A Homolog of Voltage-Gated Ca 2+ Channels Stimulated by Depletion of Secretory Ca 2+ in Yeast

Abstract

ABSTRACT In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca 2+ ) from secretory organelles. CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca 2+ 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 cerevisiae employs a CCE-like mechanism to refill Ca 2+ stores within the secretory pathway. Mutants lacking Pmr1p, a conserved Ca 2+ pump in the secretory pathway, exhibit higher rates of Ca 2+ influx relative to wild-type cells due to the stimulation of a high-affinity Ca 2+ uptake system. Stimulation of this Ca 2+ uptake system was blocked in pmr1 mutants by expression of mammalian SERCA pumps. The high-affinity Ca 2+ uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca 2+ transporters that competed with Pmr1p for substrate. A screen for yeast mutants specifically defective in the high-affinity Ca 2+ uptake system revealed two genes, CCH1 and MID1 , previously implicated in Ca 2+ 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 Ca 2+ are supplied to Pmr1p to sustain secretion and growth. Expression of Cch1p and Mid1p was not affected in pmr1 mutants. 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 Ca 2+ channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.