An overview of genes involved in the SDS sensitivity of yeast cells
To obtain a global view of genes involved in the sensitivity of yeast cells to 0.03% SDS, we screened the yeast diploid nonessential gene deletion library. 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), indicating that they possess a weakened cell membrane or cell wall. 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). We showed that mutants for genes related to the functions of metabolism and ellular 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. Notably, 11 out of 63 nonessential vacuolar protein sorting (VPS) genes in the genome of S. cerevisiae being identified . Namely, mutants for VPS1, VPS16, VPS20, VPS24, VPS25, VPS33, VPS36, VPS38, VPS51, VPS63, and VPS64 were identified being sensitive to 0.03% SDS (Table 1; Fig. 1). 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]. The V1 complex is composed of at least eight subunits (A-H) encoded by eight VMA (Vacuolar Membrane ATPase) genes: VMA1, VMA2, VMA4, VMA5, VMA7, VMA8, VMA10 and VMA13, respectively. The V0 complex is composed of at least five subunits (a, c, d, c’ and c’’) encoded by six VMA genes: VPH1, STV1, VMA3, VMA6, VMA11, and VMA16, respectively. Three genes VMA12, VMA21 and VMA22 encode proteins that are required for the biogenesis of a functional V-ATPase . V1 subcomplex and V0 subcomplex are responsible for ATP hydrolysis and proton translocation, respectively. Mutants for the VMA genes showed growth defects in response to oxidative stress, such as H2O2 . 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 [20, 21], respectively. VMA3 encodes the subunit c of the V0 complex . VMA21 is not an actual component of the V-ATPase complex, but encodes proteins functioned in the assembly of the V-ATPase . 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 15 genes identified in our study that are involved in cell cycle and DNA processing. SLX5 and SLX8 encode the subunit of Slx5-Slx8 ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL) complex [24-26]. 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 . Three genes, MSH1, FYV6 and XRS2, encode three proteins required for the DNA repair process [27-29], has been identified in this study. The other six genes, EAF1, ARP5, RSC1, RSC2, DCC1 and CTF4 associated with chromatin modification, remodeling and cohesion [30-34], are all required for SDS tolerance. Interestingly, SIT4 and PHO85 coding for a serine/threonine-protein phosphatase and a cyclin-dependent kinase, respectively, were both screened in our study. The serine/threonine-protein phosphatase Sit4 controls the processes of mitochondrial function, lifespan, cellular traffic, cell growth and cell cycle progression by regulating the phosphorylation levels of many proteins [35, 36], while 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 SIT4 or PHO85 cause a decreased resistance to oxidative stress, chemicals, toxin, utilization of carbon and nitrogen [37-41]. In addition, we have identified two genes, NEM1 and CDC50, which are required for normal nuclear envelope morphology and sporulation, or cell division, respectively [42, 43]. 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). Previously, Aro1 catalyzes steps 2 through 6 in the biosynthesis of chorismate, which is a precursor to aromatic amino acids ; 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 [44-46]. 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 [47, 48]. These results confirmed the previous findings that tryptophan exhibited protection from membrane disruptions and thus conferred resistance to SDS stress .
Oxidative stress is involved in SDS tolerance
Since SDS had been confirmed to induce the oxidative stress response , 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 3: Figure S2). Of these 108 SDS-sensitive mutants, 85 mutants accumulated significantly higher intracellular ROS levels under SDS stress compared with wild-type cells (Additional file 3: Figure S2), while the rest 23 mutants accumulated similar or lower intracellular ROS levels when treated with SDS compared with wild type cells (Additional file 3: Figure S2). Interestingly, six mutants for ARG82, TRP5, GRR1, MSH1, LAS21, and YNL296W, accumulated lower intracellular ROS levels when treated with 0.015% SDS than without SDS (Additional file 3: Figure S2). It suggested that these six genes might not be directly involved in the regulation of intracellular ROS levels under SDS stress.
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.
SDS generates oxidative stress by regulating the expression of genes involved in redox homeostasis
It was reported that many of the oxidative stress scavenging genes could be induced by SDS stress in a DNA microarray analysis . 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 SDS (Fig. 4), while no significant difference in the expression levels of TRR1 or TRX 2 were observed when treated with or without SDS (Additional file 4: Figure S3). 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). 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.