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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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Possible transport mechanisms accounting for the cellular accumulation of Cl− in pH 7.0 (A) or pH 4.0 (B) media containing 10 μM Cl−. The elevated cellular Cl− content is proposed to be a consequence of two processes. (1)Influx across the plasma membrane via a high affinity Cl− transporter (HACT), which is regulated by a mechanism that includes Yhl008c (depicted here on the plasma membrane, but the actual cellular location is not known). The Cl− gradient across the plasma membrane is higher at extracellular pH 4.0 than at pH 7, consistent with H+–Cl− cotransport across the plasma membrane. The dashed arrow represents downhill efflux of Cl− through a pathway that is unknown but must be very slow in a low Cl− medium (Fig. 5 B). (2) Sequestration of Cl− in the vacuole or prevacuolar compartment by a process that is powered by the V-ATPase (Vma), with Cl− transport (probably as Cl−/H+ exchange; see text) through Gef1p, and the pH gradient modulated by Nhx1p.
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fig9: Possible transport mechanisms accounting for the cellular accumulation of Cl− in pH 7.0 (A) or pH 4.0 (B) media containing 10 μM Cl−. The elevated cellular Cl− content is proposed to be a consequence of two processes. (1)Influx across the plasma membrane via a high affinity Cl− transporter (HACT), which is regulated by a mechanism that includes Yhl008c (depicted here on the plasma membrane, but the actual cellular location is not known). The Cl− gradient across the plasma membrane is higher at extracellular pH 4.0 than at pH 7, consistent with H+–Cl− cotransport across the plasma membrane. The dashed arrow represents downhill efflux of Cl− through a pathway that is unknown but must be very slow in a low Cl− medium (Fig. 5 B). (2) Sequestration of Cl− in the vacuole or prevacuolar compartment by a process that is powered by the V-ATPase (Vma), with Cl− transport (probably as Cl−/H+ exchange; see text) through Gef1p, and the pH gradient modulated by Nhx1p.

Mentions: The work described here has shown that S. cerevisiae maintains total cellular Cl− within a narrow range even if extracellular [Cl−] is varied >10,000 fold. The Cl− distributions and fluxes indicate the existence of a Cl− transporter with a very high apparent affinity for Cl− at acid extracellular pH. The activation of this transporter depends on a Cl−-sensing mechanism, one component of which may be the formate-nitrite transporter homologue Yhl008cp. The experiments also provide evidence that there are steep Cl− gradients across the membranes of vacuoles or prevacuolar vesicles in S. cerevisiae grown in low Cl− media. These gradients are dependent on the vacuolar H+-ATPase, the yeast CLC Gef1p, and, to a lesser extent, the cation–H+ exchanger Nhx1p. A model based on these findings is in Fig. 9.


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)

Possible transport mechanisms accounting for the cellular accumulation of Cl− in pH 7.0 (A) or pH 4.0 (B) media containing 10 μM Cl−. The elevated cellular Cl− content is proposed to be a consequence of two processes. (1)Influx across the plasma membrane via a high affinity Cl− transporter (HACT), which is regulated by a mechanism that includes Yhl008c (depicted here on the plasma membrane, but the actual cellular location is not known). The Cl− gradient across the plasma membrane is higher at extracellular pH 4.0 than at pH 7, consistent with H+–Cl− cotransport across the plasma membrane. The dashed arrow represents downhill efflux of Cl− through a pathway that is unknown but must be very slow in a low Cl− medium (Fig. 5 B). (2) Sequestration of Cl− in the vacuole or prevacuolar compartment by a process that is powered by the V-ATPase (Vma), with Cl− transport (probably as Cl−/H+ exchange; see text) through Gef1p, and the pH gradient modulated by Nhx1p.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279172&req=5

fig9: Possible transport mechanisms accounting for the cellular accumulation of Cl− in pH 7.0 (A) or pH 4.0 (B) media containing 10 μM Cl−. The elevated cellular Cl− content is proposed to be a consequence of two processes. (1)Influx across the plasma membrane via a high affinity Cl− transporter (HACT), which is regulated by a mechanism that includes Yhl008c (depicted here on the plasma membrane, but the actual cellular location is not known). The Cl− gradient across the plasma membrane is higher at extracellular pH 4.0 than at pH 7, consistent with H+–Cl− cotransport across the plasma membrane. The dashed arrow represents downhill efflux of Cl− through a pathway that is unknown but must be very slow in a low Cl− medium (Fig. 5 B). (2) Sequestration of Cl− in the vacuole or prevacuolar compartment by a process that is powered by the V-ATPase (Vma), with Cl− transport (probably as Cl−/H+ exchange; see text) through Gef1p, and the pH gradient modulated by Nhx1p.
Mentions: The work described here has shown that S. cerevisiae maintains total cellular Cl− within a narrow range even if extracellular [Cl−] is varied >10,000 fold. The Cl− distributions and fluxes indicate the existence of a Cl− transporter with a very high apparent affinity for Cl− at acid extracellular pH. The activation of this transporter depends on a Cl−-sensing mechanism, one component of which may be the formate-nitrite transporter homologue Yhl008cp. The experiments also provide evidence that there are steep Cl− gradients across the membranes of vacuoles or prevacuolar vesicles in S. cerevisiae grown in low Cl− media. These gradients are dependent on the vacuolar H+-ATPase, the yeast CLC Gef1p, and, to a lesser extent, the cation–H+ exchanger Nhx1p. A model based on these findings is in Fig. 9.

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