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ABSTRACT: The genome of the fission yeast Schizosaccharomyces pombe lacks sequence homologs to ath1 genes coding for acid trehalases in other yeasts or filamentous fungi. However, acid trehalase activity is present at the spore stage in the life cycle of the fission yeast. The enzyme responsible for this activity behaves as a surface enzyme covalently linked to the spore cell walls in both wild-type and ntp1 mutant strains devoid of neutral trehalase. Lytic treatment of particulated cell wall fractions allowed the solubilization of the enzyme into an active form. We have characterized this soluble enzyme and found that its kinetic parameters, optimum pH and temperature, thermal denaturation and salt responses are closely similar to other conventional acid trehalases. Hence, this rather unusual enzyme can be recognized as acid trehalase by its biochemical properties although it does not share genetic homology with other known acid trehalases. The potential role of such acid trehalase in the mobilization of trehalose is discussed.
Microbiological Research 05/2007; 164(3):304-11. · 2.31 Impact Factor
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ABSTRACT: The Wis1p-Sty1p mitogen-activated protein kinase cascade is a major signalling system in the fission yeast Schizosaccharomyces pombe for a wide range of stress responses. It is known that trehalose functions as a protective metabolite to counteract deleterious effects of environmental stresses. Herein it is reported that the expression of genes related to trehalose metabolism in S. pombe, ntp1(+) (neutral trehalase) and tps1(+) [trehalose-6-phosphate (T6P) synthase], is partially regulated by the Sty1p kinase under salt-induced osmotic stress and conditions of slight oxidative stress and is fully dependent on this kinase under severe oxidative stress. This control is carried out through transcription factors Atf1p/Pcr1p during osmotic stress and through Pap1p during exposure to low levels of oxidative stress. However, all three transcription factors are needed for gene expression under conditions of extreme oxidative stress. In addition, a role for Sty1p in the modulation of post-transcriptional activation of trehalase mediated by Pka1p/Sck1p kinases, as well as in the activity of T6P synthase under such stressful conditions has been demonstrated. These results reveal a novel dual action of the Wis1p-Sty1p pathway in the regulation of trehalose metabolism in fission yeast.
Microbiology 08/2003; 149(Pt 7):1745-52. · 3.06 Impact Factor
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ABSTRACT: We have characterized an open reading frame of 2,454 bp on chromosome I of Schizosaccharomyces pombe as the gene encoding trehalose-6P phosphatase (tpp1(+)). Disruption of tpp1(+) caused in vivo accumulation of trehalose-6P upon heat shock and prevented cell growth at 37 to 40 degrees C. Accumulation of trehalose-6P in cells bearing a chromosomal disruption of the tpp1(+) gene and containing a plasmid with tpp1(+) under the control of the thiamine-repressible promotor correlated with tpp1(+) repression. The level of tpp1(+) mRNA rose upon heat shock, osmostress, or oxidative stress and was negatively controlled by cyclic AMP-dependent protein kinase activity. Expression of tpp1(+) during oxidative or osmotic stress, but not during heat shock, was under positive control by the wis1-sty1 (equivalent to phh1 and spc1) mitogen-activated protein kinase pathway. Analysis of Tpp1 protein levels suggests that the synthesis of trehalose-6P phosphatase may also be subjected to translational or posttranslational control.
Journal of Bacteriology 11/2000; 182(20):5880-4. · 3.83 Impact Factor
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ABSTRACT: Addition of hydrogen peroxide, menadione, or plumbagin to growing cultures of the fission yeast Schizosaccharomyces pombe increased trehalase activity. The effect was inhibited only slightly in the presence of cycloheximide, indicating that the stimulation of trehalase triggered by oxidative stress is mostly due to posttranscriptional activation. Northern blot analysis of trehalase mRNA level revealed that oxidative stress also induces a moderate rise in transcription of trehalase. Mutants disrupted in genes encoding elements of the mitogen-activated protein kinase (MAPK) cascade showed a reduced increase in trehalase activity upon oxidative challenge, which was coincident with a block in transcription of trehalase. Taken together, the results support the idea that the enhancement of trehalase by oxidative stress is due to enzyme activation (via the Pka1/Sck1 phosphorylation pathway) and induction of trehalase mRNA (via the MAPK signaling pathway). In spite of the trehalase increase, a net accumulation of trehalose was noticed during the oxidative stress.
Fungal Genetics and Biology 12/1998; 25(2):79-86. · 3.74 Impact Factor
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ABSTRACT: We have cloned and sequenced the ntp1+ gene that codes for neutral trehalase in the fission yeast Schizosaccharomyces pombe. The ntp1+ gene product (Ntp1p) showed a 45-55% identity with neutral trehalases from other yeasts at the amino acid sequence level. However, in clear contrast to other neutral yeast trehalases so far characterized (which show two cAMP phospho-sites), only one consensus site for cAMP-dependent protein phosphorylation was found in Ntp1p. Northern blot hybridization experiments demonstrated that the Wis-Phh1/Sty1 MAP kinase cascade regulates ntp1+ expression during osmostress.
Biochimica et Biophysica Acta 12/1998; 1443(1-2):225-9. · 4.66 Impact Factor
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ABSTRACT: Cells of Schizosaccharomyces pombe disrupted in the tps1+ gene, which encodes trehalose-6P synthase, were unable to increase trehalase activity in response to the addition of glucose or nitrogen source. Moreover, in contrast to normal cells, Deltatps1 cells did not increase trehalase activity by heat shock. Overexpression of tps1+ in cells devoid of trehalose-6P synthase restored the ability to increase trehalase after addition of nutrients or by heat shock. In glucose-repressed cells, which are normally refractory to the activation of trehalase by glucose, overexpression of tps1+ enabled the cells to increase trehalase activity upon addition of the sugar. Northern hybridisations were used to determine the level of mRNA for trehalase in normal and Deltatps1 cells. Transcription for trehalase was not significantly altered upon addition of glucose or nitrogen source, but increased markedly in heat-shocked cells even though trehalase activity remained unchanged in Deltatps1 cells. These findings provide evidence for a role of trehalose-6P synthase in the signalling pathway causing post-transcriptional activation of neutral trehalase induced by nutrients or heat shock. However, trehalase increased in Deltatps1 cells under hypertonic conditions suggesting the existence in Schiz. pombe of a distinct regulatory mechanism for enhancement of trehalase, specifically triggered by osmostress.
Biochimica et Biophysica Acta 09/1998; 1381(3):271-8. · 4.66 Impact Factor
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ABSTRACT: Exposure of cells of Schizosaccharomyces pombe to heat shock or osmotic upshift results in an increased level of neutral trehalase activity, which is responsible for hydrolysis of intracellular trehalose. We constructed S. pombe mutants lacking neutral trehalase activity by gene replacement at the newly defined ntp1+ locus. Analysis of these mutants revealed that a twofold increase in trehalose accumulation, enhanced acquired thermoresistance, and marked salt tolerance characterized their ability to grow in liquid and solid media. Analysis of the expression of the trehalase gene under heat shock and osmotic upshift revealed the transcriptional activation of ntp1+ in response to both stresses.
Journal of Bacteriology 04/1998; 180(5):1342-5. · 3.83 Impact Factor
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ABSTRACT: In Saccharomyces cerevisiae and other yeasts the activity of regulatory trehalases increases in response to the addition of glucose and to thermal changes in the extracellular medium. We have performed an screening on the extent of this response among different representative yeast species and the results show that this ability is displayed only by a few members of the Saccharomycetaceae family. However, all yeasts examined contain a gene related to that coding for regulatory trehalase in S. cerevisiae. This finding reveals that the operational distinction between regulatory and nonregulatory trehalase in yeasts is not a property of the enzyme by itself but relays on the expression of accompanying mechanisms able to modulate trehalase activity.
Microbiología (Madrid, Spain) 01/1998; 13(4):481-8.
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ABSTRACT: Trehalase activity is markedly enhanced upon addition of glucose and a nitrogen source to cells of the fission yeast Schizosaccharomyces pombe. This increase corresponds to a post-translational activation of the enzyme, which is controlled by cAMP-dependent and cAMP-independent pathways. Recent work has shown that overexpression of SCK1 in Schiz. pombe is able to suppress mutations that result in reduced Pka1 (cAMP-dependent protein kinase A activity, suggesting that Sck1 (suppressor of loss of cAMP-dependent protein kinase) might be a functional analogue of Pka1 in the fission yeast. Here, an analysis of the possible role of Sck1 in the activation of trehalase triggered by glucose and a nitrogen source is reported in cells that were deficient in either Pka1, Sck1 or both protein kinases. The results showed that, except in repressed cells, Sck1 probably mediates a cAMP-independent activation of trehalase following the signal(s) triggered by glucose and the nitrogen source. The absence of functional Sck1 in depressed cells renders trehalase insensitive to activation by glucose and the nitrogen source even in the presence of Pka1, indicating that the Sck1-dependent, cAMP-independent pathway is the main signalling pathway controlling trehalase activation under derepression conditions. It is proposed that, during the activation of trehalase induced by glucose or a nitrogen source, the cAMP-Pka1 activation pathway previously characterized is to some extent parallel to this newly described one which includes Sck1 as phosphorylating enzyme. Neither of these two pathways, however, plays a key role in the heat-induced increase in trehalase activity.
Microbiology 08/1997; 143 ( Pt 7):2457-63. · 3.06 Impact Factor
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ABSTRACT: Exposure of repressed growing cultures of Schizosaccharomyces pombe to various extracellular concentrations of NaCl, sorbitol or glycerol resulted in a reversible increase in neutral trehalase activity which was maintained while the cells were in the presence of high environmental osmolarity. Treatment of osmo-stress-induced trehalase by phosphatase lead to a decreased activity indicating that the active enzyme is phosphorylated. The stress response following the osmotic shock required protein synthesis and was independent of the cAMP-dependent protein kinase pathway. Cells disrupted for wis] or phh1 (identical to sty1 and spc1), which encode members of the mitogen-activated protein kinase (MAPK) cascade, showed that the osmo-stress-induced increase in trehalase markedly diminished. In contrast, the heat shock-induced increase in trehalase remained unchanged in these cells. Taken together, the data suggest that the elevation of trehalase activity in Schiz. pombe under conditions of high osmolarity is due to de novo synthesis of the enzyme and that this process is modulated through a MAPK signal transduction pathway as part of the physiological response to the osmotic stress. The wisl-phhl MAPK cascade, however, does not appear to form part of the mechanism underlaying the increase in trehalase after heat stress.
Biochimica et Biophysica Acta 07/1997; 1357(1):41-8. · 4.66 Impact Factor
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ABSTRACT: Heat sensitivity at 48 degrees C was determined in log-phase cultures of control and pka1-disrupted cells of the fission yeast Schizosaccharomyces pombe grown at 25 degrees C. Cells devoid of protein kinase A exhibited a considerable heat-shock resistance as compared to control cells. Addition of cAMP to control cells prompted a further decrease in viability during heat shock. This effect was not observed with pka1-disrupted cells, suggesting that cAMP-dependent phosphorylation is involved in modulation of the heat-shock response. When control or pka1-disrupted cells were grown at 25 degrees C and then shifted to 37 degrees C they acquired thermo-tolerance to a subsequent treatment at 48 degrees C both in the absence and in the presence of exogenous cAMP. Inhibition of protein synthesis during the adaptive treatment did not block the development of thermo-tolerance. However, the arrest in translation significantly prevented trehalose accumulation in control cells but only slightly affected trehalose increase in pka1-disrupted cells. These data indicate that heat resistance may be established in growing cells of S. pombe by at least two independent post-translational mechanisms: a decrease in cAMP-dependent protein phosphorylation and a hitherto unknown process which may be independent of trehalose accumulation.
Current Genetics 03/1997; 31(2):112-8. · 2.56 Impact Factor
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ABSTRACT: Trehalase activity was markedly enhanced in Zygosaccharomyces rouxii upon exposure of the cells to a heat shock. The increase in trehalase was independent of rapid changes in the intracellular concentration of cAMP and was not blocked by inhibitors of protein synthesis. Trehalase activated in vivo by heat shock was deactivated in vitro by phosphatase, suggesting that heat stress triggers a cAMP-independent signalling pathway that includes the activation of trehalase by phosphorylation of the enzyme protein. The addition to these cells before heating of either glycerol or other polyols produced a significant decrease in the heat-shock induced activation of trehalase. However, the trehalose content in cells heat-shocked in the presence of polyols did not increase significantly, indicating that these compounds may also influence the synthesis of the disaccharide.
Biochemistry and molecular biology international 03/1996; 38(1):43-50.
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ABSTRACT: Cells of the fission yeast Schizosaccharomyces pombe were permeabilized by treatment with toluene-ethanol. The permeabilized cells lost the bulk of the internal trehalose pool while most of the alkaline phosphatase, invertase, alpha-glucosidase, or neutral trehalase activities located inside the cells remained unaffected. This system was used as an in situ assay to determine the involvement of trehalose in enzyme protection during thermal treatments. The addition of trehalose to suspensions of permeabilized cells resulted in a sugar-dependent thermoprotection of the internal marker enzymes. This approach demonstrates that in whole cells of the fission yeast trehalose plays a physiological role as a protective molecule against thermal denaturation of cellular enzymes.
Canadian Journal of Microbiology 11/1995; 41(10):936-41. · 1.36 Impact Factor
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ABSTRACT: Resting cells of the fission yeast Schizosaccharomyces pombe, suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen tannophilus, the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.
FEMS Microbiology Letters 11/1995; 132(3):229-32. · 2.04 Impact Factor
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ABSTRACT: Schizosaccharomyces pombe cells carrying a disruption in the PKA1 gene, that encodes the catalytic subunit of cAMP-dependent protein kinase (PKA), lacked the glucose- and nitrogen-source-induced activation of trehalase at stationary-phase but rised trehalase activity in response to these compounds during the exponential phase of growth. Treatment by phosphatase of either glucose- or nitrogen-source-activated trehalase resulted in trehalase deactivation suggesting that phosphorylation of the enzyme protein occurs during activation. These data indicate that in growing cells of this yeast the mechanism responsible for the activation of trehalase can be independent of interactions with free catalytic subunits of PKA and related to a signaling pathway involving a type of protein kinase different from PKA.
FEBS Letters 08/1995; 367(3):263-6. · 3.54 Impact Factor
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ABSTRACT: Derepressed cells of Candida utilis suspended in buffer exhibited both a transient cAMP-mediated signal and a marked activation of cytoplasmic trehalase when supplemented with glucose. Nitrogen sources or protein synthesis inhibitors, as well as protonophores or uncouplers, were also able to cause trehalase stimulation in derepressed cells even in the absence of the sugar. The increase in trehalase activity caused by nitrogen sources or protein synthesis inhibitors was not accompanied by changes in cAMP levels. Moreover, acridine orange inhibited both the cAMP signal and the glucose-induced activation of trehalase without affecting the increase in trehalase activity caused by nitrogen sources or protein synthesis inhibitors. These results suggest that cAMP is not involved as second messenger in the signal for trehalase stimulation induced by the latter compounds. By contrast, the addition of glucose to repressed cells suspended in buffer failed to cause the cAMP-mediated glucose signal and sugar-induced trehalase activation. No significant changes in either trehalase activity or cAMP concentration were observed upon addition to these cells of asparagine, cycloheximide, anisomycin or other agents, including protonophores and uncouplers. However, heat treatment of repressed cultures resulted in a moderate increase in trehalase activity with negligible change in cAMP levels, whereas such an effect was not observed in derepressed cultures. The thermally induced increase in trehalase activity was dependent on de novo protein synthesis and required the presence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)
Microbiology 04/1995; 141 ( Pt 3):679-86. · 3.06 Impact Factor
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ABSTRACT: Cells of Zygosaccharomyces rouxii suspended in buffer responded to the addition of both fermentable and nonfermentable sugars by increasing trehalase activity. This response was preceded by a cyclic-AMP (cAMP) signal. In contrast to previous findings in other yeast species, the glucose-induced trehalase activation is not repressed by glucose in these cells. The protonophore 2,4-dinitrophenol also triggered a transient activation of trehalase, but this response was not accompanied by an increase in cAMP. However, nitrogen sources, protein-synthesis inhibitors and the respiratory inhibitor sodium azide did not induce activation of trehalase. Incubation of cell extracts with ATP and cAMP produced an in vitro activation of trehalase suggesting that the enzyme may be stimulated in vivo by phosphorylation. The above results support the existence in Z. rouxii of both cAMP-dependent and cAMP-independent phosphorylation pathways which share trehalase as a molecular target. These activation pathways are markedly different in many respects from those induced in yeasts like Saccharomyces cerevisiae, Candida utilis, and Schizosaccharomyces pombe suggesting a species-specific design in the signal transduction systems involved in trehalase activation.
Mycological Research.
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ABSTRACT: In the fission yeast Schizosaccharomyces pombe the Wak1p/Win1p-Wis1p-Sty1p stress-activated protein kinase (SAPK) pathway relays environmental signals to the transcriptional machinery and modulates gene expression via a cascade of protein phosphorylation. Cells of S. pombe subjected to cold shock (transfer from 28 degrees C to 15 degrees C) transiently activated the Sty1p mitogen-activated protein kinase (MAPK) by phosphorylation. Induction of this response was completely abolished in cells disrupted in the upstream response regulator Mcs4p. The cold-triggered Sty1p activation was partially dependent on Wak1p MAPKKK and fully dependent on Wis1p MAPKK suggesting that the signal transmission follows a branched pathway, with the redundant MAPKKK Win1p as alternative transducer to Wis1p, which subsequently activates the effector Sty1p MAPK. Also, the bZIP transcription factor Atf1p became phosphorylated in a Sty1p-dependent way during the cold shock and this phosphorylation was found responsible for the increased expression of gpd1+, ctt1+, tps1+ and ntp1+ genes. Strains deleted in transcription factors Atf1p or Pcr1p were unable to grow upon incubation at low temperature whereas those disrupted in any member of the SAPK pathway were able to do so. These data reveal that S. pombe responds to cold by inducing the SAPK pathway. However, such activation is dispensable for yeast growth in cold conditions, supporting that the presence of Atf1/Pcr1 heterodimers, rather than an operative SAPK pathway, is critical to ensure yeast growth at low temperature by an as yet undefined mechanism.
Eur J Biochem. 269(20):5056-65.
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ABSTRACT: In the fission yeast Schizosaccharomyces pombe the Wak1p/Win1p-Wis1p-Sty1p stress-activated protein kinase (SAPK) pathway relays environmental signals to the transcriptional machinery and modulates gene expression via a cascade of protein phosphorylation. Cells of S. pombe subjected to cold shock (transfer from 28 degrees C to 15 degrees C) transiently activated the Sty1p mitogen-activated protein kinase (MAPK) by phosphorylation. Induction of this response was completely abolished in cells disrupted in the upstream response regulator Mcs4p. The cold-triggered Sty1p activation was partially dependent on Wak1p MAPKKK and fully dependent on Wis1p MAPKK suggesting that the signal transmission follows a branched pathway, with the redundant MAPKKK Win1p as alternative transducer to Wis1p, which subsequently activates the effector Sty1p MAPK. Also, the bZIP transcription factor Atf1p became phosphorylated in a Sty1p-dependent way during the cold shock and this phosphorylation was found responsible for the increased expression of gpd1+, ctt1+, tps1+ and ntp1+ genes. Strains deleted in transcription factors Atf1p or Pcr1p were unable to grow upon incubation at low temperature whereas those disrupted in any member of the SAPK pathway were able to do so. These data reveal that S. pombe responds to cold by inducing the SAPK pathway. However, such activation is dispensable for yeast growth in cold conditions, supporting that the presence of Atf1/Pcr1 heterodimers, rather than an operative SAPK pathway, is critical to ensure yeast growth at low temperature by an as yet undefined mechanism.
Eur J Biochem. 269(20):5056-65.
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ABSTRACT: In the fission yeast Schizosaccharomyces pombe the Wak1p/Win1p-Wis1p-Sty1p stress-activated protein kinase (SAPK) pathway relays environmental signals to the transcriptional machinery and modulates gene expression via a cascade of protein phosphorylation. Cells of S. pombe subjected to cold shock (transfer from 28 degrees C to 15 degrees C) transiently activated the Sty1p mitogen-activated protein kinase (MAPK) by phosphorylation. Induction of this response was completely abolished in cells disrupted in the upstream response regulator Mcs4p. The cold-triggered Sty1p activation was partially dependent on Wak1p MAPKKK and fully dependent on Wis1p MAPKK suggesting that the signal transmission follows a branched pathway, with the redundant MAPKKK Win1p as alternative transducer to Wis1p, which subsequently activates the effector Sty1p MAPK. Also, the bZIP transcription factor Atf1p became phosphorylated in a Sty1p-dependent way during the cold shock and this phosphorylation was found responsible for the increased expression of gpd1+, ctt1+, tps1+ and ntp1+ genes. Strains deleted in transcription factors Atf1p or Pcr1p were unable to grow upon incubation at low temperature whereas those disrupted in any member of the SAPK pathway were able to do so. These data reveal that S. pombe responds to cold by inducing the SAPK pathway. However, such activation is dispensable for yeast growth in cold conditions, supporting that the presence of Atf1/Pcr1 heterodimers, rather than an operative SAPK pathway, is critical to ensure yeast growth at low temperature by an as yet undefined mechanism.
Eur J Biochem. 269(20):5056-65.
