YEAST

The

VOL. 7: 575-587 (1991)

Control of Trehalose Biosynthesis in Saccharom yces cerevisiae: Evidence for a Catabolite Inactivation and Repression of Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase JEAN FRANCOIS, MARIA-JOSE NEVES AND HENRI-GERY HERS

Lahorutoire de Chimie Physiologique, UniversitP Catholique de Louvain and International Institute of Cellular and Molrculur Pathology. UCL. 7539. Avenue Hippocrate. 7S, 8-1200 Bruxelles, Belgium

Received I September 1990; revised 5 March 1991

During diauxic growth of yeast in glucose-rich medium, the accumulation of trehalose started well after complete cxhaustion of glucose from the medium. The accumulation of the disaccharide was concomitant with a resumption of cell growth on the ethanol accumulated in the medium, but not with a degradation of glycogen which occurred as soon as glucosc had been consumed. In contrast, in a mutant deficient in phosphoenolpyruvate carboxykinase, the synthesis of trehalose coincided exactly with the degradation of glycogen. Upon inoculation of stationary phase wild-type cells into a glucose medium, the activities of trehalose-6-phosphate (Tre6P) synthase and Tre6P phosphatase dropped in parallel to reach only 15% of their initial values after 3 h, and only recovered their original values as cells re-entered stationary phase. In the presence of cycloheximide, the decrease in Tre6P synthase and Tre6P phosphatase activities was restricted to 5 M % , the remaining decrease being inhibited by the drug. Furthermore, the reappearance of the enzyme activities following transfer ofcells to an acetate medium was blocked by cycloheximide. It was also shown that loss of activity of these two enzymes required a combination of metabolizable sugars together with a nitrogen source. Low activities of Tre6P synthase and Tre6P phosphatase were measured in mutants with increased adenylate cyclase activity (RAST'"'""''' mutants). Moreover, derepression of these enzymes at the approach of stationary phase was prevented in apde2 mutant when it wascultivated in the presence of exogenouscyclic nucleotide. The mechanism of this effect is not clear, but may involve a transcriptional regulation by cAMP of the genes encoding these proteins.

KEY WORDS Yeast; trehalose metabolism; catabolite inactivation and repression.

INTRODUCTION The occurrence of trehalose, a non-reducing disac- charide. in fungi is well documented (Thevelein, 1984). In Saccharomyces cerevisiae, the accumu- lation of trehalose appears generally to be associ- ated with periods of reduced growth, such as during starvation of cells for nitrogen, phosphate or sul- phur (Lillie and Pringle, 1980), as well as during the stationary phase of growth on glucose (Lillie and Pringle, 1980; Panek and Mattoon, 1977; FranCois P t al., 1987). Conversely, resumption of growth by adding the appropriate nutrient to the deficient medium or induction of growth by resuspending resting cells into a fresh glucose medium results in a rapid mobilization of trehalose (Thevelein, 1984).

Several lines of evidence suggest that, in yeast, trehalose mobilization is regulated by a CAMP-

0749 503X:91/06057S13 $06.50 0 1991 by John Wiley & Sons Ltd

dependent phosphorylation of the neutral trehalase (Thevelein, 1984). Indeed, addition of glucose to stationary-phase yeast causes a simultaneous transient increase in cAMP levels and an activation of trehalase (van der Plaat and van Solingen, 1974; FranCois el al., 1984; Thevelein and Beullens, 1985). Incubation of crude extracts or of purified trehalase with CAMP-dependent protein kinase, cAMP and ATP-Mg results in a ten-fold increase in trehalase activity (Uno e t al., 1983; Dellamora-Ortiz ef al., 1986; Thevelein and Beullens, 1985). Mutants deficient in the regulatory subunit of the CAMP- dependent protein kinase showed low levels of trehalose and high activity of trehalase (Uno et al., 1983). Another trehalase, distinct from the first one by an activity optimal at pH 5.0, also exists in yeast. This enzyme is not under the control of CAMP and

576 J. FRANCOIS, M.-J. NEVES AND H.-G. HERS

Yeast strains andgrowth conditions The following strains were used: a wild type

S288C (MATa SUC2 ma1 melga12 CUPl) from the Yeast Genetic Stock Center (Berkeley, CA, U.S.A.); PUK-3B (MATapck 1 ura3 adel), kindly provided by Dr C. Gancedo, Madrid, Spain); and the strains JC482 (MATa ura3 leu2 hisl), JC302-26B (MATa ura3 leu2 his4 ras2-.530::LEU2), LRA81 ( M A Ta ura3 leu2 his4 RAS2ala'8val'9 ) and RW301 (MATa trpl pde2:: TRP1) from K. Tatchell (Raleigh, NC, U.S.A.). The cells were grown at 28°C on a 2% glucose, 2% bacto-peptone and 1% yeast extract medium (YEPD or glucose-rich medium) with vigorous agitation. Approximately 10 h after exhaustion of glucose from the medium, cells were harvested by centrifugation, washed once with sterile water and reinoculated at approximately 1.5 mg wet weight/ml (or 2 x lo7 cells/ml) in either the same medium (see Figures 1 4 , 6 and 7) or in a sodium succinate buffer, 50 mM, pH 5.5 (Figure 5). In this latter condition, the cells were preincubated at 28°C for 60 min before addition of the various compounds as indicated in Figure 5. When used, bacto-peptone, yeast extract, potassium phosphate and NH4C1 were added to the yeast suspension as ten-fold concentrated aqueous solution adjusted to pH 5.5 with NaOH or HCI.

its role in the degradation of trehalose remains to be established (Thevelein, 1984).

While much work has been done on trehalose mobilization in S . cerevisiae, little is known about the regulation of its biosynthesis. Trehalose-6- phosphate (Tre6P) synthase and Tre6P phospha- tase, the two enzymes involved in the synthesis of trehalose, have recently been characterized (Vandercammen et al., 1989; Londesborough and Vuorio, 199 I). Their activities co-elute using several chromatographic procedures, suggesting that they are part of a single bifunctional protein. In addition, contrary to the claim of Panek et al. (1987), neither the activity of Tre6P synthase nor that of Tre6P phosphatase appears to be regulated by intercon- version of two forms through phosphorylation/ dephosphorylation. However, regulation of these enzymes, either through allosteric effects or catabo- lite inactivation and/or repression, must occur in order to explain why glucose-repressed yeast have a much lower capacity for trehalose accumulation than do glucose-derepressed cells when resuspended in a nitrogen-free glucose medium (Panek, 1975; Panek and Mattoon, 1977; Panek et al., 1980). Moreover, it is unclear whether the glucosyl units required for the synthesis of trehalose in the absence of glucose come from the degradation of glycogen (Lillie and Pringle, 1980; Franqois et al., 1987) or are supplied by gluconeogenesis (Grba et al., 1975; Panek, 1975).

The present work was undertaken to clarify these problems by determining, under various growth and incubation conditions of yeast, the concen- trations of trehalose, glycogen, key metabolites and the activities of the enzymes involved in trehalose metabolism.

MATERIALS AND METHODS Materials

14C]G1c 1 P was purchased from the Radiochemical Centre(Amersham, U.K.). UDP-U-'4C] glucoseand I4C]Tre6P were synthesized as described previously (Vandercammen et al., 1989). Glucose and mannose were obtained from Fluka (Switzerland); xylose, 2- deoxyglucose, dithiothreitol and PMSF (phenyl- methylsulfonyl fluoride) were from Janssen Chimica (Belgium). Fructose, trehalose, 2,4-dinitrophenol and other chemicals of analytical grade were from Merck. Yeast extract, bactopeptone and yeast nitrogen base were purchased from Difco (U.S.A.). Cycloheximide, auxiliary enzymes and other bio- chemicals were from Boehringer (F.R.G.).

Measurement of trehalose, glycogen and other metabolites

Samples were collected as described in (Franqois et al., 1984). Glycogen (Franqois et al., 1987) was assayed in alkaline extracts prepared by mixing frozen samples (50mg wet weight) in 1 ml of hot 0.25 M-Na,CO, and further incubated at 90°C for 2 h with occasional shaking. Trehalose was assayed in the same alkaline extracts treated as follows: after only 30min of incubation at 90"C, 0.2ml of the alkaline suspension was withdrawn and centrifuged for 5 min at 1000 x g. The supernatant was then adjusted to pH 5.5 with 1 M-acetic acid and brought to a final volume of 0.5 ml with 0.1 M-Na acetate, pH 5.5. Trehalose was then determined as described previously (Vandercammen et al., 1990). Extraction for determination of metabolites was performed according to Franqois et al. (1984). Glucose-6- phosphate and fructose-6-phosphate (Lang and Michal, 1974), UDP-glucose (Strominger et al., 1957) and ATP (Lamprecht and Trautschold, 1974) were measured by published procedures. Concen- trations of metabolites are expressed as mol/g wet weight.

THE CONTROL OF TREHALOSE BIOSYNTHESIS IN SACCHAROMYCES CEREWSZAE 577

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Figure 1. Changes in the concentrations of glycogen, trehalose and related metabolites and enzymes during the growth of a wild-type strain on glucose. The strain S288C was grown in culture medium containing 2% glucose, 1% yeast extract and 2% bacto-peptone (YEPD medium). Note that the vegetative growth is expressed as amount of protein per ml of culture. Tre6Psyn = Tre6P synthase and Tre6Ppase = Tre6P phosphatase.

Time since inoculation (h)

Enzymatic assays

Cell samples (about 100mg wet weight) were collected on nitrocellulose filters (diameter 45 mm, pore size 0.45 p ~ ) as described previously (FranCois et al., 1984). The extracts were prepared in a solution containing 100 mM-KCl, 1 mM-PMSF, 1 mM-dithiothreitol, 2 mM-EDTA, 20 pg/ml anti- pain and 20m~-Hepes, pH 7.1 according to Vandercammen et al. (1989). Trehalase (FranCois et al., 1984), fructose-l,6-bisphosphatase (FBPase- 1 Gancedo, 197 11) and phosphoenolpyruvate car- boxykinase (PEPCK Gancedo and Schwerzmann, 19761) wereassayedasdescribed. Tre6P synthaseand Tre6P phosphatase were measured by the radio- chemicalmethodsdescribed by Vandercammenet al. (1989), except that the concentration of UDP-U- ''C]glucose and that of I4C]Tre6P were 0.8 and 0 . 3 m ~ , respectively. One unit is the amount of enzyme that catalyses the conversion of 1 pmol substrate in 1 min under the conditions of the assay.

Other methods

The concentration of protein was determined by the method of Bradford (1976) with bovine

immunoglobulin G as a standard. For the esti- mation of total cellular protein, a sample of the yeast suspension was diluted five-fold in cold water and centrifuged for 5 min at 1000 x g. The super- natant was decanted and the pellet digested in 1 M- NaOH for 2 hat 90°C. After cooling, the suspension was centrifuged for 5 min at 5000 x g and an aliquot of the clear supernatant was taken for the protein assay. Glucose (Huggett and Nixon, 1957) and ethanol (Cornell and Veech, 1983) were assayed in the culture medium by published procedures. Results shown are representative of at least three different experiments which yielded similar results.

RESULTS Changes in the concentrations of glycogen, trehalose and of their related metabolites and of enzymes during growth on glucose

As illustrated in Figure 1A and B, the wild-type strain S288C grown to stationary phase in YEPD medium contained approximately 80 and 30 pmol glucose equivalents of trehalose and glycogen, respectively. When this strain was inoculated in the same medium at a cell density of 1-5 mg/ml (approxi- mately 2 x lo7 cells/ml), trehalose was completely

578 J. FRANCOIS, M.-J. NEVES AND H.-G. HERS

occurred later than that of the trehalose-synthesizing enzymes and was concomitant with the start of trehalose synthesis.

Using a minimal medium (SD) or a synthetic com- plete medium containing glucose (Sherman et al., 1986), the changes in trehalose and trehalose synthe- sizing enzymes during growth were qualitatively similar to those reported in Figure 1 (data not shown). In contrast, when glucose was replaced by a gluconeogenic source, such as ethanol, the activities of Tre6P synthase and Tre6P phosphatase were diminished by only 30% during the first hour of growth and then remained at a steady-state value of 60 and 16 mU/mg protein, respectively, whereas trehalase was maintained at a low level of 1 mU/mg protein throughout the growth. Interestingly, both trehalose and glycogen accumulated in parallel during the growth on ethanol at a similar rate of approximately 50 nmol glucose units incorporated/ min per g cells (results not illustrated).

degraded within2 hatarate thatcouldexceed2 pmol glucose produced/min per g wet weight, whereas the concentration of glycogen was slightly increased before declining at a similarly rapid rate (Figure IB). The concentration of these two carbo- hydrates remained very low (below 2 pmol glucose equivalents/g) during the exponential phase (between 2 and 5 h) and, in agreement with previous results (Lillie and Pringle, 1980; Franqois et al., 1987), glycogen accumulated and reached a maxi- mum of 40 pmol/g wet weight shortly before exhaustion of glucose from the medium, being then rapidly degraded. Remarkably, the 50% decrease in glycogen concentration occurring then over a period of 2 h was not accompanied by any accumu- lation of trehalose. The synthesis of the disaccharide was actually delayed until the resumption of growth on the accumulated ethanol in the medium. This event occurred in parallel with a renewed accumulation of glycogen.

Other phenomena recorded during the growth of yeast are also shown in Figure 1C. The concen- tration of hexose monophosphate, which was initially close to 0.2 pmol/g, rose rapidly and con- comitantly with the early degradation of trehalose and glycogen to reach six times this value at 3 h, and then came down to its initial value at the end of the log phase; a slight rebound was noted at 8 h, corre- sponding to the second period of glycogenolysis. The concentration of UDP-glucose decreased two- fold during the first 2 h, and increased again when both glycogen and trehalose were actively synthe- sized. The concentration of ATP was close to 1 pmol/g during the whole experimental period (data not shown).

The activities of Tre6P synthase and Tre6P phos- phatase decreased in parallel by about three-fold during the first 2 h after inoculation of the cells, while practically no cell growth occurred (Figure 1D). In some experiments, however (see Figures 3 and 4), the decrease in phosphatase activity was more rapid than that of the synthase. These two activities then remained at about a sixth of their initial value during the exponential growth and increased again to their initial value as cells approached the stationary phase (between 6 and 8 h). It was also observed that trehalase (see Figure 5B) and FBPase-1 (see also Figure 3C) were respect- ively activated and inactivated soon after the inocu- lation into fresh medium. FBPase-1 disappeared below the level of detection but reappeared after complete disappearance of glucose from the medium. The recovery of this enzyme activity

Coincidence of glycogen degradation with accumu- lation of trehalose in a yeast mutant deficient in PEPCK

The type of experiment shown in Figure 1 was also performed with a mutant deficient in PEPCK activity. Results of this investigation are reported in Figure 2. As observed with wild-type cells, glycogen began to accumulate when approximately half of the glucose concentration initially present in the medium remained to be consumed, and reached a maximum of 120 pmol/g wet weight as cells entered stationary phase (Figure 2B). The degradation of the polysaccharide which ensued was accompanied by an accumulation of trehalose, both phenomena occurring at rates of approximately 100 nmol of glu- coseunits liberated and incorporated per min and per g of wet cells. Remarkably, the synthesis of trehalose started some hours before complete exhaustion of glucose. As expected, no carbohydrate synthesis occurred at the expense of ethanol.

Figure 2C also shows the variations in metabolite concentrations and enzyme activities during growth of the mutant. Hexose monophosphate levels were similar to those observed in wild-type cells, while the concentration of UDP-glucose exhibited a transient rise concomitant with the arrest of glycogen synthe- sis and decreased subsequently to its initial value, when trehalose was actively synthesized. The con- centration of ATP, however, decreased steadily along the exponential phase of growth to reach a minimum value of 0.25 pmol/g wet weight at the

THE CONTROL OF TREHALOSE BIOSYNTHESIS IN SACCHAROMYCES CEREVISIAE 579

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Figure 2. Changes in the concentrations of glycogen, trehalose and related metabolites and enzymes during the growth of a yeast mutant deficient in phosphoenolpyruvate carboxykinase (PEPCK) on glucose. General procedure and abbreviations were as in Figure I , except that the strain used was PUK-3B, which is deficient in PEPCK.

onset of stationary phase and remained at this low level. As observed in a wild-type strain, Tre6P syn- thase and Tre6P phosphatase activities were low early in the exponential phase of growth (Figure 2D). They then increased rapidly, precisely at the time that trehalose started to accumulate. FBPase-1 was derepressed later, when glucose in the medium was exhausted and, as expected, PEPCK was undetectable.

Studies on the phenomenon of loss of Tre6P synthase and TredP phosphatase activities

Eflect of cycloheximide The inactivation of Tre6P synthase, Tre6P phosphatase and, for the sake of comparison, FBPase 1 (see also: Gancedo, 1971; Lenz and Holzer, 1980), occurring during the first hours after inoculation of the wild-type strain S288C into the glucose medium is shown in greater detail in Figure 3. These inactivations were particu- larly rapid during the first hour of incubation, reaching two- to three-fold7 whereas the increase in protein content did not exceed 30%. Remarkably, none of these events was clearly affected by the presence of 20 pg/ml cycloheximide, a potent pro-

tein synthesisinhibitor (Schindler and Davies, 1974). However, upon prolonged incubation, this inhibitor prevented the further two-to three-fold decrease in Tre6P synthase and Tre6P phosphatase activities as well as theequivalent increaseincell growth, asdeter- mined by the measurement of total protein. In agree- ment with previous works (Gancedo, 1971), it also prevented the complete proteolytic degradation of FBPase 1.

When cells that had been incubated in a glucose- rich medium for either 10 or 120 min were removed from the culture medium by filtration and then transferred to an acetate-containing, glucose-free growth medium, the activities of Tre6P synthase, Tre6P phosphatase (Figure 4A and B) and FBPase 1 (results not shown) were progressively and roughly in parallel restored to their initial values within 6 h. Remarkably, this recovery was prevented by the presence of 20 pg/ml of cycloheximide, but only after 20min of incubation in the presence of the inhibitor. In contrast, deactivation of trehalase, which is known to be caused by dephosphorylation of the enzyme (Uno et al., 1983), was not affected by cycloheximide at both times of transfer of cells to the gluconeogenic medium (Figure 4C).

580 J. FRANCOIS, M.-J. NEWS AND H.-G. HERS

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Figure 3. Effect of cycloheximide on the activities of Tre6P synthase, Tre6P phosphatase and FBPase-1 in yeast incubated in a glucose-rich medium. Strain and general procedure were as in Figure I, except that the medium was supplemented with- out (open symbols) or with (closed symbols) 20pg/ml of cycloheximide.

Factors triggering the inactivation-repression process Because a decrease in the activities of Tre6P syn-

thase and Tre6P phosphatase had not been observed upon addition of glucose alone to a sus- pension of yeast incubated in a buffered medium (Vandercammen et al., 1989), we investigated the influence of other components of the medium (yeast extract, bacto-peptone) on the levels of trehalose and on the activities of the trehalose-metabolizing enzymes, when added either alone or together with glucose to a suspension of yeast. This experiment, illustrated in Figure 5, confirmed the previous observation (Thevelein and Beullens, 1985) that

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Figure 4. Effect of the removal of the glucose medium on the activities of Tre6P synthase. Tre6P phosphatase and trehalase in yeast. General procedure was as in Figure I , except that after 10 and I20 min (arrows), 200 ml of the cell suspension was filtered on a nitrocellulose filter (50 mm diameter. 0.45 p~ pore size). The cell cake on the filter was washed twice with 20ml of water and then resuspended in 100 ml of a medium containing 0.67% yeast nitrogen base and 1% sodium acetate, in the absence (open symbols) or in the presence (closed symbols) of20 pg/ml of cycloheximide.

addition of glucose alone causes a transient acti- vation of trehalase as well as a partial trehalose mobilization followed by a resynthesis of the disac- charide. Under this condition, the activities of Tre6P synthase and Tre6P phosphatase were not significantly modified (Figure 5C). Bacto-peptone and yeast extract, each added alone to the cell sus- pension, were without significant effect on these par- ameters, except that a slight and transient activation

THE CONTROL OF

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TREHALOSE BIOSYNTHFSIS IN SACCHAROMYCES CEREVISIA E 58 1

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Figure 5 . Effect of addition of glucose in the absence or in the presence of bacto-peptone on the concentration of trehalose and on the activities of Tre6P synthase, Tre6P phosphatase and trehalase in a suspension of yeast. The strain S288C was incubated in a SOmM-sodium succinate buffer, pH 5 5 . When used, glucose or bacto-peptone were added at a final concentration of 2%.

of trehalase was recorded after addition of yeast extract (not shown). This effect was probably due to the small amount ofglucose that contaminates yeast extract (approx 5 pmols glucose/g yeast extract).

Glucose combined with yeast extract (not shown) or with bacto-peptone (Figure 5 ) caused a complete degradation of trehalose which was not followed by a resynthesis of the disaccharide. Under that con- dition, trehalase was activated at a same or even higher level than after addition ofglucose alone, and the activity of this enzyme remained high for a longer time. Tre6P synthase and Tre6P phosphatase activities decreased progressively to reach only 15% of their initial value after 2 h of incubation. During this period, the total cellular protein had been increased by only 2.5-fold (not illustrated).

In order to know whether the decrease of Tre6P synthase and Tre6P phosphatase activities required the metabolization of glucose and was energy dependent, the experiments shown in Figure I were repeated with other glycolytic substrates, inert sugars or glucose analogues. When fructose or mannose were used instead of glucose, results were very similar to those observed in the presence of glucose. When the cells were incubated in a culture

medium containing either xylose, an inert sugar that enters the cell via the glucose transporter (Heredia et al., 1968), or 2-deoxyglucose, which depletes ATP from cells (Eraso and Gancedo, 1985), cells con- sumed their endogenous trehalose at a rate two- to three-fold lower than that in the presence of glucose. This slower rate of trehalose mobilization was related to a slower activation of trehalase. Under these latter conditions, the activities of Tre6P syn- thase and Tre6P phosphatase were not modified, although they were barely (30%) decreased in the presence of xylose (results not shown).

Tre6P synthase and Tre6 P phosphatase activities in mutants aflected in the cAMP pathway

Because of increasing evidence that cAMP could be involved in the repression of some enzymes by glucose (Bissinger et al., 1989; Cherry et al., 1989; Ramosand Cirillo, 1989), Tre6Psynthase and Tre6P phosphatase activities were determined in mutants with a disrupted (rau2) or altered (RAS2"'a'x"a''~ RAS2 gene, whose product is involved in the control of yeast adenylate cyclase (reviewed in Gibbs and Marshall, 1989). The ras2 and RAS2"Xva''9 mutant

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J. FRANCOIS, M.-J. N E W AND H.-G. HERS

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Figure 6. Concentration of trehalose and activities of Tre6P synthase, Tre6P phosphatase and trehalase inrasmutantsincubatdinaglucose-rich medium. General procedures wereasin Figure 1,except that the strains used were JC482 (wild type), LRA8 I strain) and JC302-26B (ras2 strain).

strains have, respectively a lower and a higher activity ofcyclase and thus lower and higher levels of cAMP than those of their isogenic control strain (Toda ef al., 1985). In confirmation of previous works (Fraenkel, 1985; Tatchell ef al., 1985; Toda el al., 1985), these two strains exhibited, respect- ively, higher and undetectable concentrations of trehalose as compared to the control strain (Figure 6A). Activities of Tre6P synthase and Tre6P phos- phatase in the strain carrying the ras2 disruption were similar to those of the isogenic strain but they were five-fold lower in the RASTJ"'"a'19 trans- formed cells (Figure 6C and D). Conversely, this latter strain displayed an initial activity of trehalase which was five-fold higher than that of the ras2 mutant and of the wild-type (Figure 6B). Upon inoculation into a fresh culture medium, trehalase was rapidly increased in the control and in the ras2 strains but not in the RAS2U'u18va11Y- transformed cells. Furthermore, the low Tre6P synthase and Tre6P phosphatase activities measured in these cells were not further decreased after 4 h of incubation. In contrast, the changes of activity of these enzymes in the ras2 mutant were roughly similar to those of the wild-type, although Tre6P synthase appeared to decrease less between 2 and 4 h. Similar experiments

were repeated with a bycl mutant, which contains a disruption of the gene encoding the regulatory sub- unit of the CAMP-dependent protein kinase (reviewed in Gibbs and Marshall, 1989); results comparable to those obtained with the RAS2U'''Bvu''Y transformed cells, i.e. very low Tre6P synthase and Tre6P phosphatase, and high trehalase activities, were found (results not illustrated).

Taken together, these results indicate that cAMP may somehow affect the activity of Tre6P synthase and Tre6P phosphatase. To test more directly the effect of CAMP on the activity of these enzymes, we used a pde2 mutant, which contains a disruption of the gene encoding the high-affinity phosphodiester- ase (PDE2). Because of this mutation, this strain was found to be able to incorporate exogenous cAMP (Wilson and Tatchell, personal communi- cation). Indeed, when this mutant iscultivated in the presence of exogenous nucleotide, its intracellular concentration of cAMP can be raised more than 20-fold, and as a consequence of this dramatic increase, the cells display roperties similar to those of bycl and RASP'a'8va'9 mutants, i.e. they are unable to accumulate reserve carbohydrate and are extremely sensitive to heat shock (Wilson and Tatchell, personal communication). As shown in

THE CONTROL OF TREHALOSE BIOSYNTHESIS IN SACCHAROMYCES CEREVISIAE 583

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Figure 7. Changes in trehalose levels and in the activities of trehalose metabolizing enzymes during growth of a pde2 mutant in a glucose-rich medium in the absence (open symbols) or in the presence (closed symbols) of CAMP. General procedures were as in Figure I , except that the strain used was RW301. which contains a disruption of the gene encoding the low K, phosphodiesterase. CAMP was added to the culture medium at a finalconcen- tration of4 mM either at time 0 (black circles), at the log phase (filled triangle) or at the stationary phase (filled squares) of the growth.

Figure 7. the activation of trehalase, loss of Tre6P synthase and Tre6P phosphatase activities and the degradation of trehalose that occurred after inocu- lation of cells were not significantly affected when the culture medium was supplemented with 4 mM- CAMP. As cells approached stationary phase, trehalase became inactive and the activities ofTre6P synthase and Tre6P phosphatase increased to their initial values. These changes as well as the accumu- lation of trehalose during the stationary phase of growth were strongly impaired when cAMP was added to yeast either at the beginning or at the mid- log phase of growth. In contrast, the nucleotide was without effect on these parameters when it was added to cells in the stationary phase of growth. I t is also interesting to note that the optical density

reached by yeast culture in stationary phase was significantly reduced when cAMP was added to the medium either at the beginning or at the log phase of the growth.

DISCUSSION In the absence of glucose, irehalose can be formed at the expense ofglycogen or from gluconeogenic precursors

The data presented in Figures 1 and 2 clearly show that both gluconeogenesis and glycogenolysis can provide the glucosyl units for the synthesis of trehalose during the stationary phase of growth, after that glucose has been exhausted from the medium. Which carbon source is used depends on

584 J. FRANCOIS, M.-J. NEVES AND H . G . HERS

within 1 h after inoculation of yeast in the glucose- rich medium was about 2.5-fold higher than the increase in total protein during the same period of time, it cannot be accounted for only in terms of repression of enzyme synthesis and dilution of the pre-existing activity by growth. In addition, the fact that the reappearance of enzyme activities after transfer of cells to a growth medium deprived of glucose was prevented by cycloheximide indicates that this reactivation requires protein synthesis and cannot be explained by a reversible interconversion between two forms, one active, the other inactive, of the two enzymes, as suggested previously (Panek et al., 1987) and as occurs in the case of trehalase (see Figure 4C).

The further two- to three-fold decrease in the activities of Tre6P synthase and Tre6P phosphatase observed during the next 2 h of incubation was more likely the result of a repression of enzyme synthesis, since the extent of this change was comparable to that of cell growth and also because this decrease was prevented by cycloheximide. However, this explanation does not apply to the effect of cyclo- heximide to block the irreversible inactivation of FBPase-1, since this phenomenon is due to a proteo- lytic degradation of that enzyme (Funayama et al., 1980), in which the role of protein synthesis is still poorly understood (Gancedo, 1971; Gancedo and Schwerzmann, 1976). It is also worth noting that, in our hands, cycloheximide had no detectable effect either on protein synthesis or on enzyme activities within the first 20 to 30min after its addition to the medium. A similar latency in the effect of the inhibitor was also apparent in the experiments by Gancedo (1971), who reported for FBPase 1 results very similar to those shown in Figure 4 for Tre6P synthase and Tre6P phosphatase; it might be explained by a slow penetration of the drug into the cells.

The repression of Tre6P synthase and Tre6P phos- phataseduring growth on glucose is also indicated by the rapid resynthesis of these enzymes which, inter- estingly, occurred before complete exhaustion of glucose from the medium. This indicates that, in contrast to FBPase- 1 which reappeared only after glucose had been consumed (see Figure l), Tre6P synthase and Tre6P phosphatase are not under for- mal glucose repression and that, as discussed below, factors in addition to glucose are involved in the mechanism of inactivation-repression of these latter two enzymes. Moreover, this mechanism of inactivation-repression does not give rise to a com- plete disappearance of Tre6P synthase and Tre6P

both the culture medium and the strain used. When a wild-type strain was grown on a glucose-rich medium (YEPD), the accumulation of the disac- charide began just after the derepression of FBPase-1, which opened gluconeogenesis, at a time when glycogen accumulation was also active. Remarkably, no synthesis of trehalose occurred earlier, during the phase of glycogen degradation, despite the fact that Tre6P synthase and Tre6P phos- phatase had already been reactivated. This suggests that the glucose units derived from glycogen cannot be used for the synthesis of trehalose because they serve as an energy source for the respiratory adap- tation of yeast as suggested by others (Beck and von Meyenburg, 1968; Labbe-Bois et al., 1973; Lillie and Pringle, 1980) and in agreement with a simul- taneous decrease in the concentration of glucose- 6-phosphate. In the strain unable to perform gluconeogenesis because it lacked PEPCK, the synthesis of trehalose started as soon as the two trehalose-synthesizing enzymes were derepressed and was independent of the derepression of FBPase- 1. In addition, this accumulation was concomitant with the degradation of glycogen, both processes occurring at similar rates, as well as with an import- ant decrease in the concentration of UDP-glucose (see Figure 2). One can assume that, under this con- dition, ethanol, which cannot be used for gluconeo- genesis, serves as a respiratory substrate, making glycogen available for trehalose synthesis.

Our results also suggest that the accumulation of trehalose was not dependent on the variations in the concentrations of UDP-glucose but more likely on those of glucose-6-phosphate, since the highest rate of trehalose synthesis was observed at the lowest UDP-glucose concentration (Figure 2). This is in agreement with the kinetic data of Tre6P synthase reported previously (Vandercammen et al., 1989), which showed a K,,, for UDP-glucose (0.5 mM) close to the intracellular concentration of this nucleotide measured in yeast (see Figures 1 and 2) and a K, for glucose-6-phosphate (3.5 mM) well below the concentration of this metabolite determined in stationary-phase cells. We have no explanation to offer for the marked decrease in ATP concentration occurring in thepckl mutant during the exponential phase of the growth.

Tre6P synthase and Tre6P phosphatase are cat- abolite inactivated and repressed during growth on glucose

Because the decrease in the activities of Tre6P synthase and Tre6P phosphatase that occurred

THE CONTROL OF TREHALOSE BIOSYNTHESIS IN SACCHAROMYCES CEREVISIAE 585

phosphatase as it is observed in the case of gluconeo- genic enzymes (Ferguson et al., 1968; Gancedo, 197 I ; Funayama et al., 1980; Haarasilta and Oura, 1975). The incomplete disappearance of these enzymes suggests either the Occurrence of a reduced enzyme synthesis in cells during growth on glucose or the presence of isoenzymes which are not subject to repression.

The low amount of Tre6P synthase and Tre6P phosphatase in exponentially growing cells can now provide an explanation for the fact that glu- cose-repressed yeast have a much lower capacity for trehalose accumulation than derepressed cells when they are resuspended in a buffered medium containing only glucose (Panek, 1975; Panek and Mattoon, 1977; Panek et al., 1980). Further- more, in most of the conditions investigated, the disappearance and the reappearance of Tre6P synthase and Tre6P phosphatase during growth were roughly parallel. This result is consist- ent with the hypothesis that both activities are born by a single bifunctional protein (Vander- cammen et al., 1989; Londesborough and Vuorio, 1991).

In considering which components in the medium caused the loss of Tre6P synthase and Tre6P phosphatase activities, it became clear to us that this phenomenon required a fermentable sugar (such as glucose, fructose or mannose) together with a nitrogen source (bacto-peptone being the most potent). In the absence of a nitro- gen source, the addition of glucose to a suspen- sion of yeast incubated in a buffered medium caused no significant change in enzyme activities as previously reported (Vandercammen et al., 1989) and as illustrated in Figure 5 . This can explain the resynthesis of trehalose after a short period of degradation resulting from the transient activation of trehalase (Thevelein, 1984 and refer- ences therein). In contrast, no resynthesis of the disaccharide was observed when glucose and a ni- trogen source were given to the yeast suspension, not only because of a more durable activation of trehalase (Thevelein and Beullens, 1985) but also because of a loss of Tre6P synthase and Tre6P phosphatase activities. The mechanism by which the nitrogen source affects the activities of the trehalose-metabolizing enzymes is still unclear (Thevelein and Beullens, 1985), but in the case of Tre6P synthase and Tre6P phosphatase, the ni- trogen source might stimulate the activity of a putative protein responsible for the inactivation of these enzymes.

Role of CAMP in the control of Tre6P synthase and Tre6P phosphatase

The low activities of Tre6P synthase and Tre6P phosphatase measured in a mutant harboring an unbridled activity of the CAMP-dependent protein kinase (resulting from a defective regulatory sub- unit), in a mutant with an increased adenylate cyclase activity (RAS2""'"'"l'9 mutant) or in a mutant able to accumulate exogenous cAMP (pde2 mutant), revealed a potential regulation of these enzymes by CAMP. These results are apparently in contradiction with those reported previously (Vandercammen et al., 1989), in which it was shown that another RAS2vu"Y mutant (strain LG39sp7) contained the same levels of Tre6P synthase and Tre6P phosphatase as the wild type. However, a more careful analysis revealed that this mutant still accumulated glycogen and trehalose at levels com- parable to the isogenic strain, suggesting that the mutation was leaky.

Several lines of evidence were provided against a direct action ofcAMP on Tre6P synthase and Tre6P phosphatase through phosphorylation of these enzymes by CAMP-dependent protein kinase (Vandercammen et al., 1989). This conclusion was confirmed in this work by showing that the loss of activity of these enzymes is irreversible. Therefore, we can propose that, as is the case with many other yeast enzymes (Ferguson et al., 1968; Funayama et al., 1980; Muller et al., 1981), the initial loss of Tre6P synthase and Tre6P phosphatase activities is due to a proteolytic degradation and is independent of CAMP. It must be recognized, however. that, because our data are based only on measurements of catalytic activities, undistinguishable results would have been observed if the enzymes were denatured or dissociated into inactive subunits. The activities of Tre6P synthase and Tre6P phosphatase are then maintained low by a repression mechanism which, by contrast, seems to be under the control of CAMP. The cyclic nucleotide could, for example, exert its effect at the transcriptional level, since such a mech- anism has recently been demonstrated for alcohol dehydrogenase I1 (Cherry et al., 1989) and for cata- lase T (Bissinger et al., 1989), two other glucose- repressible enzymes.

The observation that exogenous cAMP was unable to cause an activation of trehalase when it was added topde2 mutant cells (known to be perme- able to the nucleotide) in the stationary phase of growth deserves further comment. Because incu- bation of a crude extract prepared from stationary-

J. FRANCOIS, M.-J. N E W S A N D H.-G. HERS

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phase cells with cAMP results in a rapid activation of trehalase (Franqois et al., 1984; Uno et al., 1983; Thevelein and Beullens, 1985), it is unlikely that the lack of cAMP effect in vivo is due to an absence of trehalase or of CAMP-dependent protein kinase, but most probably due to the fact that cells in stationary phase have become less permeable to exogenous CAMP.

Physiological SigniJicance of the catabolite inactivation-repression Tre6P synthase and Tre6P phosphatase

Because the synthesis of 1 mol of trehalose from glucose requires 3 mol of ATP, whereas no ATP is produced upon hydrolysis of the disaccharide by trehalase, a futile cycle should arise during simul- taneous synthesis and degradation of trehalose. As pointed out by Holzer (1976), the rapid inactivation of enzymes should be an excellent life-saving device of cells to circumvent this problem. However, the inactivation and repression of Tre6P synthase and Tre6P phosphatase seem to be too slow to prevent a harmful depletion of ATP, unless the kinetics of the inactivated enzymes are deeply modified. Whether a futile recycling of trehalose exists in yeast incubated under the conditions used here and whether the kinetics of the partially inactivated enzymes are modified remain to be investigated.

ACKNOWLEDGEMENTS

Helpful discussions and critical reading of the manuscript by K. Tatchell, F. R. Rosenzweig and S. Thompson-Jaeger were greatly appreciated. This work was supported by the Belgian State-Prime Minister’s office-Science Policy Programming and in part by the National Institutes of Health grants DK 9235. (H.-G.H.) and CA 37702 (K. Tatchell).

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