Solubilization and characterization of a cell wall-bound trehalase from ascospores of the fission yeast Schizosaccharomyces pombe.

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.

Different roles for the stress-activated protein kinase pathway in the regulation of trehalose metabolism in Schizosaccharomyces pombe.

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.

Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe.

Spores from Schizosaccharomyces pombe contain neutral and acid trehalases. When spores from strains disrupted for ntp1(+), which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores. Further outgrowth was also reduced. Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination. Inhibition by phloridzin of the sporulation-specific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores. These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process.

Characterization of tpp1(+) as encoding a main trehalose-6P phosphatase in the fission yeast Schizosaccharomyces pombe.

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.

Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells.

Growth of Micromonospora chalcea on a defined medium containing laminarin as the sole carbon source induced the production of an extracellular enzyme system capable of lysing cells of various yeast species. Production of the lytic enzyme system was repressed by glucose. Incubation of sensitive cells with the active component enzymes of the lytic system produced protoplasts in high yield. Analysis of the enzyme composition indicated that beta(1-->3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids. The system also displayed weak chitinase and beta(1-->6) glucanase activities whilst devoid of mannanase activity. Our observations suggest that the glucan supporting the cell wall framework of susceptible yeast cells is not directly accessible to the purified endo-beta(1-->3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells. We propose that protease acts in synergy with beta(1-->3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis.

Presence of non-suppressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations.

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.

Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.

Analysis of the ntp1+ gene, encoding neutral trehalase in the fission yeast Schizosaccharomyces pombe.

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.

Trehalose-6P synthase is essential for trehalase activation triggered by glucose, nitrogen source or heat shock, but not by osmostress, in Schizosaccharomyces pombe.

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.

Characterization of mutants devoid of neutral trehalase activity in the fission yeast Schizosaccharomyces pombe: partial protection from heat shock and high-salt stress.

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.

Enhancement of neutral trehalase activity by oxidative stress in the fission yeast Schizosaccharomyces pombe.

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.

Altered processing of precursor transcripts and increased levels of the subunit I of mitochondrial cytochrome c oxidase in Syrian hamster fetal cells initiated with ionizing radiation.

Treatment of Syrian hamster fetal cells (SHFC) with ionizing radiation resulted in the establishment of 21 transformed cell lines. Relative to unirradiated controls, cells from early post-irradiation passages (p.3) showed marked morphologic alterations, increased growth rate and extended life span, and they were contact-inhibited and not tumorigenic in nude mice, although they became tumorigenic after extended passaging in culture (p. > 30). Differential mRNA display analyses of normal cells (84-3) and radiation-initiated cell lines at early passage showed that the latter contained increased steady-state levels of the precursor (4-fold) and mature (1.7-fold) transcripts of the mitochondrial (mt) gene encoding the subunit I of cytochrome c oxidase (CO I). These molecular alterations were consistently observed in 57% of the irradiated (HDR) cell lines, and were stably maintained during continuous passaging (p. > 50). Further analyses of one of these cell lines (HDR-3) demonstrated that the accumulation of CO I precursor transcripts was the result of mRNA stabilization and increased replication and/or amplification of the mt DNA. Radiation-initiated cells contained elevated levels of the CO I protein, showed a 75% reduction in cytochrome c oxidase (CO) activity, and a 5-fold increase in the concentration of hydrogen peroxide secreted into their culture medium compared with cells with no alterations in CO I mRNA processing. Our findings suggest that alterations in mt CO I processing may play a role in the neoplastic conversion of mammalian cells by ionizing radiation.

Protein kinase Sck1 is involved in trehalase activation by glucose and nitrogen source in the fission yeast Schizosaccharomyces pombe.

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.

Osmo-stress-induced changes in neutral trehalase activity of the fission yeast Schizosaccharomyces pombe.

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.

Heat-shock response in Schizosaccharomyces pombe cells lacking cyclic AMP-dependent phosphorylation.

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.

Distribution of the trehalase activation response and the regulatory trehalase gene among yeast species.

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.

Tissue-specific expression, evolutionary conservation and localization of the cph proto-oncogene on Syrian hamster chromosome X.

Treatment of Syrian hamster embryo fibroblasts with a single dose of 3-methylcholanthrene caused the activation of the transforming potential of cellular sequences (Notario et al, Oncogene 5: 1425-1430, 1990), which were subsequently isolated by cosmid rescue techniques, and further identified as a novel oncogene, termed cph because of its involvement in the carcinogenic progression of hamster embryo cells (Velasco et al, Oncogene 9: 2065-2069, 1994). We have analysed the expression of the cph proto-oncogene in adult Syrian hamster tissues by northern hybridization using cph-specific genomic probes. The three cph transcripts expressed in normal and neoplastic Syrian hamster embryo cells in culture (5.0, 3.5 and 2.0 kb) were also present in most adult tissues, although different mRNA species, most likely resulting from alternative splicing events, were expressed in testes. The highest steady-state level of cph mRNA was found in kidney, whereas cph expression was nearly undetectable in skin and skeletal muscle. Southern blot analyses of DNAs from other eucaryotic organisms were performed under moderate stringency conditions with a Syrian hamster-specific cph probe. Discrete cph-hybridizing sequences were present in genomes from yeast to mammalian species, including humans, thus demonstrating that cph is a highly conserved gene in eucaryotic evolution. Using fluorescence in situ hybridization (FISH), we have determined also the chromosomal localization of the cph proto-oncogene in the hamster genome. FISH experiments demonstrated that cph is a single copy gene, localized on the euchromatic short arm of the X chromosome, at region Xpa7. Because chromosome X is frequently involved in structural alterations in neoplastic Syrian hamster cells transformed by chemical carcinogens and oncogenic viruses, the localization of the cph locus on this chromosome supports the notion that the cph oncogene plays a role in the malignant conversion of chemically transformed hamster fibroblasts. The wide range of tissue-specific expression and species-specific distribution of cph strongly suggest that the normal function of the cph protein product(s) may be essential for metabolic processes involved in the regulation of cell proliferation and survival.

Inhibition by polyols of the heat-shock-induced activation of trehalase in the yeast Zygosaccharomyces rouxii.

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.

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger.

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.