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Journal of Bacteriology, April 1999, p. 2640-2642, Vol. 181, No. 8
Instituto de Investigaciones Biomédicas
"Alberto Sols," CSIC-UAM, 28029 Madrid, Spain
Received 7 December 1998/Accepted 1 February 1999
External cyclic AMP (cAMP) hindered the derepression of
gluconeogenic enzymes in a pde2 mutant of
Saccharomyces cerevisiae, but it did not prevent invertase
derepression. cAMP reduced nearly 20-fold the transcription driven by
upstream activation sequence (UAS1FBP1)
from FBP1, encoding fructose-1,6-bisphosphatase; it
decreased 2-fold the activation of transcription by
UAS2FBP1. Nuclear extracts from cells
derepressed in the presence of cAMP were impaired in the formation of
specific UASFBP1-protein complexes in band
shift experiments. cAMP does not appear to act through the repressing
protein Mig1. Control of FBP1 transcription through cAMP is
redundant with other regulatory mechanisms.
An increase in cyclic AMP (cAMP)
acts as a hunger signal in Escherichia coli (14)
and is required to relieve repression by glucose (20), while
in yeasts cAMP levels are highest in cells using a carbon source such
as glucose, which allows efficient growth (11). For
Schizosaccharomyces pombe it has been demonstrated that cAMP
represses fructose-1,6-bisphosphatase (FbPase) (18), whereas
it was suggested that cAMP is not involved in catabolite repression in
Saccharomyces cerevisiae (22, 23). However, more
recent work points to cAMP being able to repress the synthesis of
proteins which are normally derepressed upon glucose exhaustion (2, 3).
We have reexamined the role of cAMP in catabolite repression in
S. cerevisiae, using a strain which lacks phosphodiesterase 2 and responds to the presence of cAMP in the medium.
S. cerevisiae OL556 MATa/MAT Yeast extracts were prepared by shaking with glass beads
(1). FbPase (13), phosphoenolpyruvate
carboxykinase (27), isocitrate lyase (9), and
NAD-dependent glutamate dehydrogenase (10) were tested
spectrophotometrically. Invertase was assayed by the method of
Goldstein and Lampen (15), using whole cells. The protein
concentration was determined with the bicinchoninic acid protein assay
reagent (Pierce).
RNA was extracted with the GIBCO TRIzol reagent (6). RNA
electrophoresis in formaldehyde gels, blotting, and hybridization were
performed essentially by standard methods (4). For probes we
used a 0.82-kb EcoRV-StuI fragment of
FBP1 (+57 to +877) and a 0.37-kb
SmaI-EcoRV fragment of GLK1 (+950 to
+1318), labelled with a Pharmacia labelling kit (12).
Nuclear extracts were obtained as described previously (29);
for yeasts derepressed with cAMP, solutions included 5 mM cAMP. Band
shift assays were performed by using oligonucleotides OL1 and OL2,
which correspond to upstream activation sequence 2 from FBP1
(UAS2FBP1) and to
UAS1FBP1, respectively (30).
In a pde2 strain, cAMP in the medium blocked the
derepression of FbPase and, to a lesser degree, that of other enzymes
(Table 1). For invertase, 5 mM cAMP in
the corresponding derepression medium did not affect significantly the
degree of derepression; an activity around 300 µmol/min/g of yeast
was reached in both cases.
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Cyclic AMP Can Decrease Expression of Genes Subject
to Catabolite Repression in Saccharomyces
cerevisiae
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cdc25-5/cdc25-5 his3/his3 leu2/leu2
rca1(pde2)/rca1 TRP1/trp1 ura3/ura3,
supplied by M. Jacquet (2), was grown at 23°C in YPD (1%
yeast extract, 2% peptone, 2% dextrose) and was either collected at 2 to 3 mg (wet weight) of cells per ml (repressed cells) or derepressed by incubation at 20 mg/ml in YP-2% ethanol for the times indicated below. To derepress invertase, repressed cells were resuspended in
YP-0.05% glucose and incubated for 3 h. For isolation of
transformed strains, YNBS medium was used (2).
TABLE 1.
Effect of external cAMP on the derepression of
different enzymes
The effect of cAMP on FbPase derepression was transient; after 24 h of derepression in ethanol, FbPase activity was the same in samples with or without cAMP. Northern blotting performed after 4 or 12 h of derepression showed that at 4 h the block of transcription by cAMP was nearly complete, while after 12 h cAMP decreased the FBP1 mRNA level about threefold (Fig. 1). Since we observed that in a pde1 pde2 double mutant the effect of cAMP on FbPase expression was maintained for up to 24 h, we suggest that in the pde2 mutant derepression of the low-affinity phosphodiesterase Pde1 reduces the internal concentration of cAMP and relieves FbPase repression. The effect of cAMP on transcription was not unspecific, as shown by using GLK1, which encodes glucokinase and is repressed by glucose (17), as a control gene (Fig. 1).
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The FBP1 promoter comprises two UAS elements (24, 26,
30) and an upstream repressing sequence able to bind the
regulatory protein Mig1 (21, 25). cAMP could act by blocking
activation through the UASs or by interfering with the release of Mig1
inhibition which occurs upon glucose removal (8). To
investigate possible targets for cAMP, we used different fusions with
the reporter gene lacZ: either the complete FBP1
promoter or the UAS1 (
450 to 407) or UAS2 (507 to 489) element
was fused with lacZ. As shown in Table
2, cAMP decreased the expression of
FBP1-lacZ and also had a strong effect (over 15-fold
repression) on UAS1-lacZ; for UAS2-lacZ, the
decrease in expression was only two to threefold. To test whether cAMP
could act by converting Mig1 into a constitutive repressor, we looked
at the effect of cAMP on a pde2 strain where the
MIG1 gene had been interrupted and found that cAMP was as effective in blocking FBP1 expression in it as in the
corresponding MIG1 strain.
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To examine the effect of cAMP on the transcription factors binding the UASs, we tested by a band shift assay nuclear extracts from cells derepressed in the presence of cAMP; the specific DNA-protein complex formed with UAS2 was not observed under these conditions, and only one of the specific complexes was formed with UAS1, a pattern similar to that found with extracts from repressed cells (Fig. 2). We checked that cAMP added during the band shift assay had no effect on the formation of DNA-protein complexes; within the cell, however, cAMP could interfere with the synthesis of FBP1-activating proteins or trigger their modification, decreasing their capacity to bind in vitro to the corresponding UASs. The relevant target for the protein kinases activated by cAMP has not been identified; a candidate would be the transcription factor Cat8, required for the derepression of gluconeogenic enzymes (16, 28) and with two potential sites for phosphorylation by the cAMP-dependent protein kinases. Although the transcription factors Msn2 and Msn4 control many genes induced at the diauxic transition, they are not required for the derepression of isocitrate lyase (2), and therefore, they are probably also not involved in FBP1 transcription.
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To investigate whether cAMP may be the main trigger for catabolite repression of certain genes, we have utilized a yeast strain, derived from RS13-58A-1h (5), with a low protein kinase activity independent of cAMP levels. In S. cerevisiae JF908 (MATa ade8 his3 leu2 trp1 ura3 tpk1w tpk2::HIS3 tpk3::TRP1 bcy1::LEU2 GSY2-lacZ::URA3), provided by J. M. François, both FbPase and NAD-dependent glutamate dehydrogenase were repressed by glucose as in a wild-type strain and derepressed upon incubation in an ethanol medium. It is therefore clear that derepression of FbPase (and of other enzymes) does not depend only on changes in cAMP levels. This is consistent with the results of Yin et al. (31), which suggested that different signalling pathways were involved in the response to glucose of FBP1 and PCK1 transcription. Control by cAMP would then be at least partially redundant with other regulatory mechanisms.
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ACKNOWLEDGMENTS |
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We thank M. Jacquet for strain OL556, J. M. François (INSA, Toulouse, France) for strain JF908, and M. J. Mazón for comments on the manuscript.
This work was supported by grant PB094-0091-CO2-01 from the Dirección General de Investigación Científica y Técnica. O.Z. had a fellowship from the Spanish Plan de Formación de Personal Investigator. C.L. was the recipient of an ERASMUS student mobility grant in a program of work placement for undergraduates of the University of Huddersfield (United Kingdom).
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FOOTNOTES |
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* Corresponding author. Mailing address: Instituto de Investigaciones Biomédicas, Arturo Duperier 4, 28029 Madrid, Spain. Phone: 34-91-5854622. Fax: 34-91-5854587. E-mail: jmgancedo{at}iib.uam.es.
Present address: School of Biochemistry and Molecular Biology,
University of Leeds, Leeds LS2 9JT, United Kingdom.
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Journal of Bacteriology, April 1999, p. 2640-2642, Vol. 181, No. 8
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