ARCHIVES
OF BIOCHEMISTRY
Regulation
AND
of Energy
Relationships
between
ANITA Department OfBiochemistry, Department of Physiological
183,
BIOPHYSICS
306-316
Metabolism Catabolite Mitochondrial
D. PANEK’
in Saccharomyces
Repression, Trehalose Development’ JAMES
AND
Institute ofchemistry, Chemistry, Johns Received
(1977)
Synthesis,
and
R. MATTOON:’
Federal Unwersity Hopkins Uniwrsity, March
Cerevisiae
ofRio de Janeiro, Baltimore, Maryland
Brazil and 21218
29, 1977
Changes in trehalose accumulation and in cytochromes during diauxic growth in glucose medium were examined in a normal Saccharomyces cereuisiae strain. While no appreciable disaccharide accumulation occurred during most of the logarithmic phase, a rapid synthesis took place during the final stages. The intrinsic capacity of cells to accumulate trehalose was also determined under nonproliferating conditions, in glucose medium lacking a nitrogen source. Cells harvested at an early growth stage had a much lower trehalose accumulation capacity than cells taken after glucose was exhausted from the culture medium. A high trehalose accumulation capacity could also be obtained at any growth stage by using maltose or galactose as carbon source. Since cells grown under various conditions exhibit a correlated change in cytochrome development and in trehalose accumulation capacity, it was concluded that the level of glucose repression determines the concentration and/or state of activation of the trehalose synthetasetrehalase complex. Independent control of trehalose accumulation capacity and mitochondrial biogenesis by the level of glucose repression was shown in two ways: by demonstrating derepression of trehalose accumulation without development of cytochromes a and c in microaerobic cells, and by showing repression-dependent changes in a cytoplasmic respiration-deficient (p-j mutant, which lacked functional mitochondria. Therefore, the capacity of a cell to accumulate trehalose is not regulated solely by the supply of ATP generated by oxidative phosphorylation.
During
of a variety of inducible enzymes which are subject to catabolite repression change markedly as cells progress from logarithmic growth through diauxie. It is widely held that, at least in procaryotes, most, if not all, of these enzymatic changes are controlled by the available glucose by means of a cyclic AMP-directed regulatory system. In yeast, particular interest has been focused on transitions in levels of cytochromes and other mitochondrial enzymes which accompany changes in the glucose content of the growth medium (25). Cytochrome biosynthesis is also subject to regulation by oxygen (2, 6-81, and the relative importance of glucose-directed and oxygen-directed control has been difficult to ascertain (8, 9). Moreover, the role of cyclic AMP in mediating catabolite ef-
logarithmic
growth of cells of on glucose medium, the intracellular content of trehalose, a reserve carbohydrate, is very low. Upon depletion of glucose from the medium, yeast cells enter a brief phase of retarded growth referred to as diauxie. During this period of adaptation, substantial trehalose accumulation occurs (1). It is well known that the concentrations
Saccharomyces
cerevisiae
i This work was supported by grants U.S. Public Health Service, the National of Health (GM 158841, the University Council (Federal University of Rio de
from the Institutes Research Janeiro),
CAPES,and CNPq (Brazil). e To whom reprint requests should be sent. s Visiting Professor at the Federal University of Rio de Janeiro on leave from Johns Hopkins University. 306 Copyright All rights
0 1977 by Academic Press. of reproduction in any form
Inc. reserved.
ISSN
0003-9861
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fects in yeast is not well defined (10). Since enzymes of trehalose metabolism and mitochondria are both associated with the energy metabolism of the yeast cell, the parallel changes in mitochondrial development and in trehalose synthesis in response to changes in glucose concentrations during growth are of special interest. The object of the present study was to explore more fully the regulation of these two systems under varying conditions of repression and oxygen supply. Changes in trehalose metabolism and in mitochondria were studied simultaneously in an effort to discover new relationships governing cellular energy metabolism as it, responds to a changing environment. The present data clearly demonstrate that the capacity to accumulate trehalose during incubation of cells in a nitrogenfree medium is strongly decreased by prior logarithmic growth in glucose medium. Although parallel changes in cytochrome formation and trehalose accumulation capacity usually occur in response to various conditions of repression, cytochrome synthesis can be suppressed while trehalose synthesis is reactivated by allowing cells to enter stationary phase anaerobically. Moreover, the variation of trehalose synthesis with repression persists in yeast cells rendered respiration deficient by the p mutation. Throughout this work, the term “trehalose accumulation” is employed to emphasize the fact that in vim trehalose content is determined by relative activities of the synthetase complex and trehalase. MATERIALS
AND
METHODS
Microorgnni.sms. Saccharornyws crrcuisiac~. Strain BII, diploid, was received from the Pasteur Institute, Paris, and maintained on 1% glucose, IQ yeast extract agar. Strain BII (p: ) was prepared by growing Strain BII in medium containing 1% yeast extract. 2% peptone, 23 glucose. and 20 ~g of ethidium bromide per milliliter. After 6 h of growth at 3O”C, treated cells were plated and a single small colony was subcultured. Gmlc,th conditions. Cells were grown in a mcdium containing 1% yeast extract and a 1% carbon source (glucose, maltose, or galactosei. Aerobic cultures were grown in 100 ml of medium contained in 500-m] Erlenmcyer flasks. and incubation was at
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28°C on a rotary shaker operated at 150 rpm. Microaerobic cultures were stationary, in flasks filled to the brim. Harvested cells were washed twice with distilled water. Nonproliferating cwditions. Washed cells wercl incubated at 28°C in 0.05 M phosphate buffer. pH 6.0. in the presence of a carbon source at a proportion of 3 mg dry weight/ml. For aerobic incubations. suspensions were shaken at 150 oscillations per minute. Anal~~ticd rncfhods. Growth of cultures was folowed by turbidity measurements at 570 nm. Glucose was determined with glucose oxidase (111. and maltose and galactose were determined according to Nelson (12). Trehalose w,as extracted with 0.5 M trichloroacctic acid (13) and determined by anthrone 1141. Cytochromt~ spwtra. Cells harvested from growth media were washed with distilled water. Suspensions containing 8 or 10% wet weight of cells were made using 0.05 M phosphate buffer, pH 6.0. A suspension of dried milk solids in distilled water served as turbidity reference. Spectra were measured in a Cary 17 spectrophotometer. Reduction of cytochromes was accomplished by adding a few grains of solid sodium dithionite to the cuvette containing the cell suspension. Measurements were made at room temperature ~approximatcly 23’Ci. RESULTS
Trehalose Growth
Synthmis of Cells
during
Aerobic
When yeast cells are placed in fresh growth medium containing glucose as carbon source, the disaccharide reserve remains low during logarithmic growth. To determine whether this apparent inhibition of trehalose accumulation requires glucose per se or is primarily a consequence of the mitotic activity of the cells, a comparison of the changes in trehalose content during growth on maltose and glucose was made. The results depicted in Fig. 1 show that when maltose is the carbon source, substantial trehalose accumulation occurs continuously throughout the logarithmic growth phase. In contrast, when glucose is the substrate, no significant trehalose accumulation takes place until the end of logarithmic phase, when glucose becomes limiting. At this point, however, a large increase in trehalose content occurs. Clearly then, cell division is not the only determinant regulating trehalose content during the logarithmic phase; the nature of the substrate is also of
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key importance. It should be pointed out, however, that the cells grow more rapidly on glucose than on maltose. Trehalose ferating
Accumulation under NonproliConditions by Early-Log Cells
To determine the trehalose accumulation capacity of yeast cells without the complications which arise from parallel formation of new cell components required for growth, disaccharide formation can be measured in cells incubated under nonproliferating conditions. In fact, such conditions, in which no source of nitrogen is present, are especially favorable for obtaining active trehalose accumulation (1, 13). With this approach it could be shown that early log-phase cells taken from glucose medium have no significant capacity for trehalose accumulation, even when cell division is arrested. Consequently, the lack of accumulated trehalose in cells growing in glucose cannot be attributed solely to the fact that cell division is rapid in growth medium containing this substrate. As shown in Fig. 2, the intrinsic capacity of yeast to accumulate trehalose, as measured in a nitrogen-free medium, is substantial in maltose-grown, log-phase cells, while trehalose content increases only slightly in glucose-grown cells harvested during early logarithmic growth and incubated under the same nonproliferating conditions. Effects of Carbon Source on Cytochromes of Logarithmic-Phase Yeast Cells Yeast Strain BII grows even more slowly on galactose medium than it does on maltose medium. Figure 2 shows that growth on either carbohydrate favors trehalose accumulation. Figure 3 compares the cytochrome spectra of early-log BII cells grown on the three fermentable carbon sources: glucose, maltose, and galactose. Galactose-grown cells exhibit substantial levels of all cytochromes, while cells from glucose medium are strongly repressed, having much lower concentrations of cytochromes b and c and no detectable cytochrome a. When maltose is used, cytochrome levels are slightly less than
FIG. 1. Trehalose accumulation during growth of Strain BII. The dashed lines correspond to the growth of cells and the solid lines correspond to trehalose accumulation. 0, cells grown on 1% yeast extract, 1% glucose; 0, cells grown on 1% yeast extract, 1% maltose.
OW 0
60 MINUTES
FIG. 2. Trehalose accumulation by early-log cells incubated under nonproliferating conditions. The cells were pregrown aerobically for 4 h (log phase) in media containing 1% of the corresponding carbohydrate and 1% yeast extract and then transferred to aerobic nonproliferating conditions in the presence of the same carbon source. Each incubation mixture contained 21 pmol of glucose and 3 mg of cells (dry weight) per milliliter. 0, early-log cells pregrown on glucose; A, early-log cells pregrown on maltose; n, early-log cells pregrown on galactose.
those observed with galactose, but repression is clearly less than that obtained with glucose. Trehalose Accumulation Cytoch.rome Spectra Transitions
and Changes in during Diauxic
The data in Fig. 4 show that BII cells grown on glucose until the carbohydrate is exhausted exhibit an increased capacity to accumulate trehalose, which, in this case, is comparable to that observed with cells
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S. cerevisiae
Effects of Oxygen Limitation on Trehalose Accumulation and Cytochrome Synthesis
I 560
5;O5AO
5:O
6hO
6:O
6;O
nm FIG. 3. Effects of different fermentable carbon sources on cytochrome spectra of cells harvested during logarithmic growth. Growth media contained 1% yeast extract and 1% carbohydrate. An inoculum of 0.25 pg/ml (dry weight) was used. The following yields (milligram per milliliter) of logphase cells were obtained: in glucose, 0.75; in maltose, 0.93; in galactose, 0.46. Spectra were determined using 10% (wet weight/volume) suspensions of washed cells in 0.05 M phosphate buffer, pH 7.0, using a dried milk suspension as turbidity reference. Cytochromes were reduced with dithionite.
grown to the same stage on the less repressive substrates, maltose and galactose. The result of this increase in accumulation capacity can be observed in growing cells at the end of the first logarithmic phase as a dramatic increase in trehalose content, as shown in Fig. 5 (inset). Figure 5 shows that during this same period a derepression of cytochrome biosynthesis occurs. Cytochrome content continues to increase during diauxie and during the subsequent growth on accumulated ethanol. It can be seen that cytochrome a can already be detected at point B, 45 min after glucose has been exhausted from the culture medium.
The parallel changes observed in the activity of the trehalose accumulation system and the concentrations of cytochromes suggest the possibility that trehalose accumulation is directly dependent upon the activity of mitochondria in producing ATP. Such a dependence was indicated in a previous study which showed that formation of trehalose from endogenous substrates did not occur in cells previously grown under microaerobic conditions (12). However, the data in Figs. 6 and 7 clearly demonstrate that fully developed mitochondria are not essential for trehalose accumulation provided cells are released from glucose repression. By growing cells under microaerobic conditions until glucose is exhausted from the medium, the capacity to accumulate trehalose can be increased markedly, in spite of the fact that the oxygen limitation abolished the synthesis of both cytochromes a and c (Fig. 7). Nevertheless, even though these cytochromes are not detectable, oxygen does stimulate trehalose formation during incubation under nonproliferating conditions. 20.
0 0
I 30 min
I 60
FIG. 4. Trehalose accumulation in derepressed cells incubated under nonproliferating conditions. Cells were pregrown in media containing 1% of the corresponding carbohydrate and 1% yeast extract until the carbohydrate was exhausted. Cells were then transferred to nonproliferating conditions in phosphate buffer containing: (0) 7 pmol of glucose/3 mg of cells, (A) 3.5 firno1 of maltose/3 mg of cells, and (W 7 pmol of galactose/3 mg of cells. Each reaction mixture contains 3 mg of cells/ml.
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FIG. 5. Changes in cytochromes and trehalose content during the diauxic transition in Strain BII. Samples were withdrawn for spectral determinations at points lettered A, B, C, and D. The corresponding spectra are indicated by the same letters. The dashed line indicates growth (ASi,,) and the solid line indicates the cellular trehalose content in milligrams per gram, dry weight. Spectra were determined as in Fig. 3. The time at which external glucose was exhausted from the growth medium is indicated by the vertical arrows.
MATTOON
plied entirely by fermentation. Glucose repression in the BII pm strain was varied in two ways: through normal utilization of glucose during growth and by replacing glucose with the less-repressing substrate, maltose. The level of cytochrome c in pm cells provides a useful in viva indicator of the degree of glucose repression, as shown in Fig. 8. Cells harvested during logarithmic growth (mid-log) on glucose exhibit a relatively weak cytochrome c band near 550 nm, while cells harvested after glucose has been completely utilized contain slightly more cytochrome c, indicating a lower degree of glucose repression. When maltose is substituted for glucose, derepression of cytochrome c biosynthesis is quite marked; both mid-log- and stationary-phase cells exhibit intense absorption bands at 550 nm. Log-phase cells grown on either carbohydrate exhibit a weak absorption band, in the 580- to 590-nm range. This band has disappeared when the stationary phase is reached. A shoulder near 560 nm is observed in all four spectra. As shown in Fig. 9, mutation to the p state does not eliminate the glucose effect
The nature of this oxygen effect is unknown, since no significant increase in cytochromes occurred during the course of a 30-min aerobic incubation of these microaerobically pregrown cells, as shown by the spectra in Fig. 7. Glucose Effect on Trehalose Synthesis Cytoplasmic Petite Mutant
in a
Additional evidence that the effects of glucose on trehalose formation are not secondary consequences of glucose-mediated changes in the capacity of mitochonclria to produce ATP was obtained with a p- mutant of Strain BII. Since the p- mutant lacks both cytochrome a and cytochrome b, no oxidative phosphorylation is possible, and the energy for ATP production is sup-
0+T~-- :o 0
60
mln FIG. 6. Trehalose accumulation under nonproliferating conditions by cells pregrown under reduced oxygen tension. Cells were grown under microaerobit conditions in the presence of 5% glucose until glucose was exhausted, and then cells were transferred to phosphate buffer containing 21 pmol of glucose/3 mg of cells. l , aerobic nonproliferating conditions; C, microaerobic nonproliferating conditions.
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15-
/ min / /
METABOLISM
IN
chromes are highly repressed. It should be stressed, however, that this effect may be quite indirect, and its mechanism remains to be studied. It may be pertinent to note that the p- mutant grows more slowly than its p+ counterpart. The data in Fig. 10 illustrate how derepression affects trehalose accumulation during the growth of BII pm cells. In maltose medium, where cells are derepressed even in the logarithmic phase (Fig. 8-, trehalose accumulates continuously during growth. In glucqse medium, in contrast, significant trehalose accumulation does not occur until the final stages of logarithmic growth, when some derepression becomes evident. Comparison of Fig. 10 with Fig. 1 reveals that the general relationships between derepression and trehalose accumulation found in p- cells
550 I
n
FIG. 7. Cytochrome spectra of cells grown under microaerobic conditions and incubated under aerobic nonproliferating conditions for various times. Cells were grown under the microaerobic conditions described for Fig. 6. After growth, cells were washed and suspended in 0.05 M phosphate buffer, pH 6.0, to a final concentration of 3 mg dry weight/ml. Suspensions were shaken aerobically at 28°C and sampled at the indicated times for determination of trehalose and cytochrome spectra. Spectra were measured as described for Fig. 3, except that 8% suspensions were used.
on the trehalose accumulation capacity. Under nonproliferating conditions, the less-repressed, glucose-grown, stationaryphase cells accumulate trehalose more readily than the repressed, mid-log cells of Strain BII p-. The data in Fig. 9 also illustrate a surprising effect of the p- mutation on trehalose accumulation. When p+ and p- cells harvested during log phase are compared, the rate of trehalose accumulation is much greater in the p- strain than in the pi strain. Therefore, the presence of functional mitochondrial DNA appears to influence the regulation of trehalose metabolism even when mitochondrial cyto-
311
S. cereczsiae
M-Slot --)
//
-G-Log
FIG. 8. Repression of cytochrome c in Strain BII py by glucose and depression by growth on maltose. The carbon source used for growth is indicated by G and M for 1% glucose and 1% maltose, respectively. Log indicates that cells were collected in the middle of the logarithmic phase, and Stat. indicates that they were harvested in the stationary phase, after the carbohydrate had been exhausted from the medium. Spectra were determined with 10% suspensions, as indicated for Fig. 3.
312
PANEK 3o
-CHANGE T;R;.i$E (mg
20
0
AND
IN
/ gcells)
-
15
30 MINUTES
45
60
FIG. 9. Effect of glucose repression on trehalose accumulation capacity of normal and petite strains. Strains BII p+ and BII pym were grown in medium containing 1% yeast extract and 1% glucose. Samples were collected during the middle of the logarithmic growth phase and after glucose was exhausted from the medium, as in Fig. 8. Trehalose accumulation was measured under nonproliferating conditions. Initial trehalose concentrations were 3.4, 5.6, 10.8, and 12.8 mg/g (dry weight) of cells for BII p+ mid-log, BII p2- mid-log, BII p+ diauxic, and BII p2 stationary cells, respectively. The initial glucose concentration was 21 mM.
are essentially the same as those observed in p+ cells. Clearly, then, glucose-mediated changes in the capacity of yeast cells to accumulate trehalose cannot be explained solely by changes in oxidative phosphorylation. DISCUSSION
Cellular regulatory systems are most readily observed when cells are undergoing physiological transitions. A very important period of transition during the growth of yeast under natural circumstances and in the leavening process occurs when growing cells exhaust their supply of glucose and undergo adaptation to accumulated ethanol. This period of transition is characterized by a pause in cell division commonly known as diauxie. A great variety of changes must occur during the diauxic shift from fermentative to aerobic metabolism. To carry out aerobic
MATTOON
growth on ethanol, enzymes of the glyoxylate cycle, of gluconeogenesis, and of porphyrin biosynthesis as well as a battery of mitochondrial enzymes must be synthesized or activated during diauxie. Obviously, yeast must possess a regulatory system which coordinates this complex process. Moreover, since the synthesis of the new enzymes needed for growth on ethanol requires ATP, some provision must be made for supplying enough energy to ensure that the shift from fermentation to ethanol oxidation is completed. Therefore, a critical event during the period of diauxie is the development of oxidative phosphorylation. If external glucose is exhausted before this mitochondrial function appears, the trehalose and glycogen reserves represent the only significant source of energy available for driving the synthetic reactions required to develop oxidative phosphorylation. If trehalose and glycogen are to serve such a purpose during the diauxic transformation, it would be necessary for the cell to have a means of ensuring the presence of an intracellular reserve of these carbohydrates before extracellular glucose falls to zero. In fact, the data presented in Fig. 1 show that significant trehalose accumulation does occur before the diauxic pause is reached. We propose, therefore, the tentative hypothesis that strain BII possesses a regulatory system which releases the trehalose accumulation system from the glucose effect (repression and/or inhibition) when exter-
I
40
FIG. 10. Trehalose accumulation during growth of respiration-deficient Strain BII pym on glucose and maltose. The dashed lines correspond to the growth of cells and the solid lines correspond to trehalose accumulation. 0, cells grown on 1% yeast extract, 1% glucose; 0, cells grown on 1% yeast extract, 1% maltose.
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nal glucose falls below a critical level. Moreover, this proposed regulatory system operates in the presence of an adequate supply of nitrogen and all other essential nutrients. It also operates in a p- strain, which completely lacks oxidative phosphorylation (Fig. 10). The level of trehalose in yeast depends upon the relative concentrations of the enzymes involved in its biosynthesis and hydrolysis and upon the intracellular supply of substrates. It is beyond the scope of the present investigation to determine the quantitative changes in each of the various enzymes during growth. However, by using nonproliferating conditions it has been possible to assess growth-dependent changes in the poise of the metabolic system which controls trehalose levels. This approach is designed to avoid limitations in substrate supply so that the observed trehalose accumulation will reflect the concentration of the biosynthetic enzyme complex, andlor its state of activation, relative to the hydrolytic activity of trehalose. The absence of amino nitrogen is of critical importance. It has been known for many years (13) that yeast cells can convert extracellular glucose to trehalose with high efficiency under conditions of nitrogen starvation, whereas trehalose accumulation during growth in medium containing similar glucose concentrations is absent or nearly so. Although we have used the term “trehalose accumulation” throughout this work, we favor the hypothesis that glucose-dependent changes in disaccharide accumulation capacity, measured under nonproliferating conditions, are primarily due to increased trehalose synthesis capacity. This view is supported by the fact that trehalose content declines slowly, if at all, after extracellular glucose is completely consumed. Although the exact mechanism which limits trehalose accumulation in proliferating cells is unknown, an important factor may be the supply of UTP. Actively growing cells require large amounts of UTP to produce new RNA, while in resting cells this UTP should be available, at least potentially, for the production of UDPG,
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one of the two substrates of trehalose phosphate synthetase. By using nonproliferating conditions one avoids possible limitation of trehalose accumulation resulting from competition for UTP by RNA synthesis. In fact, Rothman and Cabib (16) observed a relatively high concentration of intracellular UDPG, which changed only slightly with time, in yeast fermenting glucose in nitrogen-free medium. These investigators have also shown that omission of nitrogen source is important in maintaining a substantial level of the other trehalose phosphate synthetase substrate, glucose 6-phosphate. Addition of NH,+ ion to fermenting yeast suspensions caused a marked drop in the intracellular concentration of this metabolite. Because of the marked suppression of trehalose accumulation by nitrogen source, lack of trehalose in growing cells could lead to the erroneous conclusion that an enzyme of the biosynthetic complex was completely repressed or inactivated by the carbon source or one of its catabolites. For example, as Fig. 10 shows, no significant accumulation of trehalose occurred in cells of BII p- during most of the period of logarithmic growth in glucose medium. Nevertheless, cells harvested midway through this period possess substantial capacity to accumulate trehalose when they are tested under nonproliferating conditions, as shown in Fig. 9. Although the trehalose synthetase-enzyme complex may be considered constitutiue, the data presented here clearly demonstrate that the response of the disaccharide accumulation system of yeast strain BII to various conditions of catabolite repression resembles that of the inducible cytochrome system. However, the observation of parallel behavior in the two enzymatic systems does not prove that biosynthesis of any of the enzymes required for trehalose formation is in fact subject to direct catabolite repression. The data do establish the existence of a regulatory system which modulates trehalose accumulation in response to glucose and other carbohydrate substrates. It seems very unlikely that allosteric regulation of trehalose phosphate synthetase or trehalose by
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PANEK AND MATTOON
metabolites could be the sole means of control, since glucose-repressed cells fail to produce more than limited amounts of trehalose even under nonproliferating conditions. In contrast, derepressed cells exhibit a substantial capacity for trehalose accumulation even in the presence of adequate supplies of amino nitrogen and other growth factors (Figs. 1 and 10). The effects of repression on the enzyme complex itself can best be compared by using nonproliferating conditions, where differences in substrate supply and probably allosteric effector levels are minimized. Thus, from Fig. 9, we can conclude unambiguously that under identical assay conditions the capacity for trehalose accumulation by glucoserepressed cells is much lower than that of derepressed cells. It is highly probable that these differences represent changes in the actual concentration of one or more of the essential enzymes, in the catalytic form of the enzyme(s), or both. The chemostat experiments of Kuenzi and Fiechter (17) provide some interesting comparisons. When these workers grew their yeast strain at dilution rates higher than 0.24 h-l, using glucose as the limiting nutrient, the respiratory quotient was greater than unity, and alcohol appeared in the medium. It is very likely that cells grown under these fermentative conditions experience some degree of glucose repression. Therefore, the extremely low trehalose content which these investigators observed in their fermentative cells is probably a manifestation of the same glucose effect which is described in the present work. Trehalose content increased markedly as dilution rates were decreased below 0.24 h-l, where cells grew completely aerobically. It was also shown that fermenting chemostat cultures could be induced to produce more trehalose by using nitrogen limitation. This experiment is analogous to our experiment using nonproliferating conditions. The observation that the trehalose content of such nitrogen-limited fermenting cultures was much less than that of glucose-limited cultures is in agreement with out observation that, under nonproliferating conditions, midlog, glucose-grown cells store trehalose
much less rapidly than late-log cells do. It should be pointed out, however, that the aerobic cells grown in the chemostat have fully functioning mitochondria, so they are not in the same physiological state as our late-log cells from batch cultures. Serious consideration may be given to the possibility that some of the enzymes of trehalose metabolism can occur in two alternative forms with different kinetic properties. Alternative forms of yeast glydesignated D and I, cogen synthetase, have already been described by RothmanDenes and Cabib (16). The D form, which is dependent upon relatively high concentrations of glucose 6-phosphate for its activity, is found in log-phase cells, while the I (independent) form predominates in cells isolated from stationary-phase cultures. The I form appears to be converted into the D form by a protein phosphorylation reaction. Although interconvertible forms of trehalose phosphate synthetase have not yet been described in yeast, Killick and Wright (19) reported a “masked” form of this enzyme in extracts of the slime mold, Dictyostetium discoideum. These investigators (20) have also presented evidence that this enzyme is activated in uivo during the late stages of fungal development. However, in a recent paper, Alexander and Sussman (21) have challenged some of the findings of Killick and Wright. Perhaps differences in strains, culture conditions, and assay methods are responsible for the discrepancies. Wyatt (22) has described apparent allosteric effects of glucose 6-phosphate and trehalose on trehalose-phosphate synthetase activity in crude extracts from insects. Unfortunately, these properties were partially lost during purification. The observed variations in trehalose synthesis with level of glucose repression suggest the possibility that 3’,5’-cyclic AMP may be involved in regulating the metabolism of the disaccharide. Cyclic AMP has been shown to be a modulator of catabolite repression in bacteria (20-23). We have tested the effects of 3’,5’-cyclic AMP on trehalose accumulation using conditions similar to those described by Fang and Butow (29). Although proto-
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plasts from derepressed cells synthesized trehalose actively, those prepared from glucose-repressed, early-log cells did not accumulate significant trehalose even when 1.2 mM cyclic AMP was added to the incubation medium (data not shown). These negative results do not necessarily exclude a role of cyclic AMP, since other possible factors such as limited entry of nucleotide or slow protein synthesis in protoplasts may be considered. In other work, van Soligen and van der Plaat (30) studied a cryptic form of trehalase which appears to be activated by a protein phosphorylation mechanism, An activating protein fraction was stimulated to a limited extent by cyclic AMP, but the role of this nucleotide remains uncertain, since its activating effect diminished during protein purification. It is pertinent to note that three cyclic AMP-independent protein kinase activities have been described in yeast (31). It may be concluded, therefore, that the role of cyclic AMP in modulating catabolite repression in yeast remains to be clarified (10, 24-31). If future studies should in fact demonstrate the existence of interconvertible forms of enzymes of trehalose metabolism in yeast, it would be of particular interest to determine the effects of NH,+ ions on these forms and their interconversions. Although in the above discussion we have considered trehalose synthetase as a constitutive enzyme, the possibility remains that it is in fact inducible, just as cytochromes are inducible in S. cerevisiae. Lacking evidence for the existence of inactive forms of enzymes of this system in glucose-repressed cells, we believe the question of inducibility remains an open one. While there is no doubt that ATP generated by mitochondrial oxidative phosphorylation can provide energy for trehalose synthesis, the glucose-dependent inactivation of trehalose synthesis reported here cannot be explained merely by the fact that mitochondrial ATP is virtually unvailable in glucose-repressed cells. When cytochrome biosynthesis is prevented by limiting the oxygen supply, the cells still exhibit the capacity to accumulate the di-
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saccharide, provided glucose repression is eliminated by allowing cells to grow to a glucose-limited stage. It remains to be determined whether mitochondrial components other than cytochromes undergo similar derepression in the absence of oxygen. The conclusion that repression-dependent changes in trehalose accumulation are not just secondary consequences of concurrent changes in mitochondrial oxidative phosphorylation is fully confirmed by the studies with the p- mutant of Strain BII. These results, taken together, establish that two components of yeast energy metabolism, the storage of trehalose and the biogenesis of mitochondria, are both subject to independent control by a system responding to the glucose concentration of the medium. Demonstration of this independence of control opens the possibility that trehalose may provide some of the energy needed to drive mitochondrial biogenesis and/or other endergonic reactions during the diauxic transition from fermentation to oxidative metabolism. ACKNOWLEDGMENTS Dr. Mattoon wishes to thank B. K. Wesley Copeland, The National Academy of Sciences (U.S.), CNPq (Brazil), and Dr. H. K. Sanders for providing administrative assistance. We are grateful for the expert technical assistance provided by Edilson Bernardes, Arnaldo Miceli, and Richard Gottal. We also thank Sophie Neufeld, Sara Thomson, and Shirley Metzger for their aid in preparing the manuscript. REFERENCES 1. PANEK,
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(1962)
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B., SLONIMSKI, P. P., YOTSLJYANAGI, TAVLITZKI, J. (1956) C.R. Lab. Carls-
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AND
A. W., and Smillie, R. M., eds.), pp. 346-359, North Holland, Amsterdam/London. SOMLO, M., AND FUKUHARA, H. (1965) Biochem. Biophys. Res. Commun. 19, 587-591. ROGERS, P. J., AND STEWART, P. R. (1973) J. Bacterial. 115, 88-97. SUOMALAINEN, H., MURMINEN, T., AND OURA, E. (1973) in Progress in Industrial Microbiology (Hockenhull, D. J. D., ed.), Vol. 12, pp. 109-167, Churchill Livingstone, Edinburgh/ London. POLAKIS, E. S., BARTLEY, W., AND MEEK, G. A. (1965) Biochem. J. 97, 293-302. SCHAMHART, D. H. J., TEN BERGE, A. M. A., AND VAN DER POLL, K. W. (1975) J. Racterio~. 121, 747-752. RAABO, E., AND TERKILDSEN, T. C. (1960) Stand. J. Clin. Lab. Invest. 12, 402-407. NELSON, N. (1944) J. Biol. Chem. 153,375-380. TREVELYAN, W. E., AND HARRISON, J. S. (1956) Rio&em. J. 62, 177-183. BRIN, M. (1966) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 9, pp. 506-514, Academic Press, New York. PANEK, A. D. (1975) Eur. J. Appl. Microbial. 2, 39-45. ROTHMAN, L. M., ANDCABIB, E. (1969)Biochemistry 8, 3332-3341. K~~ENZI, M. T., AND FEICHTER, A. (1972) Arch. Mikrobiol. 84, 254-265.
MATTOON 18. ROTHMAN-DENES, L. M., AND CABIB, E. (1971) Biochemistry 10, 1236-1242. 19. KILLICK, K. A., AND WRIGHT, B. E. (1972) J. Bid. Chm. 247. 2967-2969. 20. KILLICK, K. A., AND WRIGHT. B. E. (1975)Arth. Biochem. Biophys. 150, 634-643. 21. ALEXANDER, S., AND SUSSMAN. M. (1975) DC,vclop. Bid. 46. 211-215. 22. WYATT, G. R. (1967) A~LTL~. Insect Physiol. 1, 287-360. 23. PASTAN, I., AND PERLMAN, R. L. (19701 Scirncc 169. 339-344. 24. DE~ROMBRUGGHE. B., VARMUS. H. E., PERLMAN, R. L.. AND PASTAN. I. H. (197O)Biochcm. Biophys. Rrs. Commun. 38, 894-901. 25. KUWANO, M.. AND SCHLESSINGER, D. (1970) Proc. Net. Acad. Sci. USA 66, 1466152. 26. ROBISON, G. A., BUTCHER. R. W., AND SUTHERLAND. E. W. (1971) Cyclic AMP, pp. 422-455. Academic Press, New York/London. 27. VAN WIJK. R.. AND KONIJN. T. M. (1971) FERS Lett. 13, 184-186. 28. SCHLANDERER, G.. ANDDELLWEG, H. (1974)Eur. J. Aiochwr 49, 305-316. 29. FANG. M., AND BUTOW. R. A. 11970) Biochem. Biophy~. Res. C’onmurr II, 1579-1583. 30. VAN SOLINGEN. P.. AND VAN DER PLAAT, J. B. 11975) Brochrm. Biophys. RKS. C~,mmun. 62, 553-560. 31. TAKAI, Y., YAMAMURA, H., AND NISHIZUKA. Y. 119741 J. Biol. C’hrm. 249, 530-535.
OF BIOCHEMISTRY
Regulation
AND
of Energy
Relationships
between
ANITA Department OfBiochemistry, Department of Physiological
183,
BIOPHYSICS
306-316
Metabolism Catabolite Mitochondrial
D. PANEK’
in Saccharomyces
Repression, Trehalose Development’ JAMES
AND
Institute ofchemistry, Chemistry, Johns Received
(1977)
Synthesis,
and
R. MATTOON:’
Federal Unwersity Hopkins Uniwrsity, March
Cerevisiae
ofRio de Janeiro, Baltimore, Maryland
Brazil and 21218
29, 1977
Changes in trehalose accumulation and in cytochromes during diauxic growth in glucose medium were examined in a normal Saccharomyces cereuisiae strain. While no appreciable disaccharide accumulation occurred during most of the logarithmic phase, a rapid synthesis took place during the final stages. The intrinsic capacity of cells to accumulate trehalose was also determined under nonproliferating conditions, in glucose medium lacking a nitrogen source. Cells harvested at an early growth stage had a much lower trehalose accumulation capacity than cells taken after glucose was exhausted from the culture medium. A high trehalose accumulation capacity could also be obtained at any growth stage by using maltose or galactose as carbon source. Since cells grown under various conditions exhibit a correlated change in cytochrome development and in trehalose accumulation capacity, it was concluded that the level of glucose repression determines the concentration and/or state of activation of the trehalose synthetasetrehalase complex. Independent control of trehalose accumulation capacity and mitochondrial biogenesis by the level of glucose repression was shown in two ways: by demonstrating derepression of trehalose accumulation without development of cytochromes a and c in microaerobic cells, and by showing repression-dependent changes in a cytoplasmic respiration-deficient (p-j mutant, which lacked functional mitochondria. Therefore, the capacity of a cell to accumulate trehalose is not regulated solely by the supply of ATP generated by oxidative phosphorylation.
During
of a variety of inducible enzymes which are subject to catabolite repression change markedly as cells progress from logarithmic growth through diauxie. It is widely held that, at least in procaryotes, most, if not all, of these enzymatic changes are controlled by the available glucose by means of a cyclic AMP-directed regulatory system. In yeast, particular interest has been focused on transitions in levels of cytochromes and other mitochondrial enzymes which accompany changes in the glucose content of the growth medium (25). Cytochrome biosynthesis is also subject to regulation by oxygen (2, 6-81, and the relative importance of glucose-directed and oxygen-directed control has been difficult to ascertain (8, 9). Moreover, the role of cyclic AMP in mediating catabolite ef-
logarithmic
growth of cells of on glucose medium, the intracellular content of trehalose, a reserve carbohydrate, is very low. Upon depletion of glucose from the medium, yeast cells enter a brief phase of retarded growth referred to as diauxie. During this period of adaptation, substantial trehalose accumulation occurs (1). It is well known that the concentrations
Saccharomyces
cerevisiae
i This work was supported by grants U.S. Public Health Service, the National of Health (GM 158841, the University Council (Federal University of Rio de
from the Institutes Research Janeiro),
CAPES,and CNPq (Brazil). e To whom reprint requests should be sent. s Visiting Professor at the Federal University of Rio de Janeiro on leave from Johns Hopkins University. 306 Copyright All rights
0 1977 by Academic Press. of reproduction in any form
Inc. reserved.
ISSN
0003-9861
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fects in yeast is not well defined (10). Since enzymes of trehalose metabolism and mitochondria are both associated with the energy metabolism of the yeast cell, the parallel changes in mitochondrial development and in trehalose synthesis in response to changes in glucose concentrations during growth are of special interest. The object of the present study was to explore more fully the regulation of these two systems under varying conditions of repression and oxygen supply. Changes in trehalose metabolism and in mitochondria were studied simultaneously in an effort to discover new relationships governing cellular energy metabolism as it, responds to a changing environment. The present data clearly demonstrate that the capacity to accumulate trehalose during incubation of cells in a nitrogenfree medium is strongly decreased by prior logarithmic growth in glucose medium. Although parallel changes in cytochrome formation and trehalose accumulation capacity usually occur in response to various conditions of repression, cytochrome synthesis can be suppressed while trehalose synthesis is reactivated by allowing cells to enter stationary phase anaerobically. Moreover, the variation of trehalose synthesis with repression persists in yeast cells rendered respiration deficient by the p mutation. Throughout this work, the term “trehalose accumulation” is employed to emphasize the fact that in vim trehalose content is determined by relative activities of the synthetase complex and trehalase. MATERIALS
AND
METHODS
Microorgnni.sms. Saccharornyws crrcuisiac~. Strain BII, diploid, was received from the Pasteur Institute, Paris, and maintained on 1% glucose, IQ yeast extract agar. Strain BII (p: ) was prepared by growing Strain BII in medium containing 1% yeast extract. 2% peptone, 23 glucose. and 20 ~g of ethidium bromide per milliliter. After 6 h of growth at 3O”C, treated cells were plated and a single small colony was subcultured. Gmlc,th conditions. Cells were grown in a mcdium containing 1% yeast extract and a 1% carbon source (glucose, maltose, or galactosei. Aerobic cultures were grown in 100 ml of medium contained in 500-m] Erlenmcyer flasks. and incubation was at
METABOLISM
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307
28°C on a rotary shaker operated at 150 rpm. Microaerobic cultures were stationary, in flasks filled to the brim. Harvested cells were washed twice with distilled water. Nonproliferating cwditions. Washed cells wercl incubated at 28°C in 0.05 M phosphate buffer. pH 6.0. in the presence of a carbon source at a proportion of 3 mg dry weight/ml. For aerobic incubations. suspensions were shaken at 150 oscillations per minute. Anal~~ticd rncfhods. Growth of cultures was folowed by turbidity measurements at 570 nm. Glucose was determined with glucose oxidase (111. and maltose and galactose were determined according to Nelson (12). Trehalose w,as extracted with 0.5 M trichloroacctic acid (13) and determined by anthrone 1141. Cytochromt~ spwtra. Cells harvested from growth media were washed with distilled water. Suspensions containing 8 or 10% wet weight of cells were made using 0.05 M phosphate buffer, pH 6.0. A suspension of dried milk solids in distilled water served as turbidity reference. Spectra were measured in a Cary 17 spectrophotometer. Reduction of cytochromes was accomplished by adding a few grains of solid sodium dithionite to the cuvette containing the cell suspension. Measurements were made at room temperature ~approximatcly 23’Ci. RESULTS
Trehalose Growth
Synthmis of Cells
during
Aerobic
When yeast cells are placed in fresh growth medium containing glucose as carbon source, the disaccharide reserve remains low during logarithmic growth. To determine whether this apparent inhibition of trehalose accumulation requires glucose per se or is primarily a consequence of the mitotic activity of the cells, a comparison of the changes in trehalose content during growth on maltose and glucose was made. The results depicted in Fig. 1 show that when maltose is the carbon source, substantial trehalose accumulation occurs continuously throughout the logarithmic growth phase. In contrast, when glucose is the substrate, no significant trehalose accumulation takes place until the end of logarithmic phase, when glucose becomes limiting. At this point, however, a large increase in trehalose content occurs. Clearly then, cell division is not the only determinant regulating trehalose content during the logarithmic phase; the nature of the substrate is also of
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key importance. It should be pointed out, however, that the cells grow more rapidly on glucose than on maltose. Trehalose ferating
Accumulation under NonproliConditions by Early-Log Cells
To determine the trehalose accumulation capacity of yeast cells without the complications which arise from parallel formation of new cell components required for growth, disaccharide formation can be measured in cells incubated under nonproliferating conditions. In fact, such conditions, in which no source of nitrogen is present, are especially favorable for obtaining active trehalose accumulation (1, 13). With this approach it could be shown that early log-phase cells taken from glucose medium have no significant capacity for trehalose accumulation, even when cell division is arrested. Consequently, the lack of accumulated trehalose in cells growing in glucose cannot be attributed solely to the fact that cell division is rapid in growth medium containing this substrate. As shown in Fig. 2, the intrinsic capacity of yeast to accumulate trehalose, as measured in a nitrogen-free medium, is substantial in maltose-grown, log-phase cells, while trehalose content increases only slightly in glucose-grown cells harvested during early logarithmic growth and incubated under the same nonproliferating conditions. Effects of Carbon Source on Cytochromes of Logarithmic-Phase Yeast Cells Yeast Strain BII grows even more slowly on galactose medium than it does on maltose medium. Figure 2 shows that growth on either carbohydrate favors trehalose accumulation. Figure 3 compares the cytochrome spectra of early-log BII cells grown on the three fermentable carbon sources: glucose, maltose, and galactose. Galactose-grown cells exhibit substantial levels of all cytochromes, while cells from glucose medium are strongly repressed, having much lower concentrations of cytochromes b and c and no detectable cytochrome a. When maltose is used, cytochrome levels are slightly less than
FIG. 1. Trehalose accumulation during growth of Strain BII. The dashed lines correspond to the growth of cells and the solid lines correspond to trehalose accumulation. 0, cells grown on 1% yeast extract, 1% glucose; 0, cells grown on 1% yeast extract, 1% maltose.
OW 0
60 MINUTES
FIG. 2. Trehalose accumulation by early-log cells incubated under nonproliferating conditions. The cells were pregrown aerobically for 4 h (log phase) in media containing 1% of the corresponding carbohydrate and 1% yeast extract and then transferred to aerobic nonproliferating conditions in the presence of the same carbon source. Each incubation mixture contained 21 pmol of glucose and 3 mg of cells (dry weight) per milliliter. 0, early-log cells pregrown on glucose; A, early-log cells pregrown on maltose; n, early-log cells pregrown on galactose.
those observed with galactose, but repression is clearly less than that obtained with glucose. Trehalose Accumulation Cytoch.rome Spectra Transitions
and Changes in during Diauxic
The data in Fig. 4 show that BII cells grown on glucose until the carbohydrate is exhausted exhibit an increased capacity to accumulate trehalose, which, in this case, is comparable to that observed with cells
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METABOLISM
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S. cerevisiae
Effects of Oxygen Limitation on Trehalose Accumulation and Cytochrome Synthesis
I 560
5;O5AO
5:O
6hO
6:O
6;O
nm FIG. 3. Effects of different fermentable carbon sources on cytochrome spectra of cells harvested during logarithmic growth. Growth media contained 1% yeast extract and 1% carbohydrate. An inoculum of 0.25 pg/ml (dry weight) was used. The following yields (milligram per milliliter) of logphase cells were obtained: in glucose, 0.75; in maltose, 0.93; in galactose, 0.46. Spectra were determined using 10% (wet weight/volume) suspensions of washed cells in 0.05 M phosphate buffer, pH 7.0, using a dried milk suspension as turbidity reference. Cytochromes were reduced with dithionite.
grown to the same stage on the less repressive substrates, maltose and galactose. The result of this increase in accumulation capacity can be observed in growing cells at the end of the first logarithmic phase as a dramatic increase in trehalose content, as shown in Fig. 5 (inset). Figure 5 shows that during this same period a derepression of cytochrome biosynthesis occurs. Cytochrome content continues to increase during diauxie and during the subsequent growth on accumulated ethanol. It can be seen that cytochrome a can already be detected at point B, 45 min after glucose has been exhausted from the culture medium.
The parallel changes observed in the activity of the trehalose accumulation system and the concentrations of cytochromes suggest the possibility that trehalose accumulation is directly dependent upon the activity of mitochondria in producing ATP. Such a dependence was indicated in a previous study which showed that formation of trehalose from endogenous substrates did not occur in cells previously grown under microaerobic conditions (12). However, the data in Figs. 6 and 7 clearly demonstrate that fully developed mitochondria are not essential for trehalose accumulation provided cells are released from glucose repression. By growing cells under microaerobic conditions until glucose is exhausted from the medium, the capacity to accumulate trehalose can be increased markedly, in spite of the fact that the oxygen limitation abolished the synthesis of both cytochromes a and c (Fig. 7). Nevertheless, even though these cytochromes are not detectable, oxygen does stimulate trehalose formation during incubation under nonproliferating conditions. 20.
0 0
I 30 min
I 60
FIG. 4. Trehalose accumulation in derepressed cells incubated under nonproliferating conditions. Cells were pregrown in media containing 1% of the corresponding carbohydrate and 1% yeast extract until the carbohydrate was exhausted. Cells were then transferred to nonproliferating conditions in phosphate buffer containing: (0) 7 pmol of glucose/3 mg of cells, (A) 3.5 firno1 of maltose/3 mg of cells, and (W 7 pmol of galactose/3 mg of cells. Each reaction mixture contains 3 mg of cells/ml.
310
PANEK
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FIG. 5. Changes in cytochromes and trehalose content during the diauxic transition in Strain BII. Samples were withdrawn for spectral determinations at points lettered A, B, C, and D. The corresponding spectra are indicated by the same letters. The dashed line indicates growth (ASi,,) and the solid line indicates the cellular trehalose content in milligrams per gram, dry weight. Spectra were determined as in Fig. 3. The time at which external glucose was exhausted from the growth medium is indicated by the vertical arrows.
MATTOON
plied entirely by fermentation. Glucose repression in the BII pm strain was varied in two ways: through normal utilization of glucose during growth and by replacing glucose with the less-repressing substrate, maltose. The level of cytochrome c in pm cells provides a useful in viva indicator of the degree of glucose repression, as shown in Fig. 8. Cells harvested during logarithmic growth (mid-log) on glucose exhibit a relatively weak cytochrome c band near 550 nm, while cells harvested after glucose has been completely utilized contain slightly more cytochrome c, indicating a lower degree of glucose repression. When maltose is substituted for glucose, derepression of cytochrome c biosynthesis is quite marked; both mid-log- and stationary-phase cells exhibit intense absorption bands at 550 nm. Log-phase cells grown on either carbohydrate exhibit a weak absorption band, in the 580- to 590-nm range. This band has disappeared when the stationary phase is reached. A shoulder near 560 nm is observed in all four spectra. As shown in Fig. 9, mutation to the p state does not eliminate the glucose effect
The nature of this oxygen effect is unknown, since no significant increase in cytochromes occurred during the course of a 30-min aerobic incubation of these microaerobically pregrown cells, as shown by the spectra in Fig. 7. Glucose Effect on Trehalose Synthesis Cytoplasmic Petite Mutant
in a
Additional evidence that the effects of glucose on trehalose formation are not secondary consequences of glucose-mediated changes in the capacity of mitochonclria to produce ATP was obtained with a p- mutant of Strain BII. Since the p- mutant lacks both cytochrome a and cytochrome b, no oxidative phosphorylation is possible, and the energy for ATP production is sup-
0+T~-- :o 0
60
mln FIG. 6. Trehalose accumulation under nonproliferating conditions by cells pregrown under reduced oxygen tension. Cells were grown under microaerobit conditions in the presence of 5% glucose until glucose was exhausted, and then cells were transferred to phosphate buffer containing 21 pmol of glucose/3 mg of cells. l , aerobic nonproliferating conditions; C, microaerobic nonproliferating conditions.
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15-
/ min / /
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IN
chromes are highly repressed. It should be stressed, however, that this effect may be quite indirect, and its mechanism remains to be studied. It may be pertinent to note that the p- mutant grows more slowly than its p+ counterpart. The data in Fig. 10 illustrate how derepression affects trehalose accumulation during the growth of BII pm cells. In maltose medium, where cells are derepressed even in the logarithmic phase (Fig. 8-, trehalose accumulates continuously during growth. In glucqse medium, in contrast, significant trehalose accumulation does not occur until the final stages of logarithmic growth, when some derepression becomes evident. Comparison of Fig. 10 with Fig. 1 reveals that the general relationships between derepression and trehalose accumulation found in p- cells
550 I
n
FIG. 7. Cytochrome spectra of cells grown under microaerobic conditions and incubated under aerobic nonproliferating conditions for various times. Cells were grown under the microaerobic conditions described for Fig. 6. After growth, cells were washed and suspended in 0.05 M phosphate buffer, pH 6.0, to a final concentration of 3 mg dry weight/ml. Suspensions were shaken aerobically at 28°C and sampled at the indicated times for determination of trehalose and cytochrome spectra. Spectra were measured as described for Fig. 3, except that 8% suspensions were used.
on the trehalose accumulation capacity. Under nonproliferating conditions, the less-repressed, glucose-grown, stationaryphase cells accumulate trehalose more readily than the repressed, mid-log cells of Strain BII p-. The data in Fig. 9 also illustrate a surprising effect of the p- mutation on trehalose accumulation. When p+ and p- cells harvested during log phase are compared, the rate of trehalose accumulation is much greater in the p- strain than in the pi strain. Therefore, the presence of functional mitochondrial DNA appears to influence the regulation of trehalose metabolism even when mitochondrial cyto-
311
S. cereczsiae
M-Slot --)
//
-G-Log
FIG. 8. Repression of cytochrome c in Strain BII py by glucose and depression by growth on maltose. The carbon source used for growth is indicated by G and M for 1% glucose and 1% maltose, respectively. Log indicates that cells were collected in the middle of the logarithmic phase, and Stat. indicates that they were harvested in the stationary phase, after the carbohydrate had been exhausted from the medium. Spectra were determined with 10% suspensions, as indicated for Fig. 3.
312
PANEK 3o
-CHANGE T;R;.i$E (mg
20
0
AND
IN
/ gcells)
-
15
30 MINUTES
45
60
FIG. 9. Effect of glucose repression on trehalose accumulation capacity of normal and petite strains. Strains BII p+ and BII pym were grown in medium containing 1% yeast extract and 1% glucose. Samples were collected during the middle of the logarithmic growth phase and after glucose was exhausted from the medium, as in Fig. 8. Trehalose accumulation was measured under nonproliferating conditions. Initial trehalose concentrations were 3.4, 5.6, 10.8, and 12.8 mg/g (dry weight) of cells for BII p+ mid-log, BII p2- mid-log, BII p+ diauxic, and BII p2 stationary cells, respectively. The initial glucose concentration was 21 mM.
are essentially the same as those observed in p+ cells. Clearly, then, glucose-mediated changes in the capacity of yeast cells to accumulate trehalose cannot be explained solely by changes in oxidative phosphorylation. DISCUSSION
Cellular regulatory systems are most readily observed when cells are undergoing physiological transitions. A very important period of transition during the growth of yeast under natural circumstances and in the leavening process occurs when growing cells exhaust their supply of glucose and undergo adaptation to accumulated ethanol. This period of transition is characterized by a pause in cell division commonly known as diauxie. A great variety of changes must occur during the diauxic shift from fermentative to aerobic metabolism. To carry out aerobic
MATTOON
growth on ethanol, enzymes of the glyoxylate cycle, of gluconeogenesis, and of porphyrin biosynthesis as well as a battery of mitochondrial enzymes must be synthesized or activated during diauxie. Obviously, yeast must possess a regulatory system which coordinates this complex process. Moreover, since the synthesis of the new enzymes needed for growth on ethanol requires ATP, some provision must be made for supplying enough energy to ensure that the shift from fermentation to ethanol oxidation is completed. Therefore, a critical event during the period of diauxie is the development of oxidative phosphorylation. If external glucose is exhausted before this mitochondrial function appears, the trehalose and glycogen reserves represent the only significant source of energy available for driving the synthetic reactions required to develop oxidative phosphorylation. If trehalose and glycogen are to serve such a purpose during the diauxic transformation, it would be necessary for the cell to have a means of ensuring the presence of an intracellular reserve of these carbohydrates before extracellular glucose falls to zero. In fact, the data presented in Fig. 1 show that significant trehalose accumulation does occur before the diauxic pause is reached. We propose, therefore, the tentative hypothesis that strain BII possesses a regulatory system which releases the trehalose accumulation system from the glucose effect (repression and/or inhibition) when exter-
I
40
FIG. 10. Trehalose accumulation during growth of respiration-deficient Strain BII pym on glucose and maltose. The dashed lines correspond to the growth of cells and the solid lines correspond to trehalose accumulation. 0, cells grown on 1% yeast extract, 1% glucose; 0, cells grown on 1% yeast extract, 1% maltose.
REGULATION
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nal glucose falls below a critical level. Moreover, this proposed regulatory system operates in the presence of an adequate supply of nitrogen and all other essential nutrients. It also operates in a p- strain, which completely lacks oxidative phosphorylation (Fig. 10). The level of trehalose in yeast depends upon the relative concentrations of the enzymes involved in its biosynthesis and hydrolysis and upon the intracellular supply of substrates. It is beyond the scope of the present investigation to determine the quantitative changes in each of the various enzymes during growth. However, by using nonproliferating conditions it has been possible to assess growth-dependent changes in the poise of the metabolic system which controls trehalose levels. This approach is designed to avoid limitations in substrate supply so that the observed trehalose accumulation will reflect the concentration of the biosynthetic enzyme complex, andlor its state of activation, relative to the hydrolytic activity of trehalose. The absence of amino nitrogen is of critical importance. It has been known for many years (13) that yeast cells can convert extracellular glucose to trehalose with high efficiency under conditions of nitrogen starvation, whereas trehalose accumulation during growth in medium containing similar glucose concentrations is absent or nearly so. Although we have used the term “trehalose accumulation” throughout this work, we favor the hypothesis that glucose-dependent changes in disaccharide accumulation capacity, measured under nonproliferating conditions, are primarily due to increased trehalose synthesis capacity. This view is supported by the fact that trehalose content declines slowly, if at all, after extracellular glucose is completely consumed. Although the exact mechanism which limits trehalose accumulation in proliferating cells is unknown, an important factor may be the supply of UTP. Actively growing cells require large amounts of UTP to produce new RNA, while in resting cells this UTP should be available, at least potentially, for the production of UDPG,
METABOLISM
IN S. cereuisiae
313
one of the two substrates of trehalose phosphate synthetase. By using nonproliferating conditions one avoids possible limitation of trehalose accumulation resulting from competition for UTP by RNA synthesis. In fact, Rothman and Cabib (16) observed a relatively high concentration of intracellular UDPG, which changed only slightly with time, in yeast fermenting glucose in nitrogen-free medium. These investigators have also shown that omission of nitrogen source is important in maintaining a substantial level of the other trehalose phosphate synthetase substrate, glucose 6-phosphate. Addition of NH,+ ion to fermenting yeast suspensions caused a marked drop in the intracellular concentration of this metabolite. Because of the marked suppression of trehalose accumulation by nitrogen source, lack of trehalose in growing cells could lead to the erroneous conclusion that an enzyme of the biosynthetic complex was completely repressed or inactivated by the carbon source or one of its catabolites. For example, as Fig. 10 shows, no significant accumulation of trehalose occurred in cells of BII p- during most of the period of logarithmic growth in glucose medium. Nevertheless, cells harvested midway through this period possess substantial capacity to accumulate trehalose when they are tested under nonproliferating conditions, as shown in Fig. 9. Although the trehalose synthetase-enzyme complex may be considered constitutiue, the data presented here clearly demonstrate that the response of the disaccharide accumulation system of yeast strain BII to various conditions of catabolite repression resembles that of the inducible cytochrome system. However, the observation of parallel behavior in the two enzymatic systems does not prove that biosynthesis of any of the enzymes required for trehalose formation is in fact subject to direct catabolite repression. The data do establish the existence of a regulatory system which modulates trehalose accumulation in response to glucose and other carbohydrate substrates. It seems very unlikely that allosteric regulation of trehalose phosphate synthetase or trehalose by
314
PANEK AND MATTOON
metabolites could be the sole means of control, since glucose-repressed cells fail to produce more than limited amounts of trehalose even under nonproliferating conditions. In contrast, derepressed cells exhibit a substantial capacity for trehalose accumulation even in the presence of adequate supplies of amino nitrogen and other growth factors (Figs. 1 and 10). The effects of repression on the enzyme complex itself can best be compared by using nonproliferating conditions, where differences in substrate supply and probably allosteric effector levels are minimized. Thus, from Fig. 9, we can conclude unambiguously that under identical assay conditions the capacity for trehalose accumulation by glucoserepressed cells is much lower than that of derepressed cells. It is highly probable that these differences represent changes in the actual concentration of one or more of the essential enzymes, in the catalytic form of the enzyme(s), or both. The chemostat experiments of Kuenzi and Fiechter (17) provide some interesting comparisons. When these workers grew their yeast strain at dilution rates higher than 0.24 h-l, using glucose as the limiting nutrient, the respiratory quotient was greater than unity, and alcohol appeared in the medium. It is very likely that cells grown under these fermentative conditions experience some degree of glucose repression. Therefore, the extremely low trehalose content which these investigators observed in their fermentative cells is probably a manifestation of the same glucose effect which is described in the present work. Trehalose content increased markedly as dilution rates were decreased below 0.24 h-l, where cells grew completely aerobically. It was also shown that fermenting chemostat cultures could be induced to produce more trehalose by using nitrogen limitation. This experiment is analogous to our experiment using nonproliferating conditions. The observation that the trehalose content of such nitrogen-limited fermenting cultures was much less than that of glucose-limited cultures is in agreement with out observation that, under nonproliferating conditions, midlog, glucose-grown cells store trehalose
much less rapidly than late-log cells do. It should be pointed out, however, that the aerobic cells grown in the chemostat have fully functioning mitochondria, so they are not in the same physiological state as our late-log cells from batch cultures. Serious consideration may be given to the possibility that some of the enzymes of trehalose metabolism can occur in two alternative forms with different kinetic properties. Alternative forms of yeast glydesignated D and I, cogen synthetase, have already been described by RothmanDenes and Cabib (16). The D form, which is dependent upon relatively high concentrations of glucose 6-phosphate for its activity, is found in log-phase cells, while the I (independent) form predominates in cells isolated from stationary-phase cultures. The I form appears to be converted into the D form by a protein phosphorylation reaction. Although interconvertible forms of trehalose phosphate synthetase have not yet been described in yeast, Killick and Wright (19) reported a “masked” form of this enzyme in extracts of the slime mold, Dictyostetium discoideum. These investigators (20) have also presented evidence that this enzyme is activated in uivo during the late stages of fungal development. However, in a recent paper, Alexander and Sussman (21) have challenged some of the findings of Killick and Wright. Perhaps differences in strains, culture conditions, and assay methods are responsible for the discrepancies. Wyatt (22) has described apparent allosteric effects of glucose 6-phosphate and trehalose on trehalose-phosphate synthetase activity in crude extracts from insects. Unfortunately, these properties were partially lost during purification. The observed variations in trehalose synthesis with level of glucose repression suggest the possibility that 3’,5’-cyclic AMP may be involved in regulating the metabolism of the disaccharide. Cyclic AMP has been shown to be a modulator of catabolite repression in bacteria (20-23). We have tested the effects of 3’,5’-cyclic AMP on trehalose accumulation using conditions similar to those described by Fang and Butow (29). Although proto-
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OF
ENERGY
plasts from derepressed cells synthesized trehalose actively, those prepared from glucose-repressed, early-log cells did not accumulate significant trehalose even when 1.2 mM cyclic AMP was added to the incubation medium (data not shown). These negative results do not necessarily exclude a role of cyclic AMP, since other possible factors such as limited entry of nucleotide or slow protein synthesis in protoplasts may be considered. In other work, van Soligen and van der Plaat (30) studied a cryptic form of trehalase which appears to be activated by a protein phosphorylation mechanism, An activating protein fraction was stimulated to a limited extent by cyclic AMP, but the role of this nucleotide remains uncertain, since its activating effect diminished during protein purification. It is pertinent to note that three cyclic AMP-independent protein kinase activities have been described in yeast (31). It may be concluded, therefore, that the role of cyclic AMP in modulating catabolite repression in yeast remains to be clarified (10, 24-31). If future studies should in fact demonstrate the existence of interconvertible forms of enzymes of trehalose metabolism in yeast, it would be of particular interest to determine the effects of NH,+ ions on these forms and their interconversions. Although in the above discussion we have considered trehalose synthetase as a constitutive enzyme, the possibility remains that it is in fact inducible, just as cytochromes are inducible in S. cerevisiae. Lacking evidence for the existence of inactive forms of enzymes of this system in glucose-repressed cells, we believe the question of inducibility remains an open one. While there is no doubt that ATP generated by mitochondrial oxidative phosphorylation can provide energy for trehalose synthesis, the glucose-dependent inactivation of trehalose synthesis reported here cannot be explained merely by the fact that mitochondrial ATP is virtually unvailable in glucose-repressed cells. When cytochrome biosynthesis is prevented by limiting the oxygen supply, the cells still exhibit the capacity to accumulate the di-
METABOLISM
315
IN S. cereuisiae
saccharide, provided glucose repression is eliminated by allowing cells to grow to a glucose-limited stage. It remains to be determined whether mitochondrial components other than cytochromes undergo similar derepression in the absence of oxygen. The conclusion that repression-dependent changes in trehalose accumulation are not just secondary consequences of concurrent changes in mitochondrial oxidative phosphorylation is fully confirmed by the studies with the p- mutant of Strain BII. These results, taken together, establish that two components of yeast energy metabolism, the storage of trehalose and the biogenesis of mitochondria, are both subject to independent control by a system responding to the glucose concentration of the medium. Demonstration of this independence of control opens the possibility that trehalose may provide some of the energy needed to drive mitochondrial biogenesis and/or other endergonic reactions during the diauxic transition from fermentation to oxidative metabolism. ACKNOWLEDGMENTS Dr. Mattoon wishes to thank B. K. Wesley Copeland, The National Academy of Sciences (U.S.), CNPq (Brazil), and Dr. H. K. Sanders for providing administrative assistance. We are grateful for the expert technical assistance provided by Edilson Bernardes, Arnaldo Miceli, and Richard Gottal. We also thank Sophie Neufeld, Sara Thomson, and Shirley Metzger for their aid in preparing the manuscript. REFERENCES 1. PANEK,
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