Two Distinct Pathways for Trehalose Assimilation in the Yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.     

New insights into trehalose metabolism by Saccharomyces cerevisiae: NTH2 encodes a functional cytosolic trehalase, and deletion of TPS1 reveals Ath1p-dependent trehalose mobilization

In the yeast Saccharomyces cerevisiae, the synthesis of endogenous trehalose is catalyzed by a trehalose synthase complex, TPS, and its hydrolysis relies on a cytosolic/neutral trehalase encoded by NTH1. In this work, we showed that NTH2, a paralog of NTH1, encodes a functional trehalase that is implicated in trehalose mobilization. Yeast is also endowed with an acid trehalase encoded by ATH1 and an H+/trehalose transporter encoded by AGT1, which can together sustain assimilation of exogenous trehalose. We showed that a tps1 mutant defective in the TPS catalytic subunit cultivated on trehalose, or on a dual source of carbon made of galactose and trehalose, accumulated high levels of intracellular trehalose by its Agt1p-mediated transport. The accumulated disaccharide was mobilized as soon as cells entered the stationary phase by a process requiring a coupling between its export and immediate extracellular hydrolysis by Ath1p. Compared to what is seen for classical growth conditions on glucose, this mobilization was rather unique, since it took place prior to that of glycogen, which was postponed until the late stationary phase. However, when the Ath1p-dependent mobilization of trehalose identified in this study was impaired, glycogen was mobilized earlier and faster, indicating a fine-tuning control in carbon storage management during periods of carbon and energy restriction.     

The Arabidopsis thaliana trehalase is a plasma membrane-bound enzyme with extracellular activity

The lack of trehalose accumulation in most plant species has been partly attributed to the presence of an active trehalase. Although trehalose synthesis enzymes are thought to be cytosolic, and previous studies have indicated that trehalase activity is extracellular, the exact location of the enzyme has not yet been established in plant cell. We present evidence that the yet uncharacterised full-length Arabidopsis trehalase is a plasma membrane-bound protein, probably anchored to the membrane through a predicted N-terminal membrane spanning domain. The full-length AtTRE1, when expressed in yeast can functionally substitute for the extracellularly active trehalase Ath1p, by sustaining the growth of an ath1 null mutant strain on trehalose and at pH 4.8. We further demonstrate that AtTRE1 expressed in yeast is plasma membrane-bound as in plant cell. In light of these findings, the regulation of plant cell endogenous trehalose by trehalase is discussed.     

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.     

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.     

The transmembrane domain of acid trehalase mediates ubiquitin-independent multivesicular body pathway sorting

Trehalose serves as a storage source of carbon and plays important roles under various stress conditions. For example, in many organisms trehalose has a critical function in preserving membrane structure and fluidity during dehydration/rehydration. In the yeast Saccharomyces cerevisiae, trehalose accumulates in the cell when the nutrient supply is limited but is rapidly degraded when the supply of nutrients is renewed. Hydrolysis of trehalose in yeast depends on neutral trehalase and acid trehalase (Ath1). Ath1 resides and functions in the vacuole; however, it appears to catalyze the hydrolysis of extracellular trehalose. Little is known about the transport route of Ath1 to the vacuole or how it encounters its substrate. Here, through the use of various trafficking mutants we showed that this hydrolase reaches its final destination through the multivesicular body (MVB) pathway. In contrast to the vast majority of proteins sorted into this pathway, Ath1 does not require ubiquitination for proper localization. Mutagenesis analyses aimed at identifying the unknown targeting signal revealed that the transmembrane domain of Ath1 contains the information sufficient for its selective sequestration into MVB internal vesicles.     

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.     

Extracellular trehalose utilization by Saccharomyces cerevisiae

Two haploid strains of Saccharomyces cerevisiae viz. MATalpha and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.     

Extracellular trehalose utilization by Saccharomyces cerevisiae

Two haploid strains of Saccharomyces cerevisiae viz. MATα and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.     

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose

Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae . A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.     

Enhanced freeze tolerance of baker’s yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker’s yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301_TPS1 overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301_TPS1 were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301_TPS1 was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker’s yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker’s yeast.     

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.     

Response to oxidative stress caused by H(2)O(2) in Saccharomyces cerevisiae mutants deficient in trehalase genes

The role of trehalose as cell protector against oxidative stress induced by H(2)O(2) has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted. The addition of low H(2)O(2) concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation. This pattern was reversed after a more severe H(2)O(2) treatment that caused drastic cell killing. The most severe phenotype corresponded to the Delta nth1 mutant. Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex. The behavior of the double-disruptant Delta ath1Delta nth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H(2)O(2). Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis. Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge.     

Thermotolerance and trehalose accumulation induced by heat shock in yeast cells of Candida albicans

Candida albicans yeast cells growing exponentially on glucose are extremely sensitive to severe heat shock treatments (52.5°C for 5 min). When these cultures were subjected to a mild temperature preincubation (42°C), they became thermotolerant and displayed higher resistance to further heat stress. The intracellular content of trehalose was very low in exponential cells, but underwent a marked increase upon non-lethal heat exposure. The accumulation of trehalose is likely due to heat-induced activation of the trehalose-6-phosphate synthase complex, whereas the external trehalase remained practically unmodified. After a temperature reversion shift (from 42°C to 28°C), the pool of trehalose was rapidly mobilized without any concomitant change in trehalase activity. These results support an important role of trehalose in the mechanism of acquired thermotolerance in C. albicans and seem to exclude the external trehalase as a key enzyme in this process.     

Characterization of AGT1 encoding a general α-glucoside transporter from Saccharomyces

Molecular genetic analysis is used to characterize the AGT1 gene encoding an α-glucoside transporter. AGT1 is found in many Saccharomyces cerevisiae laboratory strains and maps to a naturally occurring, partially functional allele of the MAL1 locus. Agt1p is a highly hydrophobic, postulated integral membrane protein. It is 57% identical to Mal61p, the maltose permease encoded at MAL6 , and is also a member of the 12 transmembrane domain superfamily of sugar transporters. Like Mal61p, Agt1p is a high-affinity, maltose/proton symporter, but Mal61p is capable of transporting only maltose and turanose, while Agt1p transports these two α-glucosides as well as several others including isomaltose, α-methylglucoside, maltotriose, palatinose, trehalose and melezitose. AGT1 expression is maltose inducible and induction is mediated by the Mal-activator. The sequence of the upstream region of AGT1 is identical to that of the maltose-inducible MAL61 gene over a 469 bp region containing the UAS_MAL but the 315 bp sequence immediately upstream of AGT1 shows no significant homology to the sequence immediately upstream of MAL61 . The evolutionary origin of the MAL1 allele to which AGT1 maps and the relationship of AGT1 to other α-glucoside fermentation genes is discussed.     

Trehalase immobilization on aminopropyl glass for analytical use

Trehalase is the enzyme which hydrolyzes the disaccharide trehalose into two alpha-D-glucose molecules. In this article, we present the immobilization of trehalase on aminopropyl glass particles. The enzyme was extracted from Escherichia coli Mph2, a strain harboring the pTRE11 plasmid, which contains the trehalase gene. The partially purified enzyme had a specific activity of 356 U/mg and could be used for quantifying trehalose in the presence of sucrose, maltose, lactose, starch, and glycogen. Partially purified trehalase was immobilized by covalent coupling with retention of its catalytic activity. The support chosen for the majority of the experiments reported was aminopropyl glass, although spherisorb-5NH(2) and chitin were also tested. The immobilized enzyme was assayed continuously for 40 h, at pH 6.0 and 30 degrees C, and no release of enzyme molecules was detected during this procedure. The best condition found for storing the enzyme-support complex was at 4 degrees C in the presence of 25 mM sodium maleate, containing 7 mM beta-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), and 50% glycerol. The enzyme under these conditions was stable, retaining approximately 100% of its initial activity for at least 28 days. The immobilized enzyme can be employed to detect trehalose molecules in micromolar concentration. The optimum pH value found was 4.5 and the K(m) app. 4.9 x 10(-3) M trehalose at pH 4.6 and 30 degrees C, with V(max) of 5.88 mumol glucose . min.(-1), as calculated by a Lineweaver-Burk plot. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 33-39, 1997.     

Trehalase of Escherichia coli. Mapping and cloning of its structural gene and identification of the enzyme as a periplasmic protein induced under high osmolarity growth conditions

Escherichia coli can use the nonreducing disaccharide trehalose as a sole source of carbon and energy. Trehalose transport into the cell is mediated via the phosphotransferase system, and a mutant depleted in the nonspecific proteins enzyme I, HPr, and enzyme IIIGlc of this system was not only unable to grow on glucose or mannitol but also was strongly reduced in its ability to grow on trehalose. A pseudorevertant (PPA69) of such a deletion mutant was isolated that could again grow on glucose but not on mannitol. This revertant could now also use trehalose as a carbon source due to a constitutive galactose permease. PPA69 was subjected to Tn10 insertional mutagenesis, and a mutant (UE5) was isolated that no longer could use trehalose as a carbon source but could still grow on glucose. UE5 lacked a periplasmic trehalase that was present in PPA69. P1-mediated transduction of this Tn10 insertion (treA::Tn10) into a pts+ wild-type strain (MC4100) had no effect on the ability of MC4100 to grow on trehalose but resulted in loss of the periplasmic trehalase activity. The Tn10 insertion was mapped at 26 min on the E. coli linkage map and was 3% cotransducible with trp, in the order treA::Tn10, trp, cys. Trehalase activity in MC4100 was not induced by growth in the presence of trehalose but increased by about 10-fold when 0.6 M sucrose was added to minimal growth medium. Using the in vivo mini-Mu cloning system and growth on trehalose as selection, we cloned the treA gene. A 9-kilobase EcoRI fragment containing treA was subcloned into pBR322. Strains carrying this plasmid (pTRE5) contained about 100-fold higher periplasmic trehalase activity than PPA69 or MC4100. Using polyacrylamide gel electrophoresis, we found a protein of molecular weight 58,000 among the periplasmic proteins of the pTRE5-carrying strain that was absent in UE5. This protein was purified by ammonium sulfate precipitation and DEAE-Sepharose ion-exchange chromatography and contained all the trehalase activity. Minicells containing the treA+ plasmid produced, in addition to three other proteins, the 58,000-dalton protein. Thus, the plasmid carries the structural gene for the periplasmic trehalase and not just a gene involved in the regulation of the enzyme.