Regulation of the cAMP Level in the Yeast Saccharomyces cerevisiae: the Glucose-induced cAMP Signal Is Not Mediated by a Transient Drop in the Intracellular pH

Abstract

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1-2min and induces a CAMPdependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.

Reversible Association between the V1and V0 Domains of Yeast Vacuolar H+-ATPase Is an Unconventional Glucose-Induced Effect

“…A small decrease of the ATP levels was observed within the first minute of glucose readdition, followed by slow restoration of the intracellular ATP pool between 5 and 20 min (Table 3). A transient and rapid drop of ATP levels within 30 s of glucose addition to glucose-derepressed yeast cells has been previously detected by 31 P nuclear magnetic resonance spectroscopy and attributed to formation of sugar phosphates (63). As shown in Table 3, following glucose addition, yeast cells showed kinetics of ATP recovery almost indistinguishable from kinetics for the recovery in YEP, although the mechanism of recovery is almost certainly different.…”

Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

“…At first, rather unspecific mechanisms such as transient plasma membrane depolarization (23,31) and transient intracellular acidification (10,36,46) were proposed. However, these hypotheses were shown to be wrong, and other rather unspecific mechanisms, such as increased energy supply, were also excluded (17,41,42). On the other hand, it was confirmed that drastic lowering of the intracellular pH in yeast cells strongly stimulates in vivo cAMP synthesis (41).…”

“…(23,31) and transient intracellular acidification (10,36,46) were proposed. However, these hypotheses were shown to be wrong, and other rather unspecific mechanisms, such as increased energy supply, were also excluded (17,41,42 …”

Stress induces depletion of Cdc25p and decreases the cAMP producing capability in Saccharomyces cerevisiae

“…Cell extracts were prepared as previously described by Thevelein et al (1987) with the following modifications: cells were harvested by filtration and cAMP was extracted with 1 ml 15 % perchloric acid with three freeze-thaw cycles in liquid nitrogen. After centrifugation, the supernatant was neutralized by 1?5 M potassium carbonate/1 M acetic acid buffer.…”