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Department of Genetics and Microbiology, Facultad de Biología, University of Murcia, 30071 Murcia, Spain
Correspondence
M. Gacto
maga{at}um.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Studies in vitro and in vivo have confirmed the exceptional properties of this sugar in protecting yeast cells under extreme conditions (Crowe et al., 1984; Singer & Lindquist, 1998). In the fission yeast Schizosaccharomyces pombe synthesis of trehalose is a two-step process that includes the intermediate synthesis of trehalose 6-phosphate (T6P) by T6P synthase, encoded by the tps1+ gene (Blázquez et al., 1994), and its subsequent dephosphorylation to trehalose by T6P phosphatase, encoded by the tpp1+ gene (Franco et al., 2000). On the other hand, hydrolysis of trehalose to glucose is catalysed by the enzyme neutral trehalase, encoded by the ntp1+ gene (Soto et al., 1998).
Biosynthesis and mobilization of trehalose are subject to various regulatory controls. The stress-activated protein kinase (SAPK) pathway, a member of the mitogen-activated protein kinase (MAPK) cascades originally described in metazoans, transduces signals to the nucleus, resulting in new patterns of gene expression, and is critical for the sensing and response of S. pombe cells to a variety of changes in the external environment (Warbrick & Fantes, 1991; Millar et al., 1995; Shiozaki & Russell, 1995; Kato et al., 1996). The central element of the SAPK cascade in S. pombe is the MAPK Sty1p (also known as Spc1p or Phh1p), which is highly homologous to mammalian p38 kinase and becomes activated by a similar range of stresses (Millar et al., 1995; Shiozaki & Russell, 1995; Degols et al., 1996). Different transcription factors function downstream of the Sty1p MAPK cascade, among which Atf1p, Pcr1p and Pap1p have been characterized extensively (Wilkinson et al., 1996; Watanabe & Yamamoto, 1996; Toone et al., 1998). It has been shown previously that the mRNA level of ntp1+ and tps1+ genes rises when S. pombe cells undergo thermal, osmotic or oxidative stresses (Degols et al., 1996; Fernández et al., 1997a, 1998; Cansado et al., 1998; Soto et al., 1998) and that this increase appears to be under the control of the SAPK pathway, since expression of both genes is severely impaired in S. pombe strains deleted in the sty1+ gene (Degols et al., 1996; Fernández et al., 1998; Soto et al., 1998). To assess further the extent of this regulatory control in the stress-induced expression of ntp1+ and tps1+ we decided to analyse the role of the transcriptional factors reported to be the downstream targets of the Sty1p MAPK. In the course of these studies we also revealed a role for Sty1p in the post-transcriptional modulation of trehalase mediated by Pka1p/Sck1p and in the activity of T6P synthase upon stress.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. They were routinely grown with shaking at 28 °C in YES medium (Moreno et al., 1991) supplemented with adenine, leucine, histidine or uracil (100 mg l-1) depending on the requirements for each particular strain. Solid media were made by the addition of 2 % (w/v) bacto-agar.
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Stress treatments, RNA isolation and hybridization.
Yeast cultures grown to an OD600 of 0·7–1 at 28 °C were subjected to either saline osmotic stress (0·75 M NaCl) or oxidative stress (0·75 or 5 mM H2O2). At different times, the cells from 30 ml culture were collected and harvested by centrifugation at 4 °C. Total RNA preparations from cold-shocked strains were obtained as described by Moreno et al. (1991) and resolved through 1·5 % agarose-formaldehyde gels. Northern (RNA) hybridization analyses were performed as described by Soto et al. (1998). Probes for tps1+ and ntp1+ were prepared as reported previously (Fernández et al., 1997a; Cansado et al., 1998). An approximately 900 bp fragment of the leu1+ gene was amplified by PCR (Cansado et al., 1998) and used to probe for leu1+ mRNA as an internal standard for the amount of RNA loaded in each lane. To establish quantitative conclusions, the level of mRNAs was quantified in a Phosphorimager (Molecular Dynamics) and compared with the internal control (leu1+ mRNA).
Site-directed mutagenesis at the ntp1+ promoter and S. pombe transformation.
The mutation at the putative cAMP response (CRE) site (consensus sequence TGACGTAG at position -567) in the ntp1+ promoter was created by the overlap extension method with the use of PCR (Higuchi et al., 1988). Two separate amplification reactions were performed with plasmid pBura-ntp1+ as template with the use of a first pair of primers, PRO52 (5'-TCCGCTCGAGATCGGTTAGTTCAGAGTC-3'; the XhoI site is underlined) and ATFM-3 (5'-TTACTCAATGAGGTAGTCTACC-3'; the nucleotide substitution is indicated in bold type), and a second pair of primers, NTP3M (5'-ACTGGCATCGATTCTTCGAGT-3'; the ClaI site is underlined) and ATFM-5 (5'-GGTAGACTACCTCATTGAGTAA-3'; the nucleotide substitution is indicated in bold type). The two PCR products were purified by agarose gel electrophoresis, mixed and subjected again to PCR with primers PRO52 and NTP3M. PRO52 and NTP3M hybridize at positions -1092 to -1075 in the ntp1+ promoter and +541 to +561 in the ntp1+ ORF, respectively. The resultant 1·7 kbp fragment was digested with XhoI and ClaI and cloned into pBura4-ntp1+, creating plasmid pBura4-ntp1+(CRE), which contains the ntp1+ promoter mutated in the putative CRE-binding site plus the complete ntp1+ ORF flanked by ura4+ sequences.
Plasmids pBura4-ntp1+ and pBura4-ntp1+(CRE) were digested with XbaI and KpnI, and the Pntp1+-ntp1+ and Pntp1+(CRE)-ntp1+ constructs were integrated in single copy into the ura4+ locus in S. pombe strain MMT3 by transforming cells to 5-fluoroorotic acid resistance as described by Boeke et al. (1984). The homologous integration of the constructs into ura4+ was verified by Southern blot analysis.
Enzyme assays, trehalase activation and trehalose content.
Trehalase activity was assayed after cell breakage as described by Carrillo et al. (1994) and expressed as units (mg protein)-1. Activation of trehalase by oxidative or osmotic shock was carried out as indicated previously (Fernández et al., 1997a, b, 1998). Intracellular trehalose was extracted and estimated as described by Soto et al. (1999). All determinations were repeated at least three times with consistent results. Unless otherwise indicated, representative results are shown.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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sty1 (TK107) strains grown at 28 °C in YES medium and subjected to osmotic (0·75 M NaCl) stress for different times. Total RNA preparations (10 µg) were obtained, resolved through 1·5 % agarose-formaldehyde gels and capillary transferred to nylon membranes. Northern (RNA) hybridization was performed, employing probes for tps1+ or ntp1+ and for leu1+ as internal standard. Wild-type cells displayed similar induction kinetics for both ntp1+ and tps1+ genes upon stress (Fig. 1a). The existence of a similar expression pattern for both genes may appear intriguing, since they encode enzymes with opposite biological functions. However, this observation fits other findings suggesting that both synthesis and hydrolysis of trehalose occur in a coordinated way for this sugar to play a protective role (Singer & Lindquist, 1998). In sty1 cells, the stress-induced expression of these genes was strongly decreased and showed an altered response pattern (Fig. 1b). Thus, the SAPK pathway appears to be critical for the increased expression of ntp1+ and tps1+ genes during saline osmotic stress, although additional SAPK-independent mechanisms regulate gene expression and account for the modulation still observed in strains lacking a functional sty1+ gene.
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; Takeda et al., 1995). Atf1p forms heterodimers with Pcr1p and associates to, and is phosphorylated by Sty1p following different stresses (Shiozaki & Russell, 1996; Wilkinson et al., 1996). Similar to the effects observed in sty1 cells, the loss of Atf1p or Pcr1p produced both a clear delay and a relatively decreased induction of the expression of ntp1+ and tps1+ under osmotic stress that did not increase further even after prolonged periods of time (Fig. 1c, d
). The same results were obtained using the double-deleted atf1 pcr1 strain WSP672 (not shown). These data indicate that Atf1p and Pcr1p transcription factors, likely to be acting as heterodimers, are targets for Sty1p kinase in the regulation of ntp1+ and tps1+ expression in response to osmotic stress. However, the increase in gene expression remaining in atf1 and pcr1 strains of S. pombe suggests that factors other than Atf1p/Pcr1p modulate ntp1+ and tps1+ expression during osmotic stress in a Sty1p-independent manner, which highlights the complexity of the stress-induced transcriptional regulation of trehalose metabolism genes.
Pap1p is a member of the AP-1 family of yeast transcription factors which has been reported to control the expression of several genes protecting against oxidative damage (Toone et al., 1998). Experiments similar to those described above, but performed with strain TP108-3c, indicated that pap1+-deficient cells, contrary to atf1+- or pcr1+-disrupted cells, did not substantially differ from wild-type cells in their expression levels of ntp1+ and tps1+ under osmostress (not shown).
Differential involvement of Atf1p and Pap1p in the induction of ntp1+ and tps1+ expression under oxidative stress
We also analysed the induction of ntp1+ and tps1+ genes in S. pombe during oxidative stress with hydrogen peroxide. Expression of ntp1+ and tps1+ was triggered at low (0·75 mM) and high (5 mM) H2O2 concentrations in wild-type cells, with faster kinetics at low oxidative stimulus (Fig. 2a, b). In the absence of Sty1p MAPK, a smaller but reproducible rise in the expression of ntp1+ and tps1+ was evident at low H2O2 levels, whereas there was no detectable increase at high levels (Fig. 2c, d). These results demonstrate that Sty1p only partially controls ntp1+ and tps1+ expression at low H2O2 concentrations, while it is fully responsible for the induction at a high concentration of oxidative input. Moreover, as compared to wild-type cells, atf1+ disruption (and also pcr1+ deletion; not shown) provoked a decrease in gene expression at both high and low H2O2 concentrations (Fig. 2e, f). The fact that atf1 cells still show a significant H2O2-mediated increase in ntp1+ and tps1+ expression implies that one or several Sty1p-dependent transcription factors are responsible for this effect. One obvious candidate for this additional regulation is Pap1p (Toone et al., 1998). Although Pap1p is not a substrate directly phosphorylated by Sty1p MAPK, the presence of Sty1p is critical to allow its translocation from the cytoplasm to the cell nucleus during oxidative stress (Toone et al., 1998). As indicated in Fig. 2(g, h) disruption of the pap1+ gene resulted in a rather modest increase in ntp1+ and tps1+ expression, as compared to the wild-type strain, upon treatment with 0·75 or 5 mM H2O2. A search for AP-1-binding sites (consensus TTAG/CTA/CA) (Toone & Jones, 1999) resulted in the identification of two potential and identical Pap1p-binding sites on the complementary strand of the ntp1+ promoter at positions -557 and -380 (sequence TGAGTAA), and one site in the tps1+ promoter at position -1350 (sequence TTAGTAA). Simultaneous deletion of atf1+ and pap1+ genes prompted a slight induction at low levels of H2O2, but no increase in ntp1+ and tps1+ expression at high H2O2 concentrations (Fig. 2i, j). As a whole, these results indicate that the Sty1p-regulated Atf1p/Pcr1p and Pap1p transcription factors are entirely responsible for the increase of ntp1+ and tps1+ expression at high H2O2 concentrations, but not at low concentrations. Quinn et al. (2002) demonstrated that transcription of various genes encoding enzymes involved in H2O2 degradation is regulated in a dose-dependent manner, with Pap1p acting mainly at low levels of H2O2 (below 1 mM) and Atf1p primarily controlling the transcriptional response to high concentrations. Our results, however, show that both Atf1p and Pap1p are important for ntp1+ and tps1+ induction at low and high H2O2 concentrations (Fig. 2), although other Sty1p-independent factors also appear to be involved at low concentrations of the inducer. Congruent with this, we have observed some nuclear accumulation of a GFP-Pap1p fusion protein after 90 min incubation with 5 mM H2O2 (not shown). Considering that the function of ntp1+ and tps1+ gene products is not directly related to H2O2 degradation, it is tempting to speculate that the induction of different sets of genes is distinctly modulated by Atf1p and Pap1p depending on the severity of the oxidative input. Because sty1 and other downstream mutants are more sensitive to oxidative stress than wild-type cells it could be argued that decreased expression of ntp1+ and tps1+ in the mutants might be due to impaired transcription and cell death. However, maintained expression of the leu1+ gene under similar oxidative stress conditions gives compelling evidence that this is not the case. Moreover, functional transcription has been reported in sty1 cells at even higher levels of H2O2 (Quinn et al., 2002).
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have recently reported Sty1p- and Atf1p-dependent induction of ntp1+, but not of tps1+, under oxidative conditions. In our hands, however, both genes are expressed above such levels with respect to zero time in a way that is fully dependent on Sty1p at high oxidative input, but only partially dependent on this kinase at low oxidative doses. In addition, the enhanced transcription for ntp1+ and tps1+ partially relies on Atf1p at high and low inducer concentrations and appears to require Pap1p, particularly under strong oxidative conditions.
Role of the CRE motif in ntp1+ gene expression under stress
Atf1p is able to bind to CRE-like elements [CRE consensus sequence TGACGT(C/A)A] (Takeda et al., 1995; Neely & Hoffman, 2000). In our case, a close inspection of the ntp1+ regulatory sequences revealed the existence of a CRE-like element (consensus sequence TGACGTAG) at position -568 to -561 in the ntp1+ promoter, which was identical to the sequence present in the promoter of the fbp1+ gene and identified as a binding site for Atf1p/Pcr1p (Neely & Hoffman, 2000). These data support the existence of an Atf1p/Pcr1p-dependent regulation of ntp1+ expression under stress by direct binding to CRE/CRE-like elements. We confirmed this idea by site-directed mutagenesis of the DNA-binding motif in the ntp1+ promoter region. The effect of this sequence on ntp1+ transcription under stress was studied by constructing strain MVP-12, in which ntp1+ expression is regulated by an endogenous 1·1 kbp promoter carrying a base change (G to C) within the core ACGT sequence of the CRE-like element (see Methods). As shown in Fig. 3, the control strain MVP-10, which contains a wild-type non-mutated 1·1 kbp fragment of the ntp1+ promoter, displayed a pattern of ntp1+ expression under salt and oxidative stress similar to wild-type strains (see Figs 1 and 2), indicating that the main upstream activating sequences involved in the stress-induced expression of ntp1+ are located in this region. A single G to C change at the CRE-like element (strain MVP-12) caused an overall decrease in ntp1+ expression upon osmotic or oxidative stress quite similar to that found in atf1+-disrupted cells under the same conditions (Figs 1 and 2). Hence, the role for Atf1p in the regulation of ntp1+ expression under stress appears to operate through interaction with the CRE-like sequence located in the promoter. In addition to binding to CRE-like sequences, Atf1p is also specifically phosphorylated by Sty1p MAPK under salt and oxidative stresses (Wilkinson et al., 1996). Therefore, it is likely that the enhanced gene expression during salt and oxidative stresses occurs by direct binding of Atf1p in a phosphorylated form to a CRE-like motif at the ntp1+ promoter. In addition, we also detected a putative CRE element (sequence AGACGTA) in the tps1+ promoter at position -180. Although not proved, it is possible that the Atf1p-mediated expression of tps1+ may be regulated in a similar way to ntp1+. This suggestion correlates with the observation that both genes are expressed in parallel under different stresses.
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; Fernández et al., 1997a, b, 1998). We reported previously that cells disrupted in the Sty1p MAPK show a marked reduction in trehalase activity during osmostress or oxidative treatment and suggested a limited de novo synthesis of the enzyme protein under these conditions (Fernández et al., 1997b, 1998). Because Atf1p/Pcr1p and Pap1p transcription factors are the main downstream elements involved in ntp1+ expression under osmotic and oxidative stresses (Figs 1 and 2), it should be expected that the absence of these factors would markedly affect Ntp1p activation upon these stress treatments. To ascertain this assumption, S. pombe wild-type strain and sty1, atf1, pap1 or atf1 pap1 mutant strains were subjected to osmotic or oxidative stress, and neutral trehalase activity was measured at different periods of treatment. Surprisingly, atf1, pap1 and atf1 pap1 cells displayed a wild-type pattern of neutral trehalase activation whereas only sty1 cells were unable to increase neutral trehalase activity in response to high osmolarity (Fig. 4a) or oxidative stress (Fig. 4b). Since activation of trehalase by osmostress or oxidative stress relies on the function of Pka1p and Sck1p protein kinases (Fernández et al., 1997b, 1998), the above results reflect the fact that the relatively low level of trehalase activity in osmotic- and oxidative-stressed sty1 cells is due to a post-translational event rather than to decreased ntp1+ expression. Therefore, Sty1p appears to be needed not only for proper ntp1+ induction in response to these stresses (Figs 1 and 2), but also for modulating the function of Pka1p and Sck1p kinases in the stress-induced activation of neutral trehalase. In this context, loss of sty1+ function appears epistatic over Pka1p and Sck1p.
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). In line with the above results we also analysed the trehalose content in wild-type, sty1, atf1, pap1 and atf1 pap1 cells after osmotic or oxidative treatment as a measure of in vivo T6P synthase activity. As indicated in Fig. 5, the level of trehalose in sty1 cells was greatly reduced as compared to that shown in wild-type or in cells disrupted in the transcription factors. This supports that control by Sty1p also affects trehalose synthesis in a way that is similarly independent of Atf1p and Pap1p. Notably, wis1 cells, which contain undisrupted Sty1p, show the same phenotype as sty1 cells with respect to decreased trehalase activation and trehalose content upon stress (Fernández et al., 1997b, 1998), demonstrating that Sty1p phosphorylation, rather than the presence of the kinase enzyme protein, is needed for normal trehalase activation and trehalose accumulation.
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), exerts its action on the cytoplasmic enzymes trehalase and T6P synthase. This point remains unresolved but the nuclear localization of the SAPK argues against a direct interaction. In the case of trehalase regulation, one possibility is the existence of cross-talking between Sty1p and Pka1p/Sck1p. The SAPK and Pka pathways are known to antagonistically regulate different biological processes in S. pombe (Maeda et al., 1994; Caspari, 1997; Fernández et al., 1997a). At the transcriptional level, for example, both pathways regulate fbp1+ expression by favouring (SAPK) or inhibiting (Pka) the binding of the Atf1p/Pcr1p heterodimer to the fbp1+ promoter (Neely & Hoffman, 2000). It is also known that Sty1p affects pka1+ expression upon stress (Chen et al., 2003), suggesting that the inability for normal trehalase activation in cells lacking sty1+ might result in part from decreased transcription of pka1+. However, in contrast to trehalase activity, there is no evidence for regulation of T6P synthase activity by phosphorylation, leading to the idea that the effect of Sty1p on the synthase enzyme occurs by a different pathway independent of Pka1p/Sck1p protein kinases. Although we have yet to define precisely how Sty1p influences the activity of these enzymes, our findings reveal a new regulatory function for this kinase in the metabolism of trehalose during stress.
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ACKNOWLEDGEMENTS |
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for the kind supply of yeast strains. Our thanks to F. Garro for technical assistance. V. P. and A. F. are predoctoral fellows from the Fundación Séneca and the University of Murcia, Spain, respectively. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 3 February 2003;
revised 7 April 2003;
accepted 11 April 2003.
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