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A specific mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance

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Abstract

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, proliferation and stress resistance. Derepressed cells show a rapid increase in the cAMP level (within 1 min) after addition of glucose or after intracellular acidification. A specific mutation in adenylate cyclase, the enzyme that catalyzes the synthesis in cAMP, largely prevents both cAMP responses. The responsible mutation was originally called lcr1 (for lack of cAMP responses); lcr1 was later identified as allelic with CYR1/CDC35. The mutation was introduced into the CYR1 gene of a W303-1A wild type strain, which resulted in a large decrease in cAMP signalling. Furthermore, there was a strong reduction in GTP/Mg2+-stimulated but not in Mn2+-stimulated adenylate cyclase activity in isolated plasma membranes, which is consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the lcr1 mutant. Because of the absence of cAMP signalling during exponential growth on glucose, it was concluded that glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Activation of the PKA pathway is mediated by a G protein (either Ras1/Ras2 or Gpa2). Constitutive activation of the pathway by Ras2val19 or Gpa2val132 has a negative effect on glycogen and trehalose accumulation and heat shock survival. The lcr1 mutation partially suppresses this effect indicating that the target sites of the two G-proteins on adenylate cyclase might have at least a part in common.

Introduction

As in other eukaryotes, cAMP is synthesized in the yeast Saccharomyces cerevisiae by the enzyme adenylate cyclase (AC), which is encoded by the CYR1/CDC35 gene (Matsumoto et al., 1984). The yeast Ras1 and Ras2 proteins are controlling elements of AC and in their absence cAMP synthesis in vivo is insufficient for viability (Toda et al., 1985, Field et al., 1988). Other proteins that play an important role in the control of AC activity are Cdc25 and Ira1 and Ira2. cAMP controls the activity of protein kinase A (PKA).

Several targets of the cAMP-PKA pathway have been identified. High activity of the pathway causes reduced expression of ‘STRE’(Stress Responsive Element)-controlled genes, low levels of trehalose and glycogen and low heat stress resistance (this is e.g. the case during exponential growth on glucose). Only two triggers of the cAMP pathway are well established in yeast (Thevelein, 1991). The addition of glucose to derepressed yeast cells triggers within 1 min a transient increase in the cAMP level (van der Plaat, 1974). Intracellular acidification triggers an equally rapid but more pronounced and longer-lasting increase in the cAMP level (Caspani et al., 1985). Glucose does not act through an intracellular acidification effect (Thevelein et al., 1987). Contrary to the acidification effect, the glucose-induced cAMP signalling requires a G-protein coupled receptor system consisting of Gpr1–Gpa2 (Kraakman et al., 1999).

During our studies on glucose- and acidification-induced cAMP signals we have discovered that some ‘wild type’ laboratory strains (ENY.cat80 series, CEN.PK series) did not show both cAMP responses; the mutation responsible for it was called lcr1 (for lack of cAMP responses); lcr1 was identified as allelic with CYR1/CDC35 and contained a K1876M substitution near the end of the catalytic domain in adenylate cyclase.

We have replaced CYR1 in a wild type W303-1A strain, by the mutated allel lcr1. In this paper we present a comparison between the WT (W303-1A) and the isogenic lcr1 mutant for cAMP levels and cAMP signalling responses.

Section snippets

Results and discussion

In the lcr1 strain, glucose- and acidification-induced cAMP signalling were eliminated (Fig. 1) indicating that lysine1876 of adenylate cyclase is essential for cAMP responses. The strong reduction in GTP/Mg2+-stimulated but not in Mn2+-stimulated AC activity measured in isolated plasma membranes of the lcr1 mutant (Fig. 2) is consistent with the absence of signalling in vivo.

Several PKA targets have been investigated: trehalase activation (results not shown) and trehalose and glycogen content

Conclusion

The Cyr1K1876M mutation in the lcr1 mutant specifically prevents agonist-induced cAMP signalling apparently by strongly reducing G-protein activation of the enzyme rather than basal activity.

This delays glucose-induced changes in PKA targets associated with the adaptation to growth on glucose. However, it does not eliminate, nor even reduce the typical variation in PKA-controlled phenotypic properties during diauxic growth, which therefore must be due to a cAMP-signalling independent pathway.

Acknowledgements

We wish to thank Renata Wicik, Willy Verheyden and Sven Bogaerts for excellent technical assistance, Filip Rolland for measuring the cAMP responses in the CEN.PK2-1C strain and K.-D. Entian (Frankfurt), E. Boles (Düsseldorf) and O. Fasano (Palermo) for provision of yeast strains and plasmids. This work was supported by a fellowship from the Fund for Scientific Research–Flanders (Senior research assistant) to J.W. and by grants from the Fund for Scientific Research–Flanders, the Research Fund of

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