A Homolog of Voltage-Gated Ca2+Channels Stimulated by Depletion of Secretory Ca2+ in Yeast

Abstract

In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca²⁺) from secretory organelles. CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca²⁺ concentrations in the cytoplasm. The mechanism of CCE regulation is not understood because few of its essential components have been identified. We show here for the first time that the budding yeast Saccharomyces cerevisiaeemploys a CCE-like mechanism to refill Ca²⁺ stores within the secretory pathway. Mutants lacking Pmr1p, a conserved Ca²⁺ pump in the secretory pathway, exhibit higher rates of Ca²⁺ influx relative to wild-type cells due to the stimulation of a high-affinity Ca²⁺ uptake system. Stimulation of this Ca²⁺ uptake system was blocked inpmr1 mutants by expression of mammalian SERCA pumps. The high-affinity Ca²⁺ uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca²⁺ transporters that competed with Pmr1p for substrate. A screen for yeast mutants specifically defective in the high-affinity Ca²⁺ uptake system revealed two genes, CCH1 and MID1, previously implicated in Ca²⁺ influx in response to mating pheromones. Cch1p and Mid1p were localized to the plasma membrane, coimmunoprecipitated from solubilized membranes, and shown to function together within a single pathway that ensures that adequate levels of Ca²⁺ are supplied to Pmr1p to sustain secretion and growth. Expression of Cch1p and Mid1p was not affected in pmr1mutants. The evidence supports the hypothesis that yeast maintains a homeostatic mechanism related to CCE in mammalian cells. The homology between Cch1p and the catalytic subunit of voltage-gated Ca²⁺ channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.