Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast

In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.     

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

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 iannophilus , 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.     

Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with ¹²⁵I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.     

Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae

The trehalose-degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen-starved glucose-repressed yeast (Saccharomyces cerevisiae) cells. Trehalase activation is mediated by phosphorylation. Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles. Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro. To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22. Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source. Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive. After the addition of glucose or of a nitrogen source to starved cells, Pph22-overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress-regulated element-controlled genes. Deletion of the SCH9 gene encoding a protein kinase involved in nutrient-induced signal transduction restored glucose-induced trehalase activation in Pph22-overexpressing cells. Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient-induced signal transduction pathways in the absence of nutrients.     

Reversibility characteristics of glucose-induced trehalase activation associated with the breaking of dormancy in yeast ascospores

The breaking of dormancy in yeast ascospores by addition of glucose is associated with a sudden tenfold increase in the activity of trehalase. The rapid activation of trehalase is followed by a slower inactivation process which is greatly retarded in the presence of nitrogen sources and cycloheximide. When glucose is washed away from the spores after some time and the spores resuspended in glucose-free medium, the trehalase activity decreases sharply. Subsequent addition of new glucose partially reactivates the enzyme. The extent of reactivation decreases further with each subsequent activation/inactivation step. Changing the duration of the inactivation periods has no effect on this diminution of the reversibility. However, prolonging the duration of the activation step speeds up the loss of reversibility. On the other hand, addition of a nitrogen source or cycloheximide completely prevents the loss of reversibility. The results of the reversibility studies are in agreement with the phosphorylation mechanism which has been proposed for the underlying molecular process of trehalase activation. Apparently, they are also in agreement with proteolytic breakdown being responsible for the inactivation of trehalase after its initial activation. However, the effect of cycloheximide and nitrogen sources, at least in ascospores, does not appear to be due to inhibition or repression of protease synthesis, respectively, since the addition in the presence of glucose of a nitrogen source after trehalase inactivation immediately reactivates the enzyme completely.     

Molecular cloning of the neutral trehalase gene from Kluyveromyces lactis and the distinction between neutral and acid trehalases

We cloned the Kluyveromyces lactis KlNTH1 gene, which encodes neutral trehalase. It showed 65.2% and 68.5% identity at nucleotide and amino acid sequence level, respectively, with the Saccharomyces cerevisiae NTH1 gene. Multiple alignment of the predicted trehalase protein sequences from yeasts, bacteria, insects, and mammals revealed two major domains of conservation. Only the yeast trehalases displayed in an N-terminal extension two consensus sites for cAMP-dependent protein phosphorylation and a putative Ca²⁺-binding sequence. Gene disruption of the KlNTH1 gene abolished neutral trehalase activity and clearly revealed a trehalase activity with an acid pH optimum. It also resulted in a high constitutive trehalose level. Expression of the KlNTH1 gene in an S. cerevisiae nth1Δ mutant resulted in rapid activation of the heterologous trehalase upon addition of glucose to cells growing on a nonfermentable carbon source and upon addition of a nitrogen source to cells starved for nitrogen in a glucose-containing medium. In K. lactis, the same responses were observed except that rapid activation by glucose was observed only in early-exponential-phase cells. Inactivation of K. lactis neutral trehalase by alkaline phosphatase and activation by cAMP in cell extracts are consistent with control of the enzyme by cAMP-dependent protein phosphorylation. Received: 19 March 1996 / Accepted: 15 October 1996     

Trehalose mobilization during early germination ofPilobolus longipes sporangiospores

Pilobolus longipes spores were activated by either exogenous glucose or 6-deoxyglucose. Trehalose content of glucose-activated spores increased and the substrate for trehalose synthesis was exogenous glucose. Addition of 6-deoxyglucose resulted in mobilization of trehalose, with about 20% of the reserve being consumed in the first hour. Little or no change in trehalase activity occurred during spore activation. Most of the trehalase activity associated with spores could be removed by washing with phosphate buffer. This extracellular enzyme was relatively stable, had a pH optimum of 5.6 and a K_m of about 0.5 mM and was estimated to be 66,000 in molecular weight. The specific activity of the crude enzyme extracts fromP. longipes was not influenced by cAMP, but, under the same conditions, the regulatory trehalase fromSaccharomyces cerevisiae became activated. These experiments indicate that trehalase activity in germinatingP. longipes spores may not be regulated by cAMP-dependent phosphorylation. Instead, the results suggest that trehalose is mobilized by a decompartmentation process.     

Regulation of yeast trehalase by a monocyclic, cyclic AMP-dependent phosphorylation-dephosphorylation cascade system.

Mutation at the GLC1 locus in Saccharomyces cerevisiae resulted in simultaneous deficiencies in glycogen and trehalose accumulation. Extracts of yeast cells containing the glc1 mutation exhibited an abnormally high trehalase activity. This elevated activity was associated with a defective cyclic AMP (cAMP)-dependent monocyclic cascade which, in normal cells, regulates trehalase activity by means of protein phosphorylation and dephosphorylation. Trehalase in extracts of normal cells was largely in a cryptic form which could be activated in vitro by ATP . Mg in the presence of cAMP. Normal extracts also exhibited a correlated cAMP-dependent protein kinase which catalyzed incorporation of label from [gamma-32P]ATP into protamine. In contrast, cAMP had little or no additional activating effect on trehalase or on protamine phosphorylation in extracts of glc1 cells. Similar, unregulated activation of cryptic trehalase was also found in glycogen-deficient strains bearing a second, independently isolated mutant allele, glc1-2. Since trehalase activity was not directly affected by cAMP, the results indicate that the glc1 mutation results in an abnormally active protein kinase which has lost its normal dependence on cAMP. Trehalase in extracts of either normal or mutant cells underwent conversion to a cryptic form in an Mg2+-dependent, fluoride-sensitive reaction. Rates of this reversible reduction of activity were similar in extracts of mutant and normal cells. This same, unregulated protein kinase would act on glycogen synthase, maintaining it in the phosphorylated low-activity D-form. The glc1 mutants provide a novel model system for investigating the in vivo metabolic functions of a specific, cAMP-dependent protein kinase.     

Calcium, asparagine and cAMP are required for ornithine decarboxylase activation in intact Chinese hamster ovary cells.

Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca⁺⁺ and Mg⁺⁺ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity.     

Cyclic-AMP content and trehalase activation in vegetative cells and ascospores of yeast

Addition of glucose to yeast ascospores, glucose-grown vegetative cells from the stationary growth-phase or acetate-grown vegetative cells from the logarithmic growth-phase induces a rapid tenfold increase in the activity of trehalase. Trehalase activation is followed by a period of slow inactivation. It was possible to reverse the inactivation in the presence of glucose in all cell types immediately and completely by subsequent addition of a nitrogen source. This reactivation by nitrogen sources is in disagreement with proteolytic breakdown being responsible for trehalase inactivation in the presence of glucose. The addition of glucose induced in all cell types a rapid transient increase of the cellular cyclic-AMP content. In ascospores the increase of the cyclic-AMP level was about twofold, in glucose-grown stationary-phase vegetative cells four- to fivefold and in acetate-grown vegetative cells about sevenfold. Subsequent addition in the presence of glucose of a nitrogen source caused a new twofold increase of the cyclic-AMP level in ascospores. In the other two cell types however addition of a nitrogen source after the initial transient increase of the cyclic-AMP level did not produce a significant new increase. Although the data obtained for ascospores at first seemed to confirm the crucial role of the increase in the cyclic-AMP level for the activation of trehalase, the data obtained afterwards for vegetative cells indicated that it is possible to activate trehalase in yeast without a concomitant increase of the total cellular cyclic-AMP content.Abbreviations Mes 4-Morpholineethanesulfonic acid - Tris tris(hydroxymethyl)-aminomethane     

Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.     

Ammonium permease-based sensing mechanism for rapid ammonium activation of the protein kinase A pathway in yeast

In the yeast Saccharomyces cerevisiae starvation for nitrogen on a glucose-containing medium causes entrance into G0 and downregulation of all targets of the PKA pathway. Re-addition of a nitrogen source in the presence of glucose causes rapid activation of trehalase and other PKA targets. Trehalase activation upon ammonium re-supplementation is dependent on PKA activity, but not on its regulatory subunit nor is it associated with an increase in cAMP. In nitrogen-starved cells, ammonium transport and activation of trehalase are most active in strains expressing either the Mep2 or Mep1 ammonium permease, as opposed to Mep3. The non-metabolizable ammonium analogue, methylamine, also triggers activation of trehalase when transported by Mep2 but not when taken up by diffusion. Inhibition of ammonium incorporation into metabolism did not prevent signalling. Extensive site-directed mutagenesis of Mep2 showed that transport and signalling were generally affected in a similar way, although they could be separated partially by specific mutations. Our results suggest an ammonium permease-based sensing mechanism for rapid activation of the PKA pathway. Mutagenesis of Asn246 to Ala in Mep2 abolished transport and signalling with methylamine but had no effect with ammonium. The plant AtAmt1;1, AtAmt1;2, AtAmt1;3 and AtAmt2 ammonium transporters sustained transport and trehalase activation to different extents. Specific mutations in Mep2 affected the activation of trehalase differently from induction of pseudohyphal differentiation. We also show that Mep permease involvement in PKA control is different from their role in haploid invasive growth, in which Mep1 sustains and Mep2 inhibits, in a way independent of the ammonium level in the medium.     

A method to study the rapid phosphorylation-related modulation of neutral trehalase activity by temperature shifts in yeast

Heat shock enhanced the synthesis of neutral trehalase in growing cells of Saccharomyces cerevisiae, as detected by immunological methods. The activity of the enzyme was measured in extracts obtained by two methods: cells were either harvested by filtration and subsequent disruption with glass beads at 0-4 degrees C or immediately frozen with liquid nitrogen in the presence of Triton X-100, followed by thawing at 30 degrees C. The first procedure yielded artificially high activities of neutral trehalase in heat-shocked cells due to rapid (less than 1 min) activation during handling at 4 degrees C before homogenization. Activity of the enzyme in these homogenates decreased 75-90% upon a treatment with alkaline phosphatase, indicating that activation was due to phosphorylation. The second procedure yielded low trehalase activities for heat-shock treated cells, much higher activities for cells shifted back for some seconds to 27 degrees C, and very low activities again for cells shifted from 27 to 40 degrees C for a second time. Thus, permeabilization of cells following rapid freezing in Triton X-100 is a method of choice to study post-translational modulation of the neutral trehalase of S. cerevisiae by phosphorylation and dephosphorylation.     

Presence of two trehalose-6-phosphate synthase enzymes in Candida utilis

Abstract Total trehalose-6-phosphate synthase activity decreased in cell extracts from Candida utilis under conditions inducing activation of the regulatory trehalase by protein kinase catalysed phosphorylation. The synthase activity was reactivated by treatment with alkaline phosphatase revealing the presence of an enzyme whose activity is inactivated by reversible phosphorylation. The occurrence in the trehalose-6-phosphate synthase complex of a second synthase enzyme whose activity is not controlled by phosphorylation and dephosphorylation was demonstrated following gel filtration of cell extracts. The activity of the isolated enzymes was differently modified in vitro by the presence of alkaline phosphatase, ATP, glucose or protein kinase.     

Biochemical characterization of neutral trehalase activity in Saccharomyces boulardii

Lyophilized cells of the non-pathogenic yeast Saccharomyces boulardii are used in many countries for the treatment of several types of diarrhoea and other gastrointestinal diseases. Although the cells must be viable, their mechanism of action is unknown. The disaccharide trehalose is a protectant against several forms of environmental stress in yeast and is involved in maintaining cell viability. There is no information on the enzymes involved in degradation of trehalose in S. boulardii. The aim of the present study was to characterize trehalase activity in this yeast. Cells of S. boulardii grown in glucose exhibited neutral trehalase activity only in the exponential phase. Acidic trehalase was not detected in glucose medium. Cells grown in trehalose exhibited acid and neutral trehalase activities at all growth stages, particularly in the exponential phase. The optimum pH and temperature values for neutral trehalase activity were determined as 6.5 and 30 °C respectively, the half-life being approximately 3 min at 45 °C. The relative molecular mass of neutral trehalase is 80 kDa and the K _m 6.4 mM (±0.6). Neutral trehalase activity at pH 6.5 was weakly inhibited by 5 mM EDTA and strongly inhibited by ATP, as well as the divalent ions Cu⁺⁺, Fe⁺⁺ and Zn⁺⁺. Enzyme activity was stimulated by Mg⁺⁺ and Ca⁺⁺ only in the absence of cAMP. The presence of cAMP with no ion additions increased activity by 40%. This revised version was published online in July 2006 with corrections to the Cover Date.     

Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores

Incubation of Mucor rouxii sporangiospores in complex medium under aerobic conditions resulted in a transient 20-fold increase in trehalase activity. Maximum activity was reached after 15 min. Simultaneously, the cyclic AMP (cAMP) content increased approximately eightfold, reaching a maximum within 10 min. Increases in trehalase activity and cAMP content were also observed under anaerobic conditions (CO2). The extent of trehalase activation and the changes in cAMP content, during both aerobic and anaerobic incubation, varied with the medium used. Trehalase was activated in vitro by a cAMP- and ATP-dependent process. An even faster activation was obtained when cAMP was replaced by the catalytic subunit of beef heart protein kinase. The coincidence of, and the correlation between, increased cAMP contents and trehalase activities support the involvement of a cAMP-dependent phosphorylation in the in vivo regulation of trehalase activity.     

Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae

Yeast cells starved for inorganic phosphate on a glucose-containing medium arrest growth and enter the resting phase G0. We show that re-addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE-controlled genes and induction of ribosomal protein genes. Phosphate-induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the G-protein coupled receptor Gpr1 and by the glucose-phosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport-independent receptor. The non-metabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate-starved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long-term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose-dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.     

Biochemistry of spore germination in Phycomyces

Abstract The constitutionally dormant spores of Phycomyces blakesleeanus can be activated by heat shock or treatment with several monocarboxylic acids. Activation is followed first by a general stimulation of metabolism, e.g. respiration, protein-, RNA- and cell-wall synthesis, and subsequently by nuclear division and germ-tube emergence. Initial germination is not dependent on RNA synthesis and can even start without protein synthesis. The first common effect of different activating treatments is a transient rise in cyclic AMP (cAMP) content, caused by a change in phosphodiesterase activity after heat activation, and by unknown factors during activation by acids. cAMP transiently activates trehalase and glycerol-3-phosphatase in the spores. The activation of these enzymes causes a quick turnover of trehalose into glycerol. During the same period, the water status of the cells is altered so dramatically that perhaps this may explain at least part of the stimulation of metabolism in the germinating spore.     

Purification and characterization of neutral trehalase from the yeast ABYS1 mutant

Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine.