An overview of genes involved in the SDS sensitivity of yeast cells
To investigate the cellular functions required for cell growth under a surplus of SDS, a yeast library of diploid nonessential gene deletion was screened to identify genes involved in the sensitivity to SDS. The results show that 108 gene deletion mutants (2.3% of the screened 4757 gene deletion mutants) were identified as sensitive to 0.03% SDS (Fig. 1 and Table 1). The genotypes of these 108 mutants were confirmed by PCR with the forward primer derived from the promoter region of each correspondent gene and a reverse primer KanMX4-R (Additional file 1: Table S1 and Additional file 2: Figure S1) derived from the ORF region of KanMX4. The functional categories of these 108 genes are involved in metabolism (17), cell cycle and DNA processing (15), transcription (14), cellular transport, transport facilities and transport routes (28), biogenesis of cellular components (6), cellular communication / signal transduction mechanism (2), protein with binding function or cofactor requirement (structural or catalytic) (10), as well as unclassified proteins (16) (Table 1). Gene Ontology (GO) enrichment analysis result showed that these 108 SDS-sensitive genes were mainly enriched in vacuolar transport, ATP export, and endosomal transport among the top 16 GO terms in cluster groups (Additional file 3: Figure S2).
Exposure to SDS stress results in ROS generation
Since SDS had been confirmed to induce the oxidative stress response [2], we next measured the intracellular ROS levels of the 108 SDS-sensitive mutants under 0.015% SDS treatment. In the wild-type BY4743 cells, the intracellular ROS level was significantly increased under SDS stress (Fig. 2 and Additional file 4: Figure S3). Interestingly, only six mutants for ARG82, TRP5, GRR1, MSH1, LAS21, and YNL296W of these 108 SDS-sensitive mutants, accumulated lower intracellular ROS levels when treated with 0.015% SDS than without SDS (The relative ROS levels in these mutants was smaller than 1; Fig. 2 and Additional file 4: Figure S3). It suggested that the above six genes might not be directly involved in the regulation of intracellular ROS levels under SDS stress. Of these 108 SDS-sensitive mutants, 85 mutants accumulated significantly higher intracellular ROS levels under SDS stress compared with wild-type cells (Additional file 4: Figure S3B and D), indicating that these 85 mutants might respond to lower concentration of SDS and thereby accumulated higher ROS levels than wild type cells. The rest 23 mutants accumulated similar or lower intracellular ROS levels when treated with SDS compared with wild type cells, although the relative ROS levels in some of these mutants were also very high (Fig.2 and Additional file 4: Figure S3B and D). Here we showed that mutants for genes related to the functions of metabolism and cellular transport, transport facilities and transport routes were most sensitive to SDS stress (Table 1). We listed some genes as the representative genes of their categories as below.
Genes involved in cellular transport and transport routes are associated with SDS tolerance
The largest functional category of these 108 identified SDS-sensitive genes is the cellular transport, transport facilities and transport routes (Table 1), including 28 genes identified. There are 63 nonessential vacuolar protein sorting (VPS) genes in the genome of S. cerevisiae [15]. Notably, 15 mutants for VPS1, VPS16, VPS20, VPS24, VPS22, VPS23, VPS25, VPS32, VPS33, VPS36, VPS37,VPS38, VPS51, VPS63, and VPS64 were identified being sensitive to 0.03% SDS in the present study (Table 1; Fig. 1). The intracellular ROS levels of these 15 mutants were all induced by SDS stress, especially in mutants for VPS20, VPS36, VPS63 and VPS25 (Fig. 2). The results suggest that the VPS pathway involved in protein trafficking and membrane fusion plays an important role in the response of yeast cells to SDS stress.
The H+-ATPase localized in the membrane of vacuole (V-ATPase) is composed of the catalytic V1 subcomplex and the proton-translocating membrane V0 subcomplex, playing crucial roles in the organelles acidification and other intracellular activities [16, 17]. In this study, four mutants for VMA3, VMA5, VMA13, and VMA21 were sensitive to 0.03% SDS (Table 1; Fig. 1). VMA5 and VMA13 encodes the V1 complex subunit C and H [18, 19], respectively. VMA3 encodes the subunit c of the V0 complex [20]. VMA21 is not an actual component of the V-ATPase complex, but encodes proteins functioned in the assembly of the V-ATPase [21]. These results indicate that the V-ATPase is critical for S. cerevisiae cells in responding to SDS in the environment.
Mutants for genes involved in cell cycle and DNA processing render yeast cells sensitive to SDS stress
There are 14 genes identified in our study that are involved in cell cycle and DNA processing (Table 1; Fig. 1). The intracellular ROS levels in 13 mutants except the mutants for MSH1 were all increased under SDS stress, especially in mutants for MEM1, PHO85, EAF1 and XRS2 (Fig. 2). SLX5 and SLX8 encode the subunit of Slx5-Slx8 ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL) complex [22-24]. Mutants for SLX5 or SLX8 were sensitive to 0.03% SDS (Table 1 and Fig. 1), suggesting that STUbL complex is involved in SDS tolerance of yeast cells. The small SUMO-targeted ubiquitin ligase complex is a nuclear ubiquitin ligase complex that specifically targets sumoylated proteins. It is formed of homodimers or heterodimers of RING finger protein 4 family ubiquitin ligases and is conserved in eukaryotes [23]. Three genes, MSH1, FYV6 and XRS2, encode three proteins required for the DNA repair process [25-27], has been identified in this study. The other six genes, EAF1, ARP5, RSC1, RSC2, DCC1 and CTF4 associated with chromatin modification, remodeling and cohesion [28-32], are all required for SDS tolerance. The PHO85 gene, coding for a cyclin-dependent kinase Pho85, was screened in our study. The kinase Pho85 is involved in regulating the cellular responses of cell cycle progression, autophagy, response to DNA damage, phosphate and glycogen metabolism, establishment of cell polarity, as well calcium-mediated signaling. Therefore, deletion of the PHO85 cause a decreased resistance to oxidative stress, chemicals, toxin, utilization of carbon and nitrogen [33-37]. In addition, we have identified two genes, NEM1 and CDC50, which are required for normal nuclear envelope morphology and sporulation, or cell division, respectively [38, 39]. Taken together, these results suggests that SDS can affect the cell cycle and DNA processing of S. cerevisiae cells.
Genes involved in aromatic amino acid biosynthesis and SDS tolerance
We have identified mutants for five genes involved in the synthesis of aromatic amino acids, ARO1, ARO2, ARO7, TRP1 and TRP5 that were sensitive to 0.03% SDS (Table 1; Fig. 1). The intracellular ROS levels in mutants for ARO1, ARO7, TRP1 and TRP5 were all higher than that of wide type cells when the cells were treated with SDS (Additional file 4: Figure S3B and D). Previously, Aro1 catalyzes steps 2 through 6 in the biosynthesis of chorismate, which is a precursor to aromatic amino acids [23]; Aro2 catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to form chorismate; and Aro7 catalyzes the conversion of chorismate to prephenate to initiate the tyrosine/phenylalanine-specific branch of aromatic amino acid biosynthesis [40-42]. Trp1 and Trp5 involved in the synthesis of tryptophan, where Trp1 catalyzes the third step in tryptophan biosynthesis and Trp5 catalyzes the last step of tryptophan biosynthesis [43, 44]. It was reported previously that trp1-1 cells had a disadvantage in the response to SDS compared to auxotrophy for adenine, histidine, leucine or uracil when cells were grown on rich media [45]. They also showed that the cell membrane damage triggered by SDS was independent of CWI (cell wall integrity) signaling and was not a cause of tryptophan starvation. Our present results confirmed this previous findings that tryptophan exhibited protection from membrane disruptions and thus conferred resistance to SDS stress.
SDS generates oxidative stress by regulating the expression of genes involved in redox homeostasis
The relative ROS levels in 11 mutants for PRS3, TRP1, NEM1, EAF1, IKI3, CBP3, VPS20, VPS36, VPS63, VPS25, and TUS1 were all higher than that of wild-type cells (Fig. 2), indicating that these 11 genes were all important for dealing with the oxidative damage generated by SDS stress. To further confirm these results, we constructed the 11 plasmids expressing the above 11 genes in pRS316 plasmid, respectively, and then transformed them into the corresponding mutants. The growth defect of SDS-treatment mutant cells could be suppressed by introducing the expression plasmid back into the corresponding mutants (Fig. 3A), and their intracellular ROS levels were also recovered to that of the wild-type cells (Fig. 3B). Taken together, these results indicate that yeast cells lacking any of the above 11 genes are sensitive to SDS stress, leading to increased intracellular ROS levels.
It was reported that many of the oxidative stress scavenging genes could be induced by SDS stress in a DNA microarray analysis [2]. To investigate whether the deletion of genes PRS3, TRP1, NEM1, EAF1, IKI3, CBP3, VPS20, VPS36, VPS63, VPS25, and TUS1 influence the expression of genes coding for the antioxidant defenses, we tested the expression of GSH1 (glutamylcysteine synthetase), SOD1 (cooper/zinc superoxide dismutase), CTT1 (cytosolic catalase T), GPX2 (2-Cys peroxiredoxin), TRR1 (thioredoxin reductase) and TRX2 (thioredoxin 2) by quantitative real-time PCR analyses. In the wild-type cells, the expression levels of GSH1, SOD1, CTT1 and GPX2 were significantly up-regulated after treatment with 0.015% SDS (Fig. 4), while no significant difference in the expression levels of TRR1 or TRX2 were observed when treated with or without SDS (Additional file 5: Figure S4). Interestingly, both of the expression levels of SOD1 and CTT1 were reduced in the 11 mutants compared with wild type cells (Fig. 4B and 4C). In addition, the expression levels of GSH1 and GPX2 were also reduced in these mutants except the mutants for NEM1 and VPS25, or EAF1, respectively (Fig. 4A and 4D). To investigate the decreased expression of GSH1, SOD1, CTT1 and GPX2, we further analyzed the expression levels of these four genes in the wide type cells treated with 0.005% and 0.01% (Additional file 6: Figure S5). We found that the expression levels of GSH1, SOD1, CTT1 and GPX2 were induced when the SDS concentrations were 0.01 and 0.015, but remain unchanged or slightly induced when the SDS concentration was 0.005%. It suggested that the expressions of the above four genes were dependent on the concentration of SDS. Overall, our results demonstrate that the decreased expression of GSH1, SOD1, CTT1 and GPX2 might be responsible for the high intracellular ROS levels accumulated in these mutants than wide type cells.