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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) Initial 36Cl− influx as a function of concentration. Cells (sul1) were grown overnight in LCAPG and then incubated in fresh medium at either pH 4.0 or pH 7. Media were buffered with 5 mM K-citrate (initial pH 4.5, final pH 4) or 5 mM K-citrate, 10 mM bistris, 10 mM Tris (initial pH 7.6, final pH 6.8). The initial (1 min) influx of 36Cl− was measured at 30°C in LCAPG with the indicated concentration of total Cl− (0.005 mM nonradioactive Cl− plus various added amounts of 36Cl−). The solid curves through the data are rectangular hyperbolae with K1/2 = 0.018 mM, JMax = 0.092 mEq/L cell water-min (pH 4), and K1/2 = 0.30 mM, JMax = 0.261 mEq/L cell water-min (pH 7). (B) Very slow efflux of 36Cl− following resuspension of 36Cl−-loaded cells (sul1) in LCAPG medium. Overnight culture of sul1 cells in LCAPG was incubated in fresh LCAPG, 5 mM citrate, pH 4.5, and 8 μM 36Cl−. After 2 h at 30°C, cells were centrifuged and resuspended in fresh medium containing no 36Cl− and incubated at 30°C. (C) Rapid efflux of 36Cl− following addition of extracellular Cl−. Overnight culture of sul1 cells in LCAPG was prepared and incubated as in B, except that 2 mM KCl was added at the arrow. Vertical axis is logarithmic; the curve through the data represents the sum of two exponentials with 70% of the tracer efflux with rate constant 0.41/min and the remaining 30% with rate constant 0.050/min.
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fig5: (A) Initial 36Cl− influx as a function of concentration. Cells (sul1) were grown overnight in LCAPG and then incubated in fresh medium at either pH 4.0 or pH 7. Media were buffered with 5 mM K-citrate (initial pH 4.5, final pH 4) or 5 mM K-citrate, 10 mM bistris, 10 mM Tris (initial pH 7.6, final pH 6.8). The initial (1 min) influx of 36Cl− was measured at 30°C in LCAPG with the indicated concentration of total Cl− (0.005 mM nonradioactive Cl− plus various added amounts of 36Cl−). The solid curves through the data are rectangular hyperbolae with K1/2 = 0.018 mM, JMax = 0.092 mEq/L cell water-min (pH 4), and K1/2 = 0.30 mM, JMax = 0.261 mEq/L cell water-min (pH 7). (B) Very slow efflux of 36Cl− following resuspension of 36Cl−-loaded cells (sul1) in LCAPG medium. Overnight culture of sul1 cells in LCAPG was incubated in fresh LCAPG, 5 mM citrate, pH 4.5, and 8 μM 36Cl−. After 2 h at 30°C, cells were centrifuged and resuspended in fresh medium containing no 36Cl− and incubated at 30°C. (C) Rapid efflux of 36Cl− following addition of extracellular Cl−. Overnight culture of sul1 cells in LCAPG was prepared and incubated as in B, except that 2 mM KCl was added at the arrow. Vertical axis is logarithmic; the curve through the data represents the sum of two exponentials with 70% of the tracer efflux with rate constant 0.41/min and the remaining 30% with rate constant 0.050/min.

Mentions: Fig. 5 shows that cells grown in a low Cl− medium have a high affinity pathway for initial 36Cl− influx, and that the apparent Cl− affinity is increased by low extracellular pH. Cells (sul1) were grown in LCAPG medium, and the initial 36Cl− influx was measured in LCAPG plus various concentrations of 36Cl−. At extracellular pH 4, the influx vs. extracellular Cl− is described reasonably well by a hyperbola, with K1/2 ∼0.018 mM, similar to that observed previously in low Cl− YNB medium (Jennings et al., 2007). At extracellular pH 7, the K1/2 is over 10-fold higher than at pH 4.0 (Fig. 5 A). This finding suggests that H+ is cotransported with Cl−. However, the Vmax of the influx is also higher at pH 7.0 than at pH 4, indicating that the effects of pH on the kinetics of Cl− influx are not simple. Irrespective of the detailed kinetics, the 36Cl− influx at very low extracellular [Cl−] is lower at pH 7.0 than at pH 4. This is consistent with the fact that the steady-state distribution ratio is also lower at pH 7.0 (Fig. 2 D and Fig. 3 A).


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) Initial 36Cl− influx as a function of concentration. Cells (sul1) were grown overnight in LCAPG and then incubated in fresh medium at either pH 4.0 or pH 7. Media were buffered with 5 mM K-citrate (initial pH 4.5, final pH 4) or 5 mM K-citrate, 10 mM bistris, 10 mM Tris (initial pH 7.6, final pH 6.8). The initial (1 min) influx of 36Cl− was measured at 30°C in LCAPG with the indicated concentration of total Cl− (0.005 mM nonradioactive Cl− plus various added amounts of 36Cl−). The solid curves through the data are rectangular hyperbolae with K1/2 = 0.018 mM, JMax = 0.092 mEq/L cell water-min (pH 4), and K1/2 = 0.30 mM, JMax = 0.261 mEq/L cell water-min (pH 7). (B) Very slow efflux of 36Cl− following resuspension of 36Cl−-loaded cells (sul1) in LCAPG medium. Overnight culture of sul1 cells in LCAPG was incubated in fresh LCAPG, 5 mM citrate, pH 4.5, and 8 μM 36Cl−. After 2 h at 30°C, cells were centrifuged and resuspended in fresh medium containing no 36Cl− and incubated at 30°C. (C) Rapid efflux of 36Cl− following addition of extracellular Cl−. Overnight culture of sul1 cells in LCAPG was prepared and incubated as in B, except that 2 mM KCl was added at the arrow. Vertical axis is logarithmic; the curve through the data represents the sum of two exponentials with 70% of the tracer efflux with rate constant 0.41/min and the remaining 30% with rate constant 0.050/min.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279172&req=5

fig5: (A) Initial 36Cl− influx as a function of concentration. Cells (sul1) were grown overnight in LCAPG and then incubated in fresh medium at either pH 4.0 or pH 7. Media were buffered with 5 mM K-citrate (initial pH 4.5, final pH 4) or 5 mM K-citrate, 10 mM bistris, 10 mM Tris (initial pH 7.6, final pH 6.8). The initial (1 min) influx of 36Cl− was measured at 30°C in LCAPG with the indicated concentration of total Cl− (0.005 mM nonradioactive Cl− plus various added amounts of 36Cl−). The solid curves through the data are rectangular hyperbolae with K1/2 = 0.018 mM, JMax = 0.092 mEq/L cell water-min (pH 4), and K1/2 = 0.30 mM, JMax = 0.261 mEq/L cell water-min (pH 7). (B) Very slow efflux of 36Cl− following resuspension of 36Cl−-loaded cells (sul1) in LCAPG medium. Overnight culture of sul1 cells in LCAPG was incubated in fresh LCAPG, 5 mM citrate, pH 4.5, and 8 μM 36Cl−. After 2 h at 30°C, cells were centrifuged and resuspended in fresh medium containing no 36Cl− and incubated at 30°C. (C) Rapid efflux of 36Cl− following addition of extracellular Cl−. Overnight culture of sul1 cells in LCAPG was prepared and incubated as in B, except that 2 mM KCl was added at the arrow. Vertical axis is logarithmic; the curve through the data represents the sum of two exponentials with 70% of the tracer efflux with rate constant 0.41/min and the remaining 30% with rate constant 0.050/min.
Mentions: Fig. 5 shows that cells grown in a low Cl− medium have a high affinity pathway for initial 36Cl− influx, and that the apparent Cl− affinity is increased by low extracellular pH. Cells (sul1) were grown in LCAPG medium, and the initial 36Cl− influx was measured in LCAPG plus various concentrations of 36Cl−. At extracellular pH 4, the influx vs. extracellular Cl− is described reasonably well by a hyperbola, with K1/2 ∼0.018 mM, similar to that observed previously in low Cl− YNB medium (Jennings et al., 2007). At extracellular pH 7, the K1/2 is over 10-fold higher than at pH 4.0 (Fig. 5 A). This finding suggests that H+ is cotransported with Cl−. However, the Vmax of the influx is also higher at pH 7.0 than at pH 4, indicating that the effects of pH on the kinetics of Cl− influx are not simple. Irrespective of the detailed kinetics, the 36Cl− influx at very low extracellular [Cl−] is lower at pH 7.0 than at pH 4. This is consistent with the fact that the steady-state distribution ratio is also lower at pH 7.0 (Fig. 2 D and Fig. 3 A).

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