Coordinated glucose-induced Ca and pH responses in yeast Saccharomyces cerevisiae.

Ca and pH homeostasis are closely intertwined and this interrelationship is crucial in the cells' ability to adapt to varying environmental conditions. To further understand this Ca-pH link, cytosolic Ca was monitored using the aequorin-based bioluminescent assay in parallel with fluorescence reporter-based assays to monitor plasma membrane potentials and intracellular (cytosolic and vacuolar) pH in yeast Saccharomyces cerevisiae. At external pH 5, starved yeast cells displayed depolarized membrane potentials and responded to glucose re-addition with small Ca transients accompanied by cytosolic alkalinization and profound vacuolar acidification. In contrast, starved cells at external pH 7 were hyperpolarized and glucose re-addition induced large Ca transients and vacuolar alkalinization. In external Ca-free medium, glucose-induced pH responses were not affected but Ca transients were abolished, indicating that the intracellular [Ca] increase was not prerequisite for activation of the two primary proton pumps, being Pma1 at the plasma membrane and the vacuolar and Golgi localized V-ATPases. A reduction in Pma1 expression resulted in membrane depolarization and reduced Ca transients, indicating that the membrane hyperpolarization generated by Pma1 activation governed the Ca influx that is associated with glucose-induced Ca transients. Loss of V-ATPase activity through concanamycin A inhibition did not alter glucose-induced cytosolic pH responses but affected vacuolar pH changes and Ca transients, indicating that the V-ATPase established vacuolar proton gradient is substantial for organelle H/Ca exchange. Finally, a systematic analysis of yeast deletion strains allowed us to reveal an essential role for both the vacuolar H/Ca exchanger Vcx1 and the Golgi exchanger Gdt1 in the dissipation of intracellular Ca.