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Chloride homeostasis in Saccharomyces cerevisiae: high affinity influx, V-ATPase-dependent sequestration, and identification of a candidate Cl- sensor.

Jennings ML, Cui J - J. Gen. Physiol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. JenningsMichaelL@uams.edu

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 ( approximately 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 approximately 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 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 approximately 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.

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Activation of high affinity Cl− transport following shift from 40 mM Cl− to low Cl− medium. (A) Cells (sul1) were grown overnight in LCAPG with no added Cl− (∼0.005 mM; •) or 40 mM added NaCl (▴), and then washed twice in cold LCAPG, resuspended in LCAPG, pH 4.5, 30°C, with 8 μM Na36Cl, and the accumulation of 36Cl− measured over the next 50 min. (B) Cells (sul1) were grown in APG + 40 mM NaCl, washed twice in cold LCAPG, and suspended at t = 0 in LCAPG, pH 4.5, 30°C. At the indicated times, 8 μM Na36Cl was added and the tracer influx measured for 1 min. Data are from two separate cell preparations.
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fig7: Activation of high affinity Cl− transport following shift from 40 mM Cl− to low Cl− medium. (A) Cells (sul1) were grown overnight in LCAPG with no added Cl− (∼0.005 mM; •) or 40 mM added NaCl (▴), and then washed twice in cold LCAPG, resuspended in LCAPG, pH 4.5, 30°C, with 8 μM Na36Cl, and the accumulation of 36Cl− measured over the next 50 min. (B) Cells (sul1) were grown in APG + 40 mM NaCl, washed twice in cold LCAPG, and suspended at t = 0 in LCAPG, pH 4.5, 30°C. At the indicated times, 8 μM Na36Cl was added and the tracer influx measured for 1 min. Data are from two separate cell preparations.

Mentions: In media containing >1 mM Cl−, S. cerevisiae should have no need for a high affinity Cl− transporter, because the cells exclude rather than accumulate Cl− (Fig. 1). To determine whether the high affinity Cl− influx is regulated, cells (sul1) were grown in either LCAPG medium or APG containing 40 mM KCl. Cells were then washed in cold LCAPG medium to remove extracellular Cl−, resuspended in LCAPG plus 8 μM 36Cl− at 30°C, and the accumulation of 36Cl− measured over the next 30 min. As shown in Fig. 7 A, the initial 36Cl− influx is lower in cells grown in 40 mM Cl−, but the final accumulation of 36Cl− is nearly independent of whether the cells had been grown in high or low Cl−. This suggests that the high affinity Cl− influx activates rapidly following exposure of cells to low Cl− medium.


Chloride homeostasis in Saccharomyces cerevisiae: high affinity influx, V-ATPase-dependent sequestration, and identification of a candidate Cl- sensor.

Jennings ML, Cui J - J. Gen. Physiol. (2008)

Activation of high affinity Cl− transport following shift from 40 mM Cl− to low Cl− medium. (A) Cells (sul1) were grown overnight in LCAPG with no added Cl− (∼0.005 mM; •) or 40 mM added NaCl (▴), and then washed twice in cold LCAPG, resuspended in LCAPG, pH 4.5, 30°C, with 8 μM Na36Cl, and the accumulation of 36Cl− measured over the next 50 min. (B) Cells (sul1) were grown in APG + 40 mM NaCl, washed twice in cold LCAPG, and suspended at t = 0 in LCAPG, pH 4.5, 30°C. At the indicated times, 8 μM Na36Cl was added and the tracer influx measured for 1 min. Data are from two separate cell preparations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279172&req=5

fig7: Activation of high affinity Cl− transport following shift from 40 mM Cl− to low Cl− medium. (A) Cells (sul1) were grown overnight in LCAPG with no added Cl− (∼0.005 mM; •) or 40 mM added NaCl (▴), and then washed twice in cold LCAPG, resuspended in LCAPG, pH 4.5, 30°C, with 8 μM Na36Cl, and the accumulation of 36Cl− measured over the next 50 min. (B) Cells (sul1) were grown in APG + 40 mM NaCl, washed twice in cold LCAPG, and suspended at t = 0 in LCAPG, pH 4.5, 30°C. At the indicated times, 8 μM Na36Cl was added and the tracer influx measured for 1 min. Data are from two separate cell preparations.
Mentions: In media containing >1 mM Cl−, S. cerevisiae should have no need for a high affinity Cl− transporter, because the cells exclude rather than accumulate Cl− (Fig. 1). To determine whether the high affinity Cl− influx is regulated, cells (sul1) were grown in either LCAPG medium or APG containing 40 mM KCl. Cells were then washed in cold LCAPG medium to remove extracellular Cl−, resuspended in LCAPG plus 8 μM 36Cl− at 30°C, and the accumulation of 36Cl− measured over the next 30 min. As shown in Fig. 7 A, the initial 36Cl− influx is lower in cells grown in 40 mM Cl−, but the final accumulation of 36Cl− is nearly independent of whether the cells had been grown in high or low Cl−. This suggests that the high affinity Cl− influx activates rapidly following exposure of cells to low Cl− medium.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. JenningsMichaelL@uams.edu

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 ( approximately 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 approximately 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 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 approximately 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.

Show MeSH