Vol. 131, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Aug. 1977, p. 564-571 Copyright ©D 1977 American Society for Microbiology
Permeability of the Cell Envelope and Osmotic Behavior in Saccharomyces cerevisiae WILFRED N. ARNOLD* AND JOHN S. LACY Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103
Received for publication 4 May 1977
Bakers' yeast (Saccharomyces cerevisiae) was equilibrated with distilled water and then packed into standardized pellets by centrifugation. The fractional space (S value) that was accessible to passive permeation was probed with a variety of mono- and divalent salts, mono- and disaccharides, polyols, substrates and products of ,-fructofuranosidase (EC 3.2.1.26) and acid phosphatase (EC 3.1.3.2), and a cross-linked polymer of sucrose (Ficoll 400). A simple but very reproducible method was developed to measure pellet volume. At the limit of zero osmolality for bathing medium, the interstitial space was 0.223 ml/ml of pellet, and the aqueous volume of cell envelopes was 0.117 ml/ml of pellet. Thus the cell envelope for this yeast, under these conditions, was approximately 15% of the total cell volume. At a finite osmolality, the space in a yeast pellet that was accessible to salt was accounted for by the sum of initial interstitial space, the volume of the cell envelopes, and the volume of water abstracted from the cells by osmosis. Plots of S value versus osmolality were linear for uncharged probes and curvilinear for all salts. When Ficoll and potassium thiocyanate were presented to the yeast in admixture, the S values for the salt increased continuously over the range of osmolality studied. However, the S values for Ficoll 400 (which did not penetrate the cell wall) were lower by an amount equilivalent to the cell envelopes; they increased in parallel with the S curve for salt up to 1.15 osmol/kg and then plateaued. The results support the concept of incipient plasmolysis at 1.15 osmol/kg, and the separation of protoplasm from the cell wall is indicated with more concentrated solutions. Such cells were still viable if slowly diluted in distilled water, but they were injured by the shock of rapid dilution. However, shocking the cells did not release f8-fructofuranosidase into the medium. The complete accessibility of salts toward killed cells was demonstrated with yeast that had been pretreated with heat, organic solvents, or glutaraldehyde. The yeast cell envelope is composed of the protoplasmic membrane, the cell wall proper, and the intervening region, which is called periplasmic space. The envelope is metabolically active (6) and is known to contain several enzymes, of which ,3-fructofuranosidase (EC 3.2.1.26) and acid phosphatase (EC 3.1.3.2) are well documented. For Saccharomyces species, the available evidence supports the hypothesis (2, 4) that these enzymes are not covalently bound but are mechanically restrained within the periplasmic space. This space, which includes invaginations in the protoplasmic membrane and irregularities in the inner aspect of the cell wall, may be subject to volume changes depending upon the cell's environment. The relatively thick cell wall is undoubtedly responsible for the yeast cell's resistance to mechanical stress, whereas the site of regulation for solute exchange with the medium resides 564
primarily in the protoplasmic membrane. Thus, yeast is capable of withstanding concentrated salt or sugar solutions and, under very different conditions, of accumulating some of these same compounds against a concentration difference (19). Conversely, the cell wall presents no real barrier to the free diffusion of small molecules and ions (19). Conway and Downey (8) developed a quantitative technique for ascertaining the fractional volume in packed yeast that is available to a test solute. About 11% ofthe total cell volume of resting cells was found to be accessible to arabinose, for example, and this region of the cell was equated with the cell envelope (8). Gerhardt and co-workers (12, 18) introduced polydisperse dextrans as probes and were able to estimate threshold pore sizes. Evidence for plasmolysis in yeast (i.e., separation of the protoplasm from the cell wall in
OSMOTIC BEHAVIOR IN S. CEREVISIAE
VOL. 131, 1977
hypertonic media) has not been established and, indeed, the architecture of even the turgid cell envelope is far from satisfactorily described (7). For these reasons we felt that a physical approach that was independent of microscopy might yield interesting and complementary results. The present work includes elaboration of the Conway technique to space measurements involving a salt and a polymer in admixture. This paper also summarizes our background studies on the behavior of the yeast cell envelope toward a variety of solutes and, in some cases, over larger concentration ranges than hitherto reported. MATERIALS AND METHODS Yeast. Bakers' yeast, which was donated by Red Star Yeast and Products Co. at weekly intervals, was stored under refrigeration. About 1 cm was removed from each surface of a 454-g cake, and the remainder was crumbled into distilled water. After being stirred for 15 min and allowed to settle for 5 min, the suspension was decanted into tubes and centrifuged. The supernatant liquid was discarded. The yeast was similarly washed once more in distilled water and then suspended to 30% (wet wt/vol) in distilled water. All operations were at 23 to 25°C, and a fresh suspension was made each working day. Chemicals. All salts and sugars were analytical grade. Pentaerythritol (2,2-bishydroxymethyl-1,3propanediol) was from Eastman Kodak Co., Rochester, N.Y. Ficoll is a synthetic copolymer of sucrose and epichlorohydrin from Pharmacia Fine Chemicals, AB, Uppsala, Sweden. Ficoll 400 has a weightaverage molecular weight of 400,000 compared with 70,000 for Ficoll 70. Conway's dilution method. Pellets were obtained by centrifugation of yeast suspensions at 11,100 x g for 10 min (standard conditions). Ten-milliliter portions of 30% (wet wt/vol) suspension were added to tared 15-ml tubes, and a swinging-bucket rotor was used. The supernatant water was decanted to waste, and tubes were inverted for 1 min. Droplets of water were removed with absorbent paper. Gross weights were determined as quickly as possible, and tubes were then capped with aluminum foil. Each yeast pellet (accurately weighed, but invariably close to 3 g) was suspended with 3 ml of a particular test solution by vortex stirring. After 15 min (standard), the tube was vortexed again and then centrifuged under standard conditions. Either 1 or 2 ml of supernatant fraction was taken for analysis. An identical volume of undiluted test solution was also analyzed. In a few instances involving very dilute test solutions, the above amounts were scaled upwards within the same proportion. The aqueous space (S) in the original pellet that is accessible to test solution and consequently lowers the solute concentration upon mixing is related to the measured parameters as follows: s
V(Ci - Cf) W,,.Cf
565
where V, is the volume of solution added, W, is the wet weight of yeast pellet, Ci is the initial concentration of test solute, and C, is the final concentration of test solute (i.e., in the supernatant fraction). The dimensions of S are milliliters per gram of wet, packed yeast. When the density (d) of the pellet is known, a secondary value S, (in milliliters per milliliter of wet, packed yeast) follows: S, = S * d. Analyses. For the majority of solutes tested, the gravimetric method (8) was found to be reliable and accurate. Small aluminum pans (6 cm in diameter and 1.5 g) were tared, charged with samples, dried overnight in a convection oven set at 95°C, cooled in a desiccator, and weighed on a balance with a sensitivity of 0.1 mg. L-Arabinose, .-rhamnose, glucose, fructose, and sucrose (after acid hydrolysis) concentrations were determined with Sumner's 3,5-dinitrosalicylic acid reagent (1). Ficoll concentrations were usually monitored gravimetrically, but when in admixture with a salt the analyses followed an anthrone procedure (5). Chloride ion was determined by titration with mercuric nitrate (17). pNitrophenol and p-nitrophenolphosphate (after acid hydrolysis) concentrations were calculated from the absorbance of p-nitrophenolate ion at 425 nm (2). Thiocyanate ion was assayed as the ferric complex by the colorimetric method of Cosby and Sumner (9). Small corrections were applied where appropriate. In the gravimetric procedure, distilled-water controls were found to contain, on the average, about 0.3 mg of extracted yeast solids per ml. In the reducing-sugar procedure, a blank was constructed by measuring reducing substances extracted from yeast by mannitol (nonreducing) at an osmolality equivalent to that of the test compound. Density of pellets. The open ends of several glass centrifuge tubes (15 ml) were ground flat on carborundum paper. Tubes were identified with symbols inscribed by a diamond pencil, and several micro cover glasses (no. 1, 22-mm square) were similarly marked. Tubes and cover glasses were washed, dried, and tared. Each tube was filled with distilled water and a cover glass was placed across the ground-glass mouth, care being taken to avoid air bubbles. Excess water was wiped off, and the filled tube was weighed. The total volume of the tube was obtained from the weight of water it contained. A pellet of cells was prepared under standard conditions and weighed as usual. The tube was then carefully charged with water and topped with a cover glass as before. The volume of added water was again determined by weight difference and, when substracted from the total volume of the tube, yielded the volume of the wet yeast pellet. On one typical batch of yeast, the mean of four separate determinations of density was 1.0668 g/ml (standard deviation = 0.0004), which indicated adequate sensitivity and reliability for the method. Water abstracted by osmosis. When yeast is mixed with a solution of higher osmolality, there is a movement of water from the cells to the medium and a decrease in cell volume. This is reflected in the relative height of yest columns obtained upon centrifugation. We used Wintrobe tubes with a uniform bore of 3 mm and a graduated length of 105 mm
566
ARNOLD AND LACY
J. BACTERIOL.
(Arthur H. Thomas Co., Philadelphia). Centrifugation was at 1,440 x g for 10 min, and readings were made after letting the tubes stand for 1 h. Centrifugation conditions obviously affect the resultant "hematocrit." Also, the "rebound" in the column of partially compressed cells after centrifugation is time dependent and apparently is influenced by the diameter-to-volume ratio of the column. The above conditions were arrived at empirically and are discussed later. If V1 is the initial volume of the yeast pellet, V2 is the volume of solution added, and h is the relative height of packed cells (expressed as a fraction of the total height). Then V1 (1 - AV) VI + V2 where AV is the decrease in pellet volume per unit initial volume. Values of AV are an approximation to the fractional volume of water abstracted by osmosis. Osmolality. Values were derived from concentrations by reference to standard tables (21) and by interpolation. Unlisted compounds or mixtures were measured on an osmometer (Advanced Instruments, Inc., Needham Heights, Mass.) by freezing-point
lowering.
Fig. 2. The straight line of best fit is described by S = 0.209 + 0.00424 C, where C is given as percent (wt/vol). Ficoll 70 at a final concentration of 3.82% yielded an S value of 0.231, which was not significantly different from the response to the larger polymer. Sugars and polyols. In general, the S values for uncharged test compounds showed a linear response to final osmolality. The curve for Larabinose is depicted in Fig. 2 and is representative of this class of compounds. Equations describing the individual lines of best fit are assembled in Table 1, together with a line describing the combined data for the sugars and polyols. KSCN. The convenience of the colorimetric assay for thiocyanate, and the similarity of the S curve for potassium thiocyanate (KSCN) (Fig. 2) with that for KCl (Fig. 1), prompted our adoption of KSCN as a reference compound. The smooth curve of Fig. 2 is drawn to the
O.81
RESULTS Potassium and sodium chlorides. The relationship between S value and osmolality exhibited by these salts is curvilinear and approaches an asymptote of 0.680 ml/g (Fig. 1). The water content of standard pellets was 0.799 (±0.007) ml/g. There is no significant difference between KCl and NaCl, and analyses based on the titration of chloride ion show good agreement with those based on the gravimetric method (Fig. 1). Ficoll. The relationship between S values and concentration (C) for Ficoll 400 is shown in
.. A9,-
-o
-
--
I0.6 L-A,*nose
0.4 00.2 _:-
0 0
--
Ficoll
--
LO 0.O5 .5 Osmolality (Osmol.s/Kg) 3 5 Concentration (% wVo)
-
2.0
2.5
30
10~~~~~~~~~~~~~~~~~~ 10
FIG. 2. Effects of osmolality on the S values for KSCN (0), L-arabinose (a), and Ficoll-400 (A).
TABLE 1. S values as a function of osmolality for unmetabolized sugars and polyols 0.82
Linear
u
0.6
o
7
Compound" (mol wt) ^
>% c
ol 'n
I 0
0
2
0
3
4
5
,
OSmdoality
(OsmolS/Kg)
FIG. 1. Effects of osmolality on the S values of KCl (0) and NaCI (A). Data points are based on analyses by the gravimetric method (open symbols) or by titration of chloride ion (O). Insert contains additional data points for osmolalities greater than 3.0.
S value" (ml/g of wet packed yeast)
correlation coefficient
0.343 + 0.134 0, L-Arabinose (150)C 0.995 Lactose (342) 0.284 + 0.214 0, 0.997 Mannitol (182) 0.309 + 0.154 0, 0.995 Pentaerythritol (136) 0.308 + 0.200 0, 0.994 Combined data 0.319 + 0.150 0, 0.956 a At least four concentrations of each compound were tested. b Equations describing the straight lines of best fit for S as a function of osmolality (0,) were computed by the least-squares method. c Two S values for L-rhamnose (164) were in good agreement with those for L-arabinose at equivalent osmolality.
OSMOTIC BEHAVIOR IN S. CEREVISIAE
VOL. 131, 1977
combined data of 10 separate trials on different batches of yeast over a 6-month period. Consistent behavior by the yeast and reliability in the method are indicated. S values for KSCN are slightly higher than those for KCl at equivalent osmolality. The difference, although consistent, was never greater than 5%. Divalent cations and anions. The S values for a variety of salts are plotted in Fig. 3. The dashed curve in Fig. 3 is the KCl line (cf. Fig. 1). In general, the S values for salts of divalent cations and anions are slightly lower than those for monovalent salts at equivalent osmolality. The activity coefficients for several ofthe divalent ions are significantly removed from unity and different from each other (21). Consequently, plots (not shown here) of S values versus molarity for the above salts were quite disparate. On the other hand, the reasonable agreement of S values on the osmolality scale (Fig. 3) argues in favor of a predominant influence of osmosis on the magnitude of S. Water abstracted by osmosis. A series of standard yeast pellets was mixed with a range of KSCN concentrations, and the S values were determined. These were converted to Sv values after ascertaining the density of standard pellets. An additional portion of each suspension was removed and subjected to packed-cell volume measurement. Calculated values for AV are listed in Table 2, together with corresponding values for S,. The magnitude of S,-AV was 0.354 on the average and was fairly uniforn (standard deviation = 0.014). This parameter is not significantly different from the S, value (0.340 + 0.006) for sugars and polyols at zero osmolality. (The latter value is the product of the intercept of the S curve [Table 1] and pellet density.) These results indicate that the in-
I~~~~~~~~~~~~~~KC
,.4 I
Is
0.2 _
0,
a. 0.5
Osmololity
& I.
1.0
(Osmolts/Kg )
2.0
2.5
crease in S value with solute concentration is primarily due to osmosis. Concurrent measurements of KSCN-accessible and Ficoll-accessible spaces. A stock solution was made to contain 2.5 M KSCN and 5% (wt/vol) Ficoll 400. Serial dilutions were made to a lower limit of one-tenth. These solutions were individually mixed with standard yeast pellets. The highest final concentration of Ficoll in these experiments was 3.45%. Compared with the companion salt, Ficoll concentrations can be neglected in calculating the osmolality of the medium. A modification was involved in one such trial in that the Ficoll concentration of test solutions was kept constant at 5% while the KSCN concentration was varied. There is good agreement among the results of all three trials (Fig. 4). The smooth line which is drawn to the KSCN data is curvilinear, and S values show a continuous increase over the range examined. The Ficoll line is parallel to the KSCN line up to an osmolality of 1.15, but thereafter there is a plateau. Condition of cells. It was important to know whether the yeast had suffered any deleterious effects. In particular, we asked whether the most concentrated salt solutions might cause measurable damage to the cell envelope. We found that the KCl-accessible space approached a maximum value of 0.680 ml/g at high osmolalities, whereas the water content of standard pellets was 0.799 (+0.007) ml/g. On the other hand, killed cells (see below) exhibited comTABLE 2. Relationship between S, values and the degree of water abstracted from yeast by osmosis Osmolality of KSCN (os-
&°
FIG. 3. Effects of osmolality on the S values of MgCl2 (0), Na2SO4 (0), MgSO4 (A), K2SO4 (V), arnd NaK tartrate (0). The dashed line represents tthe equivalent response for KC1.
AVa
Stb
(S. - AV)Y
0.048 0.098 0.138 0.169 0.239 0.278 0.309 0.342 0.354
0.410 0.447 0.473 0.549 0.591 0.639 0.667 0.682 0.699
0.362 0.349 0.335 0.380 0.352 0.361 0.358 0.340 0.345 mean = 0.354
mol/kg) 0.209 0.402 0.581 0.738 1.080 1.393 1.814 2.267 2.715
0.8 _
567
SD = 0.014 a AV is the decrease in pellet volume per unit initial volume, and is an approximation to the degree of water abstracted by osmosis. All values are in milliliters per milliliter of wet, packed yeast. b Sr is the space available to test solute (KSCN) per unit volume of initial pellet. c For a salt such as KSCN, the value of (Sv - AV) is a measure of cell envelope plus interstitial water in the original pellet. SD, Standard deviation
568
ARNOLD AND LACY
J. BACTZRIOL.
AsOf
FIQOLL
0
0.5
10
1.5
Osmolality (Osnmoes/Kg)
2.0
2.5
&,
FIG. 4. Effects of osmolality on the S values for KSCN (open symbols) and Ficoll ('filled symbols) in admixture. The different symbols rrefer to three separate experiments.
plete accessibility to salts base,d on the water content of such pellets. The methylene blue test, wvhich is a good indicator of viability when p erformed under prescribed conditions (3), reveialed a high degree of integrity even in cellsithat had been mixed with concentrated salt s olutions. In one experiment, triplicate pellets of yeast were mixed with approximately equaLl volumes of 2.5 M KSCN. Analysis on one tube revealed a final concentration of 1.534 M KSCNI and yielded an S value of 0.656, which is typiccal for this salt. The contents of the second tul be were diluted about 1,000-fold by the dropwrise addition of distilled water to the stirred suspension. An appropriate volume was the*n mixed with buffered methylene blue solutio n (3) and examined in a hemocytometer. Eigght large fields (about 40 cells each) were obs3erved, and the percentage of dead (blue-staine3d) cells was recorded. The average was 7 (- :5%), compared with 1 (+3)% for cells from thi e same batch of yeast that had only received distilled-water washes. It is worth pointing ouit that the slow dilution of cells (from a concenitrated salt medium) is mandatory for the mair ntenance of viability. When a third pellet of cel[Is was similarly treated with KSCN as above but then "shocked" by rapid dilution in 1 liter of distilled water, the fraction of dead celIls then was 41 (+6%). However, this treatmerit did not affect the status of yeast fl-fructofursinosidase. Cells that were exposed to KSCN (2.'7 osmol/kg) and then washed retained 94% of the activity of untreated controls no matter h(ow quickly they were diluted. With one batch of yeast, S vadlues were measured at four concentrations ofr KSCN (range, 0.4 to 2.7 osmol/kg) and witl i four different
presentation times. Compared with the standard time of 15 min, the S values were: (i) not significantly different at 7.5 min; (ii) about 2% higher at 30 min; and (iii) about 5% higher when the incubation was extended to 60 min. Controls. Pellets prepared from cells that had been killed by pretreatments with organic solvents (ethyl acetate or chloroform), heat, or glutaraldehyde exhibited complete accessibility to KSCN (judged by their water content). All of these pretreatments were lethal, as indicated by 100% of the cells taking the stain in the methylene blue test. Although the conclusions were clear-cut, experiments on killed material yielded S values that displayed somewhat more
variability.
The S values for killed cells were independent of external concentration. For example, with heat-killed cells (90°C/3 min), KSCN S
values of 0.701, 0.693, and 0.699 ml/g (wet, packed yeast) were obtained at osmolalities of 0.713, 1.412, and 2.889 osmol/kg, respectively. The water content of pellets of heat-killed yeast was 0.640 ml/g (wet, packed yeast). Likewise, for cells pretreated overnight with 3% glutaraldehyde in 0.1 M collidine-HCl (pH 7.0), the S values over a series of KSCN concentrations had an average of 0.707 (+0.076) ml/g (wet, packed yeast), which was close to the water content of those pellets: 0.678 (+0.001) ml/g (wet, packed yeast). Substrates of 3-fructofuranosidase and acid phosphatase. Acid phosphatase exhibits maximal activity at pH 3.8 and negligible activity at pH 7.5, which was used here to study the accessibility of conventional substrates and their corresponding products of hydrolysis (Table 3). Commercial bakers' yeast has a high concentration of f8-fructofuranosidase, and the enzyme catalyzes the hydrolysis of sucrose over a broad range of pH values. It is well known that the products, glucose and fructose, are readily absorbed by the protoplasm. To overcome both problems in the assessment of passively penetrable space, we took advantage of the inhibitory effect of uranyl acetate on ,B-fructofuranosidase as well as the glucose transport system (10). The yeast was washed twice in 10 mM uranyl acetate-acetic acid (pH 5.0), and test solutions were also fortified with the inhibitor. Preliminary trials showed that the inhibitor was effective at this concentration. The results are summarized in Table 3, along with reference values. They demonstrate that substrates and products ofboth enzymes penetrate a space that is comparable to that found for salts and nonmetabolized sugars. The one exception, p-nitrophenol, exhibits large S values, which indicate accumulation by the yeast.
OSMOTIC BEHAVIOR IN S. CEREVISIAE
VOL. 131, 1977
TABLE 3. S values for substrates and products of yeast ,B fructofuranosidase and acid phosphatase S (ml/g of wet, Compound
Sucroseb Glucoseb Fructoseb Na2, -glycerophosphatec Na2 p-nitrophenolphosphatec p-Nitrophenol pH 7.5c pH 3.8d
Concn
packed yeast) Refer-
(M)t Test
0.128
ence a
0.147
0.368 0.327
0.340 0.342
0.147
0.327
0.342
0.095
0.353
0.380
0.004
0.298
0.319
NaH2PO4
0.002 0.001 0.678
1.89 3.07 0.544
0.319 0.319 0.537
KH2PO4
0.626
0.573
0.525
a Nonmetabolized polyols or KCl at an osmolality equivalent to that of the test compound. b Yeast ,3-fructofuranosidase and sugar transport systems were inhibited by 10 mM uranyl acetateacetic acid (pH 5.0). c In the presence of 10 mM phosphate buffer (pH 7.5). d In the presence of 10 mM acetate buffer (pH 3.8).
Effects of glutaraldehyde. In addition to using pellets prepared from glutaraldehyde-fixed cells as controls, some determinations of density, volume, and water content were performed on them. A batch of fresh, washed yeast was stirred overnight in 3% glutaraldehyde at 23 to 25°C, washed in copious amounts of distilled water, and then made up to 30% (wet wt/ vol). The cell concentration was determined on a suitably diluted sample in a hemacytometer. Another batch of yeast was similarly treated, except that 0.1 M collidine-HCl (pH 7.4) was included during fixation. The results are summarized in Table 4; measurements of volume and water content are compared among equal numbers of cells for each treatment. The results demonstrate that considerable shrinkage of cells results from fixation in the glutaraldehyde-collidine mixture. Smaller changes were noted when unbuffered glutaraldehyde was employed. DISCUSSION By definition, all S values refer to initial pellets, which were equilibrated with distilled water. The magnitude of the S value depends upon the final concentration of test solute. However, extrapolation to zero concentration yields a limiting value for S that is independent of, for example, an osmotic effect on the cell. The data for Ficoll 400 (Fig. 2) and for nonme-
569
tabolized polyols (Table 1) yield linear relationships, and the limiting values of S, as concentration or osmolality approaches zero, are readily computed. A summary that is based on these values and a conversion to unit volume is given in Table 5. Only the interstitial water in a yeast pellet is accessible to the polymer Ficoll (line 1, Table 5). This value is 0.223 ml/ ml of pellet, and the space that cells occupy is 0.777 ml/ml of pellet (by difference). Relatively small molecules permeate the cell envelope and are additionally diluted by that volume (line 4, Table 5). From the data of Table 5, we calculate that the cell envelope is, on the average, 15% of the total cell volume. Conway and Downey (8) obtained a comparable estimate of 11% for their yeast.
The total space that is accessible to small molecules may be viewed as the sum of the initial interstitial water, the cell envelopes, and the water abstracted from the cells by osmosis. Our results substantiate this simple working hypothesis in that a great variety of salts, mono- and disaccharides, and polyols follow (reasonably well) the same general response curve for S value versus osmolality in contrast to molarity. The increase in S value with osmolality can be accounted for by an independent estimate of the water abstracted by osmosis (Table 2). There was no indication that increased accessibility at higher salt concentrations was due to irreversible damage of the protoplasmic memTABLE 4. Effect of glutaraldehyde on yeast Yeast pellet Water Vol (ml/100 content (g/1010 cells) cells)
Pretreatment Density (g/ml)
None 3% glutaralde-
1.067 (±0.0004) 1.085 (±0.001)
0.793 0.767
0.675 0.610
1.103 (±0.002)
0.538
0.402
hyde 3% glutaraldehyde in 0.1 M
collidine-HCl (pH 7.0)
TABLE 5. Space analysis for standard yeast pellets Specific vol Component
ml/g of pellet ml/ml of pel-
Interstitial water Cells Interstitial water and cell envelopes Cell envelopes Protoplasms
0.209 0.728 0.319
0.223 0.777 0.340
0.110 0.618
0.117 0.660
570 ARNOLD AND LACY brane. Instead, exposed cells were shown to be in good condition, as judged by the methylene blue test, and S values were not greatly affected by a fourfold increase in the presentation time. The S curves for salts were curvilinear, but showed agreement with polyols at zero osmolality and approached similar values at higher osmolalities (see Fig. 2). The intervening region in which S values, at equivalent osmolality, are higher for salts than for sugars is most likely related to the ionic environment of the cell wall, to which covalently bonded carboxyl (13) and phosphate (11) groups have been attributed. A related finding may be the demonstration of salt-induced contractions in isolated bacterial cell walls (15). It has been shown (16) that negatively charged particles (e.g., kaolinite) exhibit larger hydrogen ion and cation concentrations at their surfaces than in the surrounding medium, and absorbed enzymes are influenced by this "double layer" surrounding the particle (14). In this context, we were interested in the S values exhibited by KCl at low concentrations. No evidence of exclusion or adsorption was discernible, and good agreement between gravimetric analyses of the salt and titrimetric analyses of chloride ion was obtained (Fig. 1). Yeast cells are osmotically responsive, and their survival in distilled water depends on the ability of the cell wall to withstand a substantial turgor pressure. Numerous reports in the literature, as well as the present investigation, suggest that the envelope will initially contract as a unit when the yeast cell is placed in solutions of increasing osmolality. However, plasmolysis has not been previously reported for yeasts. With reference to Fig. 4, we suggest that incipient plasmolysis has occurred at an osmolality in the vicinity of 1.15 osmol/kg. Below this point the Ficoll-accessible space and the KSCN-assessible space increase in approximately parallel fashion, which is consistent with conjoint contraction of the cell wall and the protoplasm. Above 1.15 osmol/kg, S values for KSCN continue to increase (further contraction of protoplasm), but those for Ficoll remain constant (no further change reflected at the external surface of the cell wall). The simplest explanation is that plasmolysis has occurred. An osmolality of 1.15 osmol/kg is given by 0.637 M KCl, and it should be noted that concentrations of 0.6 to 0.8 M KCl are recommended (20) for the preparation of protoplasts from S. cerevisiae. Our results offer a plausible rationale for this empirical recommendation. It was anticipated that in higher osmolalities than that required for incipient plasmolysis,
J. BACTZRIOL.
the normalized heights of packed yeast columns would exhibit a plateau in analogous fashion to the S values for Ficoll (Fig. 4). This was not found (Table 2). Possibly, the cell walls of plasmolyzed cells are sufficiently flaccid to become severely deformed under centrifugation and thus yield falsely low hematocrits. The conditions for packed-cell volume measurement were arrived at by preliminary trials in which distilled-water suspensions of yeast were subjected to different centrifugal forces in Wintrobe tubes. Relative heights of yeast columns were recorded immediately after centrifugation and thereafter at 15-min intervals. It was desirable to match these packing characteristics with those used in the preparation of standard pellets (which had different dimensions). Once the density of the latter was determined (see Materials and Methods), we were able to predict the hematocrit by calculation. Conditions that would match this value were deemed to be a good approximation to equivalent packing. However, for cells equilibrated with test solutions, there was a progressive change in column height with post-centrifugation interval, and the rate of change was slower for the cells from the more concentrated salt solutions. These results are consistent with the above suggestion that plasmolyzed cells give falsely low hematocrits. Our results with the substrates and products of f-fructofuranosidase and acid phosphatase (Table 3) indicate that the cell envelope is accessible to all of them. This is consistent with a periplasmic locale for these enzymes and with the assessment by Sherrer et al. (18) that the cell wall presents no real barrier to the passive diffusion of molecules up to a size of approximately 620 daltons. Live yeast cells are commonly assayed for acid phosphatase activity with nitrophenol phosphate as substrate. Absorption of nitrophenol by cells (as indicated by the data of Table 3) is not a problem, because base is added at the termination of incubation and redistribution of nitrophenol to the medium is achieved. Our results indicate shrinkage by cells during glutaraldehyde fixation. The common practice of fortifying fixatives with 0.1 to 0.2 M phosphate, collidine, or cacodylate buffers is not encouraged by our cell volume measurements. Meaningful ultrastructural evidence for plasmolysis in live cells will be predicated on obtaining fixation of specimens with retention of native proportions. ACKNOWLEDGMENIS This work was supported by a grant from Research Corporation and by Public Health Service grant Al 13177 from the National Institute of Allergy and Infectious Diseases.
VOL. 131, 1977
OSMOTIC BEHAVIOR IN S. CEREVISIAE
We thank Philip Bestic for helpful discussions and Phillip Gerhardt for comments on the manuscript. 12. 1. 2. 3.
4. 5.
6. 7.
8. 9. 10.
11.
LITERATURE CITED Arnold, W. N. 1965. j3-Fructofuranosidase from grape berries. Biochim. Biophys. Acta 110:134-147. Arnold, W. N. 1972. Location of acid phosphatase and j3fructofuranosidase within yeast cell envelopes. J. Bacteriol. 112:1346-1352. Arnold, W. N. 1972. The structure of the yeast cell wall. Solubilization of a marker enzyme, p-fructofuranosidase, by the autolytic enzyme system. J. Biol. Chem. 247:1161-1169. Arnold, W. N. 1973. Volume and enzyme content of the periplasmic space in yeast. Physiol. Chem. Phys. 5:117-123. Arnold, W. N., and M. N. McLellan. 1975. Trehalose and glycogen levels during the initial stages of growth of Candida albicans. Physiol. Chem. Phys. 7:369-380. Bacon, J. S. D. 1970. Life outside the cell, p. 45-66. In W. Bartley, H. L. Kornberg, and J. R. Quayle (ed.), Essays in cell metabolism. Wiley, London. Cabib, E. 1975. Molecular aspects of yeast morphogenesis. Annu. Rev. Microbiol. 29:191-214. Conway, E. J., and M. Downey. 1950. An outermetabolic region of the yeast cell. Biochem. J. 47:347-355. Cosby, E. L., and J. B. Sumner. 1945. Rhodanese. Arch. Biochem. Biophys. 7:457-460. Demis, D. J., A. Rothstein, and R. Meier. 1954. The relationship of the cell surface to metabolism. X. The location and function of invertase in the yeast cell. Arch. Biochem. Biophys. 48:55-62. Eddy, A. A., and A. D. Rudin. 1958. The structure ofthe
13. 14.
15. 16. 17. 18. 19.
20.
21.
571
yeast cell wall. I. Identification of charged groups at the surface. Proc. R. Soc. London Ser. B 148:419-432. Gerhardt, P., and J. A. Judge. 1964. Porosity of isolated cell walls of Saccharomyces cerevisiae and Bacillus megaterium. J. Bacteriol. 87:945-951. Jayatissa, P. M., and A. H. Rose. 1976. Role of wall phosphomannan in flocculation of Saccharomyces cerevisiae. J. Gen. Microbiol. 96:165-174. McLaren, A. D., and E. F. Estermann. 1957. Influence of pH on the activity of chymotrypsin at a solid-liquid interface. Arch. Biochem. Biophys. 68:157-160. Marquis, R. E. 1968. Salt-induced contraction of bacterial cell walls. J. Bacteriol. 95:775-781. Michaels, A. S., and 0. Morelos. 1955. Polyelectrolyte adsorption by kaolinite. Ind. Eng. Chem. 47:18011809. Schales, O., and S. S. Schales. 1941. A simple and accurate method for the determination of chloride in biological fluids. J. Biol. Chem. 140:879-884. Scherrer, R., L. Louden, and P. Gerhardt. 1974. Porosity of the yeast cell wall and membrane. J. Bacteriol. 118:534-540. Suomalainen, H., and E. Oura. 1971. Yeast nutrition and solute uptake, p. 3-74. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press Inc., London. Villanueva, J. R., and I. G. Acha. 1971. Production and use of fungal protoplasts. p. 665-718. In C. Booth (ed.), Methods in microbiology, vol. 4. Academic Press Inc., London. Wolf, A. V., M. G. Brown, and P. G. Prentiss. 1971. Concentration properties of aqueous solutions: conversion tables, p. D-181-226. In R. G. Weast (ed.), Handbook of chemistry and physics, 52nd ed. CRC Press, Cleveland.
JOURNAL OF BACTERIOLOGY, Aug. 1977, p. 564-571 Copyright ©D 1977 American Society for Microbiology
Permeability of the Cell Envelope and Osmotic Behavior in Saccharomyces cerevisiae WILFRED N. ARNOLD* AND JOHN S. LACY Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103
Received for publication 4 May 1977
Bakers' yeast (Saccharomyces cerevisiae) was equilibrated with distilled water and then packed into standardized pellets by centrifugation. The fractional space (S value) that was accessible to passive permeation was probed with a variety of mono- and divalent salts, mono- and disaccharides, polyols, substrates and products of ,-fructofuranosidase (EC 3.2.1.26) and acid phosphatase (EC 3.1.3.2), and a cross-linked polymer of sucrose (Ficoll 400). A simple but very reproducible method was developed to measure pellet volume. At the limit of zero osmolality for bathing medium, the interstitial space was 0.223 ml/ml of pellet, and the aqueous volume of cell envelopes was 0.117 ml/ml of pellet. Thus the cell envelope for this yeast, under these conditions, was approximately 15% of the total cell volume. At a finite osmolality, the space in a yeast pellet that was accessible to salt was accounted for by the sum of initial interstitial space, the volume of the cell envelopes, and the volume of water abstracted from the cells by osmosis. Plots of S value versus osmolality were linear for uncharged probes and curvilinear for all salts. When Ficoll and potassium thiocyanate were presented to the yeast in admixture, the S values for the salt increased continuously over the range of osmolality studied. However, the S values for Ficoll 400 (which did not penetrate the cell wall) were lower by an amount equilivalent to the cell envelopes; they increased in parallel with the S curve for salt up to 1.15 osmol/kg and then plateaued. The results support the concept of incipient plasmolysis at 1.15 osmol/kg, and the separation of protoplasm from the cell wall is indicated with more concentrated solutions. Such cells were still viable if slowly diluted in distilled water, but they were injured by the shock of rapid dilution. However, shocking the cells did not release f8-fructofuranosidase into the medium. The complete accessibility of salts toward killed cells was demonstrated with yeast that had been pretreated with heat, organic solvents, or glutaraldehyde. The yeast cell envelope is composed of the protoplasmic membrane, the cell wall proper, and the intervening region, which is called periplasmic space. The envelope is metabolically active (6) and is known to contain several enzymes, of which ,3-fructofuranosidase (EC 3.2.1.26) and acid phosphatase (EC 3.1.3.2) are well documented. For Saccharomyces species, the available evidence supports the hypothesis (2, 4) that these enzymes are not covalently bound but are mechanically restrained within the periplasmic space. This space, which includes invaginations in the protoplasmic membrane and irregularities in the inner aspect of the cell wall, may be subject to volume changes depending upon the cell's environment. The relatively thick cell wall is undoubtedly responsible for the yeast cell's resistance to mechanical stress, whereas the site of regulation for solute exchange with the medium resides 564
primarily in the protoplasmic membrane. Thus, yeast is capable of withstanding concentrated salt or sugar solutions and, under very different conditions, of accumulating some of these same compounds against a concentration difference (19). Conversely, the cell wall presents no real barrier to the free diffusion of small molecules and ions (19). Conway and Downey (8) developed a quantitative technique for ascertaining the fractional volume in packed yeast that is available to a test solute. About 11% ofthe total cell volume of resting cells was found to be accessible to arabinose, for example, and this region of the cell was equated with the cell envelope (8). Gerhardt and co-workers (12, 18) introduced polydisperse dextrans as probes and were able to estimate threshold pore sizes. Evidence for plasmolysis in yeast (i.e., separation of the protoplasm from the cell wall in
OSMOTIC BEHAVIOR IN S. CEREVISIAE
VOL. 131, 1977
hypertonic media) has not been established and, indeed, the architecture of even the turgid cell envelope is far from satisfactorily described (7). For these reasons we felt that a physical approach that was independent of microscopy might yield interesting and complementary results. The present work includes elaboration of the Conway technique to space measurements involving a salt and a polymer in admixture. This paper also summarizes our background studies on the behavior of the yeast cell envelope toward a variety of solutes and, in some cases, over larger concentration ranges than hitherto reported. MATERIALS AND METHODS Yeast. Bakers' yeast, which was donated by Red Star Yeast and Products Co. at weekly intervals, was stored under refrigeration. About 1 cm was removed from each surface of a 454-g cake, and the remainder was crumbled into distilled water. After being stirred for 15 min and allowed to settle for 5 min, the suspension was decanted into tubes and centrifuged. The supernatant liquid was discarded. The yeast was similarly washed once more in distilled water and then suspended to 30% (wet wt/vol) in distilled water. All operations were at 23 to 25°C, and a fresh suspension was made each working day. Chemicals. All salts and sugars were analytical grade. Pentaerythritol (2,2-bishydroxymethyl-1,3propanediol) was from Eastman Kodak Co., Rochester, N.Y. Ficoll is a synthetic copolymer of sucrose and epichlorohydrin from Pharmacia Fine Chemicals, AB, Uppsala, Sweden. Ficoll 400 has a weightaverage molecular weight of 400,000 compared with 70,000 for Ficoll 70. Conway's dilution method. Pellets were obtained by centrifugation of yeast suspensions at 11,100 x g for 10 min (standard conditions). Ten-milliliter portions of 30% (wet wt/vol) suspension were added to tared 15-ml tubes, and a swinging-bucket rotor was used. The supernatant water was decanted to waste, and tubes were inverted for 1 min. Droplets of water were removed with absorbent paper. Gross weights were determined as quickly as possible, and tubes were then capped with aluminum foil. Each yeast pellet (accurately weighed, but invariably close to 3 g) was suspended with 3 ml of a particular test solution by vortex stirring. After 15 min (standard), the tube was vortexed again and then centrifuged under standard conditions. Either 1 or 2 ml of supernatant fraction was taken for analysis. An identical volume of undiluted test solution was also analyzed. In a few instances involving very dilute test solutions, the above amounts were scaled upwards within the same proportion. The aqueous space (S) in the original pellet that is accessible to test solution and consequently lowers the solute concentration upon mixing is related to the measured parameters as follows: s
V(Ci - Cf) W,,.Cf
565
where V, is the volume of solution added, W, is the wet weight of yeast pellet, Ci is the initial concentration of test solute, and C, is the final concentration of test solute (i.e., in the supernatant fraction). The dimensions of S are milliliters per gram of wet, packed yeast. When the density (d) of the pellet is known, a secondary value S, (in milliliters per milliliter of wet, packed yeast) follows: S, = S * d. Analyses. For the majority of solutes tested, the gravimetric method (8) was found to be reliable and accurate. Small aluminum pans (6 cm in diameter and 1.5 g) were tared, charged with samples, dried overnight in a convection oven set at 95°C, cooled in a desiccator, and weighed on a balance with a sensitivity of 0.1 mg. L-Arabinose, .-rhamnose, glucose, fructose, and sucrose (after acid hydrolysis) concentrations were determined with Sumner's 3,5-dinitrosalicylic acid reagent (1). Ficoll concentrations were usually monitored gravimetrically, but when in admixture with a salt the analyses followed an anthrone procedure (5). Chloride ion was determined by titration with mercuric nitrate (17). pNitrophenol and p-nitrophenolphosphate (after acid hydrolysis) concentrations were calculated from the absorbance of p-nitrophenolate ion at 425 nm (2). Thiocyanate ion was assayed as the ferric complex by the colorimetric method of Cosby and Sumner (9). Small corrections were applied where appropriate. In the gravimetric procedure, distilled-water controls were found to contain, on the average, about 0.3 mg of extracted yeast solids per ml. In the reducing-sugar procedure, a blank was constructed by measuring reducing substances extracted from yeast by mannitol (nonreducing) at an osmolality equivalent to that of the test compound. Density of pellets. The open ends of several glass centrifuge tubes (15 ml) were ground flat on carborundum paper. Tubes were identified with symbols inscribed by a diamond pencil, and several micro cover glasses (no. 1, 22-mm square) were similarly marked. Tubes and cover glasses were washed, dried, and tared. Each tube was filled with distilled water and a cover glass was placed across the ground-glass mouth, care being taken to avoid air bubbles. Excess water was wiped off, and the filled tube was weighed. The total volume of the tube was obtained from the weight of water it contained. A pellet of cells was prepared under standard conditions and weighed as usual. The tube was then carefully charged with water and topped with a cover glass as before. The volume of added water was again determined by weight difference and, when substracted from the total volume of the tube, yielded the volume of the wet yeast pellet. On one typical batch of yeast, the mean of four separate determinations of density was 1.0668 g/ml (standard deviation = 0.0004), which indicated adequate sensitivity and reliability for the method. Water abstracted by osmosis. When yeast is mixed with a solution of higher osmolality, there is a movement of water from the cells to the medium and a decrease in cell volume. This is reflected in the relative height of yest columns obtained upon centrifugation. We used Wintrobe tubes with a uniform bore of 3 mm and a graduated length of 105 mm
566
ARNOLD AND LACY
J. BACTERIOL.
(Arthur H. Thomas Co., Philadelphia). Centrifugation was at 1,440 x g for 10 min, and readings were made after letting the tubes stand for 1 h. Centrifugation conditions obviously affect the resultant "hematocrit." Also, the "rebound" in the column of partially compressed cells after centrifugation is time dependent and apparently is influenced by the diameter-to-volume ratio of the column. The above conditions were arrived at empirically and are discussed later. If V1 is the initial volume of the yeast pellet, V2 is the volume of solution added, and h is the relative height of packed cells (expressed as a fraction of the total height). Then V1 (1 - AV) VI + V2 where AV is the decrease in pellet volume per unit initial volume. Values of AV are an approximation to the fractional volume of water abstracted by osmosis. Osmolality. Values were derived from concentrations by reference to standard tables (21) and by interpolation. Unlisted compounds or mixtures were measured on an osmometer (Advanced Instruments, Inc., Needham Heights, Mass.) by freezing-point
lowering.
Fig. 2. The straight line of best fit is described by S = 0.209 + 0.00424 C, where C is given as percent (wt/vol). Ficoll 70 at a final concentration of 3.82% yielded an S value of 0.231, which was not significantly different from the response to the larger polymer. Sugars and polyols. In general, the S values for uncharged test compounds showed a linear response to final osmolality. The curve for Larabinose is depicted in Fig. 2 and is representative of this class of compounds. Equations describing the individual lines of best fit are assembled in Table 1, together with a line describing the combined data for the sugars and polyols. KSCN. The convenience of the colorimetric assay for thiocyanate, and the similarity of the S curve for potassium thiocyanate (KSCN) (Fig. 2) with that for KCl (Fig. 1), prompted our adoption of KSCN as a reference compound. The smooth curve of Fig. 2 is drawn to the
O.81
RESULTS Potassium and sodium chlorides. The relationship between S value and osmolality exhibited by these salts is curvilinear and approaches an asymptote of 0.680 ml/g (Fig. 1). The water content of standard pellets was 0.799 (±0.007) ml/g. There is no significant difference between KCl and NaCl, and analyses based on the titration of chloride ion show good agreement with those based on the gravimetric method (Fig. 1). Ficoll. The relationship between S values and concentration (C) for Ficoll 400 is shown in
.. A9,-
-o
-
--
I0.6 L-A,*nose
0.4 00.2 _:-
0 0
--
Ficoll
--
LO 0.O5 .5 Osmolality (Osmol.s/Kg) 3 5 Concentration (% wVo)
-
2.0
2.5
30
10~~~~~~~~~~~~~~~~~~ 10
FIG. 2. Effects of osmolality on the S values for KSCN (0), L-arabinose (a), and Ficoll-400 (A).
TABLE 1. S values as a function of osmolality for unmetabolized sugars and polyols 0.82
Linear
u
0.6
o
7
Compound" (mol wt) ^
>% c
ol 'n
I 0
0
2
0
3
4
5
,
OSmdoality
(OsmolS/Kg)
FIG. 1. Effects of osmolality on the S values of KCl (0) and NaCI (A). Data points are based on analyses by the gravimetric method (open symbols) or by titration of chloride ion (O). Insert contains additional data points for osmolalities greater than 3.0.
S value" (ml/g of wet packed yeast)
correlation coefficient
0.343 + 0.134 0, L-Arabinose (150)C 0.995 Lactose (342) 0.284 + 0.214 0, 0.997 Mannitol (182) 0.309 + 0.154 0, 0.995 Pentaerythritol (136) 0.308 + 0.200 0, 0.994 Combined data 0.319 + 0.150 0, 0.956 a At least four concentrations of each compound were tested. b Equations describing the straight lines of best fit for S as a function of osmolality (0,) were computed by the least-squares method. c Two S values for L-rhamnose (164) were in good agreement with those for L-arabinose at equivalent osmolality.
OSMOTIC BEHAVIOR IN S. CEREVISIAE
VOL. 131, 1977
combined data of 10 separate trials on different batches of yeast over a 6-month period. Consistent behavior by the yeast and reliability in the method are indicated. S values for KSCN are slightly higher than those for KCl at equivalent osmolality. The difference, although consistent, was never greater than 5%. Divalent cations and anions. The S values for a variety of salts are plotted in Fig. 3. The dashed curve in Fig. 3 is the KCl line (cf. Fig. 1). In general, the S values for salts of divalent cations and anions are slightly lower than those for monovalent salts at equivalent osmolality. The activity coefficients for several ofthe divalent ions are significantly removed from unity and different from each other (21). Consequently, plots (not shown here) of S values versus molarity for the above salts were quite disparate. On the other hand, the reasonable agreement of S values on the osmolality scale (Fig. 3) argues in favor of a predominant influence of osmosis on the magnitude of S. Water abstracted by osmosis. A series of standard yeast pellets was mixed with a range of KSCN concentrations, and the S values were determined. These were converted to Sv values after ascertaining the density of standard pellets. An additional portion of each suspension was removed and subjected to packed-cell volume measurement. Calculated values for AV are listed in Table 2, together with corresponding values for S,. The magnitude of S,-AV was 0.354 on the average and was fairly uniforn (standard deviation = 0.014). This parameter is not significantly different from the S, value (0.340 + 0.006) for sugars and polyols at zero osmolality. (The latter value is the product of the intercept of the S curve [Table 1] and pellet density.) These results indicate that the in-
I~~~~~~~~~~~~~~KC
,.4 I
Is
0.2 _
0,
a. 0.5
Osmololity
& I.
1.0
(Osmolts/Kg )
2.0
2.5
crease in S value with solute concentration is primarily due to osmosis. Concurrent measurements of KSCN-accessible and Ficoll-accessible spaces. A stock solution was made to contain 2.5 M KSCN and 5% (wt/vol) Ficoll 400. Serial dilutions were made to a lower limit of one-tenth. These solutions were individually mixed with standard yeast pellets. The highest final concentration of Ficoll in these experiments was 3.45%. Compared with the companion salt, Ficoll concentrations can be neglected in calculating the osmolality of the medium. A modification was involved in one such trial in that the Ficoll concentration of test solutions was kept constant at 5% while the KSCN concentration was varied. There is good agreement among the results of all three trials (Fig. 4). The smooth line which is drawn to the KSCN data is curvilinear, and S values show a continuous increase over the range examined. The Ficoll line is parallel to the KSCN line up to an osmolality of 1.15, but thereafter there is a plateau. Condition of cells. It was important to know whether the yeast had suffered any deleterious effects. In particular, we asked whether the most concentrated salt solutions might cause measurable damage to the cell envelope. We found that the KCl-accessible space approached a maximum value of 0.680 ml/g at high osmolalities, whereas the water content of standard pellets was 0.799 (+0.007) ml/g. On the other hand, killed cells (see below) exhibited comTABLE 2. Relationship between S, values and the degree of water abstracted from yeast by osmosis Osmolality of KSCN (os-
&°
FIG. 3. Effects of osmolality on the S values of MgCl2 (0), Na2SO4 (0), MgSO4 (A), K2SO4 (V), arnd NaK tartrate (0). The dashed line represents tthe equivalent response for KC1.
AVa
Stb
(S. - AV)Y
0.048 0.098 0.138 0.169 0.239 0.278 0.309 0.342 0.354
0.410 0.447 0.473 0.549 0.591 0.639 0.667 0.682 0.699
0.362 0.349 0.335 0.380 0.352 0.361 0.358 0.340 0.345 mean = 0.354
mol/kg) 0.209 0.402 0.581 0.738 1.080 1.393 1.814 2.267 2.715
0.8 _
567
SD = 0.014 a AV is the decrease in pellet volume per unit initial volume, and is an approximation to the degree of water abstracted by osmosis. All values are in milliliters per milliliter of wet, packed yeast. b Sr is the space available to test solute (KSCN) per unit volume of initial pellet. c For a salt such as KSCN, the value of (Sv - AV) is a measure of cell envelope plus interstitial water in the original pellet. SD, Standard deviation
568
ARNOLD AND LACY
J. BACTZRIOL.
AsOf
FIQOLL
0
0.5
10
1.5
Osmolality (Osnmoes/Kg)
2.0
2.5
&,
FIG. 4. Effects of osmolality on the S values for KSCN (open symbols) and Ficoll ('filled symbols) in admixture. The different symbols rrefer to three separate experiments.
plete accessibility to salts base,d on the water content of such pellets. The methylene blue test, wvhich is a good indicator of viability when p erformed under prescribed conditions (3), reveialed a high degree of integrity even in cellsithat had been mixed with concentrated salt s olutions. In one experiment, triplicate pellets of yeast were mixed with approximately equaLl volumes of 2.5 M KSCN. Analysis on one tube revealed a final concentration of 1.534 M KSCNI and yielded an S value of 0.656, which is typiccal for this salt. The contents of the second tul be were diluted about 1,000-fold by the dropwrise addition of distilled water to the stirred suspension. An appropriate volume was the*n mixed with buffered methylene blue solutio n (3) and examined in a hemocytometer. Eigght large fields (about 40 cells each) were obs3erved, and the percentage of dead (blue-staine3d) cells was recorded. The average was 7 (- :5%), compared with 1 (+3)% for cells from thi e same batch of yeast that had only received distilled-water washes. It is worth pointing ouit that the slow dilution of cells (from a concenitrated salt medium) is mandatory for the mair ntenance of viability. When a third pellet of cel[Is was similarly treated with KSCN as above but then "shocked" by rapid dilution in 1 liter of distilled water, the fraction of dead celIls then was 41 (+6%). However, this treatmerit did not affect the status of yeast fl-fructofursinosidase. Cells that were exposed to KSCN (2.'7 osmol/kg) and then washed retained 94% of the activity of untreated controls no matter h(ow quickly they were diluted. With one batch of yeast, S vadlues were measured at four concentrations ofr KSCN (range, 0.4 to 2.7 osmol/kg) and witl i four different
presentation times. Compared with the standard time of 15 min, the S values were: (i) not significantly different at 7.5 min; (ii) about 2% higher at 30 min; and (iii) about 5% higher when the incubation was extended to 60 min. Controls. Pellets prepared from cells that had been killed by pretreatments with organic solvents (ethyl acetate or chloroform), heat, or glutaraldehyde exhibited complete accessibility to KSCN (judged by their water content). All of these pretreatments were lethal, as indicated by 100% of the cells taking the stain in the methylene blue test. Although the conclusions were clear-cut, experiments on killed material yielded S values that displayed somewhat more
variability.
The S values for killed cells were independent of external concentration. For example, with heat-killed cells (90°C/3 min), KSCN S
values of 0.701, 0.693, and 0.699 ml/g (wet, packed yeast) were obtained at osmolalities of 0.713, 1.412, and 2.889 osmol/kg, respectively. The water content of pellets of heat-killed yeast was 0.640 ml/g (wet, packed yeast). Likewise, for cells pretreated overnight with 3% glutaraldehyde in 0.1 M collidine-HCl (pH 7.0), the S values over a series of KSCN concentrations had an average of 0.707 (+0.076) ml/g (wet, packed yeast), which was close to the water content of those pellets: 0.678 (+0.001) ml/g (wet, packed yeast). Substrates of 3-fructofuranosidase and acid phosphatase. Acid phosphatase exhibits maximal activity at pH 3.8 and negligible activity at pH 7.5, which was used here to study the accessibility of conventional substrates and their corresponding products of hydrolysis (Table 3). Commercial bakers' yeast has a high concentration of f8-fructofuranosidase, and the enzyme catalyzes the hydrolysis of sucrose over a broad range of pH values. It is well known that the products, glucose and fructose, are readily absorbed by the protoplasm. To overcome both problems in the assessment of passively penetrable space, we took advantage of the inhibitory effect of uranyl acetate on ,B-fructofuranosidase as well as the glucose transport system (10). The yeast was washed twice in 10 mM uranyl acetate-acetic acid (pH 5.0), and test solutions were also fortified with the inhibitor. Preliminary trials showed that the inhibitor was effective at this concentration. The results are summarized in Table 3, along with reference values. They demonstrate that substrates and products ofboth enzymes penetrate a space that is comparable to that found for salts and nonmetabolized sugars. The one exception, p-nitrophenol, exhibits large S values, which indicate accumulation by the yeast.
OSMOTIC BEHAVIOR IN S. CEREVISIAE
VOL. 131, 1977
TABLE 3. S values for substrates and products of yeast ,B fructofuranosidase and acid phosphatase S (ml/g of wet, Compound
Sucroseb Glucoseb Fructoseb Na2, -glycerophosphatec Na2 p-nitrophenolphosphatec p-Nitrophenol pH 7.5c pH 3.8d
Concn
packed yeast) Refer-
(M)t Test
0.128
ence a
0.147
0.368 0.327
0.340 0.342
0.147
0.327
0.342
0.095
0.353
0.380
0.004
0.298
0.319
NaH2PO4
0.002 0.001 0.678
1.89 3.07 0.544
0.319 0.319 0.537
KH2PO4
0.626
0.573
0.525
a Nonmetabolized polyols or KCl at an osmolality equivalent to that of the test compound. b Yeast ,3-fructofuranosidase and sugar transport systems were inhibited by 10 mM uranyl acetateacetic acid (pH 5.0). c In the presence of 10 mM phosphate buffer (pH 7.5). d In the presence of 10 mM acetate buffer (pH 3.8).
Effects of glutaraldehyde. In addition to using pellets prepared from glutaraldehyde-fixed cells as controls, some determinations of density, volume, and water content were performed on them. A batch of fresh, washed yeast was stirred overnight in 3% glutaraldehyde at 23 to 25°C, washed in copious amounts of distilled water, and then made up to 30% (wet wt/ vol). The cell concentration was determined on a suitably diluted sample in a hemacytometer. Another batch of yeast was similarly treated, except that 0.1 M collidine-HCl (pH 7.4) was included during fixation. The results are summarized in Table 4; measurements of volume and water content are compared among equal numbers of cells for each treatment. The results demonstrate that considerable shrinkage of cells results from fixation in the glutaraldehyde-collidine mixture. Smaller changes were noted when unbuffered glutaraldehyde was employed. DISCUSSION By definition, all S values refer to initial pellets, which were equilibrated with distilled water. The magnitude of the S value depends upon the final concentration of test solute. However, extrapolation to zero concentration yields a limiting value for S that is independent of, for example, an osmotic effect on the cell. The data for Ficoll 400 (Fig. 2) and for nonme-
569
tabolized polyols (Table 1) yield linear relationships, and the limiting values of S, as concentration or osmolality approaches zero, are readily computed. A summary that is based on these values and a conversion to unit volume is given in Table 5. Only the interstitial water in a yeast pellet is accessible to the polymer Ficoll (line 1, Table 5). This value is 0.223 ml/ ml of pellet, and the space that cells occupy is 0.777 ml/ml of pellet (by difference). Relatively small molecules permeate the cell envelope and are additionally diluted by that volume (line 4, Table 5). From the data of Table 5, we calculate that the cell envelope is, on the average, 15% of the total cell volume. Conway and Downey (8) obtained a comparable estimate of 11% for their yeast.
The total space that is accessible to small molecules may be viewed as the sum of the initial interstitial water, the cell envelopes, and the water abstracted from the cells by osmosis. Our results substantiate this simple working hypothesis in that a great variety of salts, mono- and disaccharides, and polyols follow (reasonably well) the same general response curve for S value versus osmolality in contrast to molarity. The increase in S value with osmolality can be accounted for by an independent estimate of the water abstracted by osmosis (Table 2). There was no indication that increased accessibility at higher salt concentrations was due to irreversible damage of the protoplasmic memTABLE 4. Effect of glutaraldehyde on yeast Yeast pellet Water Vol (ml/100 content (g/1010 cells) cells)
Pretreatment Density (g/ml)
None 3% glutaralde-
1.067 (±0.0004) 1.085 (±0.001)
0.793 0.767
0.675 0.610
1.103 (±0.002)
0.538
0.402
hyde 3% glutaraldehyde in 0.1 M
collidine-HCl (pH 7.0)
TABLE 5. Space analysis for standard yeast pellets Specific vol Component
ml/g of pellet ml/ml of pel-
Interstitial water Cells Interstitial water and cell envelopes Cell envelopes Protoplasms
0.209 0.728 0.319
0.223 0.777 0.340
0.110 0.618
0.117 0.660
570 ARNOLD AND LACY brane. Instead, exposed cells were shown to be in good condition, as judged by the methylene blue test, and S values were not greatly affected by a fourfold increase in the presentation time. The S curves for salts were curvilinear, but showed agreement with polyols at zero osmolality and approached similar values at higher osmolalities (see Fig. 2). The intervening region in which S values, at equivalent osmolality, are higher for salts than for sugars is most likely related to the ionic environment of the cell wall, to which covalently bonded carboxyl (13) and phosphate (11) groups have been attributed. A related finding may be the demonstration of salt-induced contractions in isolated bacterial cell walls (15). It has been shown (16) that negatively charged particles (e.g., kaolinite) exhibit larger hydrogen ion and cation concentrations at their surfaces than in the surrounding medium, and absorbed enzymes are influenced by this "double layer" surrounding the particle (14). In this context, we were interested in the S values exhibited by KCl at low concentrations. No evidence of exclusion or adsorption was discernible, and good agreement between gravimetric analyses of the salt and titrimetric analyses of chloride ion was obtained (Fig. 1). Yeast cells are osmotically responsive, and their survival in distilled water depends on the ability of the cell wall to withstand a substantial turgor pressure. Numerous reports in the literature, as well as the present investigation, suggest that the envelope will initially contract as a unit when the yeast cell is placed in solutions of increasing osmolality. However, plasmolysis has not been previously reported for yeasts. With reference to Fig. 4, we suggest that incipient plasmolysis has occurred at an osmolality in the vicinity of 1.15 osmol/kg. Below this point the Ficoll-accessible space and the KSCN-assessible space increase in approximately parallel fashion, which is consistent with conjoint contraction of the cell wall and the protoplasm. Above 1.15 osmol/kg, S values for KSCN continue to increase (further contraction of protoplasm), but those for Ficoll remain constant (no further change reflected at the external surface of the cell wall). The simplest explanation is that plasmolysis has occurred. An osmolality of 1.15 osmol/kg is given by 0.637 M KCl, and it should be noted that concentrations of 0.6 to 0.8 M KCl are recommended (20) for the preparation of protoplasts from S. cerevisiae. Our results offer a plausible rationale for this empirical recommendation. It was anticipated that in higher osmolalities than that required for incipient plasmolysis,
J. BACTZRIOL.
the normalized heights of packed yeast columns would exhibit a plateau in analogous fashion to the S values for Ficoll (Fig. 4). This was not found (Table 2). Possibly, the cell walls of plasmolyzed cells are sufficiently flaccid to become severely deformed under centrifugation and thus yield falsely low hematocrits. The conditions for packed-cell volume measurement were arrived at by preliminary trials in which distilled-water suspensions of yeast were subjected to different centrifugal forces in Wintrobe tubes. Relative heights of yeast columns were recorded immediately after centrifugation and thereafter at 15-min intervals. It was desirable to match these packing characteristics with those used in the preparation of standard pellets (which had different dimensions). Once the density of the latter was determined (see Materials and Methods), we were able to predict the hematocrit by calculation. Conditions that would match this value were deemed to be a good approximation to equivalent packing. However, for cells equilibrated with test solutions, there was a progressive change in column height with post-centrifugation interval, and the rate of change was slower for the cells from the more concentrated salt solutions. These results are consistent with the above suggestion that plasmolyzed cells give falsely low hematocrits. Our results with the substrates and products of f-fructofuranosidase and acid phosphatase (Table 3) indicate that the cell envelope is accessible to all of them. This is consistent with a periplasmic locale for these enzymes and with the assessment by Sherrer et al. (18) that the cell wall presents no real barrier to the passive diffusion of molecules up to a size of approximately 620 daltons. Live yeast cells are commonly assayed for acid phosphatase activity with nitrophenol phosphate as substrate. Absorption of nitrophenol by cells (as indicated by the data of Table 3) is not a problem, because base is added at the termination of incubation and redistribution of nitrophenol to the medium is achieved. Our results indicate shrinkage by cells during glutaraldehyde fixation. The common practice of fortifying fixatives with 0.1 to 0.2 M phosphate, collidine, or cacodylate buffers is not encouraged by our cell volume measurements. Meaningful ultrastructural evidence for plasmolysis in live cells will be predicated on obtaining fixation of specimens with retention of native proportions. ACKNOWLEDGMENIS This work was supported by a grant from Research Corporation and by Public Health Service grant Al 13177 from the National Institute of Allergy and Infectious Diseases.
VOL. 131, 1977
OSMOTIC BEHAVIOR IN S. CEREVISIAE
We thank Philip Bestic for helpful discussions and Phillip Gerhardt for comments on the manuscript. 12. 1. 2. 3.
4. 5.
6. 7.
8. 9. 10.
11.
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