE-lacZ reporter were increased in 48 mutants in

we examined the localization of GFP-Ire1 in wild-type BY4741 cells and measured the activity of a UPRE-lacZ reporter [21] in gene deletion mutants sensitive to both cobalt and TM, since Hac1 operates at multiple levels in addition to posttranscriptional regulation (mRNA splicing) in yeast cells [53]. GFP-ire1 displayed ER-like localization when grown in normal (non-stressed) YPD medium, but clustered into distinct 8

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Fig. 6. Phenotype and GO analysis of cobalt-tolerant deletion mutants. (A) Phenotypes of cobalt-tolerant deletion mutants. Cells of the indicated strains were grown at 30 °C in liquid YPD overnight, serially diluted 10-fold, spotted on YPD plates with or without 4 mM CoCl2, and incubated for 3–5 days at 30 °C. (B) GO analysis of cobalt-tolerant deletion mutants.

cells (Fig. S4A). Among these 37 mutants, intracellular ROS levels in nine mutant for ATC1, KAR9, MRPL15, MRPS28, PEX3, RQC1, SWR1, YBL036C and YHM2, the other 28 mutants accumulated similar to or lower than those in wild-type cells under cobalt stress (Fig. S4B). Interestingly, all 37 cobalt-resistant mutants grew as well as the wild-type strain in response to TM, suggesting their unimportant roles in maintaining the ER stress induced by cobalt stress, which also indicated that the sensitivity of the cobalt-sensitive mutants might be caused by ER stress induced by cobalt toxicity.

response to cobalt stress (Fig. S3). Thus, the cobalt sensitivity of 58 TMsensitive mutants might result in ER stress induced by cobalt stress.

3.7. Cobalt-tolerant mutations screened from this study We identified 37 cobalt-tolerant mutations in this study, mostly related to the regulation of the mitochondrial matrix, mitochondrial part, mitochondria, and mitochondrial translation and gene expression (Fig. 6A and B). Fifteen mutants (FMT1, GIS2, HXK1, ISU1, KAR9, KEL3, MRX14, PEX2, PEX3, RPS1B, RQC1, SWR1, YGL241W, YOR333C and YPR097W) accumulated higher intracellular cobalt content than wildtype cells in response to cobalt stress, while intracellular cobalt concentrations in other mutants were similar or lower than in wild-type

4. Discussion Cobalt can generate reactive oxygen species (ROS), cause DNA 9

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Fig. 7. Suggested model for the toxicity mechanisms of cobalt stress. Cells uptake the extracellular cobalt via the plasma membrane transporters Smf2, Fet4 and Pho84. Once inside the cell, cobalt generates ROS, causing DNA damage and lipid/protein oxidation, thus inducing the ER stress. The oxidative damage caused by cobalt can be alleviated by inducing the genes involved in oxidative stress scavenging.

levels than wild-type cells, respectively. GO analysis showed that the two groups shared two GO terms: regulation of biological quality and vacuolar transport and (Fig. 8A). While the two clusters of ROS level and Cobalt content-increased shared 73 genes mainly involved in regulation of biological quality, ATP export and modification-dependent macromolecule catabolic process (Fig. 8E and Fig. S5). In budding yeast, genes involved in reactive oxygen species (ROS) are regulated by the transcription factor, Yap1, which play an important role in mediating tolerance to cobalt toxicity [7]. In this study, we noticed that deletion of YAP1 gene and its 14 target genes, MNN11, ADO1, VPS51, RPL29, RAD6, EPS1, BUL1, RPL20B, TSA1, MRS4, ARV1, YIR016W, YJL107C and YOR062C [56], related to cobalt tolerance (Table S2). It is therefore possible that cobalt will activate Yap1 to alleviate the oxidative damage caused by cobalt stress through these targets. In addition to cobalt, some other metals such as Cd, Cu or Mn also induce ROS production, leading to cell death when yeast cells are exposed to these metals [57,58]. It has been reported previously that 72 of 153 cobalt-sensitive mutants (http://www.yeastgenome.org) have growth defects in culture media supplemented with other metals, such as Cadmium, Zinc, Nickel, Manganese, etc. (Fig. S6 and Table S4), including seven genes involved in the vacuole membrane (V-ATPase) and 13 genes involved in the vacuolar protein sorting (VPS). The rest 81 mutants are specific to cobalt alone. Moreover, among these 72 mutants, 57 mutants or 39 mutants accumulated higher intracellular ROS or cobalt levels in response to cobalt stress, indicating their crucial roles in maintaining the intracellular ROS or cobalt homeostasis. High levels of iron and cobalt can elevate intracellular ROS levels and lead to toxicity [2]. Excessive levels of cobalt can affect iron homeostasis and reduce the bioavailability of Fe [59]. In this study, we identified seven genes involved in iron homeostasis, including AFT1, FRA1, MRS4, FRE8, VMA3, ISU1 and IBA57 involved in regulating Fe/S

damage and induce ER stress when it accumulates to toxic levels in cells [13,23] (Fig. 7), which can lead to cell death through a number of mechanisms. In this study, we identified 153 cobalt-sensitive gene deletion mutations from a genome-scale screen in budding yeast. Among these cobalt-sensitive mutants, 76 (50%) were sensitive to 1 mΜ CoCl2 and 58 (38%) were sensitive to 1 μg/ml TM, while the intracellular cobalt or ROS levels of 101 (66%) or 112 (73%) mutants were induced under cobalt stress, respectively. In comparison with a genome-wide screening of ROS resistance reported previously, we found that 78 of the 153 cobalt-sensitive mutants were sensitive to oxidative stress (Table S3), which indicates that ROS is critical for Co toxicity. The cobalt sensitivities of these cobalt-sensitive mutants were mainly due to high intracellular cobalt and ROS levels induced by cobalt stress, and the ER stress caused by high cobalt might also contribute. The meta-enrichment analysis identified three groups of genes for which deletion resulted in sensitivity to cobalt, oxidative stress and TM, associated with vacuolar transport, regulation of biological quality, cytosolic transport, late endosome to vacuole transport, negative regulation of macromolecule biosynthetic processes, endocytosis, and positive regulation of macroautophagy (Fig. 8A). Furthermore, five significant models were obtained from the protein-protein interaction (PPI) network based on MCODE analysis in Metascape (Fig. 8B). Functional enrichment analysis showed that the top three PPI modules were significantly enriched in various categories including vacuolar proton-transporting V-type ATPase complex, proton-transporting Vtype ATPase complex, proton-transporting ATPase activity (MCODE1), the rotational mechanism, cellular bud neck, cellular bud and site of polarised growth (MCODE2), and vacuole (MCODE3; Fig. 8C and D). A total of 40 interacting genes were shared by the two oxidation- and TMsensitive clusters (Fig. 8E), including 33 genes or 20 genes for which deletions resulted in accumulation of higher intracellular ROS or cobalt 10

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Fig. 8. Visualization of meta-analysis results. (A) Heatmap of the top 20 enriched terms across three gene lists, coloured based on -log 10 p-value. (B) Protein-protein interaction (PPI) networks of three clusters. Nodes are represented by pie charts indicating their associations with each input study. The identities of gene lists are colour-coded. (C) Model PPI network originating from (B) showing the most significant interactions. (D) Functional enrichment analysis of the PPI module. GO terms are presented, and each band represents one enriched term coloured according to its - log 10 p-value. (E) Venn diagram of the indicted four clusters. A–C were generated using Metascape (http://metascape.org/gp/index.html#/main/step1).

5. Conclusions

cluster synthesis related to cobalt tolerance. The cytosolic iron concentration can be regulated by the transcription factors Aft1/Aft2 and Yap5, and iron-sulfur clusters play a critical role in controlling Aft1/2 and Yap5 activity [60]. Under low iron conditions, Aft1 and Aft2 bind to and activate the promoters of iron regulon genes, including the highaffinity iron transporters FET3, FTR1, FET5 and FTH1, the low-affinity iron transporters FET4 and SMF3, the mitochondrial iron transporter MRS4 that provides iron for FeeS cluster synthesis in mitochondria [61], the Fe/S cluster synthesis gene ISU1, as well as the various ion transporter genes such as COT1, encoding a zinc/cobalt transporter and KHA1, encoding a K+/H+ antiporter [62]. FRA1 encodes a protein that negatively regulates transcription of the iron regulon, and forms an iron-independent complex with Fra2 and the [2Fee2S] cluster-binding complexes Grx3p and Grx4p that transmits a negative signal to Aft1/2 during synthesis of mitochondrial FeeS clusters [63]. FRE8 is one of nine homologous genes involved or predicted to be involved in iron uptake [37,64]. VMA3, encoding proteolipid subunit c of the V0 domain of vacuolar H(+)-ATPase, is required for vacuolar acidification, and is important for copper and iron metal ion homeostasis. Therefore, some of the gene products required for iron homeostasis are also involved in cobalt tolerance in yeast cells.

In conclusion, 153 cobalt-sensitive and 37 cobalt-tolerant mutations were identified from a yeast diploid nonessential gene deletion library. Gene functional enrichment analysis indicated that cobalt-sensitive genes were mostly associated with the regulation of biological quality, endosome, cytosolic transport, late endosome to vacuole transport, and positive regulation of macroautophagy categories, while the cobalttolerant genes were mainly related to mitochondrial functions. The results also indicate that the sensitivities caused by cobalt are related to increased intracellular cobalt and ROS levels, as well as the ER stress induced by cobalt. Our current findings provide a global overview of genes involved in the sensitivity of S. cerevisiae cells to high levels of extracellular cobalt, and clues for a better understanding of cobalt toxicity caused by high intracellular cobalt and ROS levels, and ER stress in eukaryotic cells. Author's contribution Y.Z. and Y.D. designed the experiment. C.C., S.L. and J.L. performed the experiment. Y.Z. analyzed the dates and wrote manuscript. L.L., J.W. and Y.D. revised the manuscript. All the authors read and approved the final manuscript. 11

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Declaration of competing interest All authors declare that they have no competing interests.

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Acknowledgements

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This work was supported by grants from the National Natural Science Foundation of China (21877053, 31601564), the Natural Science Foundation of Jiangsu Province (BK20181345), the Open Foundation of Jiangsu Key Laboratory of Industrial Biotechnology (KLIB-KF201807), the Fundamental Research Funds for the Central Universities (JUSRP51705A), and the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions. The funding agencies provided financial support to generate the data, however, they played no role in the design of this study, analysis and interpretation of the data, or in writing and submitting the manuscript.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbagen.2020.129516.

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