Ca²⁺ 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 Ca²⁺-pH link, cytosolic Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ transients and vacuolar alkalinization. In external Ca²⁺-free medium, glucose-induced pH responses were not affected but Ca²⁺ transients were abolished, indicating that the intracellular [Ca²⁺] 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 Ca²⁺ transients, indicating that the membrane hyperpolarization generated by Pma1 activation governed the Ca²⁺ influx that is associated with glucose-induced Ca²⁺ transients. Loss of V-ATPase activity through concanamycin A inhibition did not alter glucose-induced cytosolic pH responses but affected vacuolar pH changes and Ca²⁺ transients, indicating that the V-ATPase established vacuolar proton gradient is substantial for organelle H⁺/Ca²⁺ exchange. Finally, a systematic analysis of yeast deletion strains allowed us to reveal an essential role for both the vacuolar H⁺/Ca²⁺ exchanger Vcx1 and the Golgi exchanger Gdt1 in the dissipation of intracellular Ca²⁺.

• PMID: 34610487
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Ma TY, Deprez MA, Callewaert G, Winderickx J

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