J. Vicente-Soler

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

Abstract Heat sensitivity at 48°C was determined in log-phase cultures of control and pka1-disrupted cells of the fission yeast Schizosaccharomyces pombe grown at 25°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°C and then shifted to 37°C they acquired thermo-tolerance to a subsequent treatment at 48°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.

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 β(1→3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids. The system also displayed weak chitinase and β(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‐β(1→3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells. We propose that protease acts in synergy with β(1→3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis.

Glucose-induced, cyclic-AMP-independent signalling pathway for activation of neutral trehalase in the fission yeast Schizosaccharomyces pombe

Summary: The addition of glucose to derepressed cells of Schizosaccharomyces pombe provokes a cAMP signal and activation of the cytoplasmic neutral trehalase. This transduction pathway does not require functional RAS protein since RAS1-disrupted cells exhibited a glucose response similar to that shown by control cells. Treatment of activated trehalase by alkaline phosphatase resulted in enzyme deactivation suggesting that trehalase may be modulated in vivo by reversible phosphorylation through cAMP-dependent protein kinase (PKA1). However, the addition of glucose to derepressed growing cells of Schiz. pombe lacking the catalytic subunit of protein kinase A (Δpka1::URA4+ strains) induced stimulation of trehalase as well as phosphorylation of the enzyme protein. This glucose-induced response was absent in PKA1-deficient cells from resting cultures. Addition of exogenous cAMP activated trehalase in normal growing cells but failed to produce any effect on trehalase in PKA1-disrupted growing cells. These results confirm the occurrence of a PKA1-dependent pathway for trehalase activation and imply the existence of another glucose-induced phosphorylation pathway capable of activating trehalase during growth by a distinct, cAMP-independent protein kinase. At least one of the upstream components playing a role in the transduction of this alternative signal is either absent or inactive in cells from stationary phase and sporulated cultures. Cells harbouring the disrupted PKA1 gene responded also to a heat-shock signal by increasing trehalase activity, thus revealing that this enzyme may be a target common to various signalling pathways in the fission yeast.

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.

Activation of cytoplasmic trehalase by cyclic-AMP-dependent and cyclic-AMP-independent signalling pathways in the yeast Candida utilis

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)

Activation of neutral trehalase by glucose and nitrogen source in Schizosaccharomyces pombe strains deficient in cAMP-dependent protein kinase activity

Schizosaccharomyces pombe cells carrying a disruption in the PKAI 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.

Lack of correlation between trehalase activation and trehalose-6 phosphate synthase deactivation in cAMP-altered mutants of Saccharomyces cerevisiae.

The rise in cAMP level that follows the addition of glucose or 2,4-dinitrophenol (DNP) to stationaryphase cells of Saccharomyces cerevisiae was accompanied by a marked activation of trehalase (3-fold increase) and a concomitant deactivation of trehalose-6 phosphate synthase (50% of the basal levels). In glucose-grown exponential cells, which are deficient in glucose-induced cAMP signalling, the addition of glucose also prompted a decrease in trehalose-6 phosphate synthase, but had no effect on trehalase activity. Mutants defective in the RAS-adenylate cyclase pathway (ras1 ras2 bcy1 strain), as well as mutants containing greatly reduced protein kinase activity either cAMP-dependent (tpkw1BCY1 strains) or cAMP-independent (tpk1w1bcy1 strains), were unable to show glucose- or DNP-induced trehalase activation but still displayed a clear decrease in trehalose-6 phosphate synthase activity upon addition of these compounds. These data suggest that the activity of trehalose-6 phosphate synthase, as opposed to that of trehalase, is not controlled by the cAMP signalling pathway “in vivo”. Trehalose-6 phosphate synthase was competitively inhibited by glucose (Ki=15 mM) and resulted unaffected by ATP in assays performed “in vitro”.

Trehalase activation induced by nutrients and metabolic inhibitors in Zygosaccharomyces rouxii

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.

The Cornerstone of Nucleic Acid-Affecting Antibiotics in Bacteria

Relatively few new antibiotics targeted against nucleic acids have been developed in the last 50 years. Rifamycins and a wide group of related microbial compounds block RNA synthesis by specific inhibition of bacterial RNA polymerase without interacting with mammalian analog enzymes. Others, such as actinomycins or doxorubicin, interfere with transcription in both bacterial and mammalian systems and may be used as antitumor drugs because the fast growth favors a higher percentage of death in malignant cells respect to normal counterparts. Another group of antibiotics interferes with DNA synthesis by acting on the bacterial DNA gyrase involved in the mechanism of replication of closed-circular DNA. Most members of this group, including aminocoumarin antibiotics, work by binding to the ATPase active site located on one subunit of the gyrase enzyme. Moreover, antibiotics effective against mammalian topoisomerases or able to prevent normal DNA distribution into daughter cells are being also used as antineoplastic drugs. In spite of the wide variety of mechanisms classically invoked to explain the primary action of antibacterial drugs, a new view is emerging whereby the killing damage behind all major classes of antibiotics appears to stem from the generation of destructive molecules that fatally damage nucleic acids through a long chain of cellular events.