Journal of General Microbiology (1988), 134, 785-790.
Printed in Great Britain
785
Regulation of Trehalase Activity during the Cell Cycle of
Saccharomyces cerevisiae
By J A A P VAN D O O R N , M A R C E L E . S C H O L T E , P I E T E R W. POSTMA,
R O E L VAN D R I E L A N D K A R E L V A N D A M *
Laboratory of Biochemistry and Biotechnology Centre, University of Amsterdam, PO Box 20151,
1000 H D Amsterdam, The Netherlands
(Received 21 September 1987)
Synchronous cultures of Saccharomyces cereuisiae prepared by selection of small unbudded cells
from an elutriating rotor were used to measure trehalase activity during the cell cycle. After the
small cells had been removed from the rotor, the remainder was used to prepare asynchronous
control cultures. Both synchronous and control cultures were studied for two cell cycles. In
asynchronous cultures the trehalase activity of crude cell lysates rose continuously. In
synchronized populations trehalase activity increased from the beginning of budding onwards.
However, around the period of cell division the enzyme activity dropped rapidly but transiently
by more than 5-fold. The same changes were found during the second budding cycle.
Measurements of invertase and glucose-6-phosphate dehydrogenase activities in the same
synchronous and asynchronous cultures revealed a continuous increase for both enzymes.
Incubation of cell lysates with CAMP-dependent protein kinase before assaying for trehalase
resulted in a 2-fold enhancement of enzyme activity in asynchronous control cultures. In
synchronized cells this treatment also led to a significant stimulation of trehalase activity, and
largely abolished the cell-cycle-dependent oscillatory pattern of enzyme activity. These results
suggest that the activity of trehalase during the cell cycle is regulated, presumably at the posttranslational level, by a p hosphorylat ion-dep hosphorylation mechanism.
INTRODUCTION
The presence in Saccharomyces cerevisiae o f the disaccharide trehalose (a-D-glycopyranosyl-aD-glucopyranose) is well documented. Trehalose appears to serve as an important storage
carbohydrate that can be mobilized in a variety of physiological processes. These include the
initiation of ascospore germination, stimulation of growth, and especially the induction of
growth in resting cells after starving of glucose, nitrogen, phosphate or sulphur (Thevelein,
1984). Under conditions of glucose limitation and ‘feed-starve’-induced partial synchrony,
Kiienzie & Fiechter (1969) found that yeast cells accumulate trehalose during the unbudded
phase. Rapid mobilization of the disaccharide took place shortly before the swelling of buds. In
all these cases fast mobilization of trehalose seems to be associated with a rapid increase in the
activity of trehalase (a,a-trehalose-1-D-glycohydrolase;
EC 3.2.1.28), the only enzyme known
to be involved in trehalose hydrolysis (Kiienzie & Fiechter, 1969; Thevelein, 1984). Trehalase
activity in S. cereuisiae is thought to be regulated by a phosphorylation-dephosphorylation
mechanism. Evidence has been provided for the involvement of CAMP-dependent protein
kinase in the activation of trehalase (Van Solingen & Van Der Plaat, 1975; Wiemken &
Schellenberg, 1982; Ortiz et al., 1983; Uno et al., 1983; Thevelein, 1984; Dellamora-Ortiz et al.,
1986). A strong correlation has been observed between the intracellular concentration of CAMP
and the level of trehalase activity: for instance, after addition of glucose to stationary phase
~~
Abbreviation: PMSF, phenylmethylsulphonyl fluoride.
0001-4420 0 1988 SGM
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J . V A N D O O R N A N D OTHERS
yeast cells (Van Der Plaat, 1974; Thevelein, 1984), and in a series of mutants altered in cAMP
metabolism (Uno et al., 1983; Tenan et al., 1985). The second regulatory component of trehalase
activity is probably a protein phosphatase which converts the active phosphorylated enzyme
back to its inactive 'cryptic' form (Ortiz et al., 1983; Thevelein, 1984).
The present study was designed to investigate the possibility that trehalase activity during the
cell cycle is regulated by a phosphorylation-dephosphorylation system. Synchronous batch
cultures of S.cerevisiae generated by selection from an elutriating rotor and grown in the
presence of excess glucose were used to follow trehalase activity during the cell cycle.
METHODS
Yeast strain and growth conditions. Saccharomyces cereuisiae DLl (MATa leu2-3 leu2-112 his3-1I his3-15 ura3251 ura3-372 ura3-328) was used in all experiments. The cells were grown in a liquid medium containing 2% (w/v)
glucose, 1% (w/v) yeast extract (Difco) and 2% (w/v) Bactopeptone (Difco). The cultures were incubated at 28 "C
in a shaking water-bath.
Synchronous and control cultures. Synchronous cultures were prepared by selecting small unbudded cells from an
exponentially growing culture in a Beckman JEdB elutriating rotor, largely as described by Creanor & Mitchison
(1979). The centrifuge (52-21) was modified for elutriation and equipped with a stroboscope assembly
by the manufacturer (Beckman). A bubble chamber was interposed between the pump (Watson Marlow) and the
rotor, and the flow rate was measured by a manostat flowmeter. A gently sonicated exponential phase culture (5 x
lo7cells ml-I) was pumped through the rotor (rotor speed 3700 r.p.m.) at a flow rate of about 10ml min-I, allowing
the yeast to accumulate in the rotor cell. During centrifugation the cells were maintained at 28 "C. When sufficient
cells ( 5 x lo9) had accumulated, prewarmed fresh medium was pumped through the rotor cell. Subsequently,
the pump speed was increased to 12-1 3 ml min-I and samples of the effluent were collected. These were examined
microscopically to check for uniform populations of small unbudded cells and the best samples were pooled to
generate a synchronous culture (175 ml; 0.5-1.0 x lo6 cells ml-l). After the small cells had been removed, the
remainder (more than 96% of the initial load) was pumped out of the rotor (flow rate 30 ml min-I) with the rotor
speed reduced to 1200 r.p.m. These cells were diluted in medium to approximately the same density as the
synchronous culture, and grown as an asynchronous control. Both synchronous and asynchronous cultures were
grown for at least two cell cycles (doubling time about 110 min) with samples (6 ml) taken every 12 min. Cells
were collected at 0 "C by centrifugation, washed several times with 5 mM-sodium phosphate buffer (pH 7.0)
containing 1 mM-phenylmethyl sulphonyl fluoride (PMSF) and 50 mM-NaF, and frozen until required for enzyme
assays. Additional samples (0.25 ml) were taken to measure cell numbers and relative changes in cell size
distribution in synchronous cultures by using a Coulter counter equipped with a multichannel analyser and 30 pm
aperture. The synchrony index was estimated according to Blumenthal & Zahler (1962). Only cultures with an
index for the first division of 0-50or higher were used for determination of enzyme activities. The synchrony index
for the second cell cycle usually varied between 0.25 and 0.30. The loss of synchrony may be largely attributed to
differences in cell size between mother and daughter cells (Vanoni et al., 1983).
Preparation of cell lysates and enzyme assays. Cell suspensions were made in 5 mM-sodium phosphate buffer,
I mM-PMSF, 50 mM-NaF (pH 7.0) (final volume 0.5 ml). To each sample 25 pl distilled water containing 30 U
lyticase was added. The suspensions were incubated for 25 min at 30 "C and finally sonicated gently. This method
led to consistent cell breakage, as judged by phase-contrast microscopy.
Activation of trehalase in uitro was done as follows. Samples (usually 150 pl) of the cell lysates were diluted with
an equal volume of a mixture containing 1 mM-PMSF, 4 mM-ATP, 9 mM-MgSO,, 50 mM-NaF, 5 mMtheophylline, 50 ~M-cAMPand 30 pU ml-I CAMP-dependent protein kinase (from rabbit muscle) in 5 mMsodium phosphate buffer (pH 7.0). The reaction mixtures were incubated at 30 "C for 30 min and used
immediately for determination of trehalase activity (see below). The degree of activation was maximal after that
time and prolonged incubation did not lead to significant increases in enzyme activity (data not shown). Basal
trehalase activity was determined in lysates treated as described above, except that cAMP and CAMP-dependent
protein kinase were omitted.
Trehalase activity was determined by measuring the glucose derived from cleavage of trehalose. Standard
assays (final volume 1-0ml) contained 1 mM-PMSF, 50 mM-NaF, 25 mwtrehalose, 75 mM-SodiUm phosphate
buffer (pH 5-6)and cell lysate. Samples were incubated for several hours at 30 "C and the reaction was stopped by
heating in a water-bath at 100 "C for 3 min. In all cases parallel controls were run to correct for spontaneous
hydrolysis of the substrate. The amount of product formed was proportional to the time of incubation. The amount
of glucose liberated was determined enzymically with hexokinase and glucose-6-phosphate dehydrogenase
(Lachenicht & Bernt, 1974). The amount of NADPH produced in this reaction was measured fluorimetrically.
Trehalase activities were expressed as nmol substrate hydrolysed h-' (ml culture)-'.
-
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Trehalase activity during the yeast cell cycle
787
The remainder of the various cell lysates was used for the determination of the activity of either invertase (p-Dfructofuranoside fructohydrolase; EC 3.2.1.26) or glucose-6-phosphatedehydrogenase (EC 1.1.1.49). The assay
mixture for invertase activity (final volume 0.6 ml) contained 1 mM-PMSF, 8 mM-NaF, 33 mM-SUCrOSe, 83 mMsodium acetate buffer (pH 4.9). The reaction was allowed to proceed for several hours at 30 "C and was terminated
by heating the samples (100 "C, 3 min). Controls were used to correct for any reducing sugar present in the sucrose
solution or in the cell lysates. The resulting glucose and fructose were determined enzymically with hexokinase,
phosphoglucose isomerase and glucose-6-phosphatedehydrogenase (Lang & Michal, 1974). Invertase activity was
expressed as nmol sucrose hydrolysed h-l (ml culture)-'. Glucose-6-phosphate dehydrogenase activity was
assessed at 25 "C by direct fluorimetric determination of the rate of NADPH formation (Bergmeyer et al., 1974).
Activities were expressed as nmol substrate converted min-' (ml culture)-'.
Chemicals. Hexokinase (EC 2.7.1.1) from yeast, glucose-6-phosphatedehydrogenase (EC 1.1.1.49) from yeast,
grade 11, phosphoglucose isomerase (EC 5.3.1.9) from yeast, ATP (disodium salt) and NADP (disodium salt)
were purchased from Boehringer. PMSF, lyticase from Arthrobacter luteus, CAMP-dependent protein kinase from
rabbit muscle, trehalose from yeast, and theophylline were obtained from Sigma. cAMP was from Serva.
RESULTS
Basal trehalase activity in a synchronous culture increased from the beginning of budding
onwards (Fig. 1). However, around cell division (presumably during mitosis and the
unbudded cell phase, as indicated by phase-contrast microscopy), enzyme activity dropped
(rapidly but transiently; always more than 5-fold) to a low level. These changes were repeated in
the second cycle, but were less pronounced. Treatment of the crude cell lysates with exogenous
cAMP and CAMP-dependent protein kinase before assaying for trehalase resulted in significant
stimulation of enzyme activity, and largely abolished the oscillatory activity pattern described
above. In asynchronous control cultures basal trehalase activity and the enhanced (about 2-fold)
enzyme activity in samples treated with cAMP and CAMP-dependent protein kinase, increased
continuously together with cell numbers (Fig. 2). These results indicate that the characteristic
r
10.0
5.0
1.0
0.5
1.36
0.1
0
1
2
3 4 5
Time (h)
6
0.68
5
0
'
0.1
0
1
2 3 4
Time (h)
Fig. 1
5
6
0.66
Fig. 2
Fig. 1. Trehalase activity in a synchronous culture of S. cereuisiae DL1. A, Basal enzyme activity; A,
enzyme activity after treatment of the cell lysates with CAMP-dependent protein kinase CAMP; 0 ,
cell numbers. The results are representative of six experiments.
+
Fig. 2. Trehalase activity in an asynchronous control culture of S . cereuisiae DL1. A, Basal enzyme
activity; A,enzyme activity after treatment of the cell lysates with CAMP-dependent protein kinase
CAMP; 0 , cell numbers. Results typical of six experiments are shown.
+
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J . V A N DOORN A N D OTHERS
I
1
2
3
4
Time (h)
5
6
1
2
I
3
4
Time (h)
I
I
5
6
Fig. 4
Fig. 3
Fig. 3. Invertase activity in S. cerevisiue DLl. A, Enzyme activity in a synchronous culture; 10 A.U.
(arbitrary units) = 6.7 nmol substrate hydrolysed h-' (ml culture)-'. B, Cell numbers in A ; 10 A.U. =
1.04 x lo6 cells (ml culture)-'. C , Enzyme activity in the corresponding asynchronous control culture; 1
A.U. = 5.0 nmol substrate hydrolysed h-' (ml culture)-'. D, Cell numbers in C ; 1 A.U. = 0.72 x lo6
cells (ml culture)-'. The results are typical of three experiments.
Fig. 4. Glucose-6-phosphate dehydrogenase activity in S. cerevisiae DL1. A, Enzyme activity in a
synchronous culture; 10 A.U. (arbitrary units) = 0.32 nmol substrate converted min-' (ml culture)-'. B,
Cell numbers in A; 10 A.U. = 0.79 x lo6 cells (mi culture)-'. C, Enzyme activity in the corresponding
asynchronous control culture; 1 A.U. = 0.18 nmol substrate converted min-' (ml culture)-*. D, Cell
numbers in C ; 1 A.U. = 0.62 x lo6 cells (ml culture)-'. The results are typical of three experiments.
pattern of unstimulated trehalase activity in synchronous populations is a genuine cell cycle
event, and is not an artefact of the synchronization.
In order to obtain additional proof that the observed pattern of trehalase activity in
synchronously growing cells is specific for this enzyme and not due to general effects induced by
the preparation of cell lysates and/or the subsequent handling of the samples, the same
synchronous and control cultures were used for measurements of the activity of either invertase
or glucose-4-phosphate dehydrogenase. Neither enzyme showed periodic steps or peaks in
activity in synchronous cultures or any signs of serious perturbations in controls.
DISCUSSION
In the past, starvation methods originally described by Williams & Scopes (1962) have
frequently been used to synchronize yeast populations. Although such procedures have the
advantage of high yield, they are not suitable for measuring sensitive cell parameters, such as
enzyme activities, because of the drastic metabolic changes imposed on the cells. Furthermore,
it is impossible to run adequate control cultures. Commonly used procedures for selecting
synchrony, whereby the yeast cells are concentrated and then centrifuged through sucrose
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Trehalase activity during the yeast cell cycle
789
gradients in tubes or zonal rotors, may also introduce artefactual deviations from the normal
cell-cycle metabolism (Creanor & Mitchison, 1979; Creanor et al., 1983). However, using
centrifugal elutriation, Creanor & Mitchison (1979) developed a selection-synchronization
technique which appears to reduce such perturbations markedly in both the synchronous and
asynchronous control cultures. We have used the elutriation technique to follow changes in
activity of trehalase, invertase and glucose-6-phosphate dehydrogenase through the cell cycle of
S. cerevisiae. As expected, in unperturbed asynchronous cultures there were no major
discontinuities in the activities of the enzymes investigated. The virtual absence of steps or
peaks in the activity pattern of invertase and glucose-6-phosphate dehydrogenase in
synchronized yeast populations suggests that these enzymes, like most of the proteins in S .
cerevisiae (Lorincz et al., 1982), are synthesized continuously throughout the cell cycle. For both
enzymes it is not known, however, whether changes in the rate of synthesis occurred during the
cell cycle, since the data were too variable to discriminate between linear or exponential activity
pat terns.
In synchronous cultures the activity pattern of trehalase differed markedly from that of
invertase and glucose-6-phosphate dehydrogenase. These periodic changes in trehalase activity
support the findings of Kuenzie & Fiechter (1969), who found that in feed-starve-induced
partially synchronous cultures of S . cerevisiae trehalose mobilization was initiated from the
beginning of budding onwards, whereas trehalose accumulation took place largely during the
unbudded phase. A reciprocal correlation was found between the trehalose content of the cells and
the specific activity of trehalase. Kiienzie & Fiechter (1969, 1972) achieved synchronous growth
in a chemostat under glucose limitation at low dilution rates. It is well-established that under
such conditions the cells contain a relatively high amount of trehalose. This probably serves as
an important energy source for bud formation, and may also be required for the synthesis of
structural carbohydrates (mannan and glucan). Apparently, the energy derived from trehalose
breakdown allows the cell to traverse the cell cycle in a constant time, independent of external
nutritional limitations (Von Meyenburg, 1969). Hence the transient changes in trehalase
activity during the cell cycle could be explained. In our study, however, the cells were grown in
complex medium with glucose in excess. It has been demonstrated that fermenting yeast cells
contain only trace amounts of trehalose (Panek, 1963). Thus, the importance of cell-cycle-related
fluctuations in trehalase activity under these conditions may be less obvious, especially since the
amount of glucose produced in trehalose degradation would be relatively small compared with
glucose taken up from the medium. Perhaps regulation of trehalase activity during the cell cycle
occurs, whether it is superfluous or not. If this is true, trehalase in S . cerevisiae may in this respect
resemble nucleoside diphosphokinase in Schizosaccharomyces pombe. In the latter yeast this
enzyme (involved in DNA synthesis) shows step increases in activity during the cell cycle. The
activity steps seem to persist with normal cell-cycle timing after a block to the DNA-division
cycle imposed in cell-cycle mutants (Creanor & Mitchison, 1986).
These observations favour post-translational modification through phosphorylation and
dephosphorylation of a constitutively synthesized enzyme as the mechanism underlying the
changes in trehalase activity during the cell cycle. In view of several previous studies in vitro
(Van Solingen & Van Der Plaat, 1975; Wiemken & Schellenberg, 1982; Ortizetal., 1983; Uno et
al., 1983; Thevelein, 1984; Dellamora-Ortiz et al., 1986), cAMP is probably involved in the
activation of trehalase during part of the cell cycle, by activating a CAMP-dependent protein
kinase and the subsequent phosphorylation of the enzyme. Studies on yeast mutants deficient in
adenylate cyclase and CAMP-dependent protein kinase have indicated that cAMP is essential
for cells to proceed through the cell cycle (Casperson et a / . , 1985). Changes in the
phosphorylation state of the trehalase protein pool during the cell cycle may also be due to
periodic changes in the activity of a protein phosphatase. The molecular basis underlying the
apparently reversible activity changes of trehalase, as well as the precise moment of the transient
decrease in enzyme activity during the cell cycle are subjects for further investigation.
We are indebted to the Department of Electron Microscopy and Molecular Cytology, University of Amsterdam,
for allowing us to use the Coulter counter and centrifugal elutriation facilities.
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J . V A N D O O R N A N D OTHERS
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