A specific mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K176M, eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance.

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.

A mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, specifically affects glucose- and acidification-induced cAMP signalling and not the basal cAMP level.

In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related 'wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+-dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+-dependent activity was strongly reduced, 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 cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.

Glucose-induced activation of plasma membrane H(+)-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis.

Addition of glucose-related fermentable sugars or protonophores to derepressed cells of the yeast Saccharomyces cerevisiae causes a 3- to 4-fold activation of the plasma membrane H(+)-ATPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP synthesis, incubation at the restrictive temperature reduced the extent of H(+)-ATPase activation. Incubation of non-temperature-sensitive strains, however, at such temperatures also caused reduction of H(+)-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H(+)-ATPase activation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H(+)-ATPase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAMP-protein kinase A signaling pathway is not required for glucose activation of the H(+)-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phosphorylating enzymes hexokinase PI and PII and glucokinase showed that activation of the H(+)-ATPase with glucose or fructose was completely dependent on the presence of a kinase able to phosphorylate the sugar. These and other data concerning the role of initial sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H(+)-ATPase have a common initiation point.

Absence of glucose-induced cAMP signaling in the Saccharomyces cerevisiae mutants cat1 and cat3 which are deficient in derepression of glucose-repressible proteins.

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal. Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level. The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3. Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase. The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance. In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all. These results indicate that repressed cells do not show a true glucose-induced cAMP signal. When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed. Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect. These data show that intracellular acidification is able to enhance the cAMP level in repressed cells.(ABSTRACT TRUNCATED AT 250 WORDS)