HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 36, 33075-33080, September 6, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/36/33075    most recent M205037200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matsumoto, T. K. Articles by Hasegawa, P. M. Search for Related Content PubMed PubMed Citation Articles by Matsumoto, T. K. Articles by Hasegawa, P. M. Social Bookmarking                      What's this?

An Osmotically Induced Cytosolic Ca²⁺ Transient Activates Calcineurin Signaling to Mediate Ion Homeostasis and Salt Tolerance of Saccharomyces cerevisiae^*

Tracie K. Matsumoto, Amanda J. Ellsmore, Stephen G. Cessna^§, Philip S. Low^¶, José M. Pardo, Ray A. Bressan, and Paul M. Hasegawa^**

From the  Center for Plant Environmental Stress Physiology, Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907-1165, the ^§ Departments of Biology and Chemistry, Eastern Mennonite University, Harrisonburg, Virginia 22802, the ^¶ Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, and  Instituto de Recursos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientificas, Sevilla 41012, Spain

Received for publication, May 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hyperosmotic stress caused by NaCl, LiCl, or sorbitol induces an immediate and short duration (~1 min) transient cytosolic Ca²⁺ ([Ca²⁺]_cyt) increase (Ca²⁺-dependent aequorin luminescence) in Saccharomyces cerevisiae cells. The amplitude of the osmotically induced [Ca²⁺]_cyt transient was attenuated by the addition of chelating agents EGTA or BAPTA, cation channel pore blockers, competitive inhibitors of Ca²⁺ transport, or mutations (cch1 or mid1) that reduce Ca²⁺ influx, indicating that Ca is a source for the transient. An osmotic pretreatment (30 min) administered by inoculating cells into media supplemented with either NaCl (0.4 or 0.5 M) or sorbitol (0.8 or 1.0 M) enhanced the subsequent growth of these cells in media containing 1 M NaCl or 2 M sorbitol. Inclusion of EGTA in the osmotic pretreatment media or the cch1 mutation reduced cellular capacity for NaCl but not hyperosmotic adaptation. The stress-adaptive effect of hyperosmotic pretreatment was mimicked by exposing cells briefly to 20 mM CaCl₂. Thus, NaCl- or sorbitol-induced hyperosmotic shock causes a [Ca²⁺]_cyt transient that is facilitated by Ca²⁺ influx, which enhances ionic but not osmotic stress adaptation. NaCl-induced ENA1 expression was inhibited by EGTA, cch1 mutation, and FK506, indicating that the [Ca²⁺]_cyt transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Exposure of cells to high salt causes both ionic and hyperosmotic stresses. In yeast, at least two signal transduction pathways are activated to regulate processes necessary for ion homeostasis and osmotic adjustment. The high osmolarity glycerol (HOG)¹ pathway is primarily responsible for the control of osmotic adaptation while the calcineurin pathway regulates ion homeostasis. The HOG pathway, which includes as a key intermediate the mitogen-activated protein (MAP) kinase Hog1p, is initiated as a consequence of hypertonic stress perception by either of two osmosensing proteins Sln1p or Sho1p (1-3). Signaling transduced through the HOG cascade activates transcription of GPD1 and of other stress-responsive genes necessary for osmotic adaptation (1, 4-6). GPD1 encodes glycerol-3-phosphate dehydrogenase, which catalyzes a critical step in the biosynthesis of glycerol, the principal osmolyte of yeast cells.

The Ca²⁺/calmodulin-dependent type 2B phosphatase calcineurin is the pivotal intermediate in this signal pathway that mediates cellular Na⁺, K⁺, and Ca²⁺ homeostasis (7, 8). Hypersaline stress causes Ca²⁺ activation of calmodulin, which in turn activates calcineurin (7, 8). Signaling through calcineurin regulates the expression of ENA1/PMR2A, which encodes the P-type ATPase that is primarily responsible for Na⁺ efflux across the plasma membrane, and of PMC1 and PMR1, which encode endomembrane-localized Ca²⁺-ATPase pumps. Calcineurin dephosphorylates the zinc finger transcription factor Crz1p/Tcn1p/Hal8p, a process that facilitates its mobilization to the nucleus (9-12). Crz1p/Tcn1p/Hal8p interacts with the calcineurin-dependent response element (CDRE) that is located in the promoters of ENA1, PMC1, PMR1, and other calcineurin-activated genes (9-12). Hog1p also activates ENA1 expression by rendering the bZIP transcriptional repressor Sko1p and the Ssn6-Tup1 co-repressor complex inactive (13, 14). Calcineurin post-transcriptionally activates the Trk1-Trk2 K⁺ transport system to the high affinity state resulting in higher K⁺/Na⁺ selective uptake and reduced Na influx (8, 15). The Hal4p/Hal5p signal pathway also activates the Trk1-Trk2 transport system high affinity transition, although the post-translational process has not been defined nor is it known whether Ca²⁺ is required for the function of this pathway (16). Calcineurin post-transcriptionally regulates Vcx1p, which together with Pmc1p and Pmr1p function to evacuate Ca²⁺ from the cytosol (17, 18).

Hypotonic shock or a combination of hypotonic shock and cold stress induces a rapid, short duration [Ca²⁺]_cyt pulse in yeast cells that is hypothesized to activate the protein kinase C cell integrity pathway (19). Recently, it was determined that hypertonic shock triggers a transient increase in [Ca²⁺]_cyt that was attributed to the function of a transient receptor potential-like channel (Yvc1p), which mediates the release of Ca²⁺ from the vacuole (20). Although Ca²⁺ is implicated as a primary effector of Na⁺ and Ca²⁺ homeostasis necessary for salt tolerance (21), the molecular entities and processes involved in salt stress-dependent Ca²⁺ activation of calmodulin and calcineurin have yet to be elucidated.

Ca²⁺-dependent yeast mating pheromone signaling has been characterized in some detail (1, 22-25). Mating pheromone causes G₁ arrest and resumption of cell cycle progression by facilitating Ca influx, which results in calmodulin activation of calcineurin (1, 22-25). Pheromone-induced Ca influx is mediated by Cch1p and Mid1p, which are plasma membrane proteins that by genetic criteria function as a single Ca²⁺ uptake system (26-29). Cch1p contains four repeated peptide domains, each with sequence similarity to the ₁ pore-forming subunit of L-type voltage-gated Ca²⁺ channels (28). Mid1p is an N-glycosylated, integral plasma membrane protein inferred to be a Ca²⁺-permeable stretch-activated nonselective cation channel (26, 28).

Herein, direct evidence is presented that Ca influx facilitates salt adaptation of yeast. Hyperosmotic shock induces a short duration transient increase in [Ca²⁺]_cyt through Cch1p-Mid1p that mediates ionic but not hyperosmotic stress tolerance. The osmotically induced [Ca²⁺]_cyt transient activates signaling through calcineurin leading to ENA1 induction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast Strains and Culture Conditions-- The strains were derived from Saccharomyces cerevisiae W303-1A (MAT a: his3 leu2 trp1 ade2 ura3) (30). The cch1::kanMX4 deletion-disruption mutation was constructed using the PCR-based gene-targeting system (31). The forward primer 5'-TGA TGA AAG GCT ACG CAG GCG TAG CTT CAG TAG TTA TAG CCG ATC atc gat gaa ttc gag ctc g-3' (S1 primer for pFA6a-MX in lowercase) and the reverse primer 5'-TCT CTG ATC CAC AGT CCG TAT AGG TTG AAT TAT CAT CAG AGG AGC atc gat gaa ttc gag ctc g-3' (S2 primer for pFA6a-MX in lowercase) were used to amplify kanMX4 from plasmid pFA6a-kanMX4 (provided by Dr. P. Philippsen, Ref. 31). Homologous recombination of the PCR product introduced a kanMX4 disruption in the CCH1 (YGR217W) open reading frame beginning at +719 bp from the translation start site to 941 bp from the translation stop site. The disrupted allele was detected using primers corresponding to sequence beginning at position 260 bp (5'-CGG GAA AAT GTA ATT TGG CAT GTC A-3') from the translation start site to +262 bp (5'-TTT TTG GTC TTG TGA AGA CTA CG-3') from the translation stop site.

The mid1::kanMX4 mutation was constructed using the forward primer 5'-GGC AAG CAC TAT TCG TGG TTT ACT GCC TAT TTA CCA CTT CTA TTC atc gat gaa ttc gag ctc g-3' and reverse primer 5'-CTA CGT ATC GTC CAA TGG ATG AAT TAC CAT CAA GGA TGA GTT ACC atc gat gaa ttc gag ctc g-3' to amplify kanMX4. Homologous recombination of the PCR product introduced a disruption in the MID1 (YNL291C) open reading frame position +55 bp from the translation start site to 45 bp from the translation stop site. The disrupted allele was detected using primers corresponding to sequence beginning at position 391 bp (5'-GGG CCT GTT CCT ACA CCT TTT ATA A-3') from the translation start site to +125 bp (5'-GCG AAC TTA TTC AGA TCT CTT CAA CCA-3') from the translation stop site. The cch1 mid1 double mutation was generated by using the PCR-based gene-targeting system and HIS3MX6 (31) in place of the kanMX4 for the mid1 mutation and was introduced into cch1::kanMX4 mutant.

Yeast cells were grown using standard procedures (32) in YPD medium (1% Difco yeast extract, 2% peptone, 2% dextrose) or in S.D. medium (0.17% Difco YNB-AA-AS, 0.5% ammonium sulfate, 2% dextrose) that was supplemented with amino acids or osmotic solutes as indicated. The pH of all media was adjusted to 6.5. For spot assays, cultures were diluted to an OD₆₀₀ of ~0.4 and then further diluted 10-, 100-, or 1000-fold (Shimadzu UV-1601). EGTA or CaCl₂ in 50 mM MES buffer (pH 6.5) solutions were added to media after autoclave sterilization.

Aequorin Luminescence Measurements and [Ca²⁺]_cyt Quantification-- Aequorin luminescence was determined with a luminometer (TD-20/20 Turner Designs, Sunnyvale, CA) following procedures used to quantify [Ca²⁺]_cyt in yeast and tobacco cells (19, 33). Cells were transformed with the 2-µ plasmid pEVP11/AEQ (provided by Dr. P. Masson, 19) containing the APOAEQUORIN gene. Leucine autotrophic cells at stationary phase were harvested by centrifugation and then resuspended in YPD medium to an OD₆₀₀ of ~0.1-0.2. Cells were grown to an OD₆₀₀ of ~0.4-0.6. The cells were concentrated about 5-fold by centrifugation and 1 µl of 590 µM coelenterazine (Biotum Inc., Fremont, CA) in absolute methanol was added to 100 µl of culture. After incubation for 30 min on a rotating drum in the dark, a cellular luminescence baseline was determined by 1 min of recordings at 5/s intervals.

Hypertonic shock was administered to yeast cells by diluting the suspension 1:1 with YPD medium containing NaCl, LiCl, or sorbitol at 2× final concentration. All inhibitory or chelating compounds were diluted in 50 mM MES (pH 6.5) and added to the cultures as indicated. EGTA and BAPTA solutions (0.5 M) were made in 50 mM MES buffer (pH 6.5). NiCl₂, MgCl₂, or GdCl₃ was dissolved in water to a concentration of 0.3 M and diluted with MES buffer (50 mM final concentration, pH 6.5) to 10- to 20-fold the concentration in the treatment solution. These chemical agents or an equal volume of 50 mM MES were added to cells immediately before the acquisition of the basal luminescence reading and in the osmotic pretreatment media. Luminescence from aequorin that remained in cells at the end of an experiment was determined after treating cells with Triton X-100 (5-10%) and CaCl₂ (2-5 M). These data were used to calculate the [Ca²⁺]_cyt using Equation 1,

(Eq. 1)

where L is the luminescence intensity at any time point and L_max is the integrated luminescence intensity (34).

Measurement of ⁴⁵Ca²⁺ Accumulation-- Procedures for determining Ca²⁺ uptake into cells were as described, with modifications (17, 28). Cells in the exponential growth phase (YPD medium) were inoculated into fresh medium (OD₆₀₀ ~0.1) that contained 1 µCi/ml of ⁴⁵Ca²⁺ (⁴⁵CaCl₂, ICN Biomedicals Inc., Costa Mesa, CA). Cells were grown for 5 h and harvested for determination of intracellular radioactivity.

Osmotic Pretreatment of Cells-- Cells in the log-growth phase (OD₆₀₀ of ~0.4-0.6) were subjected to osmotic pretreatment (30 min) by diluting the suspension 1:1 with YPD medium containing NaCl or sorbitol at 2× final concentration. Cells were collected by centrifugation (Eppendorf Centrifuge 5810) at 3000 rpm for 3 min. Cells were resuspended in growth medium to an OD₆₀₀ of ~0.2-0.3, and growth was monitored (OD₆₀₀).

ENA1-lacZ -Galactosidase Assay-- Wild type and cch1 cells were transformed with pKC201 containing an ENA1-lacZ fusion (provided by Dr. K. Cunningham, Ref. 9). Cells from three independent transformation events were grown to late log phase in S.D. medium. Cells were collected, resuspended in YPD medium to an OD₆₀₀ of ~0.1, and grown to log phase (OD₆₀₀ of ~0.4-0.6). These cells were exposed to hypertonic shock by diluting the culture 1:1 with medium containing NaCl, or 1:1 with CaCl₂ at 2× final concentration. A stock solution of 2 mg/ml FK506 (provided by Fujisawa Healthcare Inc., Deerfield, IL) that was dissolved in 90% ethanol and 10% Tween-20 was added to medium as indicated. Cells were collected after 4 h, frozen in liquid nitrogen, and processed for determination of -galactosidase activity (35). The same period of 4 h was used previously to determine calcineurin-dependent induction of FKS2, PMC1, and ENA1 expression, after stimulation by NaCl, Ca²⁺, or mating pheromone (9).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hyperosmotic Stress Induces a Transient Increase in [Ca²⁺]_cyt That Is Derived from an Extracellular Pool-- NaCl shock initiates an immediate and transient elevation in [Ca²⁺]_cyt of about 1 min in duration (Fig. 1A). Because similar Ca²⁺ transient profiles were obtained after treatment of cells with equiosmolar concentrations of NaCl, sorbitol, LiCl (Fig. 1, A-C), or Na₂SO₄ or KCl (not shown), the increase in [Ca²⁺]_cyt is caused, in a concentration-dependent manner, by hypertonic shock and not ionic stress. A new and higher [Ca²⁺]_cyt steady state is established after the initial transient (Fig. 1B). This higher [Ca²⁺]_cyt steady state may be due to continued influx from the extracellular pool, release from an internal cellular store, or limited vacuolar sequestration (36).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   NaCl-induced [Ca2+]cyt transient in yeast cells is caused by hypertonic shock that is derived from Ca influx. Yeast cells expressing reconstituted aequorin (see "Experimental Procedures") were diluted with an equal volume of fresh medium supplemented with NaCl, LiCl, or sorbitol (at 2× final concentration) to initiate the osmotic stress treatment (arrow). A, NaCl; B, NaCl or sorbitol; or C, NaCl or LiCl were added and [Ca2+]cyt were determined from aequorin luminescence measurements. The NaCl-induced [Ca2+]cyt transient is attenuated by D, divalent cations Ni2+ (NiCl2) or Mg2+ (MgCl2); E, by membrane impermeable Ca2+ chelating agents EGTA or BAPTA; or F, by the Ca2+ channel blocker Gd3+ (GdCl3). The NaCl-induced [Ca2+]cyt transient was detected in cells of wild type (W303-1A) (A-F); wild type (wt) or cch1 (G); wt, cchl, mid1, or cch1 mid1 (H). Data illustrated are from one representative experiment (minimum of three independent experiments).

The hyperosmotic stress-induced increase in [Ca²⁺]_cyt was attenuated by the inclusion of 2.5 or 5 mM NiCl₂, or by 5 mM MgCl₂ in the medium (Fig. 1D). Ni²⁺ effectively inhibits L-type Ca²⁺ channel activity of guinea pig ventricular myocytes (37), and Mg²⁺ competes with Ca²⁺ for channel conductance in tobacco cells (33). Reduction in the amplitude of the transient rise in [Ca²⁺]_cyt by Ni²⁺ and Mg²⁺ implicates the extracellular pool as the cation source.

The transient elevation in [Ca²⁺]_cyt that is triggered by 1 M NaCl was inhibited also by inclusion in the medium of the membrane impermeable, divalent cation chelating agent EGTA (Fig. 1E). Similar attenuation of the transient increase in [Ca²⁺]_cyt occurred if BAPTA, a chelating agent with 10⁵-fold higher affinity for Ca²⁺ than for Mg²⁺, was substituted for EGTA in the medium (Fig. 1E). These results also confirm that the hyperosmotically induced transient rise in [Ca²⁺]_cyt is dependent on influx across the plasma membrane of cells that are inoculated into YPD medium, which contains about ~75 µM Ca²⁺ (38).

Gd³⁺ (GdCl₃) reduced the amplitude of the hypertonic shock-induced [Ca²⁺]_cyt transient in cells, albeit incompletely (Fig. 1F). Because incubation of cells with GdCl₃ was initiated 1 min prior to addition of NaCl, it is unlikely that Gd³⁺ permeated into the cell and inhibited endomembrane localized Ca²⁺-permeable channel activity to cause the attenuation of the transient elevation in [Ca²⁺]_cyt. Gd³⁺ is an inhibitor of stretch-activated Ca²⁺ channels of yeast, plant, and animal cells (39, 40), and effectively reduces Ca transients in yeast and tobacco cells (19, 33). These results implicate the involvement of a Gd³⁺-sensitive, plasma membrane Ca⁺ channel protein, perhaps Mid1p, in the Ca influx that results in increased [Ca²⁺]_cyt (29).

cch1, mid1, or cch1 mid1 disruption/deletion mutations were generated in W303-1A cells. These cells were then transformed to express apoaequorin from pEVP11/AEQ (ADH::AEQ). Net ⁴⁵Ca²⁺ accumulation in W303-1A cch1 cells was reduced to about 60% of the level that accumulated in wild type cells (not shown). cch1 caused a similar reduction in intracellular Ca²⁺ accumulation in another yeast strain (28). The NaCl-induced [Ca²⁺]_cyt transient in cch1 or mid1 cells was substantially, and to a similar degree, lower than in wild type cells (Fig. 1, G and H). The cch1 mid1 double mutation did not cause an additive effect. The NaCl-induced transient elevation in [Ca²⁺]_cyt in cch1, mid1, and cch1 mid1 cells is comparable to that in wild type cells after treatment with 5 mM GdCl₃. It can be concluded that the hyperosmotically induced [Ca²⁺]_cyt transient is mediated predominantly by influx through Cch1p and Mid1p that are genetically functioning as a single Ca²⁺ uptake system. However, there may be contribution to the [Ca²⁺]_cyt transient through an alternative uptake system or from internal stores (20) because the elevation in [Ca²⁺]_cyt is not completely abrogated by pharmacological agents or by null mutations in the plasma membrane Ca²⁺ channel.

Ca Influx through Cch1-Mid1p Is Required for Ionic but Not Hyperosmotic Stress Adaptation-- cch1 and mid1 cells are Li⁺ sensitive (28). Furthermore, cch1 and mid1 cells are Na⁺ but not hyperosmotic sensitive (mid1 data not shown), and Na⁺ and Li⁺ sensitivities of these cells are exacerbated by EGTA or attenuated by Ca²⁺ supplementation to the media (Fig. 2). Because cells exhibit Ca²⁺-dependent growth enhancement on media supplemented with Na⁺ or Li⁺ but not with sorbitol, it is concluded that the divalent cation facilitates ionic adaptation (Fig. 2). Neither Na⁺ nor Li⁺ sensitivity, nor the modulation of sensitivity to these ions by EGTA or Ca, is additive in cch1 mid1 cells compared with cch1 or to mid1 cells (not shown). These results indicate that the hyperosmotic shock-induced transient increase in [Ca²⁺]_cyt that is mediated in part by Cch1p and Mid1p is necessary for ionic stress adaptation and that the proteins function together as an individual influx system.

View larger version (55K): [in this window] [in a new window]   Fig. 2.   Ca enhances yeast cell growth in media supplemented with NaCl (0.9 M) or LiCl (0.15 M), but not with sorbitol (1.8 M). W303-1A (wt) or cch1 cells were grown on YPD medium without or with 1 mM EGTA or with 5 mM CaCl2.

Osmotic Pretreatment Facilitates Ionic Stress Adaptation That Is Ca Transient-dependent-- Cell growth in ionic or osmotic stress media was enhanced by a 30-min hyperosmotic pretreatment (Fig. 3). Interestingly, pretreatment with 20 mM Ca²⁺ enhanced cell growth only in medium that was supplemented with NaCl (Fig. 3, B-D). Cells exposed to high [Ca²⁺]_ext exhibit an immediate and transient increase in [Ca²⁺]_cyt (not shown; Refs. 20 and 36). Hypertonic shock-induced NaCl adaptation was enhanced by Ca or attenuated by EGTA (Fig. 3C). EGTA did not affect salt adaptation when the chelating agent was added to hyperosmotic pretreatment medium 25 min (30 min total) after cell inoculation, indicating that sufficient Ca²⁺ influx had occurred by this time to mediate ionic stress adaptation (not shown). Osmotic pretreatment was substantially less effective at enhancing growth of cch1 cells compared with wild type cells in NaCl-containing medium (Fig. 3C). The reduced salt-adaptive capacity of cch1 cells was similar to that determined when wild type cells were exposed to EGTA. In contrast, growth of cch1 cells was equivalent to wild type after osmotic pretreatment and inoculation into medium with 2 M sorbitol (Fig. 3D). Together, these findings link the hyperosmotically generated [Ca²⁺]_cyt transient to ionic stress adaptation and implicate Cch1p and Mid1p as the major Ca influx determinants.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Ca influx mediates ionic but not osmotic stress tolerance. Cells were pretreated for 30 min prior to transfer into media that contained a growth inhibitory but not lethal concentration of NaCl (1 M) or sorbitol (2 M). Illustrated in A and B is the growth of wild type (W303-1A) cells. B, spot assay analysis of cell growth (YPD, 1 M NaCl or, 2 M sorbitol) after pretreatment in YPD, first in YPD (YPD), and then inoculated onto 2.5 µl of YPD containing 20 mM CaCl2 (concentration of CaCl2 same as that of CaCl2 treatment) (YPD*), CaCl2, NaCl, or sorbitol medium. C and D, growth of wild type (W303-1A) or cch1 (where indicated) cells after pretreatment and transfer into medium containing NaCl (C) or sorbitol (D). Osmotic pretreatments consisted of YPD (), YPD + 20 mM Ca2+ (), 0.5 M NaCl (wt -) and cch1 (), 0.5 M NaCl + 5 mM EGTA (), or 0.5 M NaCl + 5 mM Ca2+ () for 30 min.

The Osmotically Induced Ca Transient Activates ENA1 Expression through Calcineurin Signaling-- Monitoring of ENA1 expression using the ENA1-lacZ promoter-reporter fusion system (9) confirmed that -galactosidase activity is constitutively lower in cch1 compared with wild type cells (Fig. 4 and Ref. 28). ENA1 expression is induced by inclusion of NaCl or CaCl₂ into the medium and the induction is greater in wild type than in cch1 cells (Fig. 4). EGTA abrogates the difference in NaCl-induced ENA1 expression between wild type and cch1 cells, which indicates that Ca influx through Cch1p is required for transcriptional activation of this gene (Fig. 4). The calcineurin-specific inhibitor FK506 (9, 10) reduced ENA1-lacZ expression to near basal levels in cells that were in medium with CaCl₂ but not with NaCl. The difference may reflect the contribution of the HOG pathway to ENA1 induction that is elicited by NaCl treatment. Almost total abrogation of NaCl-induced ENA1-lacZ expression by a combination of EGTA and FK506 is evidence that an alternative Ca²⁺-dependent pathway may regulate the expression of the gene during a salt stress episode. However, ENA1 expression is potentiated substantially by calcineurin. Calcineurin-dependent nuclear translocation of the Crz1p/Tcn1p/Ha18p transcription factor, which activates ENA1 expression, occurred within 10 min in response to 200 mM Ca²⁺ or 0.8 M NaCl (12). Nuclear translocation did not occur if the Ca²⁺-chelating agent EGTA is included in the medium. Furthermore, Ca²⁺-induced CDRE-lacZ reporter expression is attenuated by FK520, another inhibitor of calcineurin (12). Together, these findings establish that the hyperosmotically induced [Ca²⁺]_cyt transient is dependent on influx through Cch1p, presumably the Cch1p-Mid1p system, and activates calcineurin, which induces ENA1 expression.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Ca2+ influx through Cch1p activates calcineurin, which induces ENA1-lacZ expression. Wild type (W303-1A, gray bars) and cch1 (white bars) cells were transformed with the plasmid pKC201 to express ENA1-lacZ (9). Cells were treated with 1 M NaCl or 20 mM CaCl2 in media without or with EGTA (10 mM) and/or FK506 (0.2 µg/ml). Values are the average for three determinations of -galactosidase activity in cells from three independent colonies.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transient Increase in Ca Mediates Ionic Adaptation-- Hyperosmotic shock induces [Ca²⁺]_cyt transients in organisms as diverse as Chara (41), Dunaliella salina (42), Anabaena (43), and Arabidopsis thaliana (44) (reviewed in Ref. 45). In plants, salt stress increases [Ca²⁺]_cyt that is presumed to facilitate adaptation (46, 47). Furthermore, plants or cultured cells that are exposed to sublethal levels of salt acquire greater stress adaptive capacity (48, 49), and tomato seedlings subjected to hyperosmotic pretreatment exhibit enhanced salt tolerance (50). The results of this study provide direct evidence that hyperosmotic stress, generated by salt or a non-ionic osmotic solute, induces a transient [Ca²⁺]_cyt increase in yeast cells, and this [Ca²⁺]_cyt transient mediates ionic but not osmotic stress adaptation. Because most salts are toxic at concentrations that are also osmotically stressful, it is possible that yeast cells detect and respond to salt stress by an osmosensing and not an ion-specific sensing mechanism. Perhaps the Sln1p or Sho1p osmosensor perceives salt stress to activate the HOG pathway for osmotic adjustment and Ca²⁺-dependent ion homeostasis pathways, similar to the calcineurin signal cascade (1).

Hyperosmotic Stress-Induced [Ca²⁺]_cyt Is Mediated by Ca Influx through Mid1p-Cch1p-- Results presented here indicate that hypertonic shock induces Ca influx through Cch1p-Mid1p. Electrophysiological studies of yeast plasma membrane patches indicate that the activity and selectivity of mechanosensitive ion channels may function in osmotic sensing and turgor regulation (40). It is not known if the activity of the L-type channel-like protein Cchlp is modulated by mechanical stress but Ca²⁺ permeability of Mid1p is stretch-activated (29). Ni²⁺, an inhibitor of L-type Ca²⁺ channel activity (37), or Gd³⁺, an inhibitor of stretch-activated Ca²⁺ channel activity (39), substantially diminishes hyperosmotically induced Ca influx into rat osteoblast-like cells (51).

Hypertonic shock also induces the release of Ca²⁺ from internal stores through the vacuolar membrane localized TRP channel-like protein Yvc1p and this contributes to elevated [Ca²⁺]_cyt (20). Release of Ca through Yvc1p requires activation by Ca²⁺ on the cytosolic side of the vacuolar membrane (52). Perhaps Ca²⁺ influx through Cch1p-Mid1p, as indicated by the results presented herein, causes the activation of Yvc1p. Our data indicate that influx through Cch1p-Mid1p mediates a hypertonic shock-induced transient increase [Ca²⁺]_cyt but do not exclude a contribution from an internal source. However, it is not possible to resolve why yvc1 cells that have a functional Cch1p-Mid1p transport system are unable to generate a [Ca²⁺]_cyt transient in response to hypertonic shock (20). Interestingly, yvc1 cells have no apparent growth defects, and overexpression of YVC1 causes Ca²⁺ but not Na⁺ sensitivity (20). Herein, we show that Ca²⁺ influx through Cch1p-Mid1p facilitates ionic adaptation (Fig. 3).

Activation of Calcineurin by a [Ca²⁺]_cyt Transient Facilitates Ion Homeostasis-- The [Ca²⁺]_cyt transient signature is integral to signal diversity that is transduced through interactions with Ca²⁺-binding proteins resulting in outputs that control specific physiological processes (45, 53-56). Because Ca²⁺ is relatively immobile in the cytosol and quickly becomes associated with Ca²⁺-binding proteins, the degree of Ca²⁺ saturation within the cell even after a substantial Ca²⁺ influx is most pronounced in microdomains that are localized near the pore of the channel through which cation influx occurs (57). Recent evidence indicates that Ca²⁺-binding proteins (calmodulin, Ca²⁺-dependent protein kinases (CDPKs), phosphatases, heterotrimeric G-proteins, etc.) and their interacting proteins are associated physically with the cytosolic-localized domains of Ca²⁺ channels (55, 58-60). Some of these proteins are implicated in channel autoregulation, and others function as pivotal components of signal transduction. Thus, both Ca²⁺ signal differentiation and propagation may result from the properties of the channel that control its gating and the Ca²⁺-binding proteins that are associated with the channel cytosolic domain(s), and are activated by the transient.

The results presented here provide direct evidence that hyperosmotic stress induces a transient increase in [Ca²⁺]_cyt that activates ENA1-lacZ expression through calcineurin signaling. Calcineurin regulates the Crz1p/Tcn1p/Hal8p transcription factor to activate ENA1 expression. The nuclear localization of this transcription factor is maintained for an extended period after a stimulus-induced transient elevation in [Ca²⁺]_cyt and perhaps this is the reason why calcineurin activation of gene expression is sustained for some period after the transient has dissipated (12). Furthermore, the new [Ca²⁺]_cyt steady state that is established may also continue signaling through calcineurin and Crz1p/Tcn1p/Hal8p that results in ENA1 transcription. NFAT activation by calcineurin is potentiated by a low but sustained level of [Ca²⁺]_cyt (reviewed in Refs. 61 and 62).

A model that describes how hyperosmotic stress induces a transient increase in [Ca²⁺]_cyt that mediates ion homeostasis and salt tolerance in yeast is illustrated in Fig. 5. This yeast paradigm may be a reasonable representation of the entities and the events that function in plants to initiate Ca²⁺-dependent signaling involved in salt adaptation (63, 64). Salt induces an increase in [Ca²⁺]_cyt (44), and Ca enhances salt tolerance of plants (46). Current understanding indicates that in plants, as in yeast, separate pathways are involved in osmotic adjustment and ion homeostasis (65, 66). Indeed, a putative two-component osmosensor that may be orthologous to Sln1p has been identified in Arabidopsis, together with entities that may comprise a MAP kinase cascade responsible for osmotic signaling (67-69). The Ca²⁺-dependent SOS (salt oversensitive) pathway of Arabidopsis may be the functional equivalent of the yeast calcineurin pathway, at least for ion homeostasis that is necessary to mediate salt tolerance (65, 66). It is reasonable to expect that activation of the SOS pathway might be initiated by a hyperosmotically induced [Ca²⁺]_cyt transient that is dependent on influx through a plasma membrane localized transport system (70).

View larger version (51K): [in this window] [in a new window]   Fig. 5.   A model depicting how hypertonic stress induces Ca2+ influx through Cch1p-Mid1p to activate calcineurin signaling that mediates ion stress tolerance. Hyperosmotic shock imposes a substantial mechanical stress/strain on the plasma membrane that potentiates Ca influx through Cch1p-Mid1p (28) and subsequent Ca2+ release from the vacuole through Yvc1p (20). The [Ca2+]cyt increase in the Cch1p-Mid1p microdomain activates calmodulin (Cmd1p) that is tethered either to the channel or its associated proteins. Cmdlp activates calcineurin that dephosphorylates Crz1p/Tcn1p/Hal8 resulting in its translocation to the nucleus where it activates ENA1 expression that facilitates ion homeostasis and salt tolerance. Ca2+-activated Cmd1p also post-transcriptionally upregulates Ena1p activity (71), and calcineurin also mediates the transition of Trk1p-Trk2p system to high affinity for K+. The vacuolar membrane Ca2+/H+ exchanger Vcx1p is a major determinant that reduces [Ca2+]cyt to the steady-state level after influx from the extracellular pool or release from internal stores (20, 36).

	ACKNOWLEDGEMENTS

We thank Dr. Patrick Masson, Dr. Kyle Cunningham, Dr. Peter Philippsen for providing the plasmids pEFP11/AEQ, pKC201, and pFA6a-kanMX(HIS3MX6) and the Fujisawa Healthcare Inc. for providing FK506. We also thank Dr. Ann Batiza for technical assistance with the yeast aqueorin system.

	FOOTNOTES

^* This work was supported by National Science Foundation Plant Genome Award DBI-9813360 (to R. A. B. and P. M. H.) and by the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust (to S. G. C.). This is journal article 16839 from the Purdue Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Center for Plant Environmental Stress Physiology, Purdue University, 1165 Horticulture Bldg., West Lafayette, IN 47907-1165. Tel.: 765-494-1316; Fax: 765-494-0391; E-mail: paul.m.hasegawa.1@purdue.edu.

Published, JBC Papers in Press, June 25, 2002, DOI 10.1074/jbc.M205037200

	ABBREVIATIONS

The abbreviations used are: HOG, high osmolarity glycerol; CDRE, calcineurin-dependent response element; MAP, mitogen-activated protein; BAPTA, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; MES, 2-[N-morpholino]ethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				L. Maresova, S. Muend, Y.-Q. Zhang, H. Sychrova, and R. Rao Membrane Hyperpolarization Drives Cation Influx and Fungicidal Activity of Amiodarone J. Biol. Chem., January 30, 2009; 284(5): 2795 - 2802. [Abstract] [Full Text] [PDF]

				J. Teng, R. Goto, K. Iida, I. Kojima, and H. Iida Ion-channel blocker sensitivity of voltage-gated calcium-channel homologue Cch1 in Saccharomyces cerevisiae Microbiology, December 1, 2008; 154(12): 3775 - 3781. [Abstract] [Full Text] [PDF]

				B. Martinac, Y. Saimi, and C. Kung Ion Channels in Microbes Physiol Rev, October 1, 2008; 88(4): 1449 - 1490. [Abstract] [Full Text] [PDF]

				S. Loukin, X. Zhou, C. Kung, and Y. Saimi A genome-wide survey suggests an osmoprotective role for vacuolar Ca2+ release in cell wall-compromised yeast FASEB J, July 1, 2008; 22(7): 2405 - 2415. [Abstract] [Full Text] [PDF]

				A. Ruiz, R. Serrano, and J. Arino Direct Regulation of Genes Involved in Glucose Utilization by the Calcium/Calcineurin Pathway J. Biol. Chem., May 16, 2008; 283(20): 13923 - 13933. [Abstract] [Full Text] [PDF]

				V. S. Anil, P. Rajkumar, P. Kumar, and M. K. Mathew A Plant Ca2+ Pump, ACA2, Relieves Salt Hypersensitivity in Yeast: MODULATION OF CYTOSOLIC CALCIUM SIGNATURE AND ACTIVATION OF ADAPTIVE Na+ HOMEOSTASIS J. Biol. Chem., February 8, 2008; 283(6): 3497 - 3506. [Abstract] [Full Text] [PDF]

				A. Ruiz and J. Arino Function and Regulation of the Saccharomyces cerevisiae ENA Sodium ATPase System Eukaryot. Cell, December 1, 2007; 6(12): 2175 - 2183. [Full Text] [PDF]

				K. Iida, J. Teng, T. Tada, A. Saka, M. Tamai, H. Izumi-Nakaseko, S. Adachi-Akahane, and H. Iida Essential, Completely Conserved Glycine Residue in the Domain III S2 S3 Linker of Voltage-gated Calcium Channel {alpha}1 Subunits in Yeast and Mammals J. Biol. Chem., August 31, 2007; 282(35): 25659 - 25667. [Abstract] [Full Text] [PDF]

				J. Yadav, S. Muend, Y. Zhang, and R. Rao A Phenomics Approach in Yeast Links Proton and Calcium Pump Function in the Golgi Mol. Biol. Cell, April 1, 2007; 18(4): 1480 - 1489. [Abstract] [Full Text] [PDF]

				A. Santos, M. del Mar Alvarez, M. S. Mauro, C. Abrusci, and D. Marquina The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin J. Biol. Chem., December 23, 2005; 280(51): 41881 - 41892. [Abstract] [Full Text] [PDF]

				D. Uccelletti, F. Farina, P. Pinton, P. Goffrini, P. Mancini, C. Talora, R. Rizzuto, and C. Palleschi The Golgi Ca2+-ATPase KlPmr1p Function Is Required for Oxidative Stress Response by Controlling the Expression of the Heat-Shock Element HSP60 in Kluyveromyces lactis Mol. Biol. Cell, October 1, 2005; 16(10): 4636 - 4647. [Abstract] [Full Text] [PDF]

				S. Noma, K. Iida, and H. Iida Polarized Morphogenesis Regulator Spa2 Is Required for the Function of Putative Stretch-Activated Ca2+-Permeable Channel Component Mid1 in Saccharomyces cerevisiae Eukaryot. Cell, August 1, 2005; 4(8): 1353 - 1363. [Abstract] [Full Text] [PDF]

				K. Maeta, S. Izawa, and Y. Inoue Methylglyoxal, a Metabolite Derived from Glycolysis, Functions as a Signal Initiator of the High Osmolarity Glycerol-Mitogen-activated Protein Kinase Cascade and Calcineurin/Crz1-mediated Pathway in Saccharomyces cerevisiae J. Biol. Chem., January 7, 2005; 280(1): 253 - 260. [Abstract] [Full Text] [PDF]

				L. Viladevall, R. Serrano, A. Ruiz, G. Domenech, J. Giraldo, A. Barcelo, and J. Arino Characterization of the Calcium-mediated Response to Alkaline Stress in Saccharomyces cerevisiae J. Biol. Chem., October 15, 2004; 279(42): 43614 - 43624. [Abstract] [Full Text] [PDF]

				V.-K. Ton and R. Rao Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases Am J Physiol Cell Physiol, September 1, 2004; 287(3): C580 - C589. [Abstract] [Full Text] [PDF]

				C. L. Lawrence, C. H. Botting, R. Antrobus, and P. J. Coote Evidence of a New Role for the High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway in Yeast: Regulating Adaptation to Citric Acid Stress Mol. Cell. Biol., April 15, 2004; 24(8): 3307 - 3323. [Abstract] [Full Text] [PDF]

				N.-H. Cheng, J. K. Pittman, J.-K. Zhu, and K. D. Hirschi The Protein Kinase SOS2 Activates the Arabidopsis H+/Ca2+ Antiporter CAX1 to Integrate Calcium Transport and Salt Tolerance J. Biol. Chem., January 23, 2004; 279(4): 2922 - 2926. [Abstract] [Full Text] [PDF]

				H. Koiwa, F. Li, M. G. McCully, I. Mendoza, N. Koizumi, Y. Manabe, Y. Nakagawa, J. Zhu, A. Rus, J. M. Pardo, et al. The STT3a Subunit Isoform of the Arabidopsis Oligosaccharyltransferase Controls Adaptive Responses to Salt/Osmotic Stress PLANT CELL, October 1, 2003; 15(10): 2273 - 2284. [Abstract] [Full Text] [PDF]

				S. S. Gupta, V.-K. Ton, V. Beaudry, S. Rulli, K. Cunningham, and R. Rao Antifungal Activity of Amiodarone Is Mediated by Disruption of Calcium Homeostasis J. Biol. Chem., August 1, 2003; 278(31): 28831 - 28839. [Abstract] [Full Text] [PDF]

				T. Tada, M. Ohmori, and H. Iida Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1 J. Biol. Chem., March 7, 2003; 278(11): 9647 - 9654. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/36/33075    most recent M205037200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matsumoto, T. K. Articles by Hasegawa, P. M. Search for Related Content PubMed PubMed Citation Articles by Matsumoto, T. K. Articles by Hasegawa, P. M. Social Bookmarking                      What's this?