c&l// c&km

(1992) 13, 615-826

0 Longman Group UK Ltd 1992

Characterization of the energy-dependent, mating factor-activated Ca*+ influx in Saccharomyces cerevisiae K.R. PRASAD and P.M. ROSOFF Departments of Pediatrics (Hematology/Oncology) and Physiology, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA Abstract The yeast mating pheromones, a and a factors, bind to specific G protein-coupled receptors in haploid cells and bring about both growth arrest in the early GI phase of the cell cycle and differentiation into mating capable cells. This induces an increase in Ca2+ influx leading to elevated intracellular calcium concentrations, which has been shown to be essential for subsequent downstream events and the mating process itself [I]. We have characterized the a factor induced increase in cellular Ca2+ in wild type S. cerevisiae and in the temperature-sensitive cell division cycle mutants cdc7 and cdc28 which are growth-arrested at the Go-G1 border at the nonpermissive temperature. We observed a 2-4 fold increase in the initial velocity of Ca2+ influx in 01factor-treated wild-type cells and in cdc7 and cdc28 cells grown at the nonpermissive temperature. Calcium influx was energy dependent, inhibited b8 membrane depolarization and slightly increased by hyperpolarization. Furthermore, Ca ’ influx was sensitive to both divalent and trivalent cations, but was unaffected by nifedipine and verapamil. These data demonstrate that budding yeast possesses a regulated Ca2+ transport mechanism, the activation of which is dependent upon exit out of the cell cycle and growth cessation. This transport mechanism has many similarities to that observed in mitogen-stimulated mammalian cells.

The role of cellular Ca2’ as a molecule involved in intracellular signalling events has been extensively Abbreviufiom : [Ca2+]i, intracellular Ca2’ concentration; n, permissive temperature; NPT, nonpermissive temperature, MES. 2-(N-morpholino) ethanesulphonic acid; EGTA. ethylene glycol bis@aminoethyl ether)-N,N,N’,N’tetraacetic acid; CCCP. carbonyl cyanide lOchloropheny1 hydrazine; 2.4DNP. 2,Cdinitmphenol; G protein, guanine nucleotide binding protein.

studied in mammalian cells. It has been shown to play an essential role in secretion, muscle contraction, neurotransmitter release, fertilization, cell division and differentiation [2]. Stimulation of many plasma membrane receptor systems leading to cell growth and proliferation results in an invariable increase in intracellular Ca2+ that has been shown to be necessary, although not sufficient, for the initiation of subsequent downstream events. Our laboratory has focussed on characterizing the influx 615

616

of Ca2’ associated with stimulation of the T lymphocyte antigen receptor complex. In T lymphocytes, like many other mitogen-stimulated cells, a rapid increase in [Ca2+]i accompanies receptor activation, This is due to both a release of Ca2’ from intracellular storage sites, mediated by inositol1,4,Nrisphosphate, and an influx via a membrane potential-sensitive Ca2+ channel [3-51. We have also shown that this channel is inhibited by lanthanides and the stilbene disulfonate, DIDS, although it is insensitive to inhibitors of voltage-gated Ca2’ channels [3,6]. Due to the lack of specific, high-affinity inhibitors of this channel, attempts to both further characterize and purify it have been frustrating. For this reason, we elected to study a potentially homologous Ca2+transport system in a genetically manipulable lower eukaryote, namely the budding yeast Saccharomyces cerevisiae. In S. cerevisiae, the mating pheromones a and a initiate the mating process of haploid cells by binding to specific receptors on cells of the opposite mating type. The receptors for a and a factors are the gene products of the STE3 and STEL?genes respectively, which are members of the rhodopsin, fi adrenergic and muscarinic acetylcholine receptor family characterized by 7 transmembrane hydrophobic domains [7]. Signal transduction via the a and a receptors, like similar receptors in higher eukaryotes, also appears to be coupled to GTP binding proteins [8-101, although the second messenger-generating system(s) which they stimulate remain undefined. Exposure of haploid cells to mating pheromones brings about rapid alterations in the pattern of gene expression [ll, 121, induces the expression of cell agglutinins [131 and arrests growth of the cells in the early GI phase [14, 151. The phenotype of the cells changes dramatically as they differentiate into cells capable of mating with cells of the opposite mating type. The growth arrest is required for successful mating and differentiation. Relatively large quantities of Ca2’ are stored in intracellular vacuoles and yeast appear to have a ve efficient mechanism to augment intracellular Ca55 levels by mobilizing stored Ca2’ 1161. However the question of whether Ca2’ is required for the growth of this organism has been debated for several years. Conclusive evidence that it is

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essential for yeast cell growth came from several recent studies [17]. It was shown that the simultaneous addition of the calcium ionophore A23187 and EGTA to yeast grown in a Ca2’ deficient synthetic dextrose medium resulted in the de letion of both extracellular and intracellular CaR leading to growth arrest However, this type of growth arrest, resulting from depletion of both intra- and extracellular calcium indicated that calcium is essential for vegetative growth of these organisms. Associated with mating pheromone stimulation is an increased influx of extracellular Ca2+ leading to elevated intracellular Ca2’ levels in either a-treated Mata cells or a-treatedMatA cells [18]. The Ca2’ influx was shown to be essential for completion of downstream events in the differentiation program [l]. The Ca2’ influx started as early as 15 min after exposure to mating factor and continued for at least 90 min. Furthermom. it was sensitive to cycloheximide, suggesting that new protein synthesis was involved [18]. That the Ca2’ influx might be associated with the initiation of differentiation was suggested by experiments showing that the temperature-sensitive mutants cdc7 and cdc24, which are arrested in early Gr phase, also show marked increase in Ca2’ influx when shifted to the non-permissive temperature [19]. It thus appears that Ca2+ plays a role in supporting both vegetative cell growth as well as possibly a signaling function in mating factor-induced growth arrest and differentiation. The transport mechanism responsible for regulated Ca2’ influx has not been previously characterized. In this paper, we have addressed the question whether the receptor-mediated increase in Ca2’ influx observed in S. cerevisiae associated with growth cessation and the onset of differentiation is similar to that seen in mitogen-stimulated higher eukaryotes. We have confirmed the prior observations of other groups and have expanded them to characterize the growth-arrest activated Ca2+ transporter in these cells. Our results demonstrate that, while not identical, the 2 transport systems are quite similar and, combined with the knowledge of their apparent functional homology, suggests that the use of changes in [Ca2+]i as an intracellular signal of growth and differentiation is evolutionarily conserved in eukaryotes.

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Materials and Methods Strains and culture conditions

The S. cerevisiae haploid strains A364A (MATa). t.s.124 (MATa cdc7-1: a, ade 1, ade 2, ura 1, his 7, lys 2, ryr 1, gal 1) and STX326-8B (MATa &28-l: a, ade 1, gal 1, lys 2, met 14, his 7, tyr I), were obtained from the Yeast Genetic Stock Center, Berkeley, CA, USA. The cells were grown as haploids in YEPD medium containing 1% yeast extract (Difco), 2% Bacto peptone (Difco) and 2% glucose (Sigma). The cdc7 and cdc28 cells were maintained in YEPD medium at 23°C and A364A was cultured at 30°C. Cell number was determined in a Coulter Model ZM Counter using a 50 pm cut-off aperture. The A364A cells were treated with a factor (3.0 @4) for 3 h as described [18]; this was sufficient to achieve complete growth arrest. The cdc7 and cdc28 strains were grown at 38°C (nonpermissive temperature) for 4 h which was sufficient for complete growth arrest. In both cases, growth arrest was monitored by counting the cell number as well as confnming the terminal phenotypes under light microscopy. Reagents

Alpha factor, valinomycin, 2,4-DNP, CCCP, verapamil and nifedipine were obtained from Sigma (St Louis, MO, USA). [45Ca]-CaClz was obtained from New England Nuclear (specific activity 732 mCi/ mmol). All other chemicals used were of reagent grade. Ca2+ accumulation

The method used to measure Ca2+ influx was essentially as described 1181. Exponentially growing A364A cells were harvested, washed twice with distilled water and suspended in fresh YEPD medium. The cell suspension (1-2 x lo7 cells/ml) was divided into 2 x 2 ml aliquots in 50 ml Falcon tubes. The final concentration of Ca” in YEPD was determined to be 220 p.M by a Ca2+-sensitive electrode (data not shown). One received a factor (3 pM, final concentration) and the other one was used as control. 4sCa2t (25 BCi) was added to each

tube and incubated at 30°C with shaking. Every 30 min, 100 pl of the culture was diluted with 6 ml of chilled 20 mM CaC12, filtered quickly on Whatman-glass microfiber GF/A filters in a Millipore vacuum fdtration apparatus and washed twice with 10 ml of the same solution. Radioactivity retained on the filters was counted in a Beckman Scintillation Counter. Velociry of Ca2’ uptake

These experiments were designed to measure the linear velocity of Ca2+ uptake in cells after prior exposure to mating factor or switch to NPT or PT. Cells were harvested, washed twice with distilled water, and resuspended in MES buffer (10 mM MES/Tris pH 6.0, 1oomM glucose) at a density of 2-4 x lo7 cells/ml. The cells were preincubated for 2-3 min at the Tequisite temperature. Ca2’ uptake was initiated by the addition of [45Ca]-CaC12 (8.0 pCi) and was followed for 6-7 min. At every time point, a 100 @ aliquot was removed, diluted with ice-cold 20 mh4 CaClz and filtered rapidly on membrane filters as described above. The radioactivity retained on the filters was counted in a Beckman Scintillation Counter. The results were plotted as cpm/106 cells against time and the initial velocity of Ca2+ uptake was obtained by linear regression analysis using either CricketGraph (Cricket Software) or KaleidaGraph (Abelbeck Software) on a Macintosh SE computer. A significant increase in Ca2+ uptake in both a factor-treated wild type cells as well as in cdc7 and cdc28 cells at However, the NPT was consistently observed. magnitude of stimulation varied between 2-5 fold increase compared to control from experiment to experiment. The data shown are representative of a typical experiment carried out at least 3 times in which qualitatively similar results were obtained.

Re.WltS Growth-associated calcium in.ux

We first wished to see if we could replicate the results of Oshumi and Anraku 1181. using different strains and a different source of a factor. Treatment

618

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Fig. 1 Effect of growth anest induced by a factor on Ca”

uptakein A364A (wild type) S. csrevisfue and shift from PT to NPT in cdc7

and cdc28 mutants.

wild type A364A cells.

described

(A)

in Materials

Ca2’ accumulation

and Methods;

in a-treated

(B) Tbe initial velocity of Ca”

velocity of Ca2’ influx in cdc7 cells at both FT (23-C) and NPT (38’C); (23°C) and NPT (38’0; experiments

performed

(FJ The effect of temperature at least three times

Ca2’ accumulation

influx in mating factor-treated

was measured wild type cells;

(D) The initial velocity of Ca2+ irdkx

using “Ca’+ (C)

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in cdc28 cells at both PT

on Ca*’ uptake in A364A wild type oells. The data shown rmz representative

of

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of ‘wild type’ A364A cells with CIfactor resulted in significantly increased 45Ca2+ influx after a lag period of about 45 min (Fig. 1A). However, a rise in intracellular 4sCa2t was observed as early as 15-30 min after addition of the a factor. This was also reflected in measurements of the velocity of uptake of Ca2+ in cells treated with 01factor for 3 h. The velocity of Ca2’ uptake in a treated cells was significantly higher than in untreated cells (Fig. 1B). These data suggest that treatment with a factor is temporally associated with an increase in cellular calcium due to an influx from an extracellular source, and confirm previously reported findings [W. We next carried out Ca2’ influx measurements in the cdc7 and cdc28 mutant strains at both the PT (23°C) and NPT (38°C). Both mutations affect proteins that are active at the early Gl phase in the yeast cell cycle termed ‘start’ 1201. The CDC7 gene encodes a putative protein ser/thr kinase [21], while that encoded by the CDC28 gene is homologous to p34cdc2 in Schizosaccharomycespombe, a protein ser/thr k.inase involved in entry into the cell cycle 1221. These mutants show normal growth at 23°C but when shifted to 38”C, their growth is arrested in early Gl phase [23]. This was confirmed by monitoring cell number and examining the terminal phenotypes under light microscopy. If regulated calcium influx was initiated by exit out of the cell cycle, as occurs when cells are treated with a factor, then we would expect that these cells would display a similar phenotype to a-treated cells. It is evident that growth arrest induced by shifting cdc7 and cdc28 cells to the NPT produced a 2-3 fold increase in the velocity of Ca2+ uptake as shown in Figure 1 (C and D). In contrast to a previous report [19], we have consistently observed elevated Ca2’ uptake in cdc28 mutant cells at the NPT. Such alterations in Ca2’ uptake were not observed in wild type cells following temperature alterations (Fig. 1E). Interestingly, the increased accumulation of Ca2’ in response to both a factor and NPT was not greater than either alone, suggesting a common saturable element in their transport pathways (data not shown). These data suggest that there is a temporal association between exit out of the cell cycle (i.e. the onset of growth arrest) and Ca2’ entry. Since the function of cx factor is to induce differentiation

619

in these cells, which must be preceded by a cessation of row& these data support a role for 2f elevated [Ca ]i in this process. Characterizationof the Ca2+ transportmechanism We next wished to determine the relationshi 2T between the growth arrest-induced increase in Ca uptake observed in yeast and that which we have previously characterized in mitogen-stimulated T cells. We therefore tested in yeast the effects of different pharmacological agents known to alter the mitogen-stimulated Ca2’ uptake in T cells. Effect of membrane potential In T cells and basophils, stimulation of either the antigen receptor or the IgE receptor activates a membrane potential-sensitive Ca2’ influx [3, 4, 241. The dependence on stability of the membrane potential distinguishes this transporter from that present in many types of electrically excitable cells in which depolarization activates the channel [25]. Like higher eukaqotes, yeast maintain a membrane potential of approximately -90 mV (negative inside) that is largely dependent upon an extracellularintracellular electrochemical gradient of Kt [26,27]. Therefore, collapse of this gradient by addition of KC1 to the medium should lead to membrane depolarization. As shown in Figure 2A, 25 mM KC1 inhibited the c1 factor-induced increase in the velocity of Ca2+ uptake, measured in wild type cells. This effect was not due to an increase in either extracellular ionic strength or osmolarity since neither choline chloride (25 n&I) nor sucrose (50 mM, data not shown), had a significant effect on the velocity of Ca2’ uptake (Fig. 2A). Similar trends were also observed in the cases of the cdc7 and cdc28 mutants as shown in Figure 2B and 2C. We have also tested the effect of valinomycin, a Kt ionophore. This agent causes hyperpolarization of the plasma membrane due to a loss in intracellular potassium and blocks mitogen-activated membrane potential-sensitive Ca2+ channels in T cells [3]. As shown in Figure 2D, valinomycin had a slight stimulatory effect on growth arrest-induced Ca2’ influx in S. cerevisiae. This was only consistently observed in the cdc7 and cdc28 cells shifted to the NFT. and not in either basal Ca2’

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Fig. 2 Effect of membrane potential on the initial velocity of Ca2’ uptake. u Ca2t uptake measurements were performed as described in Materials and Methods. The values obtained for untmated A364A cells and cdc7 and c&28 at PT wem considered as 100%. Velocity measurements made on treated cells am represented in comparison to their respective controls. Depolarization was induced by the addition of 25 mM KC1 which was compared to choline chloride (25 n&l) added 6 min prior to the addition of “Ca2+. Hyperpohuization was induced by the addition of 50 pJVlvalinomycin 10 min before the addition of 4sCa2t. (A) A364A (wild type) cells f a factor ; (B) c&7 at IT (23’C) and NIT (38T); Q cdc28 at PT (23’0 and NET (38’C); (D) Hyperpolakation induced by valinomycin. The data shown are repnz-sentativeof similar trends observed in 3 different sets of separate experiments

influx or that stimulated by treatment with CIfactor. These data demonstrate that the Ca2’ transporter activated in these cells may also be regulated to some extent by the membrane potential. However, unlike the channel stimulated by growth and differentiation factors in mammahan cells, it is not inhibited by hyperpolarization. Effect of proton uncouplers

We next addressed the question of whether the increased Ca2’ uptake was energy dependent, We therefore tested the effects of 2,4-DNP and CCCP, 2 proton uncouplers and inhibitors of oxidative

phosphorylation. Results from these experiments indicated that increased Ca2’ uptake observed in a factor-treated A364A cells and the cdc7 and cdc28 mutants at NPT were inhibited by both CCCP and 2,4-DNP (Fig. 3A, 3B). Interestingly, these agents had no or minimal effects on the basal Ca2’ influx, suggesting that the Ca2’ influx associated with growth arrest and differentiation does not contribute to basal Ca2’ homeostasis in a significant manner. It would also suggest that this process is a regulated one which is only active during these receptormediated events. These results demonstrate that the growth arrest-induced Ca2’ uptake is energy

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621

under basal conditions (Fig. 4D). All 4 cations, howeve;p’oduced profound inhibition of the stimub ated Ca uptake. These data suggest that divalent cations may compete for transport via the Ca2+ channel. It remains to be determined whether this competition is at an extracellular Ca2’ binding site, or represents actual transport via the channel, thus excluding Ca2+. The difference in the action of Ni2+ compared to other divalent cations on basal Ca2’ uptake also remains to be explained.

Fig. 3

The effect of inhibitors of oxidative phosphorylation on

uptake. CCCP (100 m A) and 2,4-DNP priorto tbe addition of 4sCa2’ and the initial velocity of Ca uptake was performed as described in Materials and Methods. The results am replesentative of similar trends observed in 3 different sets of experiments the velocity of

Ca2+

(500 pM: B) were added 6 min

dependent, while Ca2’ transport in actively proliferating ceils is not. Effec! of divalentcarions Several calcium channels have been described in a wide variety of tissues that can be inhibited by the presence of other divalent cations, such as Mg2+, Mn2+, or Cd2’ [28]. We therefore tested the effects of other divalent cations on the velocity of Ca2’ uptake, in order to assess their relative s~+ticity on growth arrest-ineed increases in Ca influx. Mn2+, Mg2+$+Cd had slight inhibitory effects oy the basal Ca flux [Fig. 4A-4c). In contrast, NI produced significant enhancement of Ca2’ influx

Effect of rrivalenrcations In T lymphocytes, La3’ (a rare earth group metal) is effective in inhibiting Ca2+ influx [3 In wild type 1 yeast cells, La3+ inhibits basal Ca ’ uptake [29]. We therefore studied the actions of La3+ and Gd3+, another lanthanide, on growth arrest-stimulated Ca”’ influx. The results are presented in Figure 5A and 5B. At a concentration of 1 pM, La3’ had a slight inhibitory effect on basal Ca2’ uptake. However, it demonstrated a much more pronounced inhibition on elevated Ca2+ uptake due to CIfactor stimulation of wild type cells or a temperature shift to the NPT in cdc7 and cdc28 cells. Gd3+ was even more effective on a molar basis than La3+ in blocking the stimulated Ca2’ influx in these cells; it was at least two orders of magnitude better than La3’ with a Ki of approximately 1 @I (data not shown). These results show that trivalent cations inhibit the yeast growth-regulated calcium channel similar to that observed in mammalian cells. Effect of voltagegated Ca2+ channel blockers Verapamil and nifedipine. two voltage-activated Ca2’ channel blockers have been extensively used to characterize Ca2’ channels in a variety of vertebrate cells [25, 301. We and others have previously reported that these agents have no effect on the mitogen-stimulated influx of Ca2+ in mammalian cells [3, 41. However, others have suggested that these compounds block Ca2” influx [31]. We tested their effects on yeast, both in a factor-treated A364A cells and the cdc7 and cdc28 mutant cells. The results are shown in Figure 6. Verapamil and nifedipine had no inhibitory effect on either basal or growth arrest-induced Cazt uptake, even at 100 pM, a concentration several orders of magnitude greater than their Ki for the dihydropyridine sensitive Ca2’

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channel of cardiac muscle r2.5, 301. These data clearly confirm the fact that the elevated Ca2+influx seen in yeast related to growth arrest is not due to activation of voltage-gated Ca2+ channels that are sensitive to these drugs.

Discussion

In this report, we have investi ated the character!+ istics of a growth-associated Ca influx mechanism in the haploid budding yeast S. cerevisiue. In these cells, physiological growth arrest occurs upon exposure to mating pheromone which initiates differentiation into a cellular phenotype capable of mating. Our goal was to identify both common and distinctive features of the yeast Ca2’ transporter as

compared with the growth factor-stimulated Ca2’ infIux mechanism found in mammalian cells. In human T lymphocytes, the latter has been noted to be essentially inactive in the unstimulated state, and inhibited by alterations in the plasma membrane potential, La3+, and the stilbene disulfonate, DIDS [3, 4, 61. In addition, it has variously been found to be either insensitive 13,41 or sensitive [31] to inhibition by verapamil and the dihydropyridine class of calcium channel blockers. The data presented in this paper demonstrate that the yeast Ca2+ transporter has some important similarities to, as well as some distinct differences from, the transporter found in some human cells. In agreement with a previous report [18], we observed a significant increase in the initial velocity of Ca2’ influx in a factor-treated, wild type A364A loo0

cd%

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cd++

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Fig. 4 The effect of divaknt

cations on the initial velocity of Ca” uptake.

MnClz (50 pM: A), MgClz (250 pM: B), Cdclz (500 JIM: C),

and NiClz (250 pM: D) were added 6 mio priorto the addition of 4’Ca2’ and the initial velocity described in Materials and Methods. Similar results were obtained in 3 different sets of experiments

of Ca2+ uptake

was performed

as

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inhibitory effect in the growth arrest-related increase in Ca2” influx in yeast in the presence of high [K’lo (Fig. 2). Valinomycin, a K’ ionophore which will hyperpolarize cells, was also shown to block the mitogen induced increase in Ca” influx in T cells 131. These observations led us to suggest that the T cell Ca2’ channel was membrane potential sensitive in that it could be inhibited by both increases and decreases in the electric field across the membrane (YP). Interestingly, in yeast cells valinomycin had a modest stimulatory effect when cdc7 and c&28 cells were shifted to the NPT, thus leading to growth arrest (Fig. 2D). This phenomenon was not observed when wild type cells were treated with a factor. Basal Ca2’ flux was not affected by changes in YP. These data suggest that while stimulated Ca2’ influx in yeast is inhibited by depolarization, it

Fig.

5

The

effect of trivalent cations on the initial velocity of

Ca” uptake. LaC% (1 pMz A) or Gda

(1 pMz B) were ad&d 6

min prior to the addition of 45Ca2+ and the initial velocity of Ca2+ uptake was performed as described in Materials and Methods. The results

shown

are representative

of 3 different

sets of

separate experiments

cells. After the removal of cI factor, the cells returned to normal levels of Ca2+influx in about 5 h (data not shown). Although the results presented in this paper with respect to cdc7 are similar to a previous report [19], the findings with cdc28 are different. In our hands we consistently observed a 2-3 fold increase in Ca2’ influx in cdc28 cells at NPT. We and others [3, 4, 32, 331 have shown that depolarization induced by high concentrations of extracellular K+ resulted in the inhibition of T cell antigen receptor-stimulated increases in Ca2’ influx in T lymphocytes. We also observed a similar

Fig. 6 The effect of voltage-gated Ca*’ chant4 velocity of Ca2’ uptake.

blocks

on the

Nifedipine (100 pMz A) or vempamil

(100 pM: B) were added 6 min prior to the addition of 4sCa2+ and the initial velocity of Ca2’ uptake was performed as described in Materials

and Methods.

The results are repssentative

different sets of separate experiments

of 3

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is not affected by hyperpolarization induced by valinomycin. A recent report indicated that valinomycin in fact augmented the already enhanced intracellular Ca2+ increase observed in an activated CD4 positive T cell clone [31], possibly secondary prolonged treatment with valinomycin leading to depletion of intracellular ATP levels. Similarly, Miyamoto and Racker [34] observed enhanced Ca ’ influx in plasma membrane vesicles of bovine heart treated with valinomycin. While this certainly may be true, studies using this agent as an inhibitor of receptor-activated calcium influx have typically added the ionophom only seconds-to-minutes prior to stimulating the cells, which is probably too short a time for significant ATP depletion to occur. The plasma membrane ATPase inhibitors N,N-dicyclohexylcarbodiimide (DCCD), diethylstilbesterol and sodium orthovanadate also were shown to cause hyperpolarizatiou in yeast cells along with a concomitant increase in the carrier-mediated Ca2+ uptake [27]. Our findings with valinomycin are similar. Unlike mammalian T cells, we observed an increase in both the basal as well as growth arrestrelated increase in Ca2’ influx when cells were exposed to valinomyciu at the NPT (cdc7 and cdc28). Thus the receptor-mediated increase in Ca2’ uptake appears to be regulated by the membrane potential. We have also observed that the a factorstimulated Ca2’ influx in S. cerevisiue is dependent on metabolic energy. This was based on the observations that both CCCP and 2,4-DNP, effective inhibitors of oxidative phosphorylation, almost completely blocked the influx of Ca2’ in cells treated with either mating pheromone or in the cdc7 and cdc28 cells exposed to the NPT (Fig. 3). Interestingly, these compounds had very little effect on the resting, basal, transmembrane Ca2’ flux, again suggesting that the stimulated flux is an active process. However, since the initial increase in [Ca2+]i does not occur for at least 15 min after exposure to either 0: factor or shift to the NRT, it is distinctly possible that the actual trausport mechanism itself does not require metabolic energy; rather, a necessary, antecedent event is energy dependent. Though there have been several published reports on the effects of various monovalent and

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divalent cations on basal Ca2’ influx in yeast [26, 351,them are virtually no reports on their effects on the growth arrest-related increase in Ca2’ influx. Our results, based on initial velocity measurements of Ca2+ influx, indicated that the divalent cations tested had au inhibitory effect on the activated Ca2’ influx. Ni2+ appeared to have opposite effects on the basal and activated Ca2’ influx and thus can be used to distinguish between these two types of Ca2+ influx. In the case of mammalian cells, similar observations have been made with respect to growth factor receptor-mediated increases in Ca2+ influx, with the most effective divalent cation inhibitor being Mn2’ [28]. Because of the paucity of reproducible electrophysiological data concerning these channels, it remains unclear what the mechanism of this inhibition may be, although competition for access to a conducting ‘pore’ is certainly a possibility. It is well-known that the trivalent cation La3+ cau be used to inhibit Ca2+channels in electrically excitable tissue 1281. This compound has also been used to study transport mechanisms in oonelectrically excitable cells. For instance, we showed that La3+effectively blocked the mitogen-stimulated increase in Ca2+ influx in T cells [3]. A mcent study showed that Gd3+,another lanthanide, inhibited a mechauosensitive Ca2’ channel in Uromyces appendiculatus, a fungus that infects bean leaf stomata 1361. This study was done using the patch clamp technique with protoplasts isolated from germ tubes. Application of pressure leads to the activation of these channels which then pump in Ca2’ (and other ions) which may be necessary for differentiation of the germ tube. We also observed that the trivalent cations La3+and Gd3+blocked the increased Ca2’ influx normally seen in a-treated A364A cells and cdc7 and cdc28 mutants grown at NRT, with Gd3’ being more effective than La3+ (Fif+ 5). Nifedipine and verapamil, voltage-gated Ca channel blockers, do not affect the mitogen activated Ca2+influx mechanism in T cells (Rosoff, unpublished observations and [37]). These observations thus support a model in which growthstimulated Ca2’ channels, which can be inhibited by membrane depolarization (rather than being activated by it), would appear to have a number of similarities in both yeast aud mammalian cells.

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Unlike mammalian cells in which growth stimulation leads to increased Ca2+ influx, growth arrest an effect of a factor on A364A wild type cells and in the cdc7 and cdc28 mutants cultured at NPT induces elevated Ca2’ influx. Though both types of Ca2’ influx are the results of different biological effects, they appear to be similar. What might be the molecular, biochemical basis of the stimulated Ca2+ influx in these cells? The reproducible tern oral delay of 15-20 min before measurable 2P Ca influx is achieved, suggests that there are a number of intermediary events that must occur prior to activation of the plasma membrane Ca2’ channel. Since 01factor binds to a G protein-linked receptor, we can assume that there are proximal signalling events that lead to downsWam responses that ultimately lead to Ca2’ influx. At this time it is unknown what second messengers are produced in response to mating factor stimulation. However, both the cdc7 and cdc28 mutations, which result in growth arrest of the cell in the ‘start’ phase of Gl of the cell cycle, produce defective protein ser/thr kinases that are crucial for the cell to enter Gl [38-421. It is known that new protein synthesis is required for the initiation of the increase in Ca2+ influx after 01factor stimulation [18]. We have also noted the effect of cycloheximide and have extended these observations to show that actinomycin D (an inhibitor of transcription) also blocked activated Ca2’ influx (Sikorski and Rosoff, unpublished observations). These data suggest that the immediate events that result from stimulation of the mating factor receptor, leading to the down-regulation of the cdc7 and cdc28 gene products, thus promoting exit out of the cell cycle, activate the transcription and translation of a gene (or genes) that is instrumental in leading to Ca2+ influx. Indeed, Stetler and Thomer have cloned a number of cDNAs from newly transcribed genes in S. cerevisiue that arise very shortly after exposure to CLfactor [ll]. Could one or more of these newly transcribed genes include one encoding a protein that constitutes either the activated calcium transport mechanism or a crucial regulatory protein that is required for its Either of these models is certainly activation? possible and is consistent with the observation that the basal activity of the channel (as measured by 45Ca2t flux) is minimal. Even though there is

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ample evidence that G proteins can regulate the activity of a number of different types of ion channels, includ- ing those conducting Ca2+ [43], it is unlikely to be so in this case, due to the time lag of activation and the necessity for new protein synthesis. Based on these studies we suggest that CI factor-treated A364A cells and cdc7 and cdc28 mutants can serve as a model to isolate and further characterize the proteins that constitute the receptor mediated Ca2’ influx mechanism.

Acknowledgements The authors wish to thank Drs Dona Chikaraishi and Kathleen Dunlap for critical reading of the manuscript. This work was supported by Research Grant BE-19 fzom the American Cancer Society. PMR is a Scholar of the Leukemia Society of America.

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Please send reprint requests to: Dr Philip M. Rosoff, Department of Pediatrics (Hematology-Oncology), New England Medical Center (Box #14), 750 Washington Street, Boston MA 02111, USA Received : 6 March 1992 Revised : 9 April 1992 Accepted : 30 April 1992