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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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Effect of low K+ and Cl− concentrations on growth of sul1, gef1, and vma1 strains of S. cerevisiae. Cells from each strain were suspended at initial A590 of 0.1 and incubated aerobically at 30°C with rotary shaking for 24 h. The medium was derived from LCAPG, with increasing amounts of Cl− added as arginine Cl−. The initial concentrations of Cl− in the media were ∼0.005, 0.035, 0.10, 0.30, 1.0, and 3.0 mM (left to right) in each group of six bars. The initial concentrations of K+ (added as KH2PO4) were 0.1, 0.3, 1.0, and 3.0 mM.
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fig4: Effect of low K+ and Cl− concentrations on growth of sul1, gef1, and vma1 strains of S. cerevisiae. Cells from each strain were suspended at initial A590 of 0.1 and incubated aerobically at 30°C with rotary shaking for 24 h. The medium was derived from LCAPG, with increasing amounts of Cl− added as arginine Cl−. The initial concentrations of Cl− in the media were ∼0.005, 0.035, 0.10, 0.30, 1.0, and 3.0 mM (left to right) in each group of six bars. The initial concentrations of K+ (added as KH2PO4) were 0.1, 0.3, 1.0, and 3.0 mM.

Mentions: Despite the fact that gef1 and vma1 cells have significantly lower cellular Cl− in LCAPG than other strains, there are at most very minor effects of low Cl− on cell growth. Fig. 4 shows the final cell density of sul1, gef1, and vma1 strains grown 24 h in APG media (final pH ∼3.5) having various concentrations of Cl− and K+. Each of the four groups of six bars represents cells grown at a given initial K+ concentration with 0.005, 0.03, 0.1, 0.3, 1, and 3 mM Cl−. As in Fig. 2, a low K+ concentration results in a lower final cell density, and the vma1 strain, as expected (Kane, 2006), grows to a lower density then the other strains. At a given K+ concentration, varying the Cl− has little effect on final cell density. In the vma1 strain the data suggest an effect of Cl− on final cell density, but there is no effect of Cl− on growth rate of exponential cultures (unpublished data). We conclude that there are at most very small effects of Cl− on growth, even in the vma1 strain, which has a lower total cellular Cl− than the other strains.


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)

Effect of low K+ and Cl− concentrations on growth of sul1, gef1, and vma1 strains of S. cerevisiae. Cells from each strain were suspended at initial A590 of 0.1 and incubated aerobically at 30°C with rotary shaking for 24 h. The medium was derived from LCAPG, with increasing amounts of Cl− added as arginine Cl−. The initial concentrations of Cl− in the media were ∼0.005, 0.035, 0.10, 0.30, 1.0, and 3.0 mM (left to right) in each group of six bars. The initial concentrations of K+ (added as KH2PO4) were 0.1, 0.3, 1.0, and 3.0 mM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279172&req=5

fig4: Effect of low K+ and Cl− concentrations on growth of sul1, gef1, and vma1 strains of S. cerevisiae. Cells from each strain were suspended at initial A590 of 0.1 and incubated aerobically at 30°C with rotary shaking for 24 h. The medium was derived from LCAPG, with increasing amounts of Cl− added as arginine Cl−. The initial concentrations of Cl− in the media were ∼0.005, 0.035, 0.10, 0.30, 1.0, and 3.0 mM (left to right) in each group of six bars. The initial concentrations of K+ (added as KH2PO4) were 0.1, 0.3, 1.0, and 3.0 mM.
Mentions: Despite the fact that gef1 and vma1 cells have significantly lower cellular Cl− in LCAPG than other strains, there are at most very minor effects of low Cl− on cell growth. Fig. 4 shows the final cell density of sul1, gef1, and vma1 strains grown 24 h in APG media (final pH ∼3.5) having various concentrations of Cl− and K+. Each of the four groups of six bars represents cells grown at a given initial K+ concentration with 0.005, 0.03, 0.1, 0.3, 1, and 3 mM Cl−. As in Fig. 2, a low K+ concentration results in a lower final cell density, and the vma1 strain, as expected (Kane, 2006), grows to a lower density then the other strains. At a given K+ concentration, varying the Cl− has little effect on final cell density. In the vma1 strain the data suggest an effect of Cl− on final cell density, but there is no effect of Cl− on growth rate of exponential cultures (unpublished data). We conclude that there are at most very small effects of Cl− on growth, even in the vma1 strain, which has a lower total cellular Cl− than the other strains.

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
Related in: MedlinePlus