Loss of regulators of vacuolar ATPase function and ceramide synthesis results in multidrug sensitivity in Schizosaccharomyces pombe.

We undertook a screen to isolate determinants of drug resistance in fission yeast and identified two genes that, when mutated, result in sensitivity to a range of structurally unrelated compounds, some of them commonly used in the clinic. One gene, rav1, encodes the homologue of a budding yeast protein which regulates the assembly of the vacuolar ATPase. The second gene, lac1, encodes a homologue of genes that are required for ceramide synthesis. Both mutants are sensitive to the chemotherapeutic agent doxorubicin, and using the naturally fluorescent properties of this compound, we found that both rav1 and lac1 mutations result in an increased accumulation of the drug in cells. The multidrug-sensitive phenotype of rav1 mutants can be rescued by up-regulation of the lag1 gene which encodes a homologue of lac1, whereas overexpression of either lac1 or lag1 confers multidrug resistance on wild-type cells. These data suggest that changing the amount of ceramide synthase activity in cells can influence innate drug resistance. The function of Rav1 appears to be conserved, as we show that SpRav1 is part of a RAVE-like complex in fission yeast and that loss of rav1 results in defects in vacuolar (H(+))-ATPase activity. Thus, we conclude that loss of normal V-ATPase function results in an increased sensitivity of Schizosaccharomyces pombe cells to drugs. The rav1 and lac1 genes are conserved in both higher eukaryotes and various pathogenic fungi. Thus, our data could provide the basis for strategies to sensitize tumor cells or drug-resistant pathogenic fungi to drugs.

Multiple pathways differentially regulate global oxidative stress responses in fission yeast.

Cellular protection against oxidative damage is relevant to ageing and numerous diseases. We analyzed the diversity of genome-wide gene expression programs and their regulation in response to various types and doses of oxidants in Schizosaccharomyces pombe. A small core gene set, regulated by the AP-1-like factor Pap1p and the two-component regulator Prr1p, was universally induced irrespective of oxidant and dose. Strong oxidative stresses led to a much larger transcriptional response. The mitogen-activated protein kinase (MAPK) Sty1p and the bZIP factor Atf1p were critical for the response to hydrogen peroxide. A newly identified zinc-finger protein, Hsr1p, is uniquely regulated by all three major regulatory systems (Sty1p-Atf1p, Pap1p, and Prr1p) and in turn globally supports gene expression in response to hydrogen peroxide. Although the overall transcriptional responses to hydrogen peroxide and t-butylhydroperoxide were similar, to our surprise, Sty1p and Atf1p were less critical for the response to the latter. Instead, another MAPK, Pmk1p, was involved in surviving this stress, although Pmk1p played only a minor role in regulating the transcriptional response. These data reveal a considerable plasticity and differential control of regulatory pathways in distinct oxidative stress conditions, providing both specificity and backup for protection from oxidative damage.

A novel pathway determining multidrug sensitivity in Schizosaccharomyces pombe.

In this study, we show that a mutation isolated during a screen for determinants of chemosensitivity in S. pombe results in loss of function of a previously uncharacterized protein kinase now named Hal4. Hal4 shares sequence homology to Hal4 and Hal5 in S. cerevisiae, and previous evidence indicates that these kinases positively regulate the major potassium transporter Trk1,2 and thereby maintain the plasma membrane potential. Disruption of this ion homeostasis pathway results in a hyperpolarized membrane and a concomitant increased sensitivity to cations. We demonstrate that a mutation in hal4+ results in hyperpolarization of the plasma membrane. In addition to the original selection agent, the hal4-1 mutant is sensitive to a variety of chemotherapeutic agents and stress-inducing compounds. Furthermore, this wider chemosensitive phenotype is also displayed by corresponding mutants in S. cerevisiae, and in a trk1deltatrk2delta double deletion mutant in S. pombe. We propose that this pathway and its role in regulating the plasma membrane potential may act as a pleiotropic determinant of sensitivity to chemotherapeutic agents.

Global transcriptional responses of fission yeast to environmental stress.

We explored transcriptional responses of the fission yeast Schizosaccharomyces pombe to various environmental stresses. DNA microarrays were used to characterize changes in expression profiles of all known and predicted genes in response to five stress conditions: oxidative stress caused by hydrogen peroxide, heavy metal stress caused by cadmium, heat shock caused by temperature increase to 39 degrees C, osmotic stress caused by sorbitol, and DNA damage caused by the alkylating agent methylmethane sulfonate. We define a core environmental stress response (CESR) common to all, or most, stresses. There was a substantial overlap between CESR genes of fission yeast and the genes of budding yeast that are stereotypically regulated during stress. CESR genes were controlled primarily by the stress-activated mitogen-activated protein kinase Sty1p and the transcription factor Atf1p. S. pombe also activated gene expression programs more specialized for a given stress or a subset of stresses. In general, these "stress-specific" responses were less dependent on the Sty1p mitogen-activated protein kinase pathway and may involve specific regulatory factors. Promoter motifs associated with some of the groups of coregulated genes were identified. We compare and contrast global regulation of stress genes in fission and budding yeasts and discuss evolutionary implications.

The Srk1 protein kinase is a target for the Sty1 stress-activated MAPK in fission yeast.

The fission yeast stress-activated Sty1/Spc1 MAPK pathway responds to a similar range of stresses as do the mammalian p38 and SAPK/JNK MAPK pathways. In addition, sty1(-) cells are sterile and exhibit a G(2) cell cycle delay, indicating additional roles of Sty1 in meiosis and cell cycle progression. To identify novel proteins involved in stress responses, a microarray analysis of the Schizosaccharomyces pombe genome was performed to find genes that are up-regulated following exposure to stress in a Sty1-dependent manner. One such gene identified, srk1(+) (Sty1-regulated kinase 1), encodes a putative serine/threonine kinase homologous to mammalian calmodulin kinases. At the C terminus of Srk1 is a putative MAPK binding motif similar to that in the p38 substrates, MAPK-activated protein kinases 2 and 3. Indeed, we find that Srk1 is present in a complex with the Sty1 MAPK and is directly phosphorylated by Sty1. Furthermore, upon stress, Srk1 translocates from the cytoplasm to the nucleus in a process that is dependent on the Sty1 MAPK. Finally, we show that Srk1 has a role in regulating meiosis in fission yeast; following nitrogen limitation, srk1(-) cells enter meiosis significantly faster than wild-type cells and overexpression of srk1(+) inhibits the nitrogen starvation-induced arrest in G(1).

Distinct roles for glutathione S-transferases in the oxidative stress response in Schizosaccharomyces pombe.

We have identified three genes, gst1(+), gst2(+), and gst3(+), encoding theta-class glutathione S-transferases (GSTs) in Schizosaccharomyces pombe. The gst1(+) and gst2(+) genes encode closely related proteins (79% identical). Our analysis suggests that Gst1, Gst2, and Gst3 all have GST activity with the substrate 1-chloro-2,4-dinitrobenzene and that Gst3 has glutathione peroxidase activity. Although Gst1 and Gst2 have no detectable peroxidase activity, all three gst genes are required for normal cellular resistance to peroxides. In contrast, each mutant is more resistant to diamide than wild-type cells. The gst1Delta, gst2Delta, and gst3Delta mutants are also more sensitive to fluconazole, suggesting that GSTs may be involved in anti-fungal drug detoxification. Both gst2(+) and gst3(+) mRNA levels increase in stationary phase, and all three gst genes are induced by hydrogen peroxide. Indeed, gst1(+), gst2(+), and gst3(+) are regulated by the stress-activated protein kinase Sty1. The Gst1 and Gst2 proteins are distributed throughout the cell and can form homodimers and Gst1-Gst2 heterodimers. In contrast, Gst3 is excluded from the nucleus and forms homodimers but not complexes with either Gst1 or Gst2. Collectively, our data suggest that GSTs have separate and overlapping roles in oxidative stress and drug responses in fission yeast.

Distinct regulatory proteins control the graded transcriptional response to increasing H(2)O(2) levels in fission yeast Schizosaccharomyces pombe.

The signaling pathways that sense adverse stimuli and communicate with the nucleus to initiate appropriate changes in gene expression are central to the cellular stress response. Herein, we have characterized the role of the Sty1 (Spc1) stress-activated mitogen-activated protein kinase pathway, and the Pap1 and Atf1 transcription factors, in regulating the response to H(2)O(2) in the fission yeast Schizosaccharomyces pombe. We find that H(2)O(2) activates the Sty1 pathway in a dose-dependent manner via at least two sensing mechanisms. At relatively low levels of H(2)O(2), a two component-signaling pathway, which feeds into either of the two stress-activated mitogen-activated protein kinase kinase kinases Wak1 or Win1, regulates Sty1 phosphorylation. In contrast, at high levels of H(2)O(2), Sty1 activation is controlled predominantly by a two-component independent mechanism and requires the function of both Wak1 and Win1. Individual transcription factors were also found to function within a limited range of H(2)O(2) concentrations. Pap1 activates target genes primarily in response to low levels of H(2)O(2), whereas Atf1 primarily controls the transcriptional response to high concentrations of H(2)O(2). Our results demonstrate that S. pombe uses a combination of stress-responsive regulatory proteins to gauge and effect the appropriate transcriptional response to increasing concentrations of H(2)O(2).