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.     

Ultradian rhythm of trehalose levels coupled to heat resistance in continuous cultures of the yeast Saccharomyces cerevisiae

Heat resistance appears to cycle in concert with energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae. To study the mechanism of this oscillation, the authors first examined if heat shock proteins (Hsps) are involved. Neither the protein levels of major Hsps nor the expression of the β-galactosidase gene as a reporter under the control of the promoter carrying heat-shock element oscillated during the metabolic oscillation. The level of trehalose in yeast cycled with the same periodicity, as did energy metabolism. This oscillation was not found in a GTS1-deleted mutant that also did not show cyclic changes in heat resistance. These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by an oscillatory expression of Hsps. The increase in trehalose began at the start of the respiro-fermentative phase and the decrease began after the elevation of the cyclic adenosine monophosphate (cAMP) level. The authors hypothesize that the synthesis of trehalose parallels the activation of the glycolytic pathway and that trehalose is degraded by trehalase activated by cAMP coupled with the metabolic oscillation in the continuous culture of yeast.     

Yeast trehalases: Two enzymes,one catalytic mission

BackgroundTrehalose is a non-reducing disaccharide highly conserved throughout evolution. In yeasts, trehalose hydrolysis is confined to the enzyme trehalase, an α-glucosidase specific for trehalose as sole substrate. Two kinds of trehalase activity exist in yeasts: neutral and acid enzymes.Scope of the reviewThis review makes a comparative survey of the main biochemical and genetic parameters, regulatory systems, tridimensional structure and catalytic mechanism of the two yeast trehalases.Major conclusionsThe yeast neutral and acid trehalases display sharp differences in biochemical features (optimum pH, Mr or amino acid sequence) physiological roles, subcellular location (cytosol vs vacuoles or cell wall) and regulatory control (phosphorylation vs catabolite repression). However, their identical specificity for trehalose is based on the presence of an (α/α)₆ toroid folding structure in the active centre and a catalytic mechanism of anomeric inversion.General significanceThis review expands our knowledge of the homology, functional features and catalytic mechanisms of α-glucosidases in yeasts. It provides a further analysis of the correlation between structures and predicted biological roles of macromolecules.     

Acid trehalase in yeasts and filamentous fungi: localization, regulation and physiological function

Yeasts and filamentous fungi are endowed with two different trehalose-hydrolysing activities, termed acid and neutral trehalases according to their optimal pH for enzymatic activity. A wealth of information already exists on fungal neutral trehalases, while data on localization, regulation and function of fungal acid trehalases have remained elusive. The gene encoding the latter enzyme has now been isolated from two yeast species and two filamentous fungi, and sequences encoding putative acid trehalase can be retrieved from available public sequences. Despite weak similarities between amino acids sequences, this type of trehalase potentially harbours either a transmembrane segment or a signal peptide at the N-terminal sequence, as deduced from domain prediction algorithms. This feature, together with the demonstration that acid trehalase from yeasts and filamentous fungi is localized at the cell surface, is consistent with its main role in the utilisation of exogenous trehalose as a carbon source. The growth on this disaccharide is in fact pretty effective in most fungi except in Saccharomyces cerevisiae. This yeast species actually exhibits a "Kluyver effect" on trehalose. Moreover, an oscillatory behaviour reminiscent of what is observed in aerobic glucose-limited continuous cultures at low dilution rate is also observed in batch growth on trehalose. Finally, the S. cerevisiae acid trehalase may also participate in the catabolism of endogenous trehalose by a mechanism that likely requires the export of the disaccharide, its extracellular hydrolysis, and the subsequent uptake of the glucose released. Based on these recent findings, we suggest to rename "acid" and "neutral" trehalases as "extracellular" and "cytosolic" trehalases, which is more adequate to describe their localization and function in the fungal cell.     

Trehalases and trehalose hydrolysis in fungi

The simultaneous presence of two different trehalose-hydrolysing activities has been recognised in several fungal species. While these enzymes, known as acid and neutral trehalases, share a strict specificity for trehalose, they are nevertheless rather different in subcellular localisation and in several biochemical and regulatory properties. The function of these apparently redundant activities in the same cell was not completely understood until recently. Biochemical and genetic studies now suggest that these enzymes may have specialised and exclusive roles in fungal cells. It is thought that neutral trehalases mobilise cytosolic trehalose, under the control of developmental programs, chemical and nutrient signals, or stress responses. On the other hand, acid trehalases appear not to mobilise cytosolic trehalose, but to act as `carbon scavenger' hydrolases enabling cells to utilise exogenous trehalose as a carbon source, under the control of carbon catabolic regulatory circuits. Although much needs to be learned about the molecular identity of trehalases, it seems that in fungi at least one class of acid trehalases evolved independently from the other trehalases.     

Ultradian rhythm of trehalose levels coupled to heat resistance in continuous cultures of the yeast Saccharomyces cerevisiae

Heat resistance appears to cycle in concert with energy metabolism in continuous culture of the yeast Saccharomyces cerevisiae. To study the mechanism of this oscillation, the authors first examined if heat shock proteins (Hsps) are involved. Neither the protein levels of major Hsps nor the expression of the β-galactosidase gene as a reporter under the control of the promoter carrying heat-shock element oscillated during the metabolic oscillation. The level of trehalose in yeast cycled with the same periodicity, as did energy metabolism. This oscillation was not found in a GTS1-deleted mutant that also did not show cyclic changes in heat resistance. These results suggest that heat resistance oscillation is induced by fluctuations in trehalose level and not by an oscillatory expression of Hsps. The increase in trehalose began at the start of the respiro-fermentative phase and the decrease began after the elevation of the cyclic adenosine monophosphate (cAMP) level. The authors hypothesize that the synthesis of trehalose parallels the activation of the glycolytic pathway and that trehalose is degraded by trehalase activated by cAMP coupled with the metabolic oscillation in the continuous culture of yeast.     

Phenotypic features of trehalase mutants in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, some studies have shown that trehalose and its hydrolysis may play an important physiological role during the life cycle of the cell. Recently, other studies demonstrated a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. We had reported lack of correlation between trehalose accumulation and increase in thermotolerance under certain conditions, suggesting that trehalose may not mediate thermotolerance [Nwaka, S., et al. (1994) FEBS Lett. 344, 225–228]. Using mutants of the trehalase genes, NTH1 and YBR0106, we have demonstrated the necessity of these genes in recovery of yeast cells after heat shock, suggesting a role of these genes in thermotolerance (Nwaka, S., Kopp, M., and Holzer, H., submitted for publication). In the present paper, we have analysed the expression of the trehalase genes under heat stress conditions and present genetic evidence for the ‘poor-heat-shock-recovery’ phenotype associated with NTH1 and YBR0106 mutants. Furthermore, we show a growth defect of neutral and acid trehalase-deficient mutants during transition from glucose to glycerol, which is probably related to the ‘poor-heat-shock-recovery’ phenomenon.     

The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae.

In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.     

Inhibition of yeast glutathione reductase by trehalose: possible implications in yeast survival and recovery from stress

Accumulation of trehalose has been implicated in the tolerance of yeast cells to several forms of stress, including heat-shock and high ethanol levels. However, yeast lacking trehalase, the enzyme that degrades trehalose, exhibit poor survival after exposure to stress conditions. This suggests that optimal cell viability also depends on the capacity to rapidly degrade the high levels of trehalose that build up under stress. Here, we initially examined the effects of trehalose on the activity of an important antioxidant enzyme, glutathione reductase (GR), from Saccharomyces cerevisiae. At 25 degrees C, GR was inhibited by trehalose in a dose-dependent manner, with 70% inhibition at 1.5M trehalose. The inhibition was practically abolished at 40 degrees C, a temperature that induces a physiological response of trehalose accumulation in yeast. The inhibition of GR by trehalose was additive to the inhibition caused by ethanol, indicating that enzyme function is drastically affected upon ethanol-induced stress. Moreover, two other yeast enzymes, cytosolic pyrophosphatase and glucose 6-phosphate dehydrogenase, showed temperature dependences on inhibition by trehalose that were similar to the temperature dependence of GR inhibition. These results are discussed in terms of the apparent paradox represented by the induction of enzymes involved in both synthesis and degradation of trehalose under stress, and suggest that the persistence of high levels of trehalose after recovery from stress could lead to the inactivation of important yeast enzymes.     

On the biochemical classification of yeast trehalases: Candida albicans contains two enzymes with mixed features of neutral and acid trehalase activities

Two enzymes endowed with trehalase activity are present in Candida albicans. The cytosolic trehalase (Ntc1p), displayed high activity in exponential phase regardless of the carbon source (glucose, trehalose or glycerol). Ntc1p activity was similar in neutral (pH 7.1) or acid (pH 4.5) conditions, strongly inhibited by ATP, weakly stimulated by divalent cations (Ca²⁺or Mn²⁺) and unaffected in the presence of cyclic AMP. The Ntc1p activity decreased in stationary phase, except in glycerol-grown cultures, but the catalytic properties did not change. In turn, the cell wall-linked trehalase (Atc1p) showed elevated activity in resting cells or in cultures growing on trehalose or glycerol. Although Atc1p is subjected to glucose repression, exhaustion of glucose in itself did not increased the activity. Significant Atc1p values could also be measured at neutral or acid pH, but Atc1p was insensitive to ATP, cyclic AMP and divalent cations. These results are in direct contrast with the current classification of yeast trehalases based on their optimum pH. They are also relevant in the light of the proposed use of trehalase inhibitors for the treatment of candidiasis.     

Function and regulation of the acid and neutral trehalases of Mucor rouxii

Two different trehalose-hydrolysing activities, known as acid or non-regulatory trehalases, and neutral or regulatory trehalases, have been recognised in a number of fungal species. The true role of these apparently redundant hydrolases remained obscure for many years. However, recent evidence suggests that neutral trehalases would be specialised in the mobilisation of cytosolic trehalose, while acid trehalases would only hydrolyse extracellular trehalose. Results obtained with Mucor rouxii, a Zygomycete initially thought to posses only neutral trehalase activity, reinforced this hypothesis. M. rouxii grows efficiently in trehalose as the sole carbon source. Trehalose-grown or carbon-starved cells exhibit a high trehalase activity of optimum pH 4.5, bound to the external surface of the cell wall, in contrast with the neutral (pH 6.5) trehalase, which occurs in the cytosol. Other differences between the neutral and the acid trehalases are the temperature optimum (35°C and 45°C, respectively) and thermal stability (half-life of 2.5 min and 12 min at 45°C, respectively). The neutral trehalase, but not the acid trehalase, is activated in vitro by cAMP-dependent phosphorylation, stimulated by Ca²⁺, and inhibited by EDTA. It shows maximal activity at germination and decreases as growth proceeds. In contrast the activity of the acid trehalase is totally repressed in glucose-grown cultures and increases upon exhaustion of the carbon source, and is strongly induced by extracellular trehalose.     

Gaining insight into the response logic of Saccharomyces cerevisiae to heat shock by combining expression profiles with metabolic pathways

Extensive alteration of gene expression and metabolic remodeling enable the budding yeast Saccharomyces cerevisiae to ensure cellular homeostasis and adaptation to heat shock. The response logic of the cells to heat shock is still not entirely clear. In this study, we combined the expression profiles with metabolic pathways to investigate the logical relations between heat shock response metabolic pathways. The results showed that the heat-stressed S. cerevisiae cell accumulated trehalose and glycogen, which protect cellular proteins against denaturation, and modulate its phospholipid structure to sustain stability of the cell wall. The TCA cycle was enhanced, and the heat shock-induced turnover of amino acids and nucleotides served to meet the extra energy requirement due to heat-induced protein metabolism and modification. The enhanced respiration led to oxidative stress, and subsequently induced the aldehyde detoxification system. These results indicated that new insight into the response logic of S. cerevisiae to heat shock can be gained by integrating expression profiles and the logical relations between heat shock response metabolic pathways.     

The involvement of topoisomerases and DNA polymerase I in the mechanism of induced thermal and radiation resistance in yeast

Either an ionizing radiation exposure or a heat shock is capable of inducing both thermal tolerance and radiation resistance in yeast. Yeast mutants, deficient in topoisomerase I, in topoisomerase II, or in DNA polymerase I, were used to investigate the mechanism of these inducible resistances. The absence of either or both topoisomerase activities did not prevent induction of either heat or radiation resistance. However, if both topoisomerase I and II activities were absent, the sensitivity of yeast to become thermally tolerant (in response to a heat stress) was markedly increased. The absence of only topoisomerase I activity (top1) resulted in the constitutive expression of increased radiation resistance equivalent to that induced by a heat shock in wild-type cells, and the topoisomerase I-deficient cells were not further inducible by heat. This heat-inducible component of radiation resistance (or its equivalent constitutive expression in top1 cells) was, in turn, only a portion of the full response inducible by radiation. The absence of polymerase I activity had no detectable effect on either response. Our results indicate that the actual systems that confer resistance to heat or radiation are independent of either topoisomerase activity or DNA polymerase function, but suggest that topoisomerases may have a regulatory role during the signaling of these mechanisms. The results of our experiments imply that maintenance of correct DNA topology prevents induction of the heat-shock response, and that heat-shock induction of a component of the full radiation resistance in yeast may be the consequence of topoisomerase I inactivation.