A Homolog of Mammalian, Voltage-Gated Calcium Channels Mediates Yeast Pheromone-Stimulated Ca²⁺ Uptake and Exacerbates the cdc1(Ts) Growth Defect

Abstract

Previous studies attributed the yeast (Saccharomyces cerevisiae) cdc1(Ts) growth defect to loss of an Mn 2؉ -dependent function. In this report we show that cdc1(Ts) temperature-sensitive growth is also associated with an increase in cytosolic Ca 2؉ . We identified two recessive suppressors of the cdc1(Ts) temperature-sensitive growth which block Ca 2؉ uptake and accumulation, suggesting that cytosolic Ca 2؉ exacerbates or is responsible for the cdc1(Ts) growth defect. One of the cdc1(Ts) suppressors is identical to a gene, MID1, recently implicated in mating pheromone-stimulated Ca 2؉ uptake. The gene (CCH1) corresponding to the second suppressor encodes a protein that bears significant sequence similarity to the pore-forming subunit (␣1) of plasma membrane, voltage-gated Ca 2؉ channels from higher eukaryotes. Strains lacking Mid1 or Cch1 protein exhibit a defect in pheromone-induced Ca 2؉ uptake and consequently lose viability upon mating arrest. The mid1⌬ and cch1⌬ mutants also display reduced tolerance to monovalent cations such as Li ؉ , suggesting a role for Ca 2؉ uptake in the calcineurin-dependent ion stress response. Finally, mid1⌬ cch1⌬ double mutants are, by both physiological and genetic criteria, identical to single mutants. These and other results suggest Mid1 and Cch1 are components of a yeast Ca 2؉ channel that may mediate Ca 2؉ uptake in response to mating pheromone, salt stress, and Mn 2؉ depletion.In eukaryotic cells, cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) fluctuates transiently to regulate such diverse processes as neurotransmitter release, muscle contraction, and T-cell activation (9, 31). Intracellular Ca 2ϩ levels are tightly regulated by numerous channels, pumps, and antiporters, such that [Ca 2ϩ ] i is normally maintained at an extremely low concentration (ϳ 100 nM) despite a 10,000-fold concentration difference across the plasma membrane (9). Eukaryotic cells utilize this gradient to generate Ca 2ϩ spikes by stimulating the opening of Ca 2ϩ channels and allowing Ca 2ϩ influx down the concentration gradient (9, 31). Among the best characterized of these entry pathways are the voltage-dependent Ca 2ϩ channels of the plasma membrane which open in response to membrane depolarization (3,9). Ca 2ϩ has been implicated in numerous processes of the budding yeast Saccharomyces cerevisiae (8,16,20,37), including stress-induced expression of ion transporter genes, bud formation, and viability upon pheromone-induced arrest. However, there is compelling evidence for a role for Ca 2ϩ in only the last of these processes (7,15,16,22). Haploid yeast cells exist as two mating types, a and ␣, that secrete mating pheromones a-and ␣-factor, respectively. In the presence of ␣-factor, a cells undergo a complex developmental process in preparation for mating. For example, pheromone-treated cells accumulate high levels of intracellular Ca 2ϩ , arrest in G 1 , and form a mating projection or shmoo (16). Cells arrested in medium lacking Ca 2ϩ lose viability within 5 h after pheromone treatment, and th...

An Osmotically Induced Cytosolic Ca2+ Transient Activates Calcineurin Signaling to Mediate Ion Homeostasis and Salt Tolerance of Saccharomyces cerevisiae

“…Net 45 (Fig. 1, G and H (28). Furthermore, cch1⌬ and mid1⌬ cells are Na ϩ but not hyperosmotic sensitive (mid1⌬ data not shown), and Na ϩ and Li ϩ sensitivities of these cells are exacerbated by EGTA or attenuated by Ca 2ϩ supplementation to the media (Fig.…”

“…4 and Ref. 28). ENA1 expression is induced by inclusion of NaCl or CaCl 2 into the medium and the induction is greater in wild type than in cch1⌬ cells (Fig.…”

“…Measurement of 45 Ca 2ϩ Accumulation-Procedures for determining Ca 2ϩ uptake into cells were as described, with modifications (17,28). Cells in the exponential growth phase (YPD medium) were inoculated into fresh medium (OD 600 ϳ0.1) that contained 1 Ci/ml of 45 Ca 2ϩ ( 45 CaCl 2 , ICN Biomedicals Inc., Costa Mesa, CA).…”

“…Pheromone-induced Ca ext 2ϩ influx is mediated by Cch1p and Mid1p, which are plasma membrane proteins that by genetic criteria function as a single Ca 2ϩ uptake system (26 -29). Cch1p contains four repeated peptide domains, each with sequence similarity to the ␣ 1 pore-forming subunit of L-type voltage-gated Ca 2ϩ channels (28). Mid1p is an N-glycosylated, integral plasma membrane protein inferred to be a Ca 2ϩ -permeable stretch-activated nonselective cation channel (26,28).…”

Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

“…In yeast, Cch1 is localized in the plasma membrane, where it forms a complex with Mid1. Both proteins have been shown to be required for the uptake of extracellular Ca 2ϩ in cells responding to mating pheromones (13,(21)(22)(23)(24).…”