Mini-reviewA specific mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance
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 ack of AMP esponses); 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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