Stimulation of Strontium Accumulation in Linoleate-Enriched Saccharomyces cerevisiae Is a Result of Reduced Sr²⁺ Efflux

Abstract

The influence of modified plasma membrane fatty acid composition on cellular strontium accumulation in Saccharomyces cerevisiae was investigated. Growth of S. cerevisiae in the presence of 1 mM linoleate (18:2) (which results in 18:2 incorporation to ∼70% of total cellular and plasma membrane fatty acids, with no effect on growth rate) yielded cells that accumulated Sr²⁺ intracellularly at approximately twice the rate of S. cerevisiae grown without a fatty acid supplement. This effect was evident over a wide range of external Sr²⁺ concentrations (25 μM to 5 mM) and increased with the extent of cellular 18:2 incorporation. Stimulation of Sr²⁺ accumulation was not evident following enrichment of S. cerevisiae with either palmitoleate (16:1), linolenate (18:3) (n-3 and n-6 isomers), or eicosadienoate (20:2) (n-6 and n-9 isomers). Competition experiments revealed that Ca²⁺- and Mg²⁺-induced inhibition of Sr²⁺ accumulation did not differ between unsupplemented and 18:2-supplemented cells. Treatment with trifluoperazine (TFP) (which can act as a calmodulin antagonist and Ca²⁺-ATPase inhibitor), at a low concentration that precluded nonspecific K⁺ efflux, increased intracellular Sr²⁺ accumulation by approximately 3.6- and 1.4-fold in unsupplemented and 18:2-supplemented cells, respectively. Thus, TFP abolished the enhanced Sr²⁺ accumulation ability of 18:2-supplemented cells. Moreover, the rate of Sr²⁺ release from Sr²⁺-loaded fatty acid-unsupplemented cells was found to be at least twice as great as that from Sr²⁺-loaded 18:2-enriched cells. The influence of enrichment with other fatty acids on Sr²⁺ efflux was variable. The results reveal an enhanced Sr²⁺ accumulation ability of S. cerevisiae following 18:2-enrichment, which is attributed to diminished Sr²⁺ efflux activity in these cells.

The interactions of microorganisms with heavy metals and radionuclides have provoked considerable interest in recent years. Much research has focused on microbial sequestration of toxic metals from the environment and subsequent metal entry into the food chain. In addition, microorganisms may have biotechnological application in metal removal processes, and they serve as useful models for investigating metal transport and toxicity at the cellular level (2, 15, 20).

The plasma membrane is a primary cellular site of metal interaction that can determine both metal uptake and toxicity (5, 15). Our recent work with the yeast Saccharomyces cerevisiae has revealed a marked influence of plasma membrane fatty acid composition on cellular metal sensitivity. Thus, Cu-induced (5) and Cd-induced (19) plasma membrane permeabilization and yeast killing were severalfold higher in cells enriched with polyunsaturated fatty acids (PUFAs); these fatty acids had no apparent effect on yeast physiology under nonstressed conditions (5). In view of the large differences in fatty acid composition between (and within) the major microbial taxonomic groupings (28, 38), in addition to changes attributable to environmental acclimation (17), these results are of considerable relevance to the toxicity of metals in the natural environment. The above findings were not attributed to altered metal transport characteristics but to enhanced metal-induced lipid peroxidation in PUFA-supplemented cells (19). Altered transport kinetics (K_m and V_max values) for cesium, a radionuclide that does not induce significant plasma membrane permeabilization (3), have been reported in linoleate (18:2)-supplemented S. cerevisiae (18). Such effects are commonly attributed to changes in the mobility and/or conformation of functional proteins in an altered membrane-lipid environment (17, 21, 35). However, the influence of linoleate supplementation on net Cs⁺ accumulation was only marginal (18). Thus, in contrast to studies of amino acid transport (23), reports to date have suggested that the uptake of inorganic metal ions in S. cerevisiae is not significantly influenced by altered plasma membrane fatty acid composition.

In this study we focused on the divalent radionuclide Sr²⁺. ⁹⁰Sr is a normal by-product of nuclear fission and can occur in the environment as a consequence of controlled or accidental release (13). In addition to the isotope’s relatively long half-life (∼29 years), Sr may be particularly persistent in the environment because of its close chemical similarity to the biologically essential divalent cation Ca²⁺. Indeed, Sr²⁺ and Ca²⁺ transport occurs via common mechanisms in S. cerevisiae (4, 32). Our objectives in this study were (i) to determine whether alterations in cellular fatty acid composition influence Sr²⁺ accumulation by S. cerevisiae and (ii) to investigate the underlying cause of any observed effects. Using our fatty acid enrichment approach (5, 18, 19), we showed that Sr²⁺ accumulation was markedly stimulated in linoleate-enriched S. cerevisiae. This stimulation was accounted for by reduced Sr²⁺ efflux activity. The results are of significance not only from an environmental perspective with regard to metal-microbe interactions but also because of the potential relevance to cellular Ca²⁺ homeostasis.

MATERIALS AND METHODS

Organism and culture conditions.

S. cerevisiae NCYC 1383 was maintained on solid YEPD medium, containing (liter⁻¹) neutralized bacteriological peptone (Difco, Detroit, Mich.), 20 g; yeast extract (Difco), 10 g; glucose, 20 g; and technical agar (Difco), 16 g. Starter cultures were inoculated from plates into 100 ml of YEPD broth (YEPD medium lacking agar) in 250-ml Erlenmeyer flasks and incubated at 25°C with rotary aeration (120 rpm). Experimental cultures in YEPD broth were inoculated from 48-h starter cultures to an initial optical density at 550 nm of ∼0.1. All YEPD broth was prepared with the nonionic surfactant Tergitol (Nonidet P-40) (Sigma, St. Louis, Mo.) to enable fatty acid solubilization; the final Tergitol concentration (after fatty acid addition [see below]) was 1% (wt/vol). Where specified, fatty acids (palmitoleate [16:1], 18:2, linolenate [18:3], or eicosadienoate [20:2]; Sigma) were added from filter-sterilized stock solutions (20 mM) prepared in 5% (wt/vol) Tergitol, to give final fatty acid concentrations in the medium of 1 mM (unless otherwise specified). Cell numbers were determined by counting at least 400 cells with an improved Neubauer hemocytometer. Cell viability was determined as numbers of CFU, as described previously (5).

Determination of fatty acid composition.

Cells were harvested by centrifugation at 1,500 × g for 4 min and washed twice with distilled deionized water at 4°C. Cells were disrupted by six 1-min periods of vortexing with 1 volume of 0.5-mm-diameter glass beads (Sigma), alternated with 1-min incubations on ice. Lipids were extracted from cell homogenates by the method of Bligh and Dyer (6). For fatty acid analysis, methyl esters were generated by acid-catalyzed esterification (2.5% [vol/vol] H₂SO₄ in dry methanol) at 70°C for 2 h. Fatty acid methyl esters were extracted with redistilled petroleum spirit (boiling point, 60 to 80°C) and subsequently analyzed by gas-liquid chromatography, as described previously (5). Heptadecanoate was used as an internal standard. Separations were routinely achieved with a 30-m Stabilwax-DA capillary column, fitted to a Perkin-Elmer Autosystem gas chromatograph. Injector and detector temperatures were 250 and 260°C, respectively, with an oven temperature set isothermally to 200°C during operation. Fatty acids were identified by comparison of their retention times with those of known standards.

Determination of strontium uptake.

Mid- to late-exponential phase cells (∼16 h) were harvested from unsupplemented or fatty acid-supplemented medium by centrifugation (1,500 × g, 4 min) and washed twice with 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer plus 1% (wt/vol) glucose (unless otherwise stated), previously adjusted to pH 5.5 with NaOH. Cells were finally suspended in MES buffer to a density of approximately 3 × 10⁷ cells ml⁻¹. Fifty-milliliter volumes of cell suspension were incubated in 125-ml Erlenmeyer flasks with rotary aeration (120 rpm). After 10 min of equilibration, Sr(NO₃)₂ was added to the desired concentration. In competition studies, Ca(NO₃)₂ or Mg(NO₃)₂ was added to the appropriate concentration immediately prior to strontium addition. Where specified, trifluoperazine (TFP) was added to cell suspensions 10 min prior to strontium addition. At specified intervals, 1-ml samples (in triplicate) were removed and layered on 300 μl of an oil mixture comprising 80% (vol/vol) silicone oil (Fluka) and 20% (vol/vol) di-isononylphthalate (Fluka) in 1.5-ml microcentrifuge tubes. After microcentrifugation to rapidly separate cells from their medium, the supernatant and oil layer were removed and cell pellets were digested with 0.5 ml of 6 M HNO₃ at 95°C for 60 min. After appropriate dilution with distilled deionized water, the strontium contents of the samples were determined with a Finnigan-Mat Sola Quadropole inductively coupled plasma emission spectroscope with reference to standard Sr solutions. Indium was used as an internal standard in all samples.

Determination of strontium efflux.

Cells were loaded with Sr by incubation in the presence of 100 μM Sr(NO₃)₂ under the conditions described above. After 90 min, Sr-loaded cells were harvested by centrifugation (1,500 × g, 4 min) and washed three times with MES buffer prior to final resuspension in MES buffer containing 100 μM Ca(NO₃)₂. Ca(NO₃)₂ was added to provide Ca²⁺ as an exchangeable cation for intracellular Sr²⁺ and to maintain external conditions that approximated those used during Sr²⁺ loading. At intervals after resuspension in buffer, samples were removed and microcentrifuged exactly as described above. The cell supernatant was removed, and after dilution, the strontium concentration was determined as described above. The Sr contents of supernatants obtained 30 s after resuspension of cells in Ca-containing buffer were subtracted from those of later samples, to eliminate Sr²⁺ release attributable to any rapid exchange with Ca²⁺ at the cell surface (4).

RESULTS

Influence of 18:2 incorporation on Sr²⁺ accumulation.

Sr²⁺ accumulation was examined in S. cerevisiae previously grown in the absence or presence of 1 mM 18:2. Enrichment with 18:2 (see Table 1) had a very marked effect on Sr²⁺ accumulation (Fig. 1 and 2). An initial rapid phase of Sr²⁺ accumulation during incubation in the presence of 50 μM Sr(NO₃)₂, which largely represented cell surface Sr binding (see below) (Fig. 1b), was similar in unsupplemented and 18:2-supplemented cells (Fig. 1a). However, Sr²⁺ accumulation in a subsequent, slower phase was considerably greater in 18:2-enriched cells than in unsupplemented cells. Thus, levels of Sr accumulated after 6 h of incubation were approximately 160 and 105 pmol of Sr (10⁶ cells)⁻¹, respectively (Fig. 1a). Moreover, by subtracting the amounts of Sr accumulated after 30 min (∼50 pmol of Sr [10⁶] cells]⁻¹), which at this stage still represented mostly Sr²⁺ binding (Fig. 1b), we estimated that the rate of Sr²⁺ accumulation during the slow phase was approximately twofold higher in 18:2-supplemented than in unsupplemented S. cerevisiae (Fig. 1a).

TABLE 1.

Fatty acid composition of S. cerevisiae enriched with a range of fatty acids^a

Fatty acid added and concn (mM)	Cellular fatty acid composition (% of total fatty acids)c
	16:0b	16:1	18:0	18:1	18:2	18:3	20:2
None	13 ± 0.4	66 ± 3.5	4.2 ± 0.9	17 ± 2.1
16:1
0.025	11 ± 0.3	72 ± 0.8	4.4 ± 0.2	12 ± 0.4
0.1	11 ± 0.4	78 ± 1.0	6.3 ± 0.8	4.6 ± 0.2
1.0	10 ± 0.4	80 ± 3.1	7.1 ± 0.1	2.8 ± 0.4
18:2
0.025	15 ± 0.2	58 ± 0.1	2.0 ± 0.2	13 ± 0.1	12 ± 0.3
0.1	17 ± 0.4	27 ± 0.4	1.5 ± 0.2	5.5 ± 0.2	50 ± 1.3
1.0	13 ± 1.4	11 ± 0.9	1.7 ± 0.2	1.4 ± 0.3	69 ± 2.7
18:3
0.025	14 ± 0.6	55 ± 1.1	2.8 ± 0.1	14 ± 0.1		15 ± 0.7
0.1	16 ± 1.2	21 ± 0.3	2.6 ± 1.0	6.8 ± 0.3		53 ± 0.2
1.0	16 ± 0.1	6.8 ± 0.1	3.0 ± 0.4	2.1 ± 0.3		72 ± 0.5
20:2
0.025	13 ± 0.2	60 ± 1.1	4.6 ± 1.0	17 ± 0.4			6.0 ± 1.0
0.1	10 ± 0.4	39 ± 0.2	3.4 ± 0.3	18 ± 0.3			29 ± 0.7
1.0	10 ± 0.3	28 ± 1.4	3.5 ± 0.1	18 ± 0.5			41 ± 2.1

^aCells were grown in YEPD medium supplemented with the specified fatty acid at the concentration indicated. The fatty acid compositions of cells from mid- to late-exponential phase cultures are shown. 

^bFatty acids are abbreviated according to convention, with the first figure showing the number of carbon atoms and the second indicating the number of double bonds. 

^cValues are means ± standard errors of the means, based on three determinations. Typical results from one of at least two independent experiments are shown. Supplemented fatty acids are emphasized in boldface type. 

FIG. 1.

Influence of linoleate enrichment on Sr²⁺ accumulation by S. cerevisiae. Cells previously grown in the presence or absence of 1 mM linoleate were incubated in MES buffer with 1% (wt/vol) glucose (unless otherwise specified) and exposed to Sr(NO₃)₂. (a) Time course of Sr²⁺ accumulation by 18:2-supplemented (○) and unsupplemented (●) S. cerevisiae in the presence of 50 μM Sr(NO₃)₂. (b) Time course of Sr²⁺ accumulation by unsupplemented S. cerevisiae in the presence (●) or absence (□) of 1% (wt/vol) glucose plus 50 μM Sr(NO₃)₂. Points are means from three replicate determinations. Standard errors of the means were all less than 10% of the values of the points. Typical results from one of at least two independent experiments are shown.

FIG. 2.

Influence of Sr²⁺ concentration on rate of Sr²⁺ accumulation by 18:2-supplemented and unsupplemented S. cerevisiae. Cells previously grown in the presence (○) or absence (●) of 1 mM linoleate were incubated in MES buffer with 1% (wt/vol) glucose and exposed to Sr(NO₃)₂. Rates of Sr²⁺ accumulation were determined between 30 and 90 min after the addition of Sr(NO₃)₂ to the indicated final concentrations. Points are means from three replicate determinations (standard errors of the means [error bars] are shown where these values exceed 10% of the values of the points). Typical results from one of at least two independent experiments are shown.

Previous evidence indicated that the rapid phase of Sr²⁺ accumulation represented passive binding to the cell surface, whereas the slower phase represented metabolism-dependent intracellular Sr²⁺ accumulation (4, 32). We tested this hypothesis by monitoring Sr²⁺ accumulation (by unsupplemented S. cerevisiae) in the absence and presence of glucose. Cell suspensions incubated under both conditions accumulated Sr²⁺ to approximately 40 to 45 pmol of Sr (10⁶ cells)⁻¹ within 5 min, the earliest time point examined (Fig. 1b). However, further incubation up to 360 min resulted in no further Sr²⁺ accumulation by cells in the absence of glucose. In contrast, glucose-supplemented cells continued to accumulate Sr²⁺ during this period, at an approximately linear rate that was lower than the rate observed during the initial 5 min (Fig. 1b). These results confirmed that the slow (post-5-min) phase of Sr²⁺ accumulation was the physiologically relevant process in our experiments.

S. cerevisiae enriched with 18:2 had higher rates of metabolism-dependent Sr²⁺ accumulation than fatty acid-unsupplemented cells over a range of Sr²⁺ concentrations (Fig. 2). Rates of Sr²⁺ accumulation were determined during the slow approximately linear accumulation phase, between 30 min (after cell surface Sr binding was well completed) and 90 min, as described previously for Cs⁺ (18). The length of the linear phase decreased with increasing Sr concentration above 50 μM (results not shown), and 90 min was selected as a suitable end point for all concentrations. Because of the range of Sr²⁺ concentrations tested, data are presented on a logarithmic scale (Fig. 2). The rate of Sr²⁺ accumulation increased almost linearly with Sr²⁺ concentration, although there was some evidence for saturation at high (>1 mM) Sr(NO₃)₂ concentrations (Fig. 2) (note that because of the complication of simultaneous Sr²⁺ efflux by these cells [see below], these results could not be used as influx data for kinetic analysis). The rate of Sr²⁺ accumulation was always higher (generally by approximately twofold) in 18:2-supplemented than in unsupplemented cells. For example, at 1 mM Sr(NO₃)₂, Sr²⁺ accumulation rates were approximately 2.3 and 5.1 pmol of Sr (10⁶ cells)⁻¹ min⁻¹ in unsupplemented and 18:2-supplemented cells, respectively (Fig. 2). No reduction in cell viability (CFU) was evident at the Sr²⁺ concentrations tested.

Influence of incorporation of a range of fatty acids on Sr²⁺ accumulation.

To determine if the above effect was specific to linoleate, S. cerevisiae was grown in the presence of a range of fatty acids (supplied at varying concentrations) and examined for Sr²⁺ accumulation. The 1 mM concentration at which we routinely supply exogenous fatty acids (5, 19) apparently exceeded saturation, as similar levels of 18:2 incorporation were evident between 0.25 and 1.0 mM 18:2 (results not shown). Thus, to give varying levels of incorporation, fatty acids were supplied at 0.025, 0.1, and 1.0 mM. As demonstrated previously for 18:2 and 18:3 (5, 19), growth of S. cerevisiae in the presence of 1 mM 16:1, 18:2, or 18:3 resulted in incorporation of the exogenous fatty acids to >65% of total cellular fatty acids in each case (Table 1). We note that 16:1 is synthesized by S. cerevisiae and comprises a major fraction of the fatty acids of cells grown in unsupplemented medium. Nevertheless, increasing exogenous 16:1 concentrations yielded cells that were increasingly enriched with 16:1. This was not the case for oleate (18:1), with which supplementation did not yield cells of increased 18:1 content (results not shown). Therefore, 18:1-supplemented cells were not tested. Cells grown in the presence of the lowest 18:2 and 18:3 concentration tested (0.025 mM) showed markedly lower enrichment with these fatty acids (10 to 15% of total fatty acids). Incorporation of 20:2 was lower than that of the other fatty acids tested, comprising between 6% (at 0.025 mM) and 41% (at 1.0 mM) of total fatty acids in 20:2-supplemented cultures. The compositions of the fatty acids synthesized by S. cerevisiae that were most affected by exogenous fatty acid incorporation were those of 16:1 (except in the case of 16:1 supplementation) and 18:1 (except in the case of 20:2 supplementation). For example, enrichment with 1 mM 18:2 or 18:3 was associated with approximately six- and ninefold reductions in 16:1 composition, respectively (Table 1). Effects of fatty acid supplementation on palmitate (16:0) and stearate (18:0) as proportions of total cellular fatty acids were less marked. However, 18:3- and 16:1-supplemented cells did display increased 16:0 and 18:0 compositions, respectively, whereas small reductions in the proportion of 18:0 were evident in 18:2-enriched cells. Enrichments with the n-6 isomer of 18:3 (γ-linolenate) and the n-9 isomer of 20:2, yielded cells with fatty acid compositions that did not differ significantly from those presented in Table 1 for the corresponding common isomers of these fatty acids (n-3 and n-6, respectively).

S. cerevisiae previously grown in the presence of different fatty acid supplements displayed differing rates of Sr²⁺ accumulation in the presence of 100 μM Sr(NO₃)₂ (Table 2). Cells supplemented with 18:2 again showed an elevated Sr²⁺ accumulation rate, which increased with the level of 18:2 incorporation, albeit nonlinearly (Table 1). Cells grown in the presence of 1 mM 18:2 accumulated Sr²⁺ at a rate ∼1.6-fold greater than that of unsupplemented cells in this experiment. In contrast to the effect of 18:2 supplementation, S. cerevisiae enriched with 16:1 or either of the 18:3 isomers tested (α-linolenate [n-3] and γ-linolenate [n-6]) showed no significant difference in Sr²⁺ accumulation compared to unsupplemented cells. Enrichment with 20:2 (both n-6 and n-9) was associated with a reduction in Sr²⁺ accumulation ability (Table 2). This effect was most marked (by approximate halving of the control rate) in cells with the greatest 20:2 content that were grown at 1 mM 20:2 (n-6) (Table 1). Stimulation of Sr²⁺ accumulation was specific to linoleate (Table 2).

TABLE 2.

Rates of Sr²⁺ accumulation by S. cerevisiae enriched with a range of fatty acids^a

Fatty acid supplement (isomer)	Sr2+ accumulation at fatty acid concn (mM) suppliedb
	0	0.025	0.1	1.0
16:1 (n-7)	0.54 ± 0.02	0.50 ± 0.01	0.49 ± 0.03	0.42 ± 0.05
18:2 (n-6)	0.54 ± 0.02	0.68 ± 0.04c	0.71 ± 0.03c	0.86 ± 0.07c
18:3 (n-3)	0.54 ± 0.02	0.56 ± 0.03	0.52 ± 0.01	0.51 ± 0.03
18:3 (n-6)	0.54 ± 0.02	0.54 ± 0.01	0.48 ± 0.03	0.50 ± 0.05
20:2 (n-6)	0.54 ± 0.02	0.46 ± 0.03	0.32 ± 0.06c	0.28 ± 0.07c
20:2 (n-9)	0.54 ± 0.02	0.44 ± 0.01c	NDd	ND

^aCells previously grown in medium supplemented with the appropriate fatty acid were incubated in MES buffer with 1% (wt/vol) glucose and exposed to 100 μM Sr(NO₃)₂. 

^bThe Sr²⁺ accumulation rate is given in pmoles (10⁶ cells)⁻¹ minute⁻¹, determined during the linear accumulation phase (see text). Values are means ± standard errors of the means, based on three determinations. Typical results from one of at least two independent experiments are shown. 

^cResult that is significantly different (P < 5.0%) from the control rate (0.54 ± 0.02). 

^dND, not determined. 

Influence of Ca²⁺ and Mg²⁺ on Sr²⁺ accumulation by 18:2-supplemented and unsupplemented S. cerevisiae.

A significant influence of membrane composition on the inhibition by competing ions of a trans-membrane uptake process, can be indicative of an altered conformational state of the appropriate transporter (1, 18). Here, the influence of Ca²⁺ and Mg²⁺ on Sr²⁺ accumulation by 18:2-supplemented and unsupplemented S. cerevisiae was compared (Table 3). The presence of Ca²⁺ or Mg²⁺, supplied at equimolar concentrations to Sr²⁺ (100 μM), was associated with clear reductions in Sr²⁺ accumulation rates by both 18:2-supplemented and unsupplemented cells. When the differing rates of uninhibited accumulation were corrected for (by normalization to 100%), it was evident that the extent of inhibition did not differ markedly between the two cell types. Thus, externally supplied Ca²⁺ caused a 53 and 61% inhibition of Sr²⁺ accumulation by 18:2-supplemented and unsupplemented cells, respectively. The presence of 100 μM Mg²⁺ was associated with respective inhibitions of 72 and 70% (Table 3). Under each incubation condition, the absolute Sr²⁺ accumulation rate was higher in 18:2-supplemented than in unsupplemented cells.

TABLE 3.

Influence of Ca²⁺ and Mg²⁺ on Sr²⁺ accumulation by S. cerevisiae^a

Competing ion (concn)	Sr2+ accumulation rate [pmol (106 cells)−1 min−1] inb:
	18:2-supplemented cells	Unsupplemented cells
None	0.90 ± 0.04	0.56 ± 0.05
Ca2+ (100 μM)	0.42 ± 0.01	0.22 ± 0.04
Mg2+ (100 μM)	0.25 ± 0.01	0.17 ± 0.01

^aCells previously grown in unsupplemented or 18:2 (1 mM)-supplemented medium were incubated in MES buffer with 1% (wt/vol) glucose and exposed to 100 μM Sr(NO₃)₂ in the absence or presence of Ca(NO₃)₂ or Mg(NO₃)₂. 

^bDetermined during the linear accumulation phase (see text). Values are means ± standard errors of the means, based on three determinations. Typical results from one of at least two independent experiments are shown. 

Influence of 18:2 enrichment on Sr²⁺ efflux.

Intracellular solute accumulation is generally the net balance of two opposing fluxes: solute influx and solute efflux. Thus, we sought to study the influence of 18:2 supplementation on Sr²⁺ influx and efflux independently. Because the transport and distribution of Sr²⁺ in yeast closely match those of Ca²⁺ (4, 32), we focused on Ca²⁺ transport systems. Our preliminary attempts to inhibit Sr²⁺ influx using the L-type Ca²⁺ channel blocker, verapamil (36), were unsuccessful. Instead, TFP (see Discussion) was used as a putative inhibitor of calmodulin-dependent Ca²⁺-ATPase activity. TFP acts as an inhibitor of plasma membrane Ca²⁺-ATPase-dependent Ca²⁺ efflux in several organisms (11, 14, 24). Whereas this effect has yet to be unequivocally demonstrated in S. cerevisiae, the observed enhancement of net Ca²⁺ accumulation in TFP-treated yeast (7, 12) is consistent with such a role. TFP at 10 μM was initially determined to give no nonspecific reduction in plasma membrane impermeability (assessed as K⁺ efflux) (7, 12). Incubation of cells in the presence of TFP during exposure to 100 μM Sr²⁺ was associated with an increase in the net Sr²⁺ accumulation rate of S. cerevisiae (Fig. 3). This effect was consistent with mediation of Sr²⁺ efflux by a Ca²⁺-ATPase in non-TFP-exposed cells (Sr²⁺ efflux from cells preloaded with Sr²⁺ also was diminished by TFP [Fig. 4a]). Moreover, the stimulatory effect of TFP on Sr²⁺ accumulation was considerably greater in unsupplemented cells than in 18:2-supplemented cells. Thus, after 6 h of incubation in the presence of TFP and Sr²⁺, cellular Sr²⁺ levels were approximately 315 and 250 pmol of Sr (10⁶ cells)⁻¹ in unsupplemented and 18:2-supplemented cells, respectively (Fig. 3). This effect was particularly marked considering that 18:2-supplemented cells again accumulated the most Sr²⁺ in the absence of TFP. By subtracting cellular Sr accumulated during the first 5 min of incubation (∼70 to 100 pmol of Sr [10⁶ cells]⁻¹), we calculated that the presence of TFP caused a 3.6-fold increase in intracellular Sr²⁺ accumulation by fatty acid-unsupplemented S. cerevisiae after 6 h but only a 1.4-fold increase in 18:2-supplemented cells. The results indicated that TFP-inhibitable Sr²⁺ efflux activity was considerably greater in unsupplemented cells than in 18:2-supplemented cells. Sr²⁺ accumulation rates in the presence of TFP, which should approximate the influx component of Sr²⁺ accumulation, were slightly greater in fatty acid-unsupplemented cells (Fig. 3).

FIG. 3.

Influence of TFP on Sr²⁺ accumulation by 18:2-supplemented and unsupplemented S. cerevisiae. Cells previously grown in the presence (circles) or absence (squares) of 1 mM linoleate were incubated in MES buffer plus 1% (wt/vol) glucose, either with (solid symbols) or without (open symbols) 10 μM TFP. Sr(NO₃)₂ was added to a final concentration of 100 μM. Points represent means from three replicate determinations (standard errors of the means [error bars] are shown where these values exceed 10% of the values of the points). Typical results from one of at least two independent experiments are shown.

FIG. 4.

Sr²⁺ efflux from 18:2-supplemented and unsupplemented S. cerevisiae. Cells previously grown in the presence (circles) or absence (squares) of 1 mM linoleate were loaded with Sr by incubation in MES buffer in the presence of 100 μM Sr(NO₃)₂ for 90 min. Sr²⁺ efflux was monitored after resuspension of Sr-loaded cells in MES buffer containing 100 μM Ca(NO₃)₂, either in the presence (solid symbols) or absence (open symbols) of 10 μM TFP. The graphs show Sr²⁺ loss as absolute cellular Sr levels (a) and a percentage of the initial cellular Sr level (b). Points represent means from three replicate determinations (standard errors of the means [error bars] are shown where these values exceed 10% of the values of the points). Typical results from one of at least two independent experiments are shown.

Because of the possible nonspecificity of TFP, we confirmed that Sr²⁺ efflux was diminished in 18:2-supplemented S. cerevisiae by using an additional approach. Thus, Sr²⁺ release was monitored in cells that had been preloaded with Sr²⁺ (Fig. 4). The initial rate of Sr²⁺ efflux (∼0 to 120 min) was found to be more than twofold greater in unsupplemented than in 18:2-supplemented cells; Sr²⁺ release rates were ∼0.28 and 0.12 pmol of Sr²⁺ (10⁶ cells)⁻¹ min⁻¹, respectively (Fig. 4a). The rate in 18:2-supplemented cells was approximately constant over the 240-min time course. However, the rate decreased markedly after 120 min in fatty acid-unsupplemented cells, which probably reflected depletion of intracellular Sr²⁺. Calculation of the approximate initial intracellular Sr level of these cells (by reference to the relative proportions of intracellular and surface-bound Sr determined from Fig. 1 and 3) was consistent with this interpretation. The rate of Sr²⁺ efflux was diminished in the presence of TFP, although some release of cellular Sr²⁺ was still evident. In agreement with the results presented in Fig. 3, the effect of TFP was particularly marked for fatty acid-unsupplemented cells, i.e., those that displayed the greater Sr²⁺ efflux in the absence of TFP (Fig. 4a). Statistical analysis by Student’s t test indicated that the effect of TFP on 18:2-supplemented cells was not significant (P > 5%).

The results shown in Fig. 4a are complicated by the nonuniformity of initial cellular Sr²⁺ levels; the rate at which a cellular solute is translocated normally varies proportionally with its concentration at the source, assuming that the solute is not saturating (40). Thus, the results described above probably underrepresent the difference in Sr²⁺ efflux between unsupplemented and 18:2-supplemented cells. To compensate for differing cellular Sr²⁺ levels, Sr²⁺ efflux is presented in Fig. 4b as a percentage of the initial amount of cellular Sr²⁺ in the two cell types. Sr²⁺ efflux was approximately 3.5-fold greater in unsupplemented than in 18:2-supplemented cells when differences in cellular Sr²⁺ were corrected for (Fig. 4b). In the presence of TFP, the slow release of cellular Sr²⁺ appeared to be slightly greater in 18:2-supplemented than in unsupplemented cells and only slightly less than that of non-TFP-exposed 18:2-supplemented cells (Fig. 4b). However, statistical analyses revealed that the latter differences were not significant (P > 5%).

Influence of enrichment with a range of fatty acids on cellular Sr²⁺ release.

Prior enrichment with 16:1, 18:3, or 20:2 (by growth in the presence of 1 mM fatty acid) also influenced the rate of Sr²⁺ release by Sr-loaded S. cerevisiae (Table 4). When expressed as a percentage of initial cellular Sr²⁺ (see above), loss of Sr²⁺ from 16:1-supplemented cells appeared to be slightly slower (about 85% of the control rate) than that from cells previously grown in the absence of a fatty acid supplement. However, this effect was not significant (P > 5%). Enrichment with 16:1 also exerted no significant effect on cellular Sr²⁺ accumulation (Table 2). Again, 18:2-supplemented cells displayed slow Sr²⁺ efflux, which was approximately 2.7-fold lower than that of unsupplemented cells in this experiment. In contrast, prior enrichment of S. cerevisiae with 18:3 and 20:2 was associated with an increased rate of Sr²⁺ loss. In these cells, Sr²⁺ release was approximately 1.6-fold higher than that of fatty acid-unsupplemented cells (Table 4). These findings contrast with the lack of effect of 18:3 supplementation on Sr²⁺ accumulation but could account for the ∼50% lower Sr²⁺ accumulation rate of 20:2-enriched cells (Table 2).

TABLE 4.

Sr²⁺ release from S. cerevisiae enriched with a range of fatty acids^a

Fatty acid supplement	% of initial Sr2+ releasedb
Unsupplemented	8.5 ± 0.4
Palmitoleate (16:1)	7.3 ± 0.6
Linoleate (18:2)	3.2 ± 0.9c
Linolenate (18:3)	14 ± 1.3c
Eicosadienoate (20:2)	14 ± 1.7c

^aCells previously grown in medium supplemented with the appropriate fatty acid were loaded with Sr by incubation in MES buffer in the presence of 100 μM Sr(NO₃)₂ for 90 min. Sr-loaded cells were then resuspended in MES buffer containing 100 μM Ca(NO₃)₂. 

^bSr²⁺ released between 30 s and 90 min (see Materials and Methods) as a percentage of initial cellular Sr levels. Values for enriched cells are means ± standard errors of the means, based on three determinations. Typical results from one of at least two independent experiments are shown. 

^cResult that is significantly different (P < 5%) from the control value (8.5% ± 0.4%). 

DISCUSSION

The ability to specifically enrich S. cerevisiae with fatty acids by simple aerobic culturing in supplemented medium (5) has many advantages (18) and has enabled the present demonstration of altered Sr²⁺ transport characteristics in fatty acid-supplemented cells. Enrichment with the selected fatty acid occurs to the same extent in plasma membranes as in whole-cell extracts (5, 19).

The marked stimulatory effect of linoleate-enrichment on Sr²⁺ accumulation by S. cerevisiae contrasted with the effects of enrichment with the other fatty acids tested and with the slight reduction in Cs⁺ influx reported previously in 18:2-enriched S. cerevisiae (18). Monovalent-cation competition experiments in the latter study provided evidence that 18:2 enrichment might cause an alteration in the conformation of the plasma membrane Cs⁺ (K⁺) transporter. In the present study, the strong inhibition of Sr²⁺ accumulation by both Ca²⁺ and Mg²⁺ was consistent with previous reports (4, 32) and with the notion of a generalized yeast divalent-cation uptake system (15, 22, 32). Moreover, the inhibitory effects of Ca²⁺ and Mg²⁺ were unaffected by 18:2 supplementation, suggesting that the altered Sr²⁺ accumulation of 18:2-enriched cells was probably not attributable to a conformational change of an influx mechanism (1, 18).

The transport and distribution of Sr²⁺ in yeast, as well as other organisms, are generally considered to occur via the same routes as those for Ca²⁺ transport and distribution (4, 7, 8, 32). The mechanism(s) of Ca²⁺ influx in S. cerevisiae is not yet clearly defined (10, 22), although the lack of effect of verapamil argues against a major role of any L-type Ca²⁺ channels in Sr²⁺ uptake. Moreover, a previous report indicated that Ca²⁺ influx in S. cerevisiae was not influenced significantly by cellular enrichment with linoleate (23). Similarly here, in experiments designed to restrict the efflux component of Sr²⁺ accumulation using TFP, Sr²⁺ uptake was only slightly lower in 18:2-supplemented than in unsupplemented cells. That TFP did inhibit Sr²⁺ efflux was supported by enhanced net rates of Sr²⁺ accumulation in TFP-treated cells, as has been reported previously for Ca²⁺ accumulation (7, 12). TFP is a well-known inhibitor of calmodulin, an activator of many Ca²⁺-ATPases (27, 37). Thus, TFP is widely employed as a specific Ca²⁺-ATPase inhibitor (for examples, see references 11, 14, and 24). Use of an appropriately low TFP concentration here averted the nonspecific effects that may arise from its application to intact cells, such as altered membrane potential and/or cation permeability (both of which are associated with altered K⁺ fluxes in yeast) (7, 12, 33). Previous evidence that active Sr²⁺ efflux in yeast occurs via a plasma membrane Ca²⁺-ATPase (32) supports the possibility that the reduced net Sr²⁺ accumulation of 18:2-supplemented cells observed here results from an effect of linoleate enrichment on plasma membrane Ca²⁺-ATPase activity. However, analysis of the yeast genome sequence has not identified a gene encoding a likely plasma membrane Ca²⁺-ATPase (9). Thus, it is possible that the present results could be linked to effects exerted at the vacuolar and/or golgi membrane Ca²⁺-ATPases of yeast; both proteins are known to play a major role in yeast Ca²⁺ homeostasis (9, 30). Further work is required to resolve these possibilities. Specific phospholipid requirements for optimal activity of membrane Ca²⁺-ATPases from higher organisms have been described previously (8, 16, 26). However, evidence for an effect of altered membrane fatty acid composition on Ca²⁺-ATPase activity has been more fragmentary and generally inconclusive (8, 31, 34). The use of a well-defined in vivo model system such as that described here (5, 18) could help yield more conclusive results in future studies.

Efflux experiments confirmed reduced rates of Sr²⁺ release from 18:2-supplemented S. cerevisiae. Furthermore, the difference in Sr²⁺ efflux observed for unsupplemented and 18:2-supplemented cells approximated the difference in net Sr²⁺ accumulation described in uptake experiments. That there was no significant effect of 16:1 enrichment on Sr²⁺ efflux was consistent with this fatty acid occurring naturally in S. cerevisiae and with the view that a natural membrane composition should be more closely tailored to the requirements of natural membrane proteins (e.g., Ca²⁺-ATPases) than a manipulated (e.g., 18:2-enriched) membrane composition (17). The degree to which Sr²⁺ release from S. cerevisiae could be influenced by fatty acid supplementation was underscored by the enhancement of Sr²⁺ loss in Sr-loaded 18:3- and 20:2-enriched cells. Because 18:3-supplemented cells maintained a normal rate of Sr²⁺ accumulation despite enhanced Sr²⁺ loss, we inferred that an 18:3-rich membrane environment is unexpectedly more favorable for yeast Sr²⁺ influx than a natural membrane environment.

The sensitivity of membrane-dependent functions (including certain ATPases) to fatty acid compositional changes has in many cases been attributed to alterations in membrane fluidity and consequent alterations in membrane protein mobility (17). The degree of membrane fatty acid unsaturation is a key determinant of membrane fluidity (17), as we have confirmed by measurements of membrane order in 18:2- and 18:3-enriched S. cerevisiae (19). However, decreased Sr²⁺ efflux with increased fatty acid unsaturation due to 18:2 supplementation contrasted with stimulation of Sr²⁺ release in 18:3-enriched cells with an even higher degree of fatty acid unsaturation. Thus, no relationship between fatty acid unsaturation index (18) and Sr²⁺ accumulation or efflux could be discerned. Membrane diameter also has been proposed to be a factor regulating membrane protein activity (21). However, average cellular fatty acyl chain lengths, calculated using values for individual fatty acid molecular lengths in conjunction with cellular fatty acid compositional determinations, again showed no relationship with the cells’ respective rates of Sr²⁺ efflux here (results not shown).

One additional and relatively nonspecific consequence of membrane enrichment with PUFAs is a tendency toward decreased membrane impermeability (17, 25). Indeed, such effects have been suggested to account for elevated passive ion fluxes across PUFA-rich microbial membranes (25). Thus, it is possible that the enhanced rates of Sr²⁺ release from 18:3- and 20:2-enriched cells reported here reflect decreased plasma membrane impermeability to Sr²⁺ in these cells. Passive trans-membrane Ca²⁺ efflux does occur under physiological conditions (22, 29). Indeed, the residual Sr²⁺ release observed in TFP-treated S. cerevisiae could represent passive membrane permeation in the absence of Ca²⁺-ATPase activity. In comparison to 18:3 and 20:2, it seems likely that the physical alterations in plasma membrane properties (e.g., fluidity) arising from 18:2 enrichment are relatively specific (Sr²⁺ efflux data for these cells did not suggest increased membrane permeability). Nevertheless, the latter changes are ones to which the Sr²⁺ (and possibly Ca²⁺) efflux mechanism appears particularly sensitive. Isomers of 18:3 and 20:2 with the same n-6 structure as 18:2 did not elicit the same effect as 18:2 on Sr²⁺ transport, suggesting that the effect was not attributable to products of cellular 18:2 metabolism. Further work is needed to determine which membrane properties are specifically responsible for linoleate-induced effects on Sr²⁺ efflux activity. Whatever such properties are, we emphasize again that enrichment with linoleate exerts no adverse effect on the growth of S. cerevisiae.

In conclusion, we have found a marked inhibitory effect of linoleate enrichment on the Sr²⁺ efflux activity of yeast. The resultant enhancement of Sr²⁺ accumulation is the first reported example of altered membrane composition giving increased physiological uptake of a metal ion in a microorganism. The results are particularly pertinent, considering the marked intra (during environmental acclimation)- and interspecies variation in microbial fatty acid composition. Therefore, the results may have important implications for microbial Sr cycling in the natural environment and for potential biological Sr removal applications (15). Furthermore, whereas this study has focused on Sr²⁺, the effects reported also could apply to Ca²⁺. In view of the fundamental role of Ca²⁺ in many aspects of cellular physiology, including signal transduction and cell cycle control (39), further exploration of the influence of membrane fatty acid enrichment on yeast Ca²⁺ homeostasis is warranted.