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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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(A–C) Effect of low extracellular K+ on the cells:medium 36Cl− ratio of cells grown in low Cl− medium. Cells (sul1 in BY4741 background) were grown 16 h in LCAPG, 2% glucose, and 36Cl− (total of 8 μM Cl−). The medium initially contained either 1 mM K+ (normal APG) or 0.3 mM K+. After aerobic growth at 30°C for 16 h, cells were centrifuged, washed once in cold water, and the [K+] (B) and [36Cl−] (C) distribution ratios were measured. The final extracellular K+ concentrations were 0.45 mM and 0.06 mM, respectively, for the media that had initially contained 1 mM and 0.3 mM K+. The total cellular K+ contents were the same in the two media (A). (D) Effect of low extracellular K+ on the cells:medium 36Cl− ratio of cells following a 2-h incubation. Cells (sul1) were grown 16 h in LCAPG medium containing 2 mM K+ and then incubated 2 h in fresh LCAPG medium containing 8 μM 36Cl− and either 0.1 mM or 1 mM K+. The media were buffered either at initial pH 4.5 with 5 mM Na-citrate or initial pH 7.6 with K-citrate, 10 mM bistris, 20 mM tris.
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fig2: (A–C) Effect of low extracellular K+ on the cells:medium 36Cl− ratio of cells grown in low Cl− medium. Cells (sul1 in BY4741 background) were grown 16 h in LCAPG, 2% glucose, and 36Cl− (total of 8 μM Cl−). The medium initially contained either 1 mM K+ (normal APG) or 0.3 mM K+. After aerobic growth at 30°C for 16 h, cells were centrifuged, washed once in cold water, and the [K+] (B) and [36Cl−] (C) distribution ratios were measured. The final extracellular K+ concentrations were 0.45 mM and 0.06 mM, respectively, for the media that had initially contained 1 mM and 0.3 mM K+. The total cellular K+ contents were the same in the two media (A). (D) Effect of low extracellular K+ on the cells:medium 36Cl− ratio of cells following a 2-h incubation. Cells (sul1) were grown 16 h in LCAPG medium containing 2 mM K+ and then incubated 2 h in fresh LCAPG medium containing 8 μM 36Cl− and either 0.1 mM or 1 mM K+. The media were buffered either at initial pH 4.5 with 5 mM Na-citrate or initial pH 7.6 with K-citrate, 10 mM bistris, 20 mM tris.

Mentions: Fig. 2 C shows the cells:medium [36Cl−] ratio in cultures grown 16 h in LCAPG medium containing 5 μM 36Cl− (plus ∼5 μM nonradioactive Cl−) and (initially) either 0.3 mM or 1 mM K+. (We found, in agreement with other laboratories [Rodriguez-Navarro and Ramos, 1984; Hess et al., 2006], that similar experiments could not be performed in YNB medium because of the toxicity of NH4+ in low K+ media). In medium initially containing 0.3 mM K+, the final cell density is only about half that in 1 mM K+, but the cellular K+ (∼150 mEq/liter cell water) is the same in both media (Fig. 2 A). The cells:medium 36Cl− ratio is lower in cells grown in low K+ medium. In four measurements on two separate cell preparations, the cells:medium 36Cl− ratio in media with 0.06 mM K+ is less than half that in the medium with 0.45 mM K+ (Fig. 2 C).


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)

(A–C) Effect of low extracellular K+ on the cells:medium 36Cl− ratio of cells grown in low Cl− medium. Cells (sul1 in BY4741 background) were grown 16 h in LCAPG, 2% glucose, and 36Cl− (total of 8 μM Cl−). The medium initially contained either 1 mM K+ (normal APG) or 0.3 mM K+. After aerobic growth at 30°C for 16 h, cells were centrifuged, washed once in cold water, and the [K+] (B) and [36Cl−] (C) distribution ratios were measured. The final extracellular K+ concentrations were 0.45 mM and 0.06 mM, respectively, for the media that had initially contained 1 mM and 0.3 mM K+. The total cellular K+ contents were the same in the two media (A). (D) Effect of low extracellular K+ on the cells:medium 36Cl− ratio of cells following a 2-h incubation. Cells (sul1) were grown 16 h in LCAPG medium containing 2 mM K+ and then incubated 2 h in fresh LCAPG medium containing 8 μM 36Cl− and either 0.1 mM or 1 mM K+. The media were buffered either at initial pH 4.5 with 5 mM Na-citrate or initial pH 7.6 with K-citrate, 10 mM bistris, 20 mM tris.
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Related In: Results  -  Collection

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fig2: (A–C) Effect of low extracellular K+ on the cells:medium 36Cl− ratio of cells grown in low Cl− medium. Cells (sul1 in BY4741 background) were grown 16 h in LCAPG, 2% glucose, and 36Cl− (total of 8 μM Cl−). The medium initially contained either 1 mM K+ (normal APG) or 0.3 mM K+. After aerobic growth at 30°C for 16 h, cells were centrifuged, washed once in cold water, and the [K+] (B) and [36Cl−] (C) distribution ratios were measured. The final extracellular K+ concentrations were 0.45 mM and 0.06 mM, respectively, for the media that had initially contained 1 mM and 0.3 mM K+. The total cellular K+ contents were the same in the two media (A). (D) Effect of low extracellular K+ on the cells:medium 36Cl− ratio of cells following a 2-h incubation. Cells (sul1) were grown 16 h in LCAPG medium containing 2 mM K+ and then incubated 2 h in fresh LCAPG medium containing 8 μM 36Cl− and either 0.1 mM or 1 mM K+. The media were buffered either at initial pH 4.5 with 5 mM Na-citrate or initial pH 7.6 with K-citrate, 10 mM bistris, 20 mM tris.
Mentions: Fig. 2 C shows the cells:medium [36Cl−] ratio in cultures grown 16 h in LCAPG medium containing 5 μM 36Cl− (plus ∼5 μM nonradioactive Cl−) and (initially) either 0.3 mM or 1 mM K+. (We found, in agreement with other laboratories [Rodriguez-Navarro and Ramos, 1984; Hess et al., 2006], that similar experiments could not be performed in YNB medium because of the toxicity of NH4+ in low K+ media). In medium initially containing 0.3 mM K+, the final cell density is only about half that in 1 mM K+, but the cellular K+ (∼150 mEq/liter cell water) is the same in both media (Fig. 2 A). The cells:medium 36Cl− ratio is lower in cells grown in low K+ medium. In four measurements on two separate cell preparations, the cells:medium 36Cl− ratio in media with 0.06 mM K+ is less than half that in the medium with 0.45 mM K+ (Fig. 2 C).

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