Chloride Homeostasis in Saccharomyces cerevisiae: High Affinity Influx, V-ATPase-dependent Sequestration, and Identification of a Candidate Cl(−) Sensor

Chloride homeostasis in Saccharomyces cerevisiae has been characterized with the goal of identifying new Cl(−) transport and regulatory pathways. Steady-state cellular Cl(−) contents (∼0.2 mEq/liter cell water) differ by less than threefold in yeast grown in media containing 0.003–5 mM Cl(−). Therefore, yeast have a potent mechanism for maintaining constant cellular Cl(−) over a wide range of extracellular Cl(−). The cell water:medium [Cl(−)] ratio is >20 in media containing 0.01 mM Cl(−) and results in part from sequestration of Cl(−) in organelles, as shown by the effect of deleting genes involved in vacuolar acidification. Organellar sequestration cannot account entirely for the Cl(−) accumulation, however, because the cell water:medium [Cl(−)] ratio in low Cl(−) medium is ∼10 at extracellular pH 4.0 even in vma1 yeast, which lack the vacuolar H(+)-ATPase. Cellular Cl(−) accumulation is ATP dependent in both wild type and vma1 strains. The initial (36)Cl(−) influx is a saturable function of extracellular [(36)Cl(−)] with K(1/2) of 0.02 mM at pH 4.0 and >0.2 mM at pH 7, indicating the presence of a high affinity Cl(−) transporter in the plasma membrane. The transporter can exchange (36)Cl(−) for either Cl(−) or Br(−) far more rapidly than SO(4) (=), phosphate, formate, HCO(3) (−), or NO(3) (−). High affinity Cl(−) influx is not affected by deletion of any of several genes for possible Cl(−) transporters. The high affinity Cl(−) transporter is activated over a period of ∼45 min after shifting cells from high-Cl(−) to low-Cl(−) media. Deletion of ORF YHL008c (formate-nitrite transporter family) strongly reduces the rate of activation of the flux. Therefore, Yhl008cp may be part of a Cl(−)-sensing mechanism that activates the high affinity transporter in a low Cl(−) medium. This is the first example of a biological system that can regulate cellular Cl(−) at concentrations far below 1 mM.