Effect of acid trehalase (ATH) on impaired yeast vacuolar activity

In this study, this protein was overexpressed in yeast cells grown on trehalose-containing medium to assess its impact on yeast vacuolar activity. ATH was confirmed to be located in both cell surface and vacuoles and the overexpression of ATH was observed to decrease vacuolar activity. Therefore, an assumption was suggested to explain this phenomenon as follows: when grown on containing trehalose medium, the ATH localization at cellular periplasm, but not the vacuole, is prioritized to utilize the extracellular trehalose for cell growth. The multivesicular body pathway (MVB pathway) via which ATH is transported into vacuoles is believed to be down-regulated to favor the accumulation of ATH at cell surface area. By extension, other vacuolar proteins travelling through MVB pathway to reach yeast vacuoles likely also suffer the down regulation. It can be concluded that acid trehalase may contribute down regulation of other vacuolar proteins through MVB pathway. This study suggests that it is a potential of acid trehalase (ATH) on impaired activity of yeast vacuolar.     

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.     

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.     

Evidence for a direct role of the Doa4 deubiquitinating enzyme in protein sorting into the MVB pathway

Degradation of various membrane proteins in the lumen of the vacuole/lysosome requires their prior sorting into the multivesicular body (MVB) pathway. In this process, ubiquitin serves as a sorting signal for most cargoes. The yeast ubiquitin hydrolase Doa4 acts late in the MVB pathway. It's role is to catalyze deubiquitination of cargo proteins prior to their sorting into the endosomal vesicles. This step rescues ubiquitin from degradation in the vacuole/lysosome, enabling it to be recycled. Accordingly, the level of monomeric ubiquitin is typically reduced in doa4 mutants. Although MVB sorting of cargo proteins is also impaired in doa4 mutants, the question of whether this defect is due solely to Doa4's role in maintaining a normal pool of ubiquitin in the cell remains open. We here show that the requirement of Doa4 for correct MVB sorting of the endocytic cargo general amino acid permease and of the biosynthetic cargo carboxypeptidase S are not because of the role of Doa4 in ubiquitin recycling. This suggests a direct role of Doa4 in MVB sorting and we show that this role depends on Doa4's catalytic activity. We propose that deubiquitination by Doa4 of cargo proteins and/or some components of the MVB sorting machinery is essential to correct sorting of cargoes into the MVB pathway.     

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.     

Trehalose and trehalase in Arabidopsis

Trehalase is ubiquitous in higher plants. So far, indications concerning its function are scarce, although it has been implicated in the detoxification of exogenous trehalose. A putative trehalase gene, T19F6.15, has been identified in the genome sequencing effort in Arabidopsis. Here we show that this gene encodes a functional trehalase when its cDNA is expressed in yeast, and that it is expressed in various plant organs. Furthermore, we present results on the distribution and activity of trehalase in Arabidopsis and we describe how inhibition of trehalase by validamycin A affects the plants response to exogenous trehalose (alpha-D-glucopyranosyl-[1, 1]-alpha-D-glucopyranoside). Trehalase activity was highest in floral organs, particularly in the anthers (approximately 700 nkat g(-1) protein) and maturing siliques (approximately 250 nkat g(-1) protein) and much lower in leaves, stems, and roots (less than 50 nkat g(-1) protein). Inhibition of trehalase in vivo by validamycin A led to the accumulation of an endogenous substance that had all the properties of trehalose, and to a strong reduction in sucrose and starch contents in flowers, leaves, and stems. Thus, trehalose appears to be an endogenous substance in Arabidopsis, and trehalose and trehalase may play a role in regulating the carbohydrate allocation in plants.     

Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu

In our previous studies, it was found that Saccharomycopsis fibuligera sdu cells could accumulate 18.0% (gg(-1)) trehalose from soluble starch in SSY medium. However, the yeast strain contained high activities of acid and neutral trehalases, which were reported to mobilize trehalose accumulated by the cells during fermentation. In order to enhance the yield of trehalose, it is necessary to remove the trehalase activities from the cells. By mutagenesis of ethylmethanesulfonate, one mutant that assimilated trehalose very slowly, but grew on other carbon sources as fast as its parent strain, was isolated. In Biostat B2 2-1 fermentation, trehalose accumulation of the mutant was much higher than that of the wild type when grown in YPS medium containing starch. The activities of acid and neutral trehalases of this mutant were much lower than those of the wild type, respectively. We think the reduction of acid and neutral trehalase activities is considered to be responsible for the increased yield of trehalose accumulated by the mutant.     

A selective autophagy pathway takes an unconventional route

Selective autophagy transports specific cytoplasmic materials into lysosomes/vacuoles. In the case of macroautophagy the selectivity is mediated by receptors, which usually link the cargos to the machinery that sequesters them into the forming autophagosome. In our recent work, we found that fission yeast Nbr1, a homolog of the mammalian macroautophagy receptor NBR1, acts together with an unconventional autophagy-associated cargo sequestration apparatus, the endosomal sorting complexes required for transport (ESCRTs), to deliver 2 hydrolytic enzymes from the cytosol to the vacuole lumen. In this pathway, which we term the Nbr1-mediated vacuolar targeting (NVT) pathway, soluble cargos transit through the multi-vesicular body (MVB), rather than the autophagosome, on their way to the vacuole. Our findings reveal a novel mode of action of macroautophagy receptors and broaden our understanding of ESCRT-mediated autophagy.     

Role of 14-3-3 proteins in the regulation of neutral trehalase in the yeast Saccharomyces cerevisiae

In higher eukaryotes, 14-3-3 proteins participate in numerous cellular processes, and carry out their function through a variety of different molecular mechanisms, including regulation of protein localization and enzyme activation. Here, it is shown that the two yeast 14-3-3 homologues, Bmh1p and Bmh2p, form a complex with neutral trehalase (Nth1p), an enzyme that is responsible for trehalose degradation and is required in a variety of stress conditions. In a purified in vitro system, either one of the two 14-3-3 yeast isoforms are necessary for complete activation of neutral trehalase (Nth1p) after phosphorylation by PKA. It is further demonstrated that Bmh1p and Bmh2p bind to the amino-terminal region of phosphorylated trehalase, thereby modulating its enzymatic activity. This work represents the first demonstration of enzyme activation mediated by 14-3-3 binding in yeast.     

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase

Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.     

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.     

Sna3 Is an Rsp5 Adaptor Protein that Relies on Ubiquitination for Its MVB Sorting

The process in which ubiquitin ( Ub ) conjugation is required for trafficking of integral membrane proteins into multivesicular bodies ( MVBs ) and eventual degradation in the lumen of lysosomes/vacuoles is well defined. However , Ub ‐independent pathways into MVBs are less understood. To better understand this process, we have further characterized the membrane protein Sna 3, the prototypical Ub ‐independent cargo protein sorted through the MVB pathway in yeast. We show that Sna 3 trafficking to the vacuole is critically dependent on Rsp 5 ligase activity and ubiquitination. We find Sna 3 undergoes Ub ‐dependent MVB sorting by either becoming ubiquitinated itself or associating with other ubiquitinated membrane protein substrates. In addition, our functional studies support a role for Sna 3 as an adaptor protein that recruits Rsp 5 to cargo such as the methionine transporter Mup 1, resulting in efficient Mup 1 delivery to the vacuole .     

Evidence for contribution of neutral trehalase in barotolerance of Saccharomyces cerevisiae

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.     

Effect of pulsed electric fields on the activity of neutral trehalase from beer yeast and RSM analysis

The trehalase activity plays an important role in extraction of trehalose from beer yeast. In this study, the effect of pulsed electric field processing on neutral trehalase activity in beer yeast was investigated. In order to develop and optimize a pulsed electric field (PEF) mathematical model for activating the neutral trehalase, we have investigated three variables, including electric field intensity (10-50 kV/cm), pulse duration (2-10 μs) and liquid-solid ratio (20-50 ml/g) and subsequently optimized them by response surface methodology (RSM). The experimental data were fitted to a second-order polynomial equation and profiled into the corresponding contour plots. Optimal condition obtained by RSM is as follows: electric field intensity 42.13 kV/cm, liquid-solid ratio 30.12 ml/g and pulse duration 5.46 μs. Under these conditions, with the trehalose decreased 8.879 mg/L, the PEF treatment had great effect on activating neutral trehalase in beer yeast cells.     

Human trehalase is a stress responsive protein in Saccharomyces cerevisiae

Three trehalases ATH1, NTH1, and NTH2 have been identified in Saccharomyces cerevisiae. ATH1, and NTH1 hydrolyze trehalose to glucose to provide energy and assist in recovery from stress. Human trehalase (TREH) is expressed in the intestine and kidney and probably hydrolyzes ingested trehalose in the intestine and acts as marker of renal tubular damage in kidney. Since trehalose is not present in circulation or kidney tubules, its renal effect suggests it has other yet unidentified actions. Here we examined the function of human trehalase in budding yeast. We constructed three yeast trehalase mutants (NTH1Δ, NTH2Δ, and ATH1Δ) and then transformed TREH into these mutants. NTH1Δ did not grow on media containing trehalose as the carbon source, and TREH did not rectify NTH1Δ dysfunction and also did not grow on trehalose medium, suggesting that TREH is not responsible for utilization of exogenous trehalose in yeast. In experiments involving exposure to heat, osmotic and oxidative stresses, NTH1Δ showed no recovery. Interestingly, ATH1Δ-TREH showed high sensitivity to all three stressors. ATH1Δ and NTH2Δ showed very low neutral trehalase activity and NTH1Δ did not show any neutral trehalase activity, and trehalose concentrations were higher. Increased neutral trehalase activity (equivalent to the wild type), reduction of trehalose content and brisk sensitivity to stressors were noted in TREH-ATH1Δ strain, but not in TREH-NTH1Δ or -NTH2Δ. Our results suggest that TREH acts as a stress-response protein in the kidney rather than involved in utilization of exogenous trehalose.     

New insights on trehalose: a multifunctional molecule

Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants, where it may serve as a source of energy and carbon. In yeast and plants, it may also serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desiccation, dehydration, heat, cold, and oxidation. Finally, in mycobacteria and corynebacteria, trehalose is an integral component of various glycolipids that are important cell wall structures. There are now at least three different pathways described for the biosynthesis of trehalose. The best known and most widely distributed pathway involves the transfer of glucose from UDP-glucose (or GDP-glucose in some cases) to glucose 6-phosphate to form trehalose-6-phosphate and UDP. This reaction is catalyzed by the trehalose-P synthase (TPS here, or OtsA in Escherichia coli ). Organisms that use this pathway usually also have a trehalose-P phosphatase (TPP here, or OtsB in E. coli) that converts the trehalose-P to free trehalose. A second pathway that has been reported in a few unusual bacteria involves the intramolecular rearrangement of maltose (glucosyl-alpha1,4-glucopyranoside) to convert the 1,4-linkage to the 1,1-bond of trehalose. This reaction is catalyzed by the enzyme called trehalose synthase and gives rise to free trehalose as the initial product. A third pathway involves several different enzymes, the first of which rearranges the glucose at the reducing end of a glycogen chain to convert the alpha1,4-linkage to an alpha,alpha1,1-bond. A second enzyme then releases the trehalose disaccharide from the reducing end of the glycogen molecule. Finally, in mushrooms there is a trehalose phosphorylase that catalyzes the phosphorolysis of trehalose to produce glucose-1-phosphate and glucose. This reaction is reversible in vitro and could theoretically give rise to trehalose from glucose-1-P and glucose. Another important enzyme in trehalose metabolism is trehalase (T), which may be involved in energy metabolism and also have a regulatory role in controlling the levels of trehalose in cells. This enzyme may be important in lowering trehalose concentrations once the stress is alleviated. Recent studies in yeast indicate that the enzymes involved in trehalose synthesis (TPS, TPP) exist together in a complex that is highly regulated at the activity level as well as at the genetic level.