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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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Cl− contents (A) and distribution ratios (B) for S. cerevisiae grown in media containing varying concentrations of Cl−. Solid symbols: cells were washed and suspended at A600 of 0.05 in media in which all but ∼5 μM Cl− had been replaced by SO4=, and Cl− was added back as Na36Cl (0.001–0.3 mM), plus, for higher concentrations, 0.7–100 mM NH4Cl, KCl, or NaCl. Suspensions were incubated aerobically 16–20 h at 30°C, and the cellular 36Cl− contents were determined as described in Materials and methods. Unless otherwise indicated the final extracellular pH was 3.5 ± 0.5. Open symbols: cells were grown in APG, washed, and incubated 2 h at 30°C in APG containing various concentrations of 36Cl− before determining cellular 36Cl− contents. The dashed line in B is the distribution ratio predicted from a Nernst distribution, with plasma membrane potential of −120 mV. The solid curves through the data have no theoretical significance. ♦: strain BY4741, YNB, 25 mM K-citrate buffer, final pH 4.4. ▾, ▴: strain BY4741, YNB. •: strain FKY282, YNB. □: SUL1 deletant (BY4741 background), 2 h, APG.
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fig1: Cl− contents (A) and distribution ratios (B) for S. cerevisiae grown in media containing varying concentrations of Cl−. Solid symbols: cells were washed and suspended at A600 of 0.05 in media in which all but ∼5 μM Cl− had been replaced by SO4=, and Cl− was added back as Na36Cl (0.001–0.3 mM), plus, for higher concentrations, 0.7–100 mM NH4Cl, KCl, or NaCl. Suspensions were incubated aerobically 16–20 h at 30°C, and the cellular 36Cl− contents were determined as described in Materials and methods. Unless otherwise indicated the final extracellular pH was 3.5 ± 0.5. Open symbols: cells were grown in APG, washed, and incubated 2 h at 30°C in APG containing various concentrations of 36Cl− before determining cellular 36Cl− contents. The dashed line in B is the distribution ratio predicted from a Nernst distribution, with plasma membrane potential of −120 mV. The solid curves through the data have no theoretical significance. ♦: strain BY4741, YNB, 25 mM K-citrate buffer, final pH 4.4. ▾, ▴: strain BY4741, YNB. •: strain FKY282, YNB. □: SUL1 deletant (BY4741 background), 2 h, APG.

Mentions: Fig. 1 A shows the cellular Cl− contents (mEq/liter cell water) as a function of extracellular [Cl−] in overnight cultures of haploid yeast strains grown aerobically in media containing 0.003–100 mM Cl−. At Cl− concentrations below 4 mM, the Cl− normally present in YNB or APG was replaced by SO4=. At higher concentrations, Cl− was added as NH4Cl, KCl, or NaCl, with indistinguishable results.


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)

Cl− contents (A) and distribution ratios (B) for S. cerevisiae grown in media containing varying concentrations of Cl−. Solid symbols: cells were washed and suspended at A600 of 0.05 in media in which all but ∼5 μM Cl− had been replaced by SO4=, and Cl− was added back as Na36Cl (0.001–0.3 mM), plus, for higher concentrations, 0.7–100 mM NH4Cl, KCl, or NaCl. Suspensions were incubated aerobically 16–20 h at 30°C, and the cellular 36Cl− contents were determined as described in Materials and methods. Unless otherwise indicated the final extracellular pH was 3.5 ± 0.5. Open symbols: cells were grown in APG, washed, and incubated 2 h at 30°C in APG containing various concentrations of 36Cl− before determining cellular 36Cl− contents. The dashed line in B is the distribution ratio predicted from a Nernst distribution, with plasma membrane potential of −120 mV. The solid curves through the data have no theoretical significance. ♦: strain BY4741, YNB, 25 mM K-citrate buffer, final pH 4.4. ▾, ▴: strain BY4741, YNB. •: strain FKY282, YNB. □: SUL1 deletant (BY4741 background), 2 h, APG.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279172&req=5

fig1: Cl− contents (A) and distribution ratios (B) for S. cerevisiae grown in media containing varying concentrations of Cl−. Solid symbols: cells were washed and suspended at A600 of 0.05 in media in which all but ∼5 μM Cl− had been replaced by SO4=, and Cl− was added back as Na36Cl (0.001–0.3 mM), plus, for higher concentrations, 0.7–100 mM NH4Cl, KCl, or NaCl. Suspensions were incubated aerobically 16–20 h at 30°C, and the cellular 36Cl− contents were determined as described in Materials and methods. Unless otherwise indicated the final extracellular pH was 3.5 ± 0.5. Open symbols: cells were grown in APG, washed, and incubated 2 h at 30°C in APG containing various concentrations of 36Cl− before determining cellular 36Cl− contents. The dashed line in B is the distribution ratio predicted from a Nernst distribution, with plasma membrane potential of −120 mV. The solid curves through the data have no theoretical significance. ♦: strain BY4741, YNB, 25 mM K-citrate buffer, final pH 4.4. ▾, ▴: strain BY4741, YNB. •: strain FKY282, YNB. □: SUL1 deletant (BY4741 background), 2 h, APG.
Mentions: Fig. 1 A shows the cellular Cl− contents (mEq/liter cell water) as a function of extracellular [Cl−] in overnight cultures of haploid yeast strains grown aerobically in media containing 0.003–100 mM Cl−. At Cl− concentrations below 4 mM, the Cl− normally present in YNB or APG was replaced by SO4=. At higher concentrations, Cl− was added as NH4Cl, KCl, or NaCl, with indistinguishable results.

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