Eur. J. Biochem. 164, 369-373 (1987) 0 FEBS 1987

Changes in the concentration of CAMP, fructose 2,6-bisphosphate and related metabolites and enzymes in Saccharomyces cerevisiae during growth on glucose Jean FRANCOIS, Pilar ERAS0 and Carlos GANCEDO Instituto de Investigaciones Biomkdicas del CSIC and Departamento de Bioquimica, Facultad de Medicina, Universidad Autonoma de Madrid

(Received September 29/December 29,1986) - EJB .86 1029

Changes in the concentration of several metabolites and enzymes related to carbohydrate metabolism were measured during the growth of Saccharomyces cerevisiae on a mineral medium containing glucose as the limiting nutrient. When about 50% of the original glucose was used the exponential phase ended and the culture entered a ‘transition’ phase before the complete exhaustion of glucose. In this transition phase several metabolic changes occurred. CAMP, that decreased along growth, reached a constant value of about 0.7 nmol/g dry weight. A pronounced drop in fructose-6-phosphate-2-kinase activity and in the concentration of fructose 2,6-bisphosphate and fructose 1,6-bisphosphate was observed accompanied by a less marked decrease in hexose monophosphates. Trehalase activity also dropped and reached a minimal value at the onset of the stationary phase when synthesis of trehalose began. Glycogen concentration and glycogen synthase activity increased sharply during the transition phase. Plasma membrane ATPase began to increase at the middle of the exponential phase and then, coincident with the glucose exhaustion, a 90% decrease in the measurable activity was observed.

Growth of Saccharomyces cerevisiae on glucose elicits a well-known pattern of enzymes and metabolites [l, 21. In the last years some observations have been reported that suggest that this pattern may not be constant during growth. For example a decrease in RNA and protein synthesis is observed well in advance of glucose exhaustion [3]. Also glycogen accumulation begins when about half of the original glucose remains in the culture irrespective of the initial concentration of the sugar [4].

Recently cAMP and fructose 2,6-bisphosphate [Fru(2,6)- P2] have received considerable attention as possible ‘organizers’ of carbohydrate metabolism in yeast [5 , 61. In order to provide more experimental data to disprove or support this idea we initiated a systematic investigation of the concentrations of these compounds and some other metab- olites and enzymes related to carbohydrate metabolism during growth of yeast on glucose. The results of our study show that exponential growth ceases when glucose is still present in the culture and that important metabolic changes occur in the transition phase between the exponential and stationary phase of growth.

MATERIALS AND METHODS Growth conditions

Saccharomyces cerevisiae X2180 was grown at 30°C in a mineral medium [7] using NaCl instead of the original sodium citrate. The pH of the medium was adjusted to 5.5 with KOH. Glucose was added after sterilization at a final concentration of 2%. The media were inoculated with yeasts grown to the stationary phase on a medium containing 2% glucose, 1% yeast extract and 1 YO bactopeptone. The initial cell concentra- tion was about lo5 cells/ml. Growth was followed by measur- ing the turbidity of the culture at 660 nm and the cor- responding dry weight was calculated from a calibration curve of dry weight versus absorbance (Fig. 1). This curve was constructed in the same conditions as the subsequent experi- ments as it has been shown that the relation between cellular parameters and turbidity is not constant [8]. Since, for practi- cal reasons, determination of metabolites, enzymatic activities and other parameters was performed in different cultures, we followed for every experiment the corresponding growth curve and the concentration of glucose in the medium.

Correspondence to C . Gancedo, Instituto de Investigaciones Bio- mtdicas del Consejo Superior de Investigaciones Cientificas, Facultad de Medicina de la Universidad Autonoma de Madrid, Arzobispo Morcillo 4, E-28029 Madrid, Spain

Abbreviations. Fru(2,6)P2, fructose 2,6-bisphosphate; Fru(l,6)- P2, fructose 1,6-bisphosphate; Glc6P, glucose 6-phosphate; Fru6P- 2-kinase, fructose-6-phosphate-2-kinase.

Enzymes. Phosphofructokinase (EC 2.7.1.1 I) ; fructose-6-phos- phate-2-kinase (EC 2.7.1.105); fructose-l,6-bisphosphatase (EC 3.1.3.11); trehalase (EC 3.2.1.28); glycogen synthase (EC 2.4.1.11); plasma membrane ATPase (EC 3.6.1.35).

Determination of Fru(2,6)P2, glycogen, cAMP and other metabolites

Samples of yeast were collected as described by Saez and Lagunas [9]. About 50 mg dry weight were collected except for the determination of Fru(2,6)P2 and glycogen, where only 20 mg were used. Extraction of yeast for determination of metabolites [except for Fru(2,6)P2 and glycogen] was performed as in [9] using trichloroacetic acid instead of perchloric acid [lo]. Extraction for determination of Fru(2,6)P2 and glycogen was as follows : the frozen sample

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'.I / 2 0

0

L ) 0.4 08 1.2 1.6 20 2r

Dry weight mg.ml-'

Fig. 1. Relation between absorbance and dry weight. Yeast was grown on a glucose minimal medium as described in Materials and Methods. Appropriate samples were taken, filtered through Millipore filters and dried until constant weight

was poured on 1 ml hot 0.25 M Na2C03 and heated in a water bath at 90°C with occasional stirring. After 15 min, 0.5 ml suspension was withdrawn and centrifuged and the supernatant was used to determine Fru(2,6)P2 as in [6]. The remaining suspension was used to determine glycogen accord- ing to Becker [l l] . The suspension was further incubated at 90°C and after 2 h 0.5 ml 0.2 M sodium acetate pH 4.8 was added and pH was adjusted to 5 with 1 M acetic acid. Then 10 p1 amyloglucosidase (500 units/ml) was added and the mixture was incubated at 37°C for 2 h. After neutralization with 3 M KHC03/KOH the glucose liberated was determined with glucose oxidase [12].

Cyclic AMP was measured with the Amersham assay kit as described in [lo]. Other metabolites were tested as described in [6].

Ethanol in the culture medium was determined as in [13]. Trehalose was determined as in [14].

Preparation of extracts and enzymatic assays

Extracts were performed as in [15]. Trehalase and fructose- 6-phosphate-2-kinase (Fru6P-2-kinase) were assayed as in [6]. For the assay of plasma membrane ATPase samples of 50 mg dry yeast were filtered from the culture medium and imme- diately dropped into liquid nitrogen. Membrane fractions were prepared as described by Serrano [I61 and ATPase was assayed as described therein.

Glycogen synthase was measured in a reaction mixture containing 50 mM Mes pH 6.5, 5 mM EDTA, 2.5 mg/ml glycogen (from Sigma type 11), 0.5 mM UDP-['4C]glucose (416 cpm/nmol), 10 mM glucose 6-phosphate and an adequate amount of extract. The final volume was 150 pl. At different times an aliquot of 25 pl was withdrawn, spotted on Whatman paper and treated as described in [17]. The reaction was linear with time and amount of enzyme. The ratio of independence is defined as the quotient of activities in the absence and in the presence of 10 mM Glc6P.

Exponential Transition Stationary phase phase phase

Time (bows)

Fig. 2. Phases of yeast growth and variation of CAMP and other culture parameters. The yeast was grown as described in Materials and Meth- ods. Growth was followed turbidimetrically and dry weight was calculated from the curve shown in Fig. 1. Glucose and ethanol in the culture and intracellular CAMP were measured as described in Materials and Methods. The three phases are separated by a dotted line in (A) and indicated by arrows in (B) and in successive figures

All enzymatic assays were performed at 30°C. 1 unit is the amount of enzyme that catalyzes the conversion of 1 Fmol substrate in 1 min under the assay conditions.

Concentration of protein was measured by the methods of Bradford [IS] or Lowry et al. [I91 with bovine immuno- globulin or serum albumin as respective standards.

UDP-['4C]glucose was from Amersham (UK), auxiliary enzymes from Sigma (MO, USA).

RESULTS Growth of the culture

Culture conditions were such that glucose was the limiting factor for growth. In the conditions used about 70% of the glucose was transformed in ethanol and cessation of growth occurred as a result of exhaustion of glucose in the medium (Fig. 2A). The growth showed three phases, the exponential one, a transition phase that occurred when about 50% of the initial glucose had been used and a stationary phase where cell proliferation was not observed. These phases have also been recently considered by Boucherie 131. The exponential phase ended when the cell density was around 1 mg dry weight/ml culture and the stationary phase began around 2 mg dry weight/ml. In between lay the transition phase that lasted for a 4- 5-h period. In the figures the boundaries be- tween the different phases are marked by arrows. Ethanol increased steadily and reached a maximum value at the initia- tion of the stationary phase (Fig. 2A). In contrast with what is observed in rich media no appreciable growth occurred in our conditions on the accumulated ethanol. The external pH decreased during the exponential phase and reached a mini- mum around 4 in the transition phase (Fig. 2B).

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14 16 I8 20 22 24 26 28 Time (hours)

Time(hcurs)

Fig. 3. Variations of Fru(2,6)P2. glycogen, trehalose and relatedsigniji- cant metabolites along growth. Yeast was growth and metabolites determined as described in Materials and Methods. (B) (*---*) Fru(l,6)PZ, (0-0) hexose monophosphates: Glc6P f Fru6P

Time (hours)

Fig. 4. Changes in the levels of Fruru6P-2-kinase, glycogen synthase and trehalase during yeast growth. Yeast was grown and enzymes determined as described in Materials and Methods. The ratio of independence (RI) for glycogen synthase is defined in Materials and Methods

Changes in the level of CAMP along growth cAMP increases in yeast upon addition of glucose [20-

21 a]. In exponentially growing cultures the intracellular levels are higher on glucose than on ethanol-grown yeasts [lo]. How- ever, the variations of these levels have not been followed along growth. Fig. 2B shows this variation. It can be seen that the concentration of cAMP decreased parallel to the disappearance of glucose during the exponential phase of growth. For technical reasons it was not possible to measure metabolites (or in other cases enzymatic activities) at cell densities lower than those appearing in the figure. When the transition phase was initiated cAMP remained at a low constant value around 0.7 nmol/g dry weight. This value did not vary significantly during the stationary phase.

:>A- \ 25 30

Time (hours I

Fig. 5. Variations in plasma membrane ATPase and glucose along growth. Growth was on the medium described in Materials and Meth- ods and enzyme and glucose determinations as described

Variations in the concentration of Fru(2,6)P2, related glycolytic metabolites and reserve carbohydrates

The concentration of Fru(2,6)P2 remained high and constant (about 12 nmol/g dry weight, 6 1M) during the exponential phase of growth but dropped during the transi- tion phase (Fig. 3A). A drop of about tenfold in Fru(1,6)P2 was also observed in this phase of growth whereas the concen- tration of hexose monophosphates decreased only by a factor of two (Fig. 3B). It should be stressed that the drop of these metabolites began when glucose was still available in the medi- um. ATP remained fairly constant during exponential growth and increased roughly twice during the transition phase, re- maining at this level during the stationary phase.

Glycogen began to accumulate at the beginning of the transition phase, reached a maximum at the entrance in the stationary phase to decrease slowly along this phase (Fig. 3 A). Trehalose did not begin to accumulate until the glucose con- centration dropped to about 0.5 mg/ml and then increased during the stationary phase coincident with the disappearance of glycogen (Fig. 3 C).

Behaviour of FrubP-2-kinase, glycogen synthase and trehalase

Fru6P-2-kinase dropped during the transition phase, whereas glycogen synthase increased thus paralleling the behaviour of their respective products (Fig. 4A). Interestingly, although glycogen synthase remained high in the stationary phase the concentration of glycogen dropped (compare with Fig. 3A). The ratio of independence of the enzyme increased until the middle of the transition phase and then decreased slowly. This indicates that the synthase becomes more depen- dent on the presence of Glc6P for its activity in this last part of the curve.

Trehalase activity (Fig. 4B) decreased during the transi- tion phase and reached a minimum during the stationary phase coincident with the observed accumulation of trehalose.

Behaviour of plasma membrane ATPase Since plasma membrane ATPase may be a major contribu-

tor to ATP expenditure and this enzyme is activated by glucose [16] we followed the ATPase activity during growth. As shown in Fig. 5, ATPase activity increased in the middle of the ex-

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ponential phase to reach a maximum in the transition phase. Then, coincident with the exhaustion of glucose, it decreased abruptly.

DISCUSSION

There is ample evidence showing that the activities of several enzymes of carbohydrate metabolism in yeast are modulated by a mechanism of phosphorylation/dephos- phorylation. Trehalase [20] and Fru6P-2-kinase [6] are acti- vated and fructose-l,6-bisphosphatase [21 a, 221 inactivated by phosphorylation by a CAMP-dependent protein kinase. Glycogen synthase appears to be also regulated by this mecha- nism [23, 241. The activity of this enzyme becomes more de- pendent of the presence of Glc6P upon phosphorylation. Plasma membrane ATPase has been shown recently to be activated by glucose and phorbol esters [16, 251 suggesting that it is also a phosphorylable enzyme.

The purpose of our work was to see if the changes in the activation state of these enzymes could be related to the concentration of cAMP along growth on glucose and if the enzyme changes could in turn account for the modification of carbohydrate metabolism.

The finding that cAMP decreased steadily during ex- ponential growth until entrance into the transition phase is surprising. It is tempting to relate this decrease with the de- crease in the concentration of external glucose. This interpre- tation would be consistent with the previous findings that the extent of the transient increase of CAMP, produced by addition of glucose to a yeast culture, is dependent on the concentration of glucose added [26].

The important change in the concentration of cAMP was not paralleled by a decrease in the activity of Fru6P-2-kinase and trehalase. Such activities, which were high during the exponential growth, presumably as a result of the activation by a CAMP-dependent protein kinase, decreased later during the transition phase when the concentration of cAMP had already reached its minimal value. This suggests that other unknown determinants regulate the phosphorylation state of these two enzymes.

Most of the metabolic changes observed occurred during the transition phase during which the concentration of glucose dropped from about 50% of its initial value to nil. During this time the hexose monophosphate concentration decreased about twofold. The decrease in the concentration of Fru(2,6)P2 is presumably a result of the inactivation of Fru6P- 2-kinase. Coincident with this decrease was an increase in the synthesis of glycogen. These results may be explained by a less active phosphofructokinase due to decreasing Fru(2,6)P2 levels [27]. This inhibition would result in a decrease of Fru(l,6)P2 and of glycolytic flux with a greater proportion of hexose monophosphate available for glycogen synthesis.

It must be stressed that the change in glycogen synthase is not only due to an activation (as indicated by an increase in the ratio of independence) but also to an increase in the amount of enzyme.

During the stationary phase, glycogen synthase was pro- gressively inactivated (ratio of independence decreased) and glycogen was degraded. In striking contrast, trehalose syn- thesis proceeded steadily during this phase. The reason why trehalose synthesis begins only when glucose has been consumed is uncertain [28, 291. A possible advantage will be the avoidance of a futile cycle between synthesis and degrada- tion. Our results indicate that trehalose synthesis is delayed until trehalase is almost inactive. It has been found that

trehalose is formed at the expense of glycogen during the drying of baker’s yeast [29]. We found that the rise of trehalose can account for 50% of the glycogen breakdown suggesting that, in the culture, a similar process may take place.

Plasma membrane ATPase showed a quite different pat- tern of activity along the growth as compared with the other interconvertible enzymes. It began to increase during the ex- ponential phase to reach the maximal activity during the transition phase and then, upon exhaustion of glucose, dropped precipitously. Ths result points to a role of sugar in the maintenance of the activity of this enzyme. Indeed it has been shown that plasma membrane ATPase is activated by glucose and that this activation is rapidly reversed upon glucose removal [16]. The fact that there was no relation between change in concentration of cAMP and in the activity of ATPase, suggests that the postulated phosphorylation is independent of CAMP. This has been pointed out by Portillo and Maz6n [25]. However, further experimental evidence is needed to clarify this point. Tuduri et al. [30] have also ob- served recently that, in yeast growing on a rich medium, a decrease by a factor of two to three in the activity of ATPase occurred during the last generation of exponential growth. However, the different conditions of growth of the culture and assay of enzymes make our results and theirs not directly comparable.

The results discussed indicate that carbohydrate metab- olism is not constant during the growth of yeast on glucose. In this context it is important to mention the behaviour of the systems of sugar transport. S. cerevisiue possesses two different glucose-transport systems, one with high affinity and another with low affinity [31]. The high-affinity component is low on 2% glucose, increases as glucose is consumed from the medium and decreases in the stationary phase, while that with low affinity is present maximally during growth on a high concentration of glucose [32]. It may be that the activity of these systems influences the intracellular concentration of cAMP and other compounds.

The key role of cAMP in carbohydrate metabolism in yeast is proven beyond doubt [33]. Its participation in the regulation of certain enzymes is stressed by the behaviour of rus mutants. The ras proteins in yeast act by activating adenykdte cyclase [34]. Strains lacking RAS2 function accumulate excessive levels of glycogen and trehalose [34,35]. Mutants RAS2 (va119, gly19), with increased cyclase activity, do not respond to starvation conditions and fail to accumulate reserve carbohydrates.

Our results, however, suggest that in addition to cAMP modulation other not yet established mechanisms control the activity of enzymes related to carbohydrate metabolism during the transition phase between the exponential and the stationary phase of growth. This interesting phase has re- ceived too little attention in the past.

The authors are indebted to Dr Juana M. Gancedo for useful discussions during this work and to her and Prof. H. G. Hers and Dr E. Van Schaftingen for criticisms on the manuscript. This work was partially supported by a grant of the Spanish Comisibn Asesora de Investigacibn Cientifica y Tkcnica. J. F. is Aspirant of the Belgian Fonds National de la Recherche Scientifique.

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