JOURNAL OF BACTERIOLOGY, Apr. 1971, p. 183-191Copyright ( 1971 American Society for Microbiology

Osmotic Regulation of Invertase Formation andSecretion by Protoplasts of Saccharomyces

S.-C. KUO AND J. 0. LAMPEN

Institute ofMicrobiology, Rutgers University, The State University ofNew Jersey,New Brunswick, NewJersey 08903

Received for publication 21 December 1970

Osmotic regulation of invertase formation and secretion by protoplasts ofSaccharomyces was examined using sorbitol, KCI, NaCl, or magnesium sulfateas the osmotic support. The synthesis and secretion of the enzyme is remarkablysensitive to the osmolarity of the supporting medium irrespective of the particularsupport employed. Invertase formation was inhibited at high osmolarity and wasmaximal at 0.65 to 0.75 osmolal, even though some leakage of the intracellularenzyme a-glucosidase and of ultraviolet (UV)-absorbing materials occurred underthese conditions. The reduction of invertase formation and secretion due to highosmolarity was eliminated promptly when protoplasts were transferred into amedium of lower osmolarity. The rate of fructose uptake and of threonine incorpo-ration into protein was decreased by high osmolarity; also reduction of invertaseformation could be partially reversed by increasing the level of sugar supplied as

energy source. Thus changes in the permeability of the plasma membrane (and pre-sumably also in its structure) are important factors in the response of protoplasts tohigh osmolarity, though certainly not the complete explanation. Protoplasts sus-pended in 0.8 M sorbitol, with 10 mm fructose as the energy source, increasedtheir invertase level 5- to 10-fold during a 2-hr incubation without substantial re-lease of a-glucosidase or UV-absorbing materials. Both the large and small formsof invertase were present in the protoplasts, but only the large form was releasedinto the medium when enzyme was being actively synthesized. Formation and secre-tion of newly formed invertase and the release of enzyme initially present were in-hibited by cycloheximide.

In the past few years, a considerable amountof work was carried out on the biochemicalproperties of the isoenzymes of yeast invertase(EC 3.2.1.26), but few studies were directly con-cerned with its synthesis and secretion into thesuspending medium (for review see reference 12).Although invertase synthesis can be investigatedwith either intact cells or protoplasts, protoplastsare particularly suitable for examination of theprocess of secretion through the plasma mem-

brane.A number of investigations on the synthesis of

proteins (enzymes) and nucleic acids by yeastprotoplasts were previously reported (3, 4, 5,10, 11, 16, 18, 23, 24). However, relatively littleis known about the relation of the osmotic sta-bility of the protoplasts to their capacity to syn-thesize and secrete enzymes. We report here thatosmotic pressure has a marked influence on themetabolic functions of protoplasts. Conditions

optimal for the synthesis and secretion of inver-tase and the effects of high osmotic pressures onsome metabolic properties of protoplasts are de-scribed.

MATERIALS AND METHODS

Yeast strains and preparation of organisms. Mutant1016 (B. S. Montenecourt, Ph.D. Thesis, Rutgers-TheState University of New Jersey, New Brunswick, 1968)derived from Saccharomyces strain 303-67 (7, 8) wasused. The production of invertase by both strains is sen-sitive to repression by a medium with relatively highhexose concentration such as 0.1 M glucose or fructose.However, mutant 1016 produces much higher levels ofinvertase at low hexose concentrations than does strain303-67. Mutant 1016 is a-glucosidase inducible andforms invertase in appreciable amounts in 0.05 M malt-ose medium. The effect of various carbon sources oninvertase formation by strains 303-67 and 1016 is illus-trated in Table 1.

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TABLE 1. Effect ofcarbon source on theformation of invertase by Saccharomyces strains 303-67 and 1016

Strain 303-67 Strain 1016Carbon Concnsource (mM) Turbiditya Invertase°(units/ Turbiditya Invertase (units/

(Klett units) 5 x I07 cells) (Klett units) 5 x I07 cells)

Glucose 10 70 15.0 59 50.5100 105 4.5 127 7.6

Fructose 10 73 18.2 58 72.5100 90 4.7 105 8.5

Mannose 10 65 14.6 63 67.0100 88 7.9 100 25.8

Maltose 5 35 3.0 25 10.050 30 5.0 120 32.8

a Vogel's medium N (reference 26, modified as in Methods) was inoculated with 107 cells per ml (Initial readingwas 35 Klett units for strain 303-67 and 31 for strain 1016). Cultures were incubated for 4 hr.

'The one-step assay of Neumann and Lampen (19) was used; units are micromoles of sucrose hydrolyzed in 30min.

Stock cultures of Saccharomyces 1016 were main-tained on 0.5% peptone - 0.3% yeast extract - 1% glu-cose medium containing 1.5% agar. The cells from oneslope were transferred into 10 ml of Vogel's medium N(26) supplemented with (per liter): 2.0 mg of inositol,0.2 mg of calcium pantothenate, 0.2 mg of pyridoxine-hydrochloride, 0.2 mg of thiamine-hydrochloride, andcontaining 0.05 M maltose (nonrepressive conditions) or0.2 M glucose (repressive conditions). The cultures wereincubated for 16 hr at 28 C on a reciprocating shaker.For preparing exponential-phase cells, 10 ml of a sta-tionary-phase culture was added to 100 ml of the samemedium, the mixture was incubated as above for 4 to 5hr until an optical density corresponding to a Klettreading (filter no. 66) of 130 to 140 (equivalent to about4 x 107 cells per ml) was reached. The cells werewashed twice with distilled water and suspended at aconcentration of 4 x 108 cells per ml [equivalent to 8mg (dry weight)].

Preparation of protoplasts. Protoplasts were obtainedfrom exponential-phase cells by a slight modification ofthe procedure described by Gascon and Lampen (7). To5 ml of yeast suspension containing 4 x 108 cells perml, the following additions were made: 0.05 Mtris(hydroxymethyl)aminomethane (Tris)-hydrochloridebuffer (pH 7.5), 1 ml; 1.2 M KCI containing 0.02 MMgSO4, 6 ml; snail enzyme, 0.5 ml; and I M 2-mercap-toethanol, 0.2 ml. The mixtures were incubated at 30 Con a reciprocating shaker (75 cycles/min with a 2.5-cmstroke); after I hr, more than 95% of the cells wereconverted into protoplasts. The protoplasts were centri-fuged at 1,000 x g for 3 min and washed twice with 5mM MgSO4 and 0.02 M phosphate buffer (pH 6.0) con-taining 0.6 M KCI or 0.8 M sorbitol.

Measurement of invertase formation and secretion byprotoplasts. Washed protoplasts were suspended at aconcentration of 5 x 107 per ml in the desired osmoticstabilizer with 10 mM fructose as the energy source un-less otherwise stated. The suspensions were incubated at30 C in a reciprocating shaker as described for thepreparation of protoplasts. For measurement of invert-

ase that is released into the medium, I ml of the incu-bation mixture was withdrawn at intervals and centri-fuged at 1,000 x g for 3 min; 0.2 ml of the supernatantwas pipetted into 0.8 ml of ice-cold water and assayedas described below. For determination of the totalamount of invertase synthesized, 0.2-ml samples wereperiodically removed from the incubation mixture. andtransferred to chilled tubes containing 0.8 ml of ice-coldwater to lyse the protoplasts. The resulting suspensionswere assayed.

Separation of the two forms of invertase. A SephadexG-200 column (2 by 47 cm) was used for separation oflarge and small invertase as described by Gascon andLampen (7). Supernatant fluids (1,000 x g, 3 min) fromthe incubation mixtures (5 x 107 protoplasts per ml)and protoplast pellets lysed by resuspension to the orig-inal volume in distilled water were filtered throughmembrane filters (0.45 gm porosity, Millipore Corp.)before gel filtration. Less than 5% of the invertase ac-tivity of either supernatant fluids or lysates was retainedby the filters. One milliliter of the filtrate was appliedto the column bed, and 3-ml fractions were eluted with0.05 M Tris-hydrochloride buffer (pH 7.5).

Invertase assay. The assay was essentially that de-scribed by Gascon and Lampen (7). One unit of invert-ase is the amount of enzyme which hydrolyzed I imole-f sucrose in 30 min at 30 C in 0.05 M sodium acetatebuffer (pH 5.0) containing 0.125 M sucrose.

Assay of a-glucosidase and UV-absorbing materials.For determination of materials released into the me-dium, I ml of the incubation mixture was taken at zerotime and after 2 hr and centrifuged at 1,000 x g for 3min; 0.2 ml of the supernatant was pipetted into 0.8 mlof ice-cold water and assayed as below.

Alpha glucosidase activity was estimated by the pro-cedure of Halvorson and Ellias (9). Samples (0.1 ml)were mixed with 2.7 ml of 0.065 M potassium phosphatebuffer (pH 7.0) and 1.0 ml of reduced glutathione solu-tion (I mg/ml). The reaction was started by adding 0.1ml of substrate (3 mg of p-nitrophenyl-a-D-glucoside).The production of p-nitrophenol was measured continu-

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OSMOTIC REGULATION OF YEAST PROTOPLASTS

ously with a Beckman DB spectrophotometer at 400nm with the cuvette compartment maintained at 30 C.A unit of enzyme is defined as the quantity releasing 1

nmole of p-nitrophenol per min at 30 C. Ultraviolet(UV) absorption was measured at 260 nm.

Estimation of amino acid incorporation into proteinfraction and fructose uptake. Protoplasts (5 x 107/ml)were incubated at 30 C in sorbitol-stabilized mediumwith 10 mM unlabeled fructose and 0.1 mM L-threonine-U-14C (0.05 AiCi/ml of suspension). At intervals, proto-plast suspension (0.8 ml) was added to I ml of 10% tri-chloroacetic acid and heated at 95 C for 30 min. Theresidues (protein fraction) were collected on membranefilters (0.45-Mm porosity) and washed twice with 5%cold trichloroacetic acid. For measuring fructose up-

take, only 10 mM fructose-U-"4C (0.1 IACi/ml of suspen-

sion) was included in the incubation mixtures. At var-

ious intervals, the protoplasts from 0.8-ml samples ofthe incubation mixtures were collected on membranefilters (Millipore Corp.) and washed twice with 4 ml ofice-cold 0.8 M KCI solution. Filters were transferreddirectly to scintillation vials containing 10 ml of Bray'ssolution (1). The radioactivity was measured in a

Packard Tri-Carb liquid scintillation spectrometer.Chemicals. All chemicals were reagent quality. Ra-

dioisotopes were purchased from New England Nu-clear, Boston, Mass. Snail enzyme (Glusulase) was ob-tained from Endo Lab Inc., New York; cycloheximidefrom Upjohn Co., Kalamazoo, Mich., and D-sorbitolfrom Nutritional Biochemical Corp., Cleveland, Ohio.

RESULTS

Effect of osmotic support on the synthesis andsecretion of invertase and leakage of intracellular

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materials. In the previous study of the distribu-tion of invertase isoenzymes in intact cells andprotoplasts of Saccharomyces, the protoplastswere prepared and maintained in 0.6 M KCl toprevent osmotic lysis (7, 8). However, we discov-ered that under these conditions some of the pro-toplasts lysed during the incubation period forformation and secretion of invertase. This led us

to examine the effect of the concentration of KCl,sorbitol, and other osmotic supports on the sta-bility of the protoplasts and on their formationand secretion of invertase.

Stability of the protoplasts was determined bymeasuring the release of an intracellular enzyme,a-glucosidase (12) and of UV-absorbing sub-stances. The results in Fig. 1 show the effect ofvarious KCI concentrations on invertase forma-tion and on release of intracellular materials. At0.2 M KCI the protoplasts were fragile and imme-diately released large amounts of invertase, a-

glucosidase, and UV-absorbing materials (Fig.lA and B). There was no net synthesis of invert-ase under these conditions (Fig. 1A). Protoplaststability was excellent in KCI concentrationshigher than 0.6 M; however, invertase synthesisappeared to be severely reduced. A KCI concen-

tration of 0.4 M (equivalent to 0.75 osmolal) was

most effective for the synthesis and secretion ofinvertase, even though lysis of some protoplastswith release of intracellular materials occurred(Fig. lB). Similar data, which will not be pre-sented in detail, were obtained when NaCl was

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MOLARITY OF KCI,MOLARFIG. 1. Formation and secretion of invertase, and release of a-glucosidase and UV-absorbing materials from

protoplasts suspended in various concentrations of KCI. Protoplasts from cells grown in 0.05 M maltose were sus-

pended at 5 x 10 per ml in the indicated concentrations of KCI and 10 mm fructose as energy source, and were

incubated at 30 C. At zero time and after 2 hr, 0.2-ml samples of the incubation mixtures or of the supernatantfluids were transferred into 0.8 ml of ice-cold water and assayed. Enzyme activity is expressed as units (micromolesof disaccharide hydrolyzed in 30 min) per milliliter of the incubation mixture or of the supernatant fluid. (A) In-

vertase activity: (-) 0 time, total activity; (A) 2 hr, total activity; (0), 0 time, supernatant fluid; (A) 2 hr, super-natant fluid. (B) a-Glucosidase and UV-absorbing materials in the supernatant fluid: (0) 0 time, a-glucosidase;(A) 2 hr a-glucosidase; (0) 0 time, absorbancy at 260 nm; (A) 2 hr, absorbancy at 260 nm. Osmolality ofKCI was

calculatedfrom freezing-point depression ofthe same concentration ofKCl (27).

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KUO AND LAMPEN

used in place of KCI. These results were surpris-ing, since the concentrations of KCI and NaCltested were not extreme and were used in manyinvestigations for stabilization of yeast proto-plasts (25).An attempt was made to see whether the re-

duction of invertase synthesis and secretion inhigh concentrations of KCI was due to specificsolute (or ionic) effects or to osmotic effects. Thesame type of experiment was therefore performedwith protoplasts suspended in various concentra-tions of sorbitol or MgSO, (Fig. 2 and 3, respec-tively). Invertase was most actively synthesizedand secreted when the protoplasts were suspendedin 0.6 M sorbitol or MgSO4 (equivalent to about0.65 osmolal), and the synthesis of invertase wasreduced if the concentration of these osmotic sta-bilizers was increased. Thus, for the four osmoticsupports tested, the optimal osmotic range forinvertase formation and secretion by the proto-plasts was about 0.65 to 0.75 osmolal. It is im-portant to note that the effectiveness of invertasesynthesis by protoplasts suspended in 0.8 M sor-bitol was only 75 to 80% of that of protoplasts in0.6 M sorbitol, but 0.8 M sorbitol gave a greaterprotection of the protoplasts with no leakage ofintracellular materials into the medium duringthe 2-hr incubation (Fig. 2). For this reason, pro-toplasts suspended in 0.8 M sorbitol solution wereconsidered to be the most suitable for the studyof invertase secretion from the cell membraneand were used in all subsequent experiments.De novo syntbesis of invertase and its secretion.

Cycloheximide is known to be active againstyeast cells, and its primary action is to inhibitcytoplasmic protein synthesis at the ribosomallevel (15, 20). To determine whether the secretionof invertase is dependent on de novo protein syn-

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thesis, protoplasts from cells grown in nonrepres-sive conditions (0.05 M maltose) were incubatedwith 10 mM fructose and various concentrationsof sorbitol as the osmotic stabilizer and in theabsence or presence of 5 ,g of cycloheximide perml. During a 2-hr incubation with fructose and0.6 M sorbitol but without cycloheximide, thelevel of invertase increased about fourfold (Fig.4A). Both synthesis and secretion of the enzymewere gradually reduced by increasing sorbitolconcentrations, but in 1.0 M sorbitol, most of theinvertase originally present was released into thesuspending medium without a significant increasein total activity. The release of invertase was alsoprevented in 1.2 M sorbitol. In the presence ofcycloheximide, invertase synthesis and secretionwere almost completely inhibited, including therelease of the original activity (Fig. 4B).Form of invertase secreted by protoplasts. It

was demonstrated that Saccharomyces cells con-tain two forms of invertase (7, 8). The large formhas a molecular weight of 270,000, approximately50% mannan, and the small form has a molecularweight of 135,000 and is devoid of carbohydrate.The large form of invertase (in the periplasm andcell wall) is released from the cells during theformation of protoplasts.

It was of interest to examine which type ofenzyme was released by protoplasts when theenzyme was being actively synthesized and se-creted. Protoplasts from cells grown in 0.05 M

maltose (nonrepressive) or 0.2 M glucose(repressive) medium were incubated for 2 hr in0.8 M sorbitol and 10 mm fructose. The invertasepresent on the protoplasts and that secreted intothe suspending medium was fractionated by gelfiltration on a Sephadex G-200 column (Fig. 5).Protoplasts from cells grown on nonrepressive

OSMOLALITY OF SORBITOL, OSMOLAL086 I.IS 1.40 0 021 042 0.66 0.66 LIS 1.40

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MOLARITY OF SORBITOL, MOLARFIG. 2. Formation and secretion of invertase and release oJ a-glucosidase and U V-absorbing materials from

protoplasts suspended in various concentrations ofsorbitol. All conditions were as described in the legend to Fig. 1,except that the osmotic support was sorbitol. Osmolality of sorbitol was measured and calculated from freezing-point depression ofthe same concentrations ofsorbitol (27).

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OSMOTIC REGULATION OF YEAST PROTOPLASTS

OSMOLALITY OF MgSO4, OSMOLAL0 0.22 043 0.63 0B4 1.08 0 0.22 0.43

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FIG. 3. Formation and secretion of invertase and release of a-glucosidase and UV-absorbing materials Jromprotoplasts suspended in various concentrations of MgSO4. All conditions were as described in the legend to Fig. 1,except that the osmotic support was MgSO4 . Osmolality ofMgSO4 was calculated from freezing-point depressionof the same concentration ofMgSO4 (27).

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FIG. 4. Effect of cycloheximide on the synthesis andsecretion of invertase by protoplasts. (A) No cyclohexi-mide added (experimental conditions as for Fig. 2); (B)cycloheximide (5 Ag/ml) added to incubation mixture atzero time. Invertase activity: (-) 0 time, total; (0) 0time, supernatant fluid; (A) 2 hr total; (A) 2 hr, super-natant fluid.

medium initially contained mainly the large form(Fig. 5A); those from repressive medium con-

tained less total activity but most of this was thesmall form (Fig. 5B). Essentially identical curves

were obtained for the extracts from protoplastsincubated for 2 hr and are not repeated in Fig. 5.Both types of protoplasts secreted only the largeform of invertase, with no detectable release ofthe small form initially predominant in the proto-plasts from cells grown in repressive medium.The results strongly support the concept that in-vertase is secreted through the plasma membraneby a specific process and not as a result of mem-brane damage.

Reversal of osmotic effect on invertase synthesisin protoplasts. It was important to learn whetherthe inhibitory effect of high osmotic pressure oninvertase synthesis is reversible. For this purpose,a suspension of protoplasts in 1.2 M sorbitol with10 mm fructose was incubated as previously de-scribed. Under these conditions, there was no netsynthesis of invertase (Fig. 6). After 60 and 120min of incubation, portions of the incubationmixture were transferred to 0.8 M sorbitol withfructose. Invertase synthesis commencedpromptly, and the total activity increased nearly10-fold in 2 hr. Surprisingly, there was no signifi-cant difference in the time course of invertasesynthesis by the protoplasts whether or not theywere preincubated in 1.2 M sorbitol. This indi-cates that the inhibitory effect of high osmolarityon enzyme synthesis is not due to irreversible al-teration of the metabolic functions of the proto-plast.Sugar uptake and amino acid incorporation by

protoplasts in various sorbitol concentrations.Since synthesis of invertase by protoplasts was

severely reduced by high osmolarity, the osmotic

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188 KUO AND LAMPEN J. BACTERIOL.

vo ually repressed, however, as the fructose concen-40 100 tration in the suspending medium was increased,even though the total level of osmotic support

(A) 0.05M Maltose grown cells (fructose and sorbitol) remained constant. TheseE 30 ~ > A>t ~ 75 fiE results are consistent with previous reports of

catabolic repression of invertase synthesis ino' 20 - 1l t - 50 ' yeast cells or protoplasts by high concentration of*0> sugar (2, 6, 7, 13, 17). Protoplasts suspended in

\\ , 1.2 M sorbitol formed little, if any, enzyme when>. 10 25 S 10 mM fructose was included in the incubation> '- mixture (Fig. 8B). Synthesis of the enzyme was4 0 tn fi | ,0 > significantly stimulated as the concentration ofw < fructose was increased (at constant osmolarity)< I'. 'I''~'I'**~' znw and with prolonged incubation. Such results

, 40 (B) 0.2M Glucose grown cellsw00 would be expected if the permeability of thez , plasma membrane had decreased in high concen-( 30 75 trations of sorbitol, as the experiments of Fig. 7zs/\ had indicated. However, repression of invertase

-J l l < synthesis occurred at higher levels of external_20 Z fructose. The results were rather surprising, sinceU)~~~~~~~~~~~~~~~~~~~c

-J t \ ' the amounts of fructose incorporated by these0 10 25"0 o~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IfS\j

c 0 6015 20 25 30 32FRACTION NQ (3ml/tube)

FIG. 5. Form of invertase produced by protoplastsfrom cells grown in repressive (0.2 M glucose) and non- E50/repressive (0.05 M maltose) media. The protoplasts were /

X107~~~~~~~~~~~~~~1suspended at 5 x 107 per ml in 0.8 M sorbitol and 10mm fructose and incubated at 30 C. Sephadex G-200 /column (2 by 47 cm) was equilibrated with 0.05 M Tris- 40_hydrochloride (pH 7.5); samples equivalent to I ml of i-.incubation mixture were added and eluted with the >same buffer. Vo, void volume of column. (0) Zero /-time, protoplast lysate; (0) 2 hr, supernatant fluid. 30

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effect on sugar uptake and protein synthesis was i 20 -determined. Both the rate of "4C-fructose uptake twand of incorporation of "C-threonine into the >protein fraction decreased significantly as the Z A Bconcentration of sorbitol increased (Fig. 7). Pro- 10toplasts in 1.2 M sorbitol incorporated less than20% of the radioactivity taken up by protoplastsin 0.6 M sorbitol. Similar results were obtained 0 lwhen the incorporation of "4C-fructose into mac- 0 60 120 180romolecules such as mannan and glucan was TIME (min)measured (unpublished data). FIG. 6. Reversibility of the osmotic inhibition of

If the inhibitory effect of high osmolarity on invertase synthesis. Protoplasts from cells grown in 0.2invertase synthesis in protoplasts is the result of a M glucose medium were suspended in 0.8 or 1.2 M sor-decreased penetration of sugar into the cells, syn- bitol in the presence or absence of 10 mm fructose andthesis of the enzyme should be restored if a high incubated at 30 C in a reciprocating shaker. At variouslevel of extracellular fructose is present. To test intervals, 0.2 ml of incubation mixture from each flaskthis hypothesis, protoplasts in 0.8 M or 1.2 M sor- was lysed in 0.8 ml of ice-cold water, and total invertasebitol were incubated with concentrations of fruc- activity was measured. Symbols: (0) 0.8 M sorbitol, 10

mm fructose; (0) 1.2 M sorbitol, 10 mm fructose. At 60tose ranging from 10 to 150 mM. Protoplasts In min (arrow A) and 120 min (arrow B), samples were0.8 M sorbitol actively synthesized enzyme when centrifuged at 1,000 x gfor 3 min and suspended in 0.8the external fructose concentration was 10 mM M sorbitol with 10 mm fructose; (A) 0.8 M sorbitol,(Fig. 8A). Formation of the enzyme was grad- fructose omitted.I

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OSMOTIC REGULATION OF YEAST PROTOPLASTS

TIME (min)

FIG. 7. Effect of various concentrations of osmotic stabilizer on fructose uptake and threonine incorporationinto protein fraction. Protoplasts were suspended at 5 x 107 per ml in various concentrations ofsorbitol containing10 Mmoles (0.1 ;MCi) of 14C-fructose per ml, or 10 ;umoles of unlabeled fructose plus 0.1 ,Amole (0.05 MCi) of 11C-threonine per ml at 30 C. Samples (0.8 ml) were removed at the times indicated and analyzed for the amount ofradioactivity incorporated. Symbols: (0) 0.6 M sorbitol; (0) 0.8 M sorbitol; (A) 1.0 M sorbitol and (A) 1.2 M sor-

bitol.

60

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FRUCTOSE CONC. (mM)

FIG. 8. Invertase formation by protoplasts in low and high osmolarity as a function offructose concentration.Experimental conditions are those described in the legend ofFig. 6, except that the protoplasts were incubated at 30C with the indicated concentrations offructose and with sorbitol added to yield a total concentration of 0.8 M (A)or 1.2 M (B). Incubation time: (0) 0 time, (0) I hr, (A) 2 hr, (A) 3 hr.

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protoplasts were less than 30% of those taken upin 0.8 M sorbitol. This suggests that the repressionof invertase formation may be initiated by an ef-fect of the hexose at an external site on the proto-plast membrane. This phenomenon is being inves-tigated further.

DISCUSSIONThe synthesis and secretion of invertase by

yeast protoplasts is remarkably sensitive to theconcentration of the agent used for osmotic stabi-lization. The inhibitory effect of several agents,such as KCI, NaCl, MgSO,, and sorbitol, wascorrelated with the osmolarity of the supportingmedium rather than the absolute concentration ofthe supporting species, indicating that osmolarityis the factor responsible for the changes observed.Although there are a number of reports on pro-tein (enzyme) and nucleic acid synthesis by proto-plasts (3, 4, 5, 10, 11, 16, 23), the conditions es-sential for maximum synthetic capacity haveseldom been established or stated. For example,in studies on protein and ribonucleic acid syn-thesis by protoplasts of a strain of S. cerevisiae,Hutchison and Hartwell (10) employed 1.0 Msorbitol to stabilize the protoplasts; total proteinincreased about sixfold during incubation for aslong as 15 hr. From the data presented here, onewould expect that the synthetic activity of theprotoplasts would have been greatly enhanced if alower osmolarity had been used, although otherfactors might also be involved.One of the few studies concerned with the ef-

fect of osmotic pressure on Saccharomyces hasshown that both intact cells and protoplasts havemechanisms for adaptation to an increase inosmolarity of the medium. Lillehoj and Otto-lenghi (14) found that cells or protoplasts of Sac-charomyces suspended in KCI or sorbitol at con-centrations as high as 2 to 3 M initially shrankand then slowly returned to normal volume. Re-turn to normal volume was not accompanied bycomplete return to normal metabolism, however,since respiratory activity, reduced in the highosmolarity medium, did not return to a normalrate. They speculated that the adaption occurs byan increase in the internal pressure of the cells.Recently, Ikeda and Ottolenghi (10th Int. Congr.Microbiol., Abstr., p. 2, 1970) have shown thatthe materials responsible for the adaption of thecells to growth in media of high osmotic pressureare trehalose and glycerol, as these two intracel-lular compounds were significantly accumulatedunder these conditions. The reduction of invertaseformation that we observed with media of highosmolarity was eliminated when the protoplastswere transferred into a medium of lower osmo-larity. There was no evidence of irreversible alter-

ation of metabolic functions; rather, the timecourse of invertase formation by the protoplastswas unchanged after they had been preincubatedin a high osmotic medium. These observations,coupled with the fact that the concentrations usedfor intact cells by Ottolenghi et al. (14) weremuch higher than those used in the present study,make correlation of the results difficult.Smith (21) found that the respiratory activity

of spheroplasts of Bacillus subtilis was lower in0.8 M sucrose medium than in 0.3 to 0.4 M su-crose, either with endogenous substrates or withadded amino acids as substrates.The observations that the rate of sugar uptake

and amino acid incorporation into protein by theyeast protoplasts was severely reduced in a me-dium of high osmolarity and that inhibition ofenzyme formation could be partially reversed byincreasing the level of external sugar and incu-bating for a longer period of time indicate thatpermeability changes are involved. It seems pos-sible that the primary effect is a structural changein the plasma membrane due to the high osmo-larity. This concept is supported by the fact thatinvertase formation was most active under os-motic conditions in which some release of intra-cellular materials occurred, with the additionalimplication that metabolic functions are at amaximum for the protoplasts when the plasmamembrane is in a loosened condition.Under the optimal conditions established in

this study, the total invertase activity of a proto-plast suspension increased 5- to 10-fold during 2hr at 30 C. Formation of invertase was inhibitedin the presence of cycloheximide, thus supportingprevious evidence that de novo synthesis of pro-tein is required (12, 22). The fact that cyclohexi-mide or a very high sorbitol level (1.2 M) com-pletely inhibited release of the invertase originallypresent suggests that the process of liberation isalso enzymatically mediated. The present studyhas also established that the secretion of invertaseis very specific; only the large form was releasedinto the suspending medium. The ability to alterthe stability and functions of protoplasts bychanges in osmolarity should prove useful forstudy of the biosynthetic relationship between thetwo forms of invertase and of the secretion of theenzyme through the plasma membrane into thesuspending medium.

ACKNOWLEDGMENTSWe thank Jane Sharkey and Jan Tkacz for their stimulating

discussions during the course of this investigation.This research was supported by Public Health Service grant

Al-04572 from the National Institute of Allergy and InfectiousDiseases.

190 J. BACTERIOL.

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OSMOTIC REGULATION OF YEAST PROTOPLASTS

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