Cytosolic Ca2+ homeostasis is a constitutive function of the V-ATPase in Saccharomyces cerevisiae

Abstract

The vacuole is the major site of intracellular Ca2+ storage in yeast and functions to maintain cytosolic Ca2+ levels within a narrow physiological range via a Ca2+ pump (Pmc1p) and a H+/Ca2+ antiporter (Vcx1p) driven by the vacuolar H+-ATPase (V-ATPase). We examined the function of the V-ATPase in cytosolic Ca2+ homeostasis by comparing responses to a brief Ca2+ challenge of a V-ATPase mutant (vma2Δ) and wild-type cells treated with the V-ATPase inhibitor concanamycin A. The kinetics of the Ca2+ response were determined using transgenic aequorin as an in vivo cytosolic Ca2+ reporter system. In wild-type cells, the V-ATPase-driven Vcx1p was chiefly responsible for restoring cytosolic Ca2+ concentrations after a brief pulse. In cells lacking V-ATPase activity, brief exposure to elevated Ca2+ compromised viability, even when there was little change in the final cytosolic Ca2+ concentration, vma2Δ cells were more efficient at restoring cytosolic [Ca2+] after a pulse than concanamycin-treated wild-type cells, suggesting long term loss of V-ATPase triggers compensatory mechanisms. This compensation was dependent on calcineurin, and was mediated primarily by Pmc1p.