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

Abstract

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 36Cl− influx is a saturable function of extracellular [36Cl−] with K1/2 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 36Cl− for either Cl− or Br− far more rapidly than SO4=, phosphate, formate, HCO3−, or NO3−. 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.