Trehalose transport in yeast cells.

Saccharomyces cerevisiae possesses a specific transporter for trehalose. Transport is low until after glucose exhaustion in stationary phase, and addition of glucose in stationary phase results in loss of transport, an event that is reversible when the glucose is removed. The effect of glucose is not competitive inhibition: the increase in trehalose transport in stationary phase requires a lag time after glucose is depleted; and recovery of transport after glucose inhibition requires protein synthesis. Finally, we provide evidence that the trehalose transporter is distinct from that for maltose.

Trehalose-transporting membrane vesicles from yeasts.

We have isolated and characterized a membrane vesicle fraction from yeasts that is capable trehalose transport. The kinetics of the transport system were similar to those seen in the intact cells. The transport depends on a transmembrane pH gradient. If the gradient is collapsed, trehalose accumulated inside the vesicles is leaked into the medium. After aging for several days the ability of the vesicles to transport was lost. However, transport was partially restored by elevating internal pH in the vesicles.

Regulation of trehalose metabolism in Saccharomyces cerevisiae mutants during temperature shifts.

Temperature shifts from 23 degrees C to 36 degrees C resulted in trehalose accumulation in Saccharomyces independently of genetic lesions in the cAMP-protein kinase cascade. In parallel, trehalose 6-phosphate synthase activity increased about 3-fold in all strains; the increase could be inhibited by cycloheximide, suggesting that protein synthesis was required. Heat shock treatment after the temperature shift led to a drastic increase in trehalose activity, and deactivation of the biosynthetic enzyme with a consequent drop in trehalose. Up to now no definite correlation between acquisition of thermotolerance and trehalose accumulation has been made.

On the determination of trehalose-6-phosphate synthase in Saccharomyces.

Trehalose-6-phosphate synthase activity was determined by colorimetric, spectrophotometric and trehalose specific assays. All methods gave comparable results thus confirming our previous findings (1) and those reported by Elander (2). Different strains and mutants of Saccharomyces were carefully re-investigated in relation with the recent claim made by Vandercammen et al. (3) that our spectrophotometric assay over-estimated the enzyme activity and that no differences exist between wild type and mutant strains. In this paper we also confirm the de-activation of the trehalose synthase complex in response to a "glucose signal", and present trehalose-6-phosphate synthase and trehalase activities in different strains measured during all phases of growth on glucose.

Effect of dimethylsulfoxide on signal transduction in mutants of Saccharomyces cerevisiae.

1. As the first part of a study of pesticide toxicity we report the effects of the solvent dimethylsulfoxide (DMSO) on signal transduction in mutants of Saccharomyces cerevisiae. 2. The enzymes of trehalose metabolism, which are activated and deactivated by a "glucose signal" and by heat shock treatment, were chosen as targets for this study. 3. DMSO was shown to be able to permeate glucose and cAMP. The effects of glucose and cAMP were enhanced by pre-incubating the cells in the presence of DMSO. 4. No effects were observed during the heat shock, suggesting that the solvent acts on the cell membrane. 5. The results suggest that DMSO may be used as a vehicle for small molecules which do not easily penetrate yeast cell membranes, thus providing a new tool for biochemical and toxicological studies.

Effect of dimethylsulfoxide on signal transduction in mutants of Saccharomyces cerevisiae

As the first part of a study of pesticide toxicity we report the effects of the solvent dimethylsulfoxide (DMSO) on signal transduction in mutants of Saccharomyces cerevisiae. The enzymes of trehalose metabolism, which are activated and deactivated by a "glucose signal" and by heat shock treatment, were chosen as targets for this study. DMSO was shown to be able to permeate glucose and cAMP. The effects of glucose and cAMP were enhanced by pre-incubating the cells in the presence of DMSO. No effects were observed during the heat shock, suggesting that the solvent acts on the cell membrane. The results suggest that DMSO may be used as a vehicle for small molecules which do not easily penetrate yeast cell membranes, thus providing a new tool for biochemical and toxicological studies

Comparative studies between the glucose-induced phosphorylation signal and the heat shock response in mutants of Saccharomyces cerevisiae.

Addition of glucose to derepressed yeast cells, as well as a heat shock treatment, trigger the phosphorylation of trehalase and of trehalose-6-phosphate synthase. In the present paper, evidence is provided for the requirement of the RAS protein in the transduction of the glucose signal. On the other hand, a heat shock at 52 degrees C for 2 min was able to produce a significant phosphorylating effect even in mutant strains deficient in the GTP binding protein. Moreover, it was shown that this treatment does not affect exclusively the cAMP-dependent protein kinase. The use of a series of mutant strains confirmed that low levels of cAMP favor thermotolerance; the role of trehalose in yeast viability is also discussed.

Determination of trehalose in biological samples by a simple and stable trehalase preparation.

A three step purification procedure for trehalase from Saccharomyces cerevisiae with a recovery of 76% of the original activity is presented. The enzyme was activated by a heat shock treatment prior to homogenization of the cells. A mutant strain deleted in SUC genes was used to avoid contamination by invertase. The lyophylized enzyme was stable for, at least, 5 months and could be used to determine trehalose in the range 25 to 500 nmol. The preparation was free of inspecific phosphatases allowing for trehalose determinations in yeast cell free extracts and in insect hemolymph.

Fructose 2,6-bisphosphate and trehalose metabolism in Saccharomyces cerevisiae.

1. A regulatory mutant of Saccharomyces (fdp) unable to activate fructose 1,6-bisphosphatase presented a normal response to the glucose and fructose signals as measured by trehalase activation, indicating that the inability of the strain to grow on these sugars is caused by a defect located beyond membrane interactions. 2. In vivo experiments with a mutant strain bearing a phosphoglucoisomerase gene (pgil-delta) deletion showed that activation of trehalase and deactivation of the tehalose-6-phosphate synthase complex occurred to the same extent whether glucose or fructose was used as signal. 3. These results suggest that fructose-2,6-bisphosphate is not involved in the interconversion of forms of the enzymes of trehalose metabolism. Furthermore, when fructose-2,6-bisphosphate was assayed on trehalose synthesizing activity using cell-free extracts and partially purified preparations of the complex, no effect was observed. 4. We conclude that regulation by cAMP fulfills the requirements for control of trehalose levels in Saccharomyces.

Fructose 2,6-bisphosphate and trehalose metabolism in Saccharomyces cerevisiae

1. A regulatory mutant of Sccharomyces (fdp) unable to activate fructose 1,6-bisphosphatase present a normal response to the glucose and fructose signals as measured by trehalase activation, indicating that the inability of the strain to grow on these sugars is caused by a defect located beyond membrane interactions. 2. In vivo experiments with a mutant strain bearing a phosphoglucoisomerase gene (pgil-delta) deletion showed that activation of trehalase and deactivation of the tehalose-6-phosphate synthase complex occurred to the same extent whether glucose or fructose was used as signal. 3. These results suggest that fructose-2,6-bisphosphate is not involved in the interconversion of forms of the enzymes of trehalose metabolism. Furthermore, when fructose-2,6-bisphosphate was assayed on trehalose synthesizing activity using cell-free extracts and partially purified preparations of the complex, no effect was observed. 4. We conclude that regulation by cAMP fulfills the requirements for control of trehalose levels in Saccharomyces

Regulation of the trehalose-6-phosphate synthase complex in Saccharomyces. I. Interconversion of forms by phosphorylation.

Trehalose-6-phosphate synthase is another example of an enzyme of carbohydrate metabolism, in Saccharomyces, which could be regulated by interconversion of forms. Deactivation was mediated both in vivo and in vitro by a cyclic AMP-dependent protein kinase. Reversibility of this process was obtained by a phosphatase treatment leading to an increase in activity. The phosphorylated, less active form of the enzyme proved to be more susceptible to activation by ATP.Mg. Mutants with well defined lesions in the cyclic AMP-dependent protein kinase system were used to corroborate our findings of a possible regulatory mechanism of trehalose-6-phosphate synthase activity by interconversion of forms.

Catabolite inactivation of trehalose synthesis during growth of yeast on maltose.

1. The effects of catabolite inactivation upon the trehalose pathway linked to maltose utilization were investigated in Saccharomyces cerevisiae. Mutant strains devoid of UDPG-trehalose synthase activity were used in this study. 2. Trehalose accumulation was also susceptible to catabolite inactivation as has been reported for the carrier protein, one of the components of the maltose system. Reversibility was only achieved when incubation with glucose did not exceed 5 min and was dependent upon protein synthesis.

Catabolite inactivation of trehalose synthesis during growth of yeast on maltose

1. The effects of catbolite inactivation upon the trehalose pathway linked to maltose utilization were investigated in Saccharomyces cerevisiae. Mutant strains devoid of UDPG-trehalose synthase activity were used in this study. 2. Trehalose accumulation was also susceptible to catabolite inactivation as has been reported for the carrier protein, one of the components of the maltose system. Reversibility was only achieved when incubation with glucose did not exceed 5 min and was dependent upon protein sunthesis