Reconstitution of ethanolic fermentation in permeabilized spheroplasts of wild-type and trehalose-6-phosphate synthase mutants of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, TPS1-encoded trehalose-6-phosphate synthase (TPS) exerts an essential control on the influx of glucose into glycolysis, presumably by restricting hexokinase activity. Deletion of TPS1 results in severe hyperaccumulation of sugar phosphates and near absence of ethanol formation. To investigate whether trehalose 6-phosphate (Tre6P) is the sole mediator of hexokinase inhibition, we have reconstituted ethanolic fermentation from glucose in permeabilized spheroplasts of the wild-type, tps1Delta and tps2Delta (Tre6P phosphatase) strains. For the tps1Delta strain, ethanol production was significantly lower and was associated with hyperaccumulation of Glu6P and Fru6P. A tps2Delta strain shows reduced accumulation of Glu6P and Fru6P both in intact cells and in permeabilized spheroplasts. These results are not consistent with Tre6P being the sole mediator of hexokinase inhibition. Reconstitution of ethanolic fermentation in permeabilized spheroplasts with glycolytic intermediates indicates additional target site(s) for the Tps1 control. Addition of Tre6P partially shifts the ethanol production rate and the metabolite pattern in permeabilized tps1Delta spheroplasts to those of the wild-type strain, but only with glucose as substrate. This is observed at a very high ratio of glucose to Tre6P. Inhibition of hexokinase activity by Tre6P is less efficiently counteracted by glucose in permeabilized spheroplasts compared to cell extracts, and this effect is largely abolished by deletion of TPS2 but not TPS1. In permeabilized spheroplasts, hexokinase activity is significantly lower in a tps2Delta strain compared to a wild-type strain and this difference is strongly reduced by additional deletion of TPS1. These results indicate that Tps1-mediated protein-protein interactions are important for control of glucose influx into yeast glycolysis, that Tre6P inhibition of hexokinase might not be competitive with respect to glucose in vivo and that also Tps2 appears to play a role in the control of hexokinase activity.     

Trehalose-6-P synthase is dispensable for growth on glucose but not for spore germination in Schizosaccharomyces pombe.

Trehalose-6-P inhibits hexokinases in Saccharomyces cerevisiae (M. A. Blázquez, R. Lagunas, C. Gancedo, and J. M. Gancedo, FEBS Lett. 329:51-54, 1993), and disruption of the TPS1 gene (formerly named CIF1 or FDP1) encoding trehalose-6-P synthase prevents growth in glucose. We have found that the hexokinase from Schizosaccharomyces pombe is not inhibited by trehalose-6-P even at a concentration of 3 mM. The highest internal concentration of trehalose-6-P that we measured in S. pombe was 0.75 mM after heat shock. We have isolated from S. pombe the tps1+ gene, which is homologous to the Saccharomyces cerevisiae TPS1 gene. The DNA sequence from tps1+ predicts a protein of 479 amino acids with 65% identity with the protein of S. cerevisiae. The tps1+ gene expressed from its own promoter could complement the lack of trehalose-6-P synthase in S. cerevisiae tps1 mutants. The TPS1 gene from S. cerevisiae could also restore trehalose synthesis in S. pombe tps1 mutants. A chromosomal disruption of the tps1+ gene in S. pombe did not have a noticeable effect on growth in glucose, in contrast with the disruption of TPS1 in S. cerevisiae. However, the disruption prevented germination of spores carrying it. The level of an RNA hybridizing with an internal probe of the tps1+ gene reached a maximum after 20 min of heat shock treatment. The results presented support the idea that trehalose-6-P plays a role in the control of glycolysis in S. cerevisiae but not in S. pombe and show that the trehalose pathway has different roles in the two yeast species.     

Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation

Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1-1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.     

Uncoupling of the glucose growth defect and the deregulation of glycolysis in Saccharomyces cerevisiae Tps1 mutants expressing trehalose-6-phosphate-insensitive hexokinase from Schizosaccharomyces pombe

In the yeast Saccharomyces cerevisiae inactivation of trehalose-6-phosphate (Tre6P) synthase (Tps1) encoded by the TPS1 gene causes a specific growth defect in the presence of glucose in the medium. The growth inhibition is associated with deregulation of the initial part of glycolysis. Sugar phosphates, especially fructose-1,6-bisphosphate (Fru1,6bisP), hyperaccumulate while the levels of ATP, Pi and downstream metabolites are rapidly depleted. This was suggested to be due to the absence of Tre6P inhibition on hexokinase. Here we show that overexpression of Tre6P (as well as glucose-6-phosphate (Glu6P))-insensitive hexokinase from Schizosaccharomyces pombe in a wild-type strain does not affect growth on glucose but still transiently enhances initial sugar phosphate accumulation. We have in addition replaced the three endogenous glucose kinases of S. cerevisiae by the Tre6P-insensitive hexokinase from S. pombe. High hexokinase activity was measured in cell extracts and growth on glucose was somewhat reduced compared to an S. cerevisiae wild-type strain but expression of the Tre6P-insensitive S. pombe hexokinase never caused the typical tps1Delta phenotype. Moreover, deletion of TPS1 in this strain expressing only the Tre6P-insensitive S. pombe hexokinase still resulted in a severe drop in growth capacity on glucose as well as sensitivity to millimolar glucose levels in the presence of excess galactose. In this case, poor growth on glucose was associated with reduced rather than enhanced glucose influx into glycolysis. Initial glucose transport was not affected. Apparently, deletion of TPS1 causes reduced activity of the S. pombe hexokinase in vivo. Our results show that Tre6P inhibition of hexokinase is not the major mechanism by which Tps1 controls the influx of glucose into glycolysis or the capacity to grow on glucose. In addition, they show that a Tre6P-insensitive hexokinase can still be controlled by Tps1 in vivo.     

The Trehalose Pathway Regulates Mitochondrial Respiratory Chain Content through Hexokinase 2 and cAMP in Saccharomyces cerevisiae

In yeast, trehalose is synthesized by a multimeric enzymatic complex: TPS1 encodes trehalose 6-phosphate synthase, which belongs to a complex that is composed of at least three other subunits, including trehalose 6-phosphate phosphatase Tps2 and the redundant regulatory subunits Tps3 and Tsl1. The product of the TPS1 gene plays an essential role in the control of the glycolytic pathway by restricting the influx of glucose into glycolysis. In this paper, we investigated whether the trehalose synthesis pathway could be involved in the control of the other energy-generating pathway: oxidative phosphorylation. We show that the different mutants of the trehalose synthesis pathway (tps1Δ, tps2Δ, and tps1,2Δ) exhibit modulation in the amount of respiratory chains, in terms of cytochrome content and maximal respiratory activity. Furthermore, these variations in mitochondrial enzymatic content are positively linked to the intracellular concentration in cAMP that is modulated by Tps1p through hexokinase2. This is the first time that a pathway involved in sugar storage, i.e. trehalose, is shown to regulate the mitochondrial enzymatic content.The control of glycolysis in the yeast Saccharomyces cerevisiae has been extensively studied. First, allosteric regulation of the irreversible steps catalyzed by phosphofructokinase (1), pyruvate kinase (review in Ref. 2), and fructose-1,6-bisphosphatase (1) has been proposed, even though the overexpression of these key enzymes does not increase the glycolytic flux (3). Other mechanisms of control have been proposed such as futile cycle activity (4) and an inhibitory effect of ATP (5). Indeed, it seems likely that the regulation of glycolysis is a complex process involving different hierarchical events leading from gene expression to the metabolic fluxes via protein levels, enzyme activities, and metabolite effects (6, 7). Among these actors, the product of the TPS1 gene has been shown to play an essential role in the control of the glycolytic pathway by restricting the influx of glucose into glycolysis (8). TPS1 encodes trehalose 6-phosphate (Tre6P)³ synthase (9–12). This enzyme is part of a multimeric protein complex composed of at least three other subunits, i.e. Tre6P phosphatase encoded by TPS2 (13) and the redundant regulatory subunits Tps3 and Tsl1 (14).A particularly intriguing finding is that tps1Δ mutants are defective not only for Tre6P synthesis but also for growth on glucose or related rapidly fermented sugars (8, 11, 15). This may be explained by an uncontrolled influx of glucose into the glycolytic pathway. This phenomenon is characterized by hyperaccumulation of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate (Fru1,6bP) (8, 16–18) and depletion of ATP, P_i, and all intermediates of glycolysis downstream of glyceraldehyde-3-phosphate dehydrogenase (19). Several mutations have been described that suppress the growth defect of tps1Δ mutants apparently by reducing sugar influx into glycolysis (16, 20) or by diverting the excess sugar phosphate into glycerol synthesis through overexpression of the GPD1-encoded NAD-dependent glycerol-3-phosphate dehydrogenase (17, 21). Reconstitution of ethanolic fermentation in permeabilized yeast spheroplasts indicated that in addition to Tre6P, the Tps1 protein itself also seems to play a role in restricting glucose influx into glycolysis (22).Whatever the mechanism by which the multimeric complex involved in trehalose synthesis controls glycolytic flux in yeast, such a regulation is associated with modification of the cellular content of sugar phosphates. Moreover, in a recent paper, we have shown that in yeast, low physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulate the respiratory flux and that this effect was strongly antagonized by Fru1,6bP (18). On the other hand, Fru1,6bP by itself is able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Taken together, these results indicate that besides the thermodynamic link between glycolysis and mitochondrial respiration (i.e. the cytosolic ATP/ADP and NADH/NAD+ ratio), a kinetic control of oxidative phosphorylation activity is exerted by the level of glycolytic sugar phosphates (18, 23). This raises the question of a possible direct or indirect regulation of oxidative phosphorylation by the trehalose synthesis pathway.     

Regulation of the yeast trehalose–synthase complex by cyclic AMP-dependent phosphorylation

Background

Trehalose is an important protectant in several microorganisms. In Saccharomyces cerevisiae, it is synthesized by a large complex comprising the enzymes Tps1 and Tps2 and the subunits Tps3 and Tsl1, showing an intricate metabolic control.

Methods

To investigate how the trehalose biosynthesis pathway is regulated, we analyzed Tps1 and Tps2 activities as well as trehalose and trehalose-6-phosphate (T6P) contents by mass spectrometry.

Results

Tsl1 deficiency totally abolished the increase in Tps1 activity and accumulation of trehalose in response to a heat stress, whereas absence of Tps3 only reduced Tps1 activity and trehalose synthesis. In extracts of heat stressed cells, Tps1 was inhibited by T6P and by ATP. Mg^2 + in the presence of cAMP. In contrast, cAMP-dependent phosphorylation did not inhibit Tps1 in tps3 cells, which accumulated a higher proportion of T6P after stress. Tps2 activity was not induced in a tps3 mutant.

Conclusion

Taken together these results suggest that Tsl1 is a decisive subunit for activity of the TPS complex since in its absence no trehalose synthesis occurred. On the other hand, Tps3 seems to be an activator of Tps2. To perform this task, Tps3 must be non-phosphorylated. To readily stop trehalose synthesis during stress recovery, Tps3 must be phosphorylated by cAMP-dependent protein kinase, decreasing Tps2 activity and, consequently, increasing the concentration of T6P which would inhibit Tps1.

General significance

A better understanding of TPS complex regulation is essential for understanding how yeast deals with stress situations and how it is able to recover when the stress is over.     

Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation

Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae. Alterations in trehalose synthesis affect carbon source-dependent growth, accumulation of glycogen and sporulation. Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins. In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components. The tps1-H223Y allele (glc6-1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild-type TPS1 because, unlike the wild-type enzyme, TPS activity in tps1-H223Y cells is not inhibited by phosphate. Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1. Furthermore, high-copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait. These results suggest that the trehalose-6-phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.     

Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. S. nodorum is a polycyclic pathogen, whereby rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate again within 2–3 weeks. As several cycles of infection are needed for a damaging infection, asexual sporulation is a critical phase of its infection cycle. A non-targeted metabolomics screen for sporulation-associated metabolites identified that trehalose accumulated significantly in concert with asexual sporulation both in vitro and in planta. A reverse-genetics approach was used to investigate the role of trehalose in asexual sporulation. Trehalose biosynthesis was disrupted by deletion of the gene Tps1, encoding a trehalose 6-phosphate synthase, resulting in almost total loss of trehalose during in vitro growth and in planta. In addition, lesion development and pycnidia formation were also significantly reduced in tps1 mutants. Reintroduction of the Tps1 gene restored trehalose biosynthesis, pathogenicity and sporulation to wild-type levels. Microscopic examination of tps1 infected wheat leaves showed that pycnidial formation often halted at an early stage of development. Further examination of the tps1 phenotype revealed that tps1 pycnidiospores exhibited a reduced germination rate while under heat stress, and tps1 mutants had a reduced growth rate while under oxidative stress. This study confirms a link between trehalose biosynthesis and pathogen fitness in S. nodorum.     

Disruption of the Candida albicans TPS1 Gene Encoding Trehalose-6-Phosphate Synthase Impairs Formation of Hyphae and Decreases Infectivity

The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30°C was indistinguishable from that of the wild type. However, at 42°C it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37°C, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42°C, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 10⁶ CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation.     

Amphotericin B induces trehalose synthesis and simultaneously activates an antioxidant enzymatic response in Candida albicans

Background

Enzymes involved in trehalose metabolism have been proposed as potential targets for new antifungals. To analyse this proposal, the susceptibility to Amphotericin B (AmB) of the C. albicans trehalose-deficient mutant tps1Δ/tps1Δ, was examined.

Methods

Determination of endogenous trehalose and antioxidant enzymatic activities as well as RT-PCR analysis in cells subjected to AmB treatments was performed.

Results

Exponential tps1Δ null cultures showed high degree of cell killing upon exposure to increasing AmB doses respect to CAI.4 parental strain. Reintroduction of the TPS1 gene restored the percentage of cell viability. AmB induced significant synthesis of endogenous trehalose in parental cells, due to the transitory accumulation of TPS1 mRNA or to the moderate activation of trehalose synthase (Tps1p) with the simultaneous deactivation of neutral trehalase (Ntc1p). Since tps1Δ/tps1Δ mutant cells are highly susceptible to acute oxidative stress, the putative antioxidant response to AmB was also measured. A conspicuous activation of catalase and glutathione reductase (GR), but not of superoxide dismutase (SOD), was observed when the two cell types were exposed to high concentrations of AmB (5 μg/ml). However, no significant differences were detected between parental and tps1Δ null strains as regards the level of activities.

Conclusions

The protective intracellular accumulation of trehalose together with the induction of antioxidant enzymatic defences are worthy mechanisms involved in the resistance of C. albicans to the fungicidal action of AmB.

General significance

The potential usefulness of trehalose synthesis proteins as an interesting antifungal target is reinforced. More importantly, AmB elicits a complex defensive response in C. albicans.     

Molecular interaction of neutral trehalase with other enzymes of trehalose metabolism in the fission yeast Schizosaccharomyces pombe.

Trehalose metabolism is an essential component of the stress response in yeast cells. In this work we show that the products of the principal genes involved in trehalose metabolism in Schizosaccharomyces pombe, tps1+ (coding for trehalose-6-P synthase, Tps1p), ntp1+ (encoding neutral trehalase, Ntp1p) and tpp1+ (that codes for trehalose-6-P phosphatase, Tpp1p), interact in vitro with each other and with themselves to form protein complexes. Disruption of the gene tps1+ blocks the activation of the neutral trehalase induced by heat shock but not by osmotic stress. We propose that this association may reflect the Tps1p-dependent requirement for thermal activation of trehalase. Data reported here indicate that following a heat shock the enzyme activity of trehalase is associated with Ntp1p dimers or trimers but not with either Ntp1p monomers or with complexes involving Tps1p. These results raise the possibility that heat shock and osmotic stress activate trehalase differentially by acting in the first case through an specific mechanism involving Tps1p-Ntp1p complexes. This study provides the first evidence for the participation of the catabolic enzyme trehalase in the structural framework of a regulatory macromolecular complex containing trehalose-6-P synthase in the fission yeast.     

Flies without Trehalose

Living organisms adapt to environmental changes through metabolic homeostasis. Sugars are used primarily for the metabolic production of ATP energy and carbon sources. Trehalose is a nonreducing disaccharide that is present in many organisms. In insects, the principal hemolymph sugar is trehalose instead of glucose. As in mammals, hemolymph sugar levels in Drosophila are regulated by the action of endocrine hormones. Therefore, the mobilization of trehalose to glucose is thought to be critical for metabolic homeostasis. However, the physiological role of trehalose as a hemolymph sugar during insect development remains largely unclear. Here, we demonstrate that mutants of the trehalose-synthesizing enzyme Tps1 failed to produce trehalose as expected but survived into the late pupal period and died before eclosion. Larvae without trehalose grew normally, with a slight reduction in body size, under normal food conditions. However, these larvae were extremely sensitive to starvation, possibly due to a local defect in the central nervous system. Furthermore, Tps1 mutant larvae failed to grow on a low-sugar diet and exhibited severe growth defects on a low-protein diet. These diet-dependent phenotypes of Tps1 mutants demonstrate the critical role of trehalose during development in Drosophila and reveal how animals adapt to changes in nutrient availability.     

A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant

Abstract A Saccharomyces cerevisiae gene for trehalose-6-phosphate synthase (TPS1) was sequenced. The gene appeared to code for a protein of 495 amino acid residues, giving the protein a molecular mass of 56 kDa. The TPS1 gene was able to restore both osmotolerance and trehalose accumulation during salt stress in an Escherichia coli strain mutated in the otsA gene encoding trehalose-6-phosphate synthase. Complementation studies with E. coli galU mutants showed that the TPS1-encoded trehalose-6-phosphate synthase is UDP-glucose-dependent. Sequence analysis and data base searches showed that TPS1 is allelic to GGS1, byp1, cif1 and fdp1 . A possible gene for trehalose-6-phosphate synthase in Methanobacterium thermoautotrophicum was identified.     

Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.

The Saccharomyces cerevisiae FPS1 gene, which encodes a channel protein belonging to the MIP family, has been isolated previously as a multicopy suppressor of the growth defect of the fdp1 mutant (allelic to GGS1/TPS1) on fermentable sugars. Here we show that overexpression of FPS1 enhances glycerol production. Enhanced glycerol production caused by overexpression of GPD1 encoding glycerol-3-phosphate dehydrogenase also suppressed the growth defect of ggs1/tps1 delta mutants, suggesting a novel role for glycerol production in the control of glycolysis. The suppression of ggs1/tps1 delta mutants by GPD1 depends on the presence of Fps1. Mutants lacking Fps1 accumulate a greater part of the glycerol intracellularly, indicating that Fps1 is involved in glycerol efflux. Glycerol-uptake experiments showed that the permeability of the yeast plasma membrane for glycerol consists of an Fps1-independent component probably due to simple diffusion and of an Fps1-dependent component representing facilitated diffusion. The Escherichia coli glycerol facilitator expressed in a yeast fps1 delta mutant can restore the characteristics of glycerol uptake, production and distribution fully, but restores only partially growth of a ggs1/tps1 delta fps1 delta double mutant on glucose. Fps1 appears to be closed under hyperosmotic stress when survival depends on intracellular accumulation of glycerol and apparently opens rapidly when osmostress is lifted. The osmostress-induced High Osmolarity Glycerol (HOG) response pathway is not required for inactivation of Fps1. We conclude that Fps1 is a regulated yeast glycerol facilitator controlling glycerol production and cytosolic concentration, and might have additional functions.     

Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock

Synthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6 P ) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1 , TPS2 , and TSL1 . Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6 P synthase (Tps1) and Tre6 P phosphatase (Tps2), while Tsl1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, Tsl1, and Tps3, a protein with high homology to Tsl1, revealed that both Tsl1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1 , TPS2 , TSL1 , and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that Tsl1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6 P synthase/phosphatase complex in exponentially growing, heat-shocked cells.     

Glucose Signaling in Yeast Is Partially Mimicked by Galactose and Does Not Require the Tps1 Protein

Glucose produces multiple effects inSaccharomyces cerevisiae,as it controls the expression of many genes and the activity of various enzymes. However, the elements involved in glucose signaling are not well characterized. In this work the capacity of galactose to bring about the same effects than glucose has been assessed. Galactose mimics glucose only partially; it is suggested that it does not interact with a “sensor” in the plasma membrane and that it produces a weaker intracellular signal than glucose. To examine whether trehalose-6P synthase (Tps1) is required to transduce the glucose signal, we have constructed atps1 hxk2/tps1 HXK2strain which, at difference of atps1strain, grows on glucose, and, at difference of atps1 hxk2strain, still possess the Hxk2 protein, possibly involved in glucose repression. From the response of this strain to glucose, we conclude that Tps1 does not play a prominent role in glucose signaling.     

The involvement of hexokinases in trehalose synthesis.

Yeast cells harboring a MAL2-8c gene accumulate trehalose during the transition phase of growth on glucose due to the presence of the ADPG-dependent trehalose 6-phosphate synthase. Under these conditions, glucokinase appeared not to provide G-6-P for trehalose synthesis and the two hexokinases seemed to act synergistically. After incubation in d-xylose, trehalose levels in these cells dropped almost in 90%, confirming the involvement of both hexokinases in the accumulation of this carbohydrate. Nevertheless, G-6-P levels appeared to be similar in all strains. Some explanations for this paradox are discussed. In stationary phase, neither of the three isoenzymes were involved in trehalose synthesis. Possibly, gluconeogenesis provides the substrate for trehalose synthesis at that stage.     

Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues

Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.     

Regulation of genes encoding subunits of the trehalose synthase complex inSaccharomyces cerevisiae: novel variations of STRE-mediated transcription control?

Saccharomyces cerevisiae cells show under suboptimal growth conditions a complex response that leads to the acquisition of tolerance to different types of environmental stress. This response is characterised by enhanced expression of a number of genes which contain so-called stress-responsive elements (STREs) in their promoters. In addition, the cells accumulate under suboptimal conditions the putative stress protectant trehalose. In this work, we have examined the expression of four genes encoding subunits of the trehalose synthase complex,GGS1/TPS1, TPS2, TPS3 andTSL1. We show that expression of these genes is coregulated under stress conditions. Like for many other genes containing STREs, expression of the trehalose synthase genes is also induced by heat and osmotic stress and by nutrient starvation, and negatively regulated by the Ras-cAMP pathway. However, during fermentative growth onlyTSL1 shows an expression pattern like that of the STRE-controlled genesCTT1 andSSA3, while expression of the three other trehalose synthase genes is only transiently down-regulated. This difference in expression might be related to the known requirement of trehalose biosynthesis for the control of yeast glycolysis and hence for fermentative growth. We conclude that the mere presence in the promoter of (an) active STRE(s) does not necessarily imply complete coregulation of expression. Additional mechanisms appear to fine tune the activity of STREs in order to adapt the expression of the downstream genes to specific requirements.