Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1999 Apr; 181(8): 2640–2642.

Cyclic AMP Can Decrease Expression of Genes Subject to Catabolite Repression in Saccharomyces cerevisiae


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/MATα 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).

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 (Table1).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.

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. (Fig.1).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. (Fig.1).1).

FIG. 1
FbPase mRNA is responsive to the presence of cAMP in the derepressing medium. S. cerevisiae OL556 (pde2) was grown in YPD and derepressed in YP-ethanol (YPEt) for 4 or 12 h in the presence or absence of 5 mM cAMP. Northern analysis was performed by loading ...

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 Table2,2, 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.

Effect of external cAMP on the expression of different fusion genes

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. (Fig.2).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.

FIG. 2
Effect of the presence of cAMP in the derepressing medium on the capacity of nuclear extracts to form specific DNA-protein complexes with UAS1FBP1 (A) and UAS2FBP1 (B). Nuclear extracts from S. cerevisiae OL556 (pde2) were prepared from repressed cells ...

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.


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).


1. Blázquez M A, Lagunas R, Gancedo C, Gancedo J M. Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases. FEBS Lett. 1993;329:51–54. [PubMed]
2. Boy-Marcotte E, Tadi D, Perrot M, Boucherie H, Jacquet M. High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae. Microbiology. 1996;142:459–467. [PubMed]
3. Boy-Marcotte E, Perrot M, Bussereau F, Boucherie H, Jacquet M. Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae. J Bacteriol. 1998;180:1044–1052. [PMC free article] [PubMed]
4. Brown T, Mackey K. Analysis of RNA by Northern and slot blot hybridization. In: Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K, editors. Current protocols in molecular biology. New York, N.Y: John Wiley and Sons; 1997. pp. 4.9.1–4.9.16.
5. Cameron S, Levin L, Zoller M, Wigler M. c-AMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in Saccharomyces cerevisiae. Cell. 1988;53:555–566. [PubMed]
6. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159. [PubMed]
7. de Mesquita J, Zaragoza O, Gancedo J M. Functional analysis of upstream activating elements in the promoter of the FBP1 gene from Saccharomyces cerevisiae. Curr Genet. 1998;33:406–411. [PubMed]
8. DeVit M J, Waddle J A, Johnston M. Regulated nuclear translocation of the Mig1 glucose repressor. Mol Biol Cell. 1997;8:1603–1618. [PMC free article] [PubMed]
9. Dixon G H, Kornberg H L. Assay methods for key enzymes of the glyoxylate cycle. Biochem J. 1959;72p:3P.
10. Doherty D. l-Glutamate dehydrogenases (yeast) Methods Enzymol. 1970;17A:850–856.
11. Eraso P, Gancedo J M. Catabolite repression in yeasts is not associated with low levels of cAMP. Eur J Biochem. 1984;141:195–198. [PubMed]
12. Feinberg A P, Vogelstein B. A technique for radiolabeling DNA fragments to high specific activity. Anal Biochem. 1983;132:6–13. [PubMed]
13. Funayama S, Gancedo J M, Gancedo C. Turnover of yeast fructose-1,6-bisphosphatase in different metabolic conditions. Eur J Biochem. 1980;109:61–66. [PubMed]
14. Gancedo J M, Mazón M J, Eraso P. Biological roles of cAMP. Similarities and differences between organisms. Trends Biochem Sci. 1985;10:210–212.
15. Goldstein A, Lampen J O. β-d-Fructofuranoside fructohydrolase from yeast. Methods Enzymol. 1975;42:504–511. [PubMed]
16. Hedges D, Proft M, Entian K-D. CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1995;15:1915–1922. [PMC free article] [PubMed]
17. Herrero P, Galíndez J, Ruiz N, Martínez-Campa C, Moreno F. Transcriptional regulation of the Saccharomyces cerevisiae HXK1, HXK2 and GLK1 genes. Yeast. 1995;11:137–144. [PubMed]
18. Hoffman C S, Winston F. Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes Dev. 1991;5:561–571. [PubMed]
19. Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983;153:163–168. [PMC free article] [PubMed]
20. Kolb A, Busby S, Buc H, Garges S, Adhya S. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993;62:749–795. [PubMed]
21. Lundin M, Nehlin J O, Ronne H. Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein MIG1. Mol Cell Biol. 1994;14:1979–1985. [PMC free article] [PubMed]
22. Matsumoto K, Uno I, Toh-e A, Ishikawa T, Oshima Y. Cyclic AMP may not be involved in catabolite repression in Saccharomyes [sic] cerevisiae: evidence from mutants capable of utilizing it as an adenine source. J Bacteriol. 1982;150:277–285. [PMC free article] [PubMed]
23. Matsumoto K, Uno I, Ishikawa T, Oshima Y. Cyclic AMP may not be involved in catabolite repression in Saccharomyces cerevisiae: evidence from mutants unable to synthesize it. J Bacteriol. 1983;156:898–900. [PMC free article] [PubMed]
24. Mercado J J, Gancedo J M. Regulatory regions in the yeast FBP1 and PCK1 genes. FEBS Lett. 1992;311:110–114. [PubMed]
25. Mercado J J, Vincent O, Gancedo J M. Regions in the promoter of the yeast FBP1 gene implicated in catabolite repression may bind the product of the regulatory gene MIG1. FEBS Lett. 1991;291:97–100. [PubMed]
26. Niederacher D, Schüller H-J, Grzesitza D, Gütlich H, Hauser H P, Wagner T, Entian K-D. Identification of UAS elements and binding proteins necessary for derepression of Saccharomyces cerevisiae fructose-1,6-bisphosphatase. Curr Genet. 1992;22:363–370. [PubMed]
27. Perea J, Gancedo C. Isolation and characterization of a mutant of Saccharomyces cerevisiae defective in phosphoenolpyruvate carboxykinase. Arch Microbiol. 1982;132:141–143. [PubMed]
28. Rahner A, Schöler A, Mertens E, Gollwitzer B, Schüller H-J. Dual influence of the yeast Cat1p (Snf1p) protein kinase on carbon source-dependent transcriptional activation of gluconeogenic genes by the regulatory gene CAT8. Nucleic Acids Res. 1996;24:2331–2337. [PMC free article] [PubMed]
29. Schneider R, Gander I, Müller U, Mertz R, Winnacker E L. A sensitive and rapid assay for nuclear factor I and other DNA-binding proteins in crude nuclear extracts. Nucleic Acids Res. 1986;14:1303–1317. [PMC free article] [PubMed]
30. Vincent O, Gancedo J M. Analysis of positive elements sensitive to glucose in the promoter of the FBP1 gene from yeast. J Biol Chem. 1995;270:12832–12838. [PubMed]
31. Yin Z, Smith R J, Brown A J P. Multiple signalling pathways trigger the exquisite sensitivity of yeast gluconeogenic mRNAs to glucose. Mol Microbiol. 1996;20:751–764. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...