In Saccharomyces cerevisiae, the ATF/CREB transcription factor Sko1 (Acr1) regulates the expression of genes induced by osmotic stress under the control of the high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway. By combining chromatin immunoprecipitation and microarrays containing essentially all intergenic regions, we estimate that yeast cells contain approximately 40 Sko1 target promoters in vivo; 20 Sko1 target promoters were validated by direct analysis of individual loci. The ATF/CREB consensus sequence is not statistically overrepresented in confirmed Sko1 target promoters, although some sites are evolutionarily conserved among related yeast species, suggesting that they are functionally important in vivo. These observations suggest that Sko1 association in vivo is affected by factors beyond the protein-DNA interaction defined in vitro. Sko1 binds a number of promoters for genes directly involved in defense functions that relieve osmotic stress. In addition, Sko1 binds to the promoters of genes encoding transcription factors, including Msn2, Mot3, Rox1, Mga1, and Gat2. Stress-induced expression of MSN2, MOT3, and MGA1 is diminished in sko1 mutant cells, while transcriptional regulation of ROX1 seems to be unaffected. Lastly, Sko1 targets PTP3, which encodes a phosphatase that negatively regulates Hog1 kinase activity, and Sko1 is required for osmotic induction of PTP3 expression. Taken together our results suggest that Sko1 operates a transcriptional network upon osmotic stress, which involves other specific transcription factors and a phosphatase that regulates the key component of the signal transduction pathway.
The peroxin Pex24p of the yeast Yarrowia lipolytica exhibits high sequence similarity to two hypothetical proteins, Yhr150p and Ydr479p, encoded by the Saccharomyces cerevisiae genome. Like YlPex24p, both Yhr150p and Ydr479p have been shown to be integral to the peroxisomal membrane, but unlike YlPex24p, their levels of synthesis are not increased upon a shift of cells from glucose- to oleic acid–containing medium. Peroxisomes of cells deleted for either or both of the YHR150w and YDR479c genes are increased in number, exhibit extensive clustering, are smaller in area than peroxisomes of wild-type cells, and often exhibit membrane thickening between adjacent peroxisomes in a cluster. Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening. Overexpression of the genes PEX25 or VPS1, but not the gene PEX11, restored the wild-type phenotype to cells deleted for one or both of the YHR150w and YDR479c genes. Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae. Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.
biogenesis; peroxin; protein similarity; open reading frame; membrane fission
The environmental niche of each fungus places distinct functional demands on the cell wall. Hence cell wall regulatory pathways may be highly divergent. We have pursued this hypothesis through analysis of Candida albicans transcription factor mutants that are hypersensitive to caspofungin, an inhibitor of beta-1,3-glucan synthase. We report here that mutations in SKO1 cause this phenotype. C. albicans Sko1 undergoes Hog1-dependent phosphorylation after osmotic stress, like its Saccharomyces cerevisiae orthologues, thus arguing that this Hog1-Sko1 relationship is conserved. However, Sko1 has a distinct role in the response to cell wall inhibition because 1) sko1 mutants are much more sensitive to caspofungin than hog1 mutants; 2) Sko1 does not undergo detectable phosphorylation in response to caspofungin; 3) SKO1 transcript levels are induced by caspofungin in both wild-type and hog1 mutant strains; and 4) sko1 mutants are defective in expression of caspofungin-inducible genes that are not induced by osmotic stress. Upstream Sko1 regulators were identified from a panel of caspofungin-hypersensitive protein kinase–defective mutants. Our results show that protein kinase Psk1 is required for expression of SKO1 and of Sko1-dependent genes in response to caspofungin. Thus Psk1 and Sko1 lie in a newly described signal transduction pathway.
In the transcriptional response of Saccharomyces cerevisiae to stress, both activators and repressors are implicated. Here we demonstrate that the ion homeostasis determinant, HAL1, is regulated by two antagonistically operating bZIP transcription factors, the Sko1p repressor and the Gcn4p activator. A single CRE-like sequence (CREHAL1) at position −222 to −215 with the palindromic core sequence TTACGTAA is essential for stress-induced expression of HAL1. Down-regulation of HAL1 under normal growth conditions requires specific binding of Sko1p to CREHAL1 and the corepressor gene SSN6. Release from this repression depends on the function of the high-osmolarity glycerol pathway. The Gcn4p transcriptional activator binds in vitro to the same CREHAL1 and is necessary for up-regulated HAL1 expression in vivo, indicating a dual control mechanism by a repressor-activator pair occupying the same promoter target sequence. gcn4 mutants display a strong sensitivity to elevated K+ or Na+ concentrations in the growth medium. In addition to reduced HAL1 expression, this sensitivity is explained by the fact that amino acid uptake is drastically impaired by high Na+ and K+ concentrations in wild-type yeast cells. The reduced amino acid biosynthesis of gcn4 mutants would result in amino acid deprivation. Together with the induction of HAL1 by amino acid starvation, these results suggest that salt stress and amino acid availability are physiologically interconnected.
Three Saccharomyces cerevisiae proteins (Yng1/YOR064c, Yng2/YHR090c, and Pho23) and two Schizosaccharomyces pombe proteins (Png1/CAA15917 and Png2/CAA21250) share significant sequence identity with the human candidate tumor suppressor p33ING1 in their C-terminal regions. The homologous regions contain PHD finger domains which have been implicated in chromatin-mediated transcriptional regulation. We show that GFP-Yng2, like human Ing1, is localized in the nucleus. Deletion of YNG2 results in several phenotypes, including an abnormal multibudded morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine. These phenotypes are suppressed by expression of either human Ing1 or S. pombe Png1, suggesting that the yeast and human proteins are functionally conserved. Yng1- and Pho23-deficient cells also share some of these phenotypes. We demonstrated by yeast two-hybrid and coimmunoprecipitation tests that Yng2 interacts with Tra1, a component of histone acetyltransferase (HAT) complexes. We further demonstrated by coimmunoprecipitation that HA-Yng1, HA-Yng2, HA-Pho23, and HA-Ing1 are associated with HAT activities in yeast. Genetic and biochemical evidence indicate that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 HAT complex. These studies suggest that the yeast Ing1-related proteins are involved in chromatin remodeling. They further suggest that these functions may be conserved in mammals and provide a possible mechanism for the human Ing1 candidate tumor suppressor.
After a sudden shift to high osmolarity, Saccharomyces cerevisiae cells respond by transiently inducing the expression of stress-protective genes. Msn2p and Msn4p have been described as two transcription factors that determine the extent of this response. In msn2 msn4 mutants, however, many promoters still show a distinct rise in transcriptional activity upon osmotic stress. Here we describe two structurally related nuclear factors, Msn1p and a newly identified protein, Hot1p (for high-osmolarity-induced transcription), which are also involved in osmotic stress-induced transcription. hot1 single mutants are specifically compromised in the transient induction of GPD1 and GPP2, which encode enzymes involved in glycerol biosynthesis, and exhibit delayed glycerol accumulation after stress exposure. Similar to a gpd1 mutation, a hot1 defect can rescue cells from inappropriately high HOG pathway activity. In contrast, Hot1p has little influence on the osmotic stress induction of CTT1, where Msn1p appears to play a more prominent role. Cells lacking Msn1p, Msn2p, Msn4p, and Hot1p are almost devoid of the short-term transcriptional response of the genes GPD1, GPP2, CTT1, and HSP12 to osmotic stress. Such cells also show a distinct reduction in the nuclear residence of the mitogen-activated protein kinase Hog1p upon osmotic stress. Thus, Hot1p and Msn1p may define an additional tier of transcriptional regulators that control responses to high-osmolarity stress.
In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p–controlled osmoinducible genes upon osmotic shock. Although plc1Δ mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock–induced recruitment of the SAGA complex. Like plc1Δ cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p–repressed promoters in plc1Δ cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p–controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.
Saccharomyces cerevisiae adapts to osmotic stress through the activation of a conserved high-osmolarity growth (HOG) mitogen-activated protein (MAP) kinase pathway. Transmission through the HOG pathway is very well understood, yet other aspects of the cellular response to osmotic stress remain poorly understood, most notably regulation of actin organization. The actin cytoskeleton rapidly disassembles in response to osmotic insult and is induced to reassemble only after osmotic balance with the environment is reestablished. Here, we show that one of three MEK kinases of the HOG pathway, Ssk2p, is specialized to facilitate actin cytoskeleton reassembly after osmotic stress. Within minutes of cells' experiencing osmotic stress or catastrophic disassembly of the actin cytoskeleton through latrunculin A treatment, Ssk2p concentrates in the neck of budding yeast cells and concurrently forms a 1:1 complex with actin. These observations suggest that Ssk2p has a novel, previously undescribed function in sensing damage to the actin cytoskeleton. We also describe a second function for Ssk2p in facilitating reassembly of a polarized actin cytoskeleton at the end of the cell cycle, a prerequisite for efficient cell cycle completion. Loss of Ssk2p, its kinase activity, or its ability to localize and interact with actin led to delays in actin recovery and a resulting delay in cell cycle completion. These unique capabilities of Ssk2p are activated by a novel mechanism that does not involve known components of the HOG pathway.
Signal transmission progresses via a series of transient protein-protein interactions and protein movements, which require diffusion within a cell packed with different molecules. Yeast Hog1, the effector protein kinase of the High Osmolarity Glycerol pathway, translocates transiently from the cytosol to the nucleus during adaptation to high external osmolarity. We followed the dynamics of osmostress-induced cell volume loss and Hog1 nuclear accumulation upon exposure of cells to different NaCl concentrations. While Hog1 nuclear accumulation peaked within five minutes following mild osmotic shock it was delayed up to six-fold under severe stress. The timing of Hog1 nuclear accumulation correlated with the degree of cell volume loss and the cells capacity to recover. Also the nuclear translocation of Msn2, the transcription factor of the general stress response pathway, is delayed upon severe osmotic stress suggesting a general phenomenon. We show by direct measurements that the general diffusion rate of Hog1 in the cytoplasm as well as its rate of nuclear transport are dramatically reduced following severe volume reduction. However, neither Hog1 phosphorylation nor Msn2 nuclear translocation were as much delayed as Hog1 nuclear translocation. Our data provide direct evidence that signaling slows down during cell volume compression, probably as a consequence of molecular crowding. Hence one purpose of osmotic adaptation is to restore optimal diffusion rates for biochemical and cell biological processes. In addition, there may be mechanisms slowing down especially Hog1 nuclear translocation under severe stress in order to prioritize Hog1 cytosolic targets.
Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. Functional complementation of the oleic acid–nonutilizing strain mut1-1 of the yeast Yarrowia lipolytica has identified the novel gene, PEX24. PEX24 encodes Pex24p, a protein of 550 amino acids (61,100 Da). Pex24p is an integral membrane protein of peroxisomes that exhibits high sequence homology to two hypothetical proteins encoded by the open reading frames YHR150W and YDR479C of the Saccharomyces cerevisiae genome. Pex24p is detectable in wild-type cells grown in glucose-containing medium, and its levels are significantly increased by incubation of cells in oleic acid–containing medium, the metabolism of which requires intact peroxisomes. pex24 mutants are compromised in the targeting of both matrix and membrane proteins to peroxisomes. Although pex24 mutants fail to assemble functional peroxisomes, they do harbor membrane structures that contain subsets of peroxisomal proteins.
Hyperosmotic stress activates an array of cellular detoxification mechanisms, including the high-osmolarity glycerol (HOG) pathway. We report here that vacuolar H+-ATPase (V-ATPase) activity helps provide osmotic tolerance in Saccharomyces cerevisiae. V-ATPase subunit genes exhibit complex haploinsufficiency interactions with HOG pathway components. vma mutants lacking V-ATPase function are sensitive to high concentrations of salt and exhibit Hog1p activation even at low salt concentrations, as demonstrated by phosphorylation of Hog1p, a shift in Hog1-green fluorescent protein localization, transcriptional activation of a subset of HOG pathway effectors, and transcriptional inhibition of parallel mitogen-activated protein kinase pathway targets. vma2Δ hog1Δ and vma3Δ pbs2Δ double mutants have a synthetic growth phenotype, poor salt tolerance, and an aberrant, hyper-elongated morphology on solid media, accompanied by activation of a filamentous response element-LacZ construct, indicating cross talk into the filamentous growth pathway. Vacuoles isolated from wild-type cells briefly exposed to salt show higher levels of V-ATPase activity, and Na+/H+ exchange in isolated vacuolar vesicles suggests a biochemical basis for the genetic interactions observed. V-ATPase activity is upregulated during salt stress by increasing assembly of the catalytic V1 sector with the membrane-bound Vo sector. Together, these data suggest that the V-ATPase acts in parallel with the HOG pathway in order to mediate salt detoxification.
Exposure of Saccharomyces cerevisiae to increases in extracellular osmolarity activates the stress-activated Hog1 mitogen-activated protein kinase (MAPK), which is essential for cell survival upon osmotic stress. Yeast cells respond to osmotic stress by inducing the expression of a very large number of genes, and the Hog1 MAPK plays a critical role in gene transcription upon stress. To understand how Hog1 controls gene expression, we designed a genetic screen to isolate new transcription factors under the control of the MAPK and identified the MEF2-like transcription factor, Smp1, as a target for Hog1. Overexpression of SMP1 induced Hog1-dependent expression of osmoresponsive genes such as STL1, whereas smp1Δ cells were defective in their expression. Consistently, smp1Δ cells displayed reduced viability upon osmotic shock. In vivo coprecipitation and phosphorylation studies showed that Smp1 and Hog1 interact and that Smp1 is phosphorylated upon osmotic stress in a Hog1-dependent manner. Hog1 phosphorylated Smp1 in vitro at the C-terminal region. Phosphorylation of Smp1 by the MAPK is essential for its function, since a mutant allele unable to be phosphorylated by the MAPK displays impaired stress responses. Thus, our data indicate that Smp1 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK. Moreover, Smp1 concentrates in the nucleus during the stationary phase, and the lack of SMP1 results in cells that lose viability in the stationary phase. Localization of Smp1 depends on HOG1, and consistently, hog1Δ cells also lose viability during this growth phase. These data suggest that Smp1 could be mediating a role for the Hog1 MAPK during the stationary phase.
The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals (J. E. Bennett, K. Izumikawa, and K. A. Marr. Antimicrob. Agents Chemother. 48:1773–1777, 2004). Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown that Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata, the open reading frame (ORF) designated CAGL0I02552g has 38.8% amino acid identity with STB5 (YHR178w) and shares an N-terminal Zn2Cys6 binuclear cluster domain and a fungus-specific transcriptional factor domain, prompting us to test for homologous function and a possible role in azole resistance. Complementation of a Δyhr178w (Δstb5) mutant with CAGL0I02552g resolved the increased sensitivity to cold, hydrogen peroxide, and caffeine of the mutant, for which reason we designated CAGl0I02552g CgSTB5. Overexpression of CgSTB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 caused a modest increase in resistance. Expression analysis found that CgSTB5 shares many transcriptional targets with CgPDR1 but, unlike the latter, is a negative regulator of pleiotropic drug resistance, including the ABC transporter genes CDR1, PDH1, and YOR1.
Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite minor activation of glycerol biosynthesis, the inhibitory effect of citric acid was not due to an osmotic shock. HOG1 negatively regulated the expression of a number of proteins in response to citric acid stress, including Bmh1p. Evidence suggests that BMH1 is induced by citric acid to counteract the effect of amino acid starvation. In addition, deletion of BMH2 rendered cells sensitive to citric acid. Deletion of the transcription factor MSN4, which is known to be regulated by Bmh1p and Hog1p, had a similar effect. HOG1 was also required for citric acid-induced up-regulation of Ssa1p and Eno2p. To counteract the cation chelating activity of citric acid, the plasma membrane Ca2+ channel, CCH1, and a functional vacuolar membrane H+-ATPase were found to be essential for optimal adaptation. Also, the transcriptional regulator CYC8, which mediates glucose derepression, was required for adaptation to citric acid to allow cells to metabolize excess citrate via the tricarboxylic acid (TCA) cycle. Supporting this, Mdh1p and Idh1p, both TCA cycle enzymes, were up-regulated in response to citric acid.
The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.
When challenged with osmotic shock, Saccharomyces cerevisiae induces hundreds of genes, despite a concurrent reduction in overall transcriptional capacity. The stress-responsive MAP kinase Hog1 activates expression of specific genes through interactions with chromatin remodeling enzymes, transcription factors, and RNA polymerase II. However, it is not clear whether Hog1 is involved more globally in modulating the cell’s transcriptional program during stress, in addition to activating specific genes. Here we show that large-scale redistribution of RNA Pol II from housekeeping to stress genes requires Hog1. We demonstrate that decreased RNA Pol II occupancy is the default outcome for highly expressed genes upon stress and that Hog1 is partially required for this effect. We find that Hog1 and RNA Pol II colocalize to open reading frames that bypass global transcriptional repression. These activation targets are specified by promoter binding of two osmotic stress-responsive transcription factors. The combination of reduced global transcription with a gene-specific override mechanism allows cells to rapidly switch their transcriptional program in response to stress.
MAP kinase; transcription; stress response; osmotic shock; Hog1
In Saccharomyces cerevisiae, the family of ATF/CREB transcriptional regulators consists of a repressor, Acr1 (Sko1), and two activators, Aca1 and Aca2. The AP-1 factor Gen4 does not activate transcription through ATF/CREB sites in vivo even though it binds these sites in vitro. Unlike ATF/CREB activators in other species, Aca1- and Aca2-dependent transcription is not affected by protein kinase A or by stress, and Aca1 and Aca2 are not required for Hog1-dependent salt induction of transcription through an optimal ATF/CREB site. Aca2 is important for a variety of biological functions including growth on nonoptimal carbon sources, and Aca2-dependent activation is modestly regulated by carbon source. Strains lacking Aca1 are phenotypically normal, but overexpression of Aca1 suppresses some defects associated with the loss of Aca2, indicating a functional overlap between Aca1 and Aca2. Acr1 represses transcription both by recruiting the Cyc8-Tup1 corepressor and by directly competing with Aca1 and Aca2 for target sites. Acr1 does not fully account for osmotic regulation through ATF/CREB sites, and a novel Hog1-dependent activator(s) that is not a bZIP protein is required for ATF/CREB site activation in response to high salt. In addition, Acr1 does not affect a number of phenotypes that arise from loss of Aca2. Thus, members of the S. cerevisiae ATF/CREB family have overlapping, but distinct, biological functions and target genes.
The RGT1 gene of Saccharomyces cerevisiae plays a central role in the glucose-induced expression of hexose transporter (HXT) genes. Genetic evidence suggests that it encodes a repressor of the HXT genes whose function is inhibited by glucose. Here, we report the isolation of RGT1 and demonstrate that it encodes a bifunctional transcription factor. Rgt1p displays three different transcriptional modes in response to glucose: (i) in the absence of glucose, it functions as a transcriptional repressor; (ii) high concentrations of glucose cause it to function as a transcriptional activator; and (iii) in cells growing on low levels of glucose, Rgt1p has a neutral role, neither repressing nor activating transcription. Glucose alters Rgt1p function through a pathway that includes two glucose sensors, Snf3p and Rgt2p, and Grr1p. The glucose transporter Snf3p, which appears to be a low-glucose sensor, is required for inhibition of Rgt1p repressor function by low levels of glucose. Rgt2p, a glucose transporter that functions as a high-glucose sensor, is required for conversion of Rgt1p into an activator by high levels of glucose. Grr1p, a component of the glucose signaling pathway, is required both for inactivation of Rgt1p repressor function by low levels of glucose and for conversion of Rgt1p into an activator at high levels of glucose. Thus, signals generated by two different glucose sensors act through Grr1p to determine Rgt1p function.
Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast (Saccharomyces cerevisiae) responds to osmotic stress by triggering G1 and G2 checkpoint delays that are dependent on the mitogen-activated protein kinase (MAPK) Hog1. The high-osmolarity glycerol (HOG) pathway is also activated by arsenite, and the hog1Δ mutant is highly sensitive to arsenite, partly due to increased arsenite influx into hog1Δ cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. Here, we found that long-term exposure to arsenite led to transient G1 and G2 delays in wild-type cells, whereas cells that lack the HOG1 gene or are defective in Hog1 kinase activity displayed persistent G1 cell cycle arrest. Elevated levels of intracellular arsenite and “cross talk” between the HOG and pheromone response pathways, observed in arsenite-treated hog1Δ cells, prolonged the G1 delay but did not cause a persistent G1 arrest. In contrast, deletion of the SIC1 gene encoding a cyclin-dependent kinase inhibitor fully suppressed the observed block of G1 exit in hog1Δ cells. Moreover, the Sic1 protein was stabilized in arsenite-treated hog1Δ cells. Interestingly, Sic1-dependent persistent G1 arrest was also observed in hog1Δ cells during hyperosmotic stress. Taken together, our data point to an important role of the Hog1 kinase in adaptation to stress-induced G1 cell cycle arrest.
The HOG mitogen-activated protein kinase pathway mediates the osmotic stress response in Saccharomyces cerevisiae, activating genes like GPD1 (glycerol phosphate dehydrogenase), required for survival under hyperosmotic conditions. Activity of this pathway is regulated by Sln1p, a homolog of the “two-component” histidine kinase family of signal transduction molecules prominent in bacteria. Sln1p also regulates the activity of a Hog1p-independent pathway whose transcriptional output can be monitored using an Mcm1p-dependent lacZ reporter gene. The relationship between the two Sln1p branches is unclear, however, the requirement for unphosphorylated pathway intermediates in Hog1p pathway activation and for phosphorylated intermediates in the activation of the Mcm1p reporter suggests that the two Sln1p branches are reciprocally regulated. To further investigate the signals and molecules involved in modulating Sln1p activity, we have screened for new mutations that elevate the activity of the Mcm1p-dependent lacZ reporter gene. We find that loss of function mutations in FPS1, a gene encoding the major glycerol transporter in yeast activates the reporter in a SLN1-dependent fashion. We propose that elevated intracellular glycerol levels in the fps1 mutant shift Sln1p to the phosphorylated state and trigger the Sln1-dependent activity of the Mcm1 reporter. These observations are consistent with a model in which Sln1p autophosphorylation is triggered by a hypo-osmotic stimulus and indicate that the Sln1p osmosensor is tied generally to osmotic balance, and may not specifically sense an external osmolyte.
Previous work has implicated the Hog1 stress-activated protein kinase (SAPK) in osmotic and oxidative stress responses in the human pathogen Candida albicans. In this study, we have characterized the role of Hog1 in mediating these and other stress responses in C. albicans. We provide evidence that a SAPK-dependent core stress response exists in this pathogen. The Hog1 SAPK is phosphorylated and it accumulates in the nucleus in response to diverse stress conditions. In addition, we have identified Hog1-regulated genes that are induced in response to stress conditions that activate Hog1. These analyses reveal both activator and repressor functions for the Hog1 SAPK. Our results also demonstrate that stress cross-protection, a classical hallmark of the core stress response, occurs in C. albicans between stresses that activate the Hog1 SAPK. Importantly, we find that the core stress response in C. albicans has adapted to the environmental niche of this human pathogen. This niche specificity is reflected by the specific environmental conditions that drive the Hog1-regulated core stress response in C. albicans and by differences in the molecular circuitry that control this response.
The resistance of Candida albicans to many stresses is dependent on the stress-activated protein kinase (SAPK) Hog1. Hence we have explored the role of Hog1 in the regulation of transcriptional responses to stress. DNA microarrays were used to characterize the global transcriptional responses of HOG1 and hog1 cells to three stress conditions that activate the Hog1 SAPK: osmotic stress, oxidative stress, and heavy metal stress. This revealed both stress-specific transcriptional responses and a core transcriptional response to stress in C. albicans. The core transcriptional response was characterized by a subset of genes that responded in a stereotypical manner to all of the stresses analyzed. Inactivation of HOG1 significantly attenuated transcriptional responses to osmotic and heavy metal stresses, but not to oxidative stress, and this was reflected in the role of Hog1 in the regulation of C. albicans core stress genes. Instead, the Cap1 transcription factor plays a key role in the oxidative stress regulation of C. albicans core stress genes. Our data show that the SAPK network in C. albicans has diverged from corresponding networks in model yeasts and that the C. albicans SAPK pathway functions in parallel with other pathways to regulate the core transcriptional response to stress.
Osmotic stress triggers the activation of the HOG (high osmolarity glycerol) pathway in Saccharomyces cerevisiae. This signaling cascade culminates in the activation of the MAPK (mitogen-activated protein kinase) Hog1. Quantitative single cell measurements revealed a discrepancy between kinase- and transcriptional activities of Hog1. While kinase activity increases proportionally to stress stimulus, gene expression is inhibited under low stress conditions. Interestingly, a slow stochastic gene activation process is responsible for setting a tunable threshold for gene expression under basal or low stress conditions, which generates a bimodal expression pattern at intermediate stress levels.
MAPK signaling; HOG pathway; cellular stress response; transcription; single cell analysis
The assembly of signaling components and transcription factors in ordered subcellular structures is increasingly implicated as an important regulatory strategy to modulate the activity of cellular pathways. Here, we document the inducible formation of sub-nuclear foci formed by two mitogen-activated protein kinases (MAPKs) in Saccharomyces cerevisiae upon hyper-osmotic stress. Specifically, we demonstrate that activation of the hyper-osmotic stress response pathway induces the mating pathway MAPK Fus3 and the filamentation pathway MAPK Kss1 to form foci in the nucleus that are organized by their shared downstream transcription factor Ste12. Foci formation of co-localized Ste12, Fus3, and Kss1 requires the kinase activity of the hyper-osmotic response MAPK Hog1 and correlates with attenuated signaling in the mating pathway. Conversely, activation of the mating pathway prevents foci formation upon subsequent hyper-osmotic stress. These results suggest that Hog1-mediated spatial localization of Fus3 and Ste12 into sub-nuclear foci could contribute to uncoupling the pheromone and osmolarity pathways, which share signaling components, under high osmolarity conditions.
Spc1, an osmotic-stress-stimulated mitogen-activated protein kinase (MAPK) homolog in the fission yeast Schizosaccharomyces pombe, is required for the induction of mitosis and survival in high-osmolarity conditions. Spc1, also known as Sty1, is activated by Wis1 MAPK kinase and inhibited by Pyp1 tyrosine phosphatase. Spc1 is most closely related to Saccharomyces cerevisiae Hog1 and mammalian p38 kinases. Whereas Hog1 is specifically responsive to osmotic stress, we report here that Spc1 is activated by multiple forms of stress, including high temperature and oxidative stress. In this regard Spc1 is more similar to mammalian p38. Activation of Spc1 is crucial for survival of various forms of stress. Spc1 regulates expression of genes encoding stress-related proteins such as glycerol-3-phosphate dehydrogenase (gpd1+) and trehalose-6-phosphate synthase (tps1+). Spc1 also promotes expression of pyp2+, which encodes a tyrosine phosphatase postulated as a negative regulator of Spc1. This proposal is supported by the finding that Spc1 associates with Pyp2 in vivo and that the amount of Spc1 tyrosine phosphorylation is lower in a Pyp2-overproducing strain than in the wild type. Moreover, the level of stress-stimulated gpd1+ expression is higher in delta pyp2 mutants than in the wild type. These findings demonstrate that Spc1 promotes expression of genes involved in stress survival and that of regulation may be commonly employed to modulate MAPK signal transduction pathways in eukaryotic species.