Eur. J. Biochem.

141, 195-198 (1984)

0 FEBS 1984

Catabolite repression in yeasts is not associated with low levels of cAMP

Pilar ERAS0 and Juana M. GANCEDO
Departamento de Bioquimica de la Facultad de Medicina de la Universidad Autonoma de Madrid; and
Instituto de Enzimologia del Consejo Superior de Investigaciones Cientificas; Madrid
(Received November 14, 1983) - EJB 83 1227

The relationship between levels of CAMP and catabolite repression in yeasts has been investigated. Strains of
Succharomyces cerevisiae, Schizosaccharomyces pomhe and Kluyuerornyces jrugilis were used. The yeasts were
grown on different carbon sources to attain various degrees of repression. Galactose repressed as much as glucose,
while maltose was less effective. Full derepression was achieved with ethanol.
The enzymes tested were fructose-bisphosphatase, malate dehydrogenase, glutamate dehydrogenase (NAD
dependent), cytochrome oxidase and isocitrate lyase (this last enzyme was found to be absent in Schizosac-
charomyces).
The levels of CAMP were 2 - 3 times higher in the repressed conditions than in the derepressed ones. It is
therefore concluded that in yeasts catabolite repression is not mediated by a lowering of the intracellular
concentration of CAMP.

Catabolite repression in yeasts is a well known pheno-
menon [I] but its underlying mechanism has not been yet
established. Since in bacteria catabolite repression is cor-
related with lowered levels of CAMP (for a review see [2]), it has
been tempting to assume that the same situation ocurred in
yeast. Indeed some early data on CAMP levels appeared to
confirm this idea [3,4] and a more recent review on the subject
agreed also with this view [5]. However several results casted
doubts on the role of CAMP in mediating catabolite repression
in yeast. Montenecourt et al. [6] did not found a clear
correlation between the cAMP level and the sensitivity of
invertase synthesis to catabolite repression. Also, in yeast
strains permeable to cAMP [7] the addition of exogenous
cAMP did not prevent glucose repression of galactokinase
synthesis [8]. Finally, it has been repeatedly shown that the
addition of glucose to a Saccharomyces cerevisiae culture
produces an increase in the levels of CAMP [9 - 1I], although
this increase was thought to be transient.
We decided therefore to reinvestigate systematically the
relationship between CAMPlevels and catabolite repression in
yeasts. Our results show that in different yeast genera cata-
bolite repression is not associated with a decreased level of
CAMP. On the contrary, the lowest levels of CAMPare found
in yeast not subject to catabolite repression.
MATERIALS A N D METHODS
Strains ojyeust and growth conditions
The following yeast strains were used : Succharomyces
cerevisiae F11 (obtained from G. Fink) and X2180, Sclzizo-
succharomyces pomhe NCYC 972h- and Kluyverornyces fru-
Enzymes. Fructose-bisphosphatase (EC 3.1.3.11); isocitrate
lyase (EC 4.1.3.1); malate dehydrogenase (EC 1.1.1.37); glutamate
dehydrogenase (NAD dependent) (EC 1.4.1.2); cytochrome oxidase
(EC 1.9.3.1).
gilis 1407 (obtained from C. Gancedo). The yeasts were grown
either on a complex medium with 1 % yeast extract and 2 %
peptone or on a mineral medium [12] using NaCl (0.25 g/l)
instead of the original sodium citrate and adjusting the initial
p H of the medium to 5.5. The different carbon sources were
added at 2 % final concentration unless otherwise indicated.
Sampling of yeast and extruction procedures
Sampling and extraction of yeast for the determination
of cAMP were performed as Saez and Lagunas [I31 except
that 6 % trichloroacetic acid was used instead of perchloric
acid. T o remove the trichloroacetic acid from the extracts HC1
was added to a final concentration of 1 0 m M and four
extractions with ethyl ether were done. The residual ether
was eliminated with a vacuum pump and the samples
were neutralized with 1 M imidazole. When indicated, the
yeast was collected by centrifugation instead of filtration and
the extraction was performed either with trichloroacetic acid
as described above or with acetic acid as described by
Schlanderer and Dellweg [4].
Assay of C A M P
The Amersham assay kit, based on the competition for
binding to a specific protein between CAMPin the sample and
a fixed quantity of labelled cAMP [I41 was used. The
recommended procedure was strictly followed. In particular
great care was taken to mantain the temperature at 0 "C during
the incubation of CAMP with the binding protein and to pipet
exact amounts of the charcoal suspension. To check if the
CAMP found was sensitive to phosphodiesterase, the pro-
cedure of Butcher and Sutherland [I51 was used. To calculate
the intracellular concentration of CAMP it was accepted that
1 g wet yeast contains 0.6 ml cell sap [16].
196
Preparation of extracts and enzyme assays
Extracts for enzyme assays were performed as described by
Funayama et al. [I71 with 20 m M imidazole/HCl p H 7.
Fructose-bisphosphatase was assayed as in [18], malate dehy-
drogenase as in [19], glutamate dehydrogenase (NAD de-
pendent) as in [20] and cytochrome oxidase as in [21] using in
all cases the same assay mixture than in the fructose bisphos-
phatase assay. Isocitrate lyase was tested as described by
Dixon and Kornberg [22].
Protein assay
Protein concentration was determined by the method of
Lowry [23] using bovine serum albumin as standard.
Glucose determination
Glucose in the medium was assayed enzymatically as
described by Bergmeyer et al. [24].
RESULTS
Validation of’ the methodology used
Published values for internal concentrations of CAMP in
yeasts differ widely. For Saccharomyces strains growing in
similar conditions the reported concentrations range from
0.4 pM [3] to 10 pM [25] while for Schizosaccharomycespombe
the values given are either less than 0.005 pM [4] or about
1 pM [26]. Although extreme values could be due to the
method used for measuring cAMP [3] or to the extraction
process [4] there are still large differences between authors that
use basically the same procedure [6,25,27,28].
It was therefore essential to check the reliability of the
method used for sampling the yeast and measuring CAMP. An
important control is to test that no cAMP is lost in the course
of the different manipulations. This was done by adding cAMP
during the extraction process and measuring the extent of
recovery. Saccharomyces cerevisiae grown in the presence of
glucose or in the presence of ethanol was used and in both cases
the recovery was between 80 % and 100 %. Another necessary
control is to check that what is measured is really CAMP. This
was done by incubating aliquots of the extracts with CAMP
phosphodiesterase. With all the yeasts used and in all the
conditions tested the amount of CAMP found after treatment
with phosphodiesterase was 20% or less of the initial value.
Levels of CAMP in the culture medium were found to be
extremely low (less than 2 nM). Contamination of the cells by
the medium had therefore a negligible effect on CAMP
determination.
To be able to compare our results with those of others we
measured cAMP levels in S.pombe using different sampling
and extraction procedures. As shown in Table 1 all methods
gave the same results. The reproducibility of the results was
good as it can be seen in Table 1 and in the corresponding
column of Table2. From the results presented we may
therefore conclude that the methodology used is reliable.
Measurement of C A M P levels in yeasts subject
to different degrees of catabolite repression
Levels of CAMP and of enzymes subject to catabolite
repression were measured in yeasts belonging to different
Table 1.Influence ofsampling andextractionprocedures on the oalues of
cAMP found
S. pomhe was grown on glucose ( 5 ”/, initial concentration) and
collected when the glucose concentration reached 2 %. Sampling and
extraction were performed as indicated (for details see Materials and
Methods). Results are average of six samples & SEM
~~ ~
Sampling Extraction cAMP found
procedure procedure
~~
PM
Filtration trichloroacetic acid 0.31 k0.02
Centrifugation trichloroacetic acid 0.30+0.02
Centrifugation acetic acid 0.34k 0.04
genera. Growth conditions were chosen so as to allow
comparisons with earlier work. The enzymes were selected as
representative of different pathways. The results are shown
in Table 2. As it can be seen the enzymes were strongly
repressed in yeast growing on glucose and collected in the
logarithmic phase of growth. The enzymes were derepressed
when the yeast was growing on ethanol, either supplied from
the beginning o r produced during glucose fermentation (glu-
cose stationary). In this last case, however, the derepression
was less marked in S. cerevisiae grown on a mineral medium.
The effect on catabolite repression of galactose or maltose was
tested in S. cerevisiae and S . pombe respectively. It can be seen
that the catabolite repression was as marked with galactose as
with glucose, while it was partially relieved by maltose, except
for fructose-bisphosphatase.
The levels of CAMP were usually 2-3-fold higher in the re-
pressed conditions than during derepression. Two exceptions
should be noted. In Kluyveromyces fragilis grown on yeast
extract/peptone/glucose until the stationary phase, there was a
full derepression of the enzymes tested without a correspond-
ing decrease in the cAMP concentration. O n the other hand in
S. cerevisiae grown on mineral medium/glucose and collected
in the stationary phase, the drop in cAMP levels was not
associated with a marked derepression of the enzymes assayed.
DISCUSSION
Our results conclusively show that in the different yeasts
tested the levels of cAMP are not lowered in conditions of
catabolite repression. These results differ from those reported
by other authors [3,4,6,27]. The reasons for this difference are
not obvious. Since we have tested a variety of strains, strain
differences are probably no explanation. In fact for Schizo-
saccharomycespombe we utilized the very strain that had been
used in earlier work [4]. Growth conditions have been also very
similar to those used previously. Moreover the results of
Table 1 indicate that the procedures for sampling and ex-
tracting the yeasts are not critical. It seems therefore that the
differences are due to the determination of CAMP itself. An
early method of determination based on the aggregation of
Dictyostelium discoideum [3] could have been subjected to
artifacts, and we have also observed that the Gilman method,
employed here and used by most authors, can yield erratic
results if it is not performed with the utmost care. The
following reasons make us confident in the reliability of our
results : we have performed careful determinations of a great
number of samples, collected in independent experiments, and
 197

Table 2. CAMP levels and activity of some enzymes subject to catabolite repression in yeasts grown in dqferent conditions
The yeasts were grown as indicated and samples taken for measuring cAMP and enzymatic activity as described in Materials and Methods.
Figures for enzymatic activity are the mean of at least two separate cultures. For cAMP the number of samples tested i s given in brackets. YP,
yeast extract/peptone medium; MM, mineral medium

Yeast Growth Stage of growth Fruc- Iso- Malate Glutamate Cyto- cAMP
species medium (amount of yeast) tose-1,6- citrate dehydro- dehydro- chrome
bisphos- lyase genase genase oxidase
phatase (NAD
dependent)

mg/ml units/g protein PM

S. cereoisiae YP-glucose early log (3 -4) 2 I 0.2 200 7 2 1 . 1 3 ~ 0 . 1 1(8)

X2180 Y P-glucose stationary (40 -45) 75 5 1500 110 18 0.40 0.01 (4)
YP-ethanol early log (3 -4) 80 10 3000 190 11 0.46 kO.08 (9)
MM-glucose early log (1) 1 0.3 300 7 7 0.77 (2)
MM-glucose late log to (9 -15) 11 30 1100 18 8 0.25 kO.08 ( 6 )
stationary
S. cerevisiae MM-glucose early log (1 -2) 2 5 0.3 340 20 7 0.71 kO.11 (9)
F11 MM-galactose early log (1 -2) 2 < 0.3
- 560 17 14 0.89T0.09 (9)
MM-glucose stationary (8) 1 1.3 400 8 32 0.32$0.02 (3)
Schizosacch. MM-glucose early log (3 -5) 1 < 0.1
- 97 15 9 0.45 5 0.05 (9)
pombe MM-maltose early log (3 -6) I I 0.1 250 45 20 0.47 T0.04 (9)
MM-glucose stationary (14-15) 79 5 0.1 3400 48 90 0.28 TO.01 (3)
K. frugilis YP-glucose early log (3 -8) 2 I0.3 740 8 30 0.47-tO.05 (17)
YP-ethanol early log (3 -4) 98 10 3500 33 100 0.28 k 0.02 (9)
YP-glucose stationary (50 - 55) 110 48 1900 35 56 0.52k0.06 (9)

attained always a good reproducibility; we have run controls levels of cAMP cause catabolite repression in yeasts. For
showing that cAMP was not lost during the manipulations and instance, in Kluyveromyces frugilis cAMP levels do not
that there were only minor contaminants insensitive to cAMP decrease at the end of the growth on glucose and there is
phosphodiesterase. nevertheless a very marked derepression of different enzymes.
With regard to the determination of enzymatic activities in The results presented show that although catabolite repres-
different conditions the following comments should be made. sion is widely extended among microorganisms the underlying
Although Van Wijk and Konijn [3] found that a-glucosidase mechanisms are not uniform. In fact, it has been shown that
and succinate dehydrogenase were derepressed during growth while in Escherichia coli cAMP is necessary for relieving
on galactose, we found no derepression of the enzymes we catabolite repression [2] this is not the case for Pseudomonas
tested except for a doubling in the activity of cytochrome [36], Bacillus [37] or Tetrahymena [38,39].
oxidase. These results, consistent with those reported earlier by A possible role for cAMP in yeast could be to control cell
Polakis and Bartley [29], indicate that galactose does not proliferation [40] and the initiation of meiosis which precedes
always relieve catabolite repression in yeast. When a culture of sporulation [41].
Saccharomyces cereuisiae on glucose reaches the stationary We are indebted to Drs C. Gdncedo and M. J. Mazon for helpful
phase the degree of derepression attained depends on the discussions and to Dr A. Sols for critical reading of the manuscript.
nitrogen source present. If there is ammonium in the medium This work was partly supported by a grant from the CAICyT.
(MM) the derepression is not very marked, a fact which had
been previously reported for fructose bisphosphatase [30] and
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