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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) Cell water:medium distribution ratio of 36Cl− in various deletant strains. Cells were grown overnight in LCAPG and then incubated 2 h at 30°C in fresh medium plus 8 μM Na36Cl. Cellular 36Cl− contents at the end of the incubation were determined as described in Materials and methods. Media were buffered with 5 mM K-citrate (initial pH 4.5, final pH 4.1; black bars), or 5 mM K-citrate, 10 mM bistris, 20 mM tris (initial pH 7.6, final pH 6.7; gray bars). Strains with deletions of the indicated genes were all in the background of BY4741. Bars labeled yhl, ybr, and ypr refer respectively to strains deleted in YHL008c, YBR235w, and YPR003c. Data represent single determinations except for bars labeled to indicate the number of determinations. Error bars are SEM. (B) Cellular Cl− contents in low Cl− media in paired experiments with control strain sul1. Cells were incubated in LCAPG media containing 8 μM 36Cl−, and cellular Cl contents were determined after sufficient time to reach a steady-state distribution (>1.5 h). Bars represent mean ± SEM of the ratio between cellular [Cl−] in the test strain (nhx1, gef1, or vma1) and control strain sul1 at either pH 4.0 (black bars) or pH 7.0 (gray bars). Results of paired Student t tests (pH 4 and pH 7.0 combined): sul1 vs. nhx1, P < 0.02 (n = 4); sul1 vs. gef1, P < 10−5, n = 12; sul1 vs. vma1, P < 0.002, n = 6. (C) Cells:medium 36Cl− distribution ratio for sul1 and vma1 cells incubated as in A and B but with 0.2 mM DNP or DNP and 20 mM 2-deoxyglucose replacing 2% glucose during the 2 h incubation with 36Cl−. The gray horizontal lines indicate a cells:medium ratio of unity.
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fig3: (A) Cell water:medium distribution ratio of 36Cl− in various deletant strains. Cells were grown overnight in LCAPG and then incubated 2 h at 30°C in fresh medium plus 8 μM Na36Cl. Cellular 36Cl− contents at the end of the incubation were determined as described in Materials and methods. Media were buffered with 5 mM K-citrate (initial pH 4.5, final pH 4.1; black bars), or 5 mM K-citrate, 10 mM bistris, 20 mM tris (initial pH 7.6, final pH 6.7; gray bars). Strains with deletions of the indicated genes were all in the background of BY4741. Bars labeled yhl, ybr, and ypr refer respectively to strains deleted in YHL008c, YBR235w, and YPR003c. Data represent single determinations except for bars labeled to indicate the number of determinations. Error bars are SEM. (B) Cellular Cl− contents in low Cl− media in paired experiments with control strain sul1. Cells were incubated in LCAPG media containing 8 μM 36Cl−, and cellular Cl contents were determined after sufficient time to reach a steady-state distribution (>1.5 h). Bars represent mean ± SEM of the ratio between cellular [Cl−] in the test strain (nhx1, gef1, or vma1) and control strain sul1 at either pH 4.0 (black bars) or pH 7.0 (gray bars). Results of paired Student t tests (pH 4 and pH 7.0 combined): sul1 vs. nhx1, P < 0.02 (n = 4); sul1 vs. gef1, P < 10−5, n = 12; sul1 vs. vma1, P < 0.002, n = 6. (C) Cells:medium 36Cl− distribution ratio for sul1 and vma1 cells incubated as in A and B but with 0.2 mM DNP or DNP and 20 mM 2-deoxyglucose replacing 2% glucose during the 2 h incubation with 36Cl−. The gray horizontal lines indicate a cells:medium ratio of unity.

Mentions: In the experiments in Fig. 3, cells were grown overnight in LCAPG medium without 36Cl−, centrifuged, and resuspended in fresh LCAPG medium, 2% glucose, plus 8 μM Na36Cl. Media were buffered either with 5 mM K-citrate (initial pH 4.5), or with 5 mM K-citrate, 10 mM bistris, 20 mM tris (initial pH 7.6). After 2 h of aerobic incubation at 30°C, 5 ml aliquots of suspension were diluted in 40 ml of ice-cold water, centrifuged, the supernatant aspirated completely, and the cellular 36Cl− determined by scintillation counting.


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) Cell water:medium distribution ratio of 36Cl− in various deletant strains. Cells were grown overnight in LCAPG and then incubated 2 h at 30°C in fresh medium plus 8 μM Na36Cl. Cellular 36Cl− contents at the end of the incubation were determined as described in Materials and methods. Media were buffered with 5 mM K-citrate (initial pH 4.5, final pH 4.1; black bars), or 5 mM K-citrate, 10 mM bistris, 20 mM tris (initial pH 7.6, final pH 6.7; gray bars). Strains with deletions of the indicated genes were all in the background of BY4741. Bars labeled yhl, ybr, and ypr refer respectively to strains deleted in YHL008c, YBR235w, and YPR003c. Data represent single determinations except for bars labeled to indicate the number of determinations. Error bars are SEM. (B) Cellular Cl− contents in low Cl− media in paired experiments with control strain sul1. Cells were incubated in LCAPG media containing 8 μM 36Cl−, and cellular Cl contents were determined after sufficient time to reach a steady-state distribution (>1.5 h). Bars represent mean ± SEM of the ratio between cellular [Cl−] in the test strain (nhx1, gef1, or vma1) and control strain sul1 at either pH 4.0 (black bars) or pH 7.0 (gray bars). Results of paired Student t tests (pH 4 and pH 7.0 combined): sul1 vs. nhx1, P < 0.02 (n = 4); sul1 vs. gef1, P < 10−5, n = 12; sul1 vs. vma1, P < 0.002, n = 6. (C) Cells:medium 36Cl− distribution ratio for sul1 and vma1 cells incubated as in A and B but with 0.2 mM DNP or DNP and 20 mM 2-deoxyglucose replacing 2% glucose during the 2 h incubation with 36Cl−. The gray horizontal lines indicate a cells:medium ratio of unity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279172&req=5

fig3: (A) Cell water:medium distribution ratio of 36Cl− in various deletant strains. Cells were grown overnight in LCAPG and then incubated 2 h at 30°C in fresh medium plus 8 μM Na36Cl. Cellular 36Cl− contents at the end of the incubation were determined as described in Materials and methods. Media were buffered with 5 mM K-citrate (initial pH 4.5, final pH 4.1; black bars), or 5 mM K-citrate, 10 mM bistris, 20 mM tris (initial pH 7.6, final pH 6.7; gray bars). Strains with deletions of the indicated genes were all in the background of BY4741. Bars labeled yhl, ybr, and ypr refer respectively to strains deleted in YHL008c, YBR235w, and YPR003c. Data represent single determinations except for bars labeled to indicate the number of determinations. Error bars are SEM. (B) Cellular Cl− contents in low Cl− media in paired experiments with control strain sul1. Cells were incubated in LCAPG media containing 8 μM 36Cl−, and cellular Cl contents were determined after sufficient time to reach a steady-state distribution (>1.5 h). Bars represent mean ± SEM of the ratio between cellular [Cl−] in the test strain (nhx1, gef1, or vma1) and control strain sul1 at either pH 4.0 (black bars) or pH 7.0 (gray bars). Results of paired Student t tests (pH 4 and pH 7.0 combined): sul1 vs. nhx1, P < 0.02 (n = 4); sul1 vs. gef1, P < 10−5, n = 12; sul1 vs. vma1, P < 0.002, n = 6. (C) Cells:medium 36Cl− distribution ratio for sul1 and vma1 cells incubated as in A and B but with 0.2 mM DNP or DNP and 20 mM 2-deoxyglucose replacing 2% glucose during the 2 h incubation with 36Cl−. The gray horizontal lines indicate a cells:medium ratio of unity.
Mentions: In the experiments in Fig. 3, cells were grown overnight in LCAPG medium without 36Cl−, centrifuged, and resuspended in fresh LCAPG medium, 2% glucose, plus 8 μM Na36Cl. Media were buffered either with 5 mM K-citrate (initial pH 4.5), or with 5 mM K-citrate, 10 mM bistris, 20 mM tris (initial pH 7.6). After 2 h of aerobic incubation at 30°C, 5 ml aliquots of suspension were diluted in 40 ml of ice-cold water, centrifuged, the supernatant aspirated completely, and the cellular 36Cl− determined by scintillation counting.

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