JOURNAL OF BACTERIOLOGY, Oct 1971, p. 451-458Copyright 0 1971 American Society for Microbiology

Vol. 108, No. IPrinted in U.S.A.

Cold Osmotic Shock in Saccharomyces cerevisiaeJ. W. PATCHING AND A. H. ROSE

Microbiology Laboratories, Bath University, Claverton Down, Bath, England

Received for publication 22 March 1971

Saccharomyces cerevisiae NCYC 366 is susceptible to cold osmotic shock. Ex-ponentially growing cells from batch cultures grown in defined medium at 30 C,after being suspended in 0.8 M mannitol containing 10 mm ethylenedia-minetetraacetic acid and then resuspended in ice-cold 0.5 mM MgCl2, accumulatedthe nonmetabolizable solutes D-glucosamine-hydrochloride and 2-aminoisobutyrateat slower rates than unshocked cells; shocked cells retained their viability. Storageof unshocked batch-grown cells in buffer at 10 C led to an increase in ability toaccumulate glucosamine, and further experiments were confined to cells grown in a

chemostat under conditions of glucose limitation, thereby obviating the need forstoring cells before use. A study was made of the effect of the different stages inthe cold osmotic shock procedure, including the osmotic stress, the chelating agent,and the cold Mg2+-containing diluent, on viability and solute-accumulating ability.Growth of shocked cells in defined medium resembled that of unshocked cells;however, in malt extract-yeast extract-glucose-peptone medium, the shocked cellshad a longer lag phase of growth and initially grew at a slower rate. Cold osmoticshock caused the release of low-molecular-weight compounds and about 6 to 8% ofthe cell protein. Neither the cell envelope enzymes, invertase, acid phosphatase andL-leucine-f,-naphthylamidase, nor the cytoplasmic enzyme, alkaline phosphatase,were released when yeast cells were subjected to cold osmotic shock.

A number of different shock treatments cause

the release from microbial cells of low- and high-molecular-weight compounds, effects which are

often accompanied by death of the cells. One ofthe first shock treatments to be studied in detailwas referred to as "cold shock," in whichsudden chilling of a dilute suspension of expo-nential-phase gram-negative bacteria in water orvery dilute buffer causes the death of many ofthe cells (6, 9, 10, 20, 34, 36, 37). Cold shock,which is manifested mainly in gram-negative (36,37) bacteria, is accompanied by release of intra-cellular amino acids and nucleotides (36). Coldshock has not been reported in yeasts (20). Themain modification to the cold shock treatmenthas consisted in incubating cells in a hyperos-molar solution of inert solute (e.g. sucrose) con-

taining ethylenediaminetetraacetic acid (EDTA)followed by centrifugation and suspending thecells in cold water containing Mg2+. This treat-ment has been termed osmotic shock; the earlierliterature on it was reviewed by Heppel ( 11).Osmotic shock, which strictly speaking should bereferred to as cold osmotic shock (40), resemblescold shock in that it causes release of low- andhigh-molecular-weight compounds, includingperiplasmic enzymes, such as acid phosphatase(27) and nucleotidases (23, 24), and certain mem-

brane-bound transport proteins (13, 28); how-ever, it does not usually lead to a decrease in theviability of the cell population. Cold osmoticshock has been demonstrated in gram-negativebacteria, especially in members of the Enterobac-teriaceae (23) and in Neurospora crassa (40).The present paper describes cold osmotic shockin a strain of Saccharomyces cerevisiae andcompares this with related phenomena in gram-negative bacteria and N. crassa. Demonstrationof cold osmotic shock in S. cerevisiae shouldfurnish a useful experimental tool for workersinterested in the physiology of solute uptake inyeast.

MATERIALS AND METHODS

Organism. The strain of S. cerevisiae NCYC 366used in this study was maintained as described pre-viously (5).

Cultures. Batch cultures of the yeast were grown in1-liter portions of medium, as described by Diamondand Rose (5), and were stirred at 30 C, as described byPatching and Rose (29). Concentration of cells in cul-ture was determined by measuring the optical density(OD) in a Hilger Spekker absorptiometer (model H760) with neutral green-gray filters and a water blank.OD measurements were related to dry weight of cellsper milliliter by a calibration curve. Cells were har-

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vested by centrifuging mid-exponential-phase cultures[0.20 to 0.25 mg (dry weight)/ml] at 1,500 x g.

Chemostat cultures were grown in a glass vessel of2-liter working volume as described by Maclennan andPirt (17) and Brown and Rose (4). The medium con-

tained, per liter: (NH4)2SO4, 2.0 g; KH2PO4, 1.0 g;MgSO4-7H2O, 25 mg; CaCl2-6H2O, 25 mg; inositol,10 mg; calcium-D-pantothenate, 1.0 mg; thiamin-hy-drochloride, 1 0 mg; pyridoxin-hydrochloride, 10 mg; D-biotin, 2 Ag. This basal medium was supplemented with2 g of glucose per liter for cultures grown at 30 C andI g per liter for cultures grown at 15 C. These concen-

trations ensured that the cultures were glucose-limited.Cells were grown at dilution rates (equal to growthrates) of 0.1 or 0.05 hr-I at 30 C or at 0.05 hr-' at 15C. The pH value of cultures was maintained at 4.5 +0.1, and the dissolved oxygen tension was maintainedat 94 mm of Hg. Cells were harvested, and the concen-

tration of cells in effluent culture was determined, asdescribed by Brown and Rose (4).

Shock treatments. The cold osmotic shock treatmentto which yeast cells were subjected was a modificationof that used with bacteria (11). Cells from a batch cul-ture or a continuous culture [containing 0.8 to 0.9 mg

(dry weight) per ml] were suspended to a concentrationof. 1.0 or 2.5 mg (dry weight) per ml in 10 mmKH,PO4 (pH 4.5; reference 31) at 30 C (stage I).Immediately after suspension, the cells were removedby centrifuging at 30 C, and the supernatant fluid was

retained for analysis. The cells were then suspended tothe same concentration in 0.8 M mannitol containing 10mM EDTA at 30 C (stage 2). As soon as the cells were

suspended, they were removed by centrifuging at 30 C,and the supernatant fluid was retained for analysis. Thecells were then suspended in 0.5 mm MgCl, at 0 C(stage 3) and again removed by centrifuging. In certainexperiments, the shock procedure was modified; thesemodifications are indicated in the text.

Analytical methods. Protein was extracted by sus-

pending cells (5 to 15 mg, dry weight) in 2 ml of I NNaOH and placing the tubes in a bath of boiling waterfor 15 min. Cell residues were separated by centrifug-ing, and the residues were extracted twice with 2-mlportions of I N NaOH. The combined extracts were

made up to 10 ml with water. Protein in cell extractsand in supernatant fluids obtained by the shock treat-ment was estimated by the method of Lowry et al. (16)with bovine plasma albumin as a standard. For mostdeterminations, the standard curve was prepared withwater as a solvent. However, with determinations ofprotein in cell extracts and in shock supernatant fluidscontaining 0.8 M mannitol + 10 mM EDTA, it was nec-essary to prepare standard curves in solutions con-

taining 0.6 N NaOH and 0.8 M mannitol + 10 mM

EDTA, since these solutes depressed color developmentin the assay. The sensitivity of the protein assay was

also decreased in the presence of mannitol + EDTA.Protein contents of cells were also determined by esti-mating the total nitrogen content by Kjeldahl digestionand nesslerization (21) after removal of nucleic acidswith hot I N perchloric acid. These results agreedclosely with values obtained by the alkali extractionmethod after allowance had been made for the contentof lipid nitrogen.

Ninhydrin-positive compounds were extracted from

cells with boiling water as described by Brown andRose (3). Contents of ninhydrin-positive compounds incell extracts and in shock supernatant fluids were deter-mined by the Spies modification (35) of the Moore andStein (22) method, with DL-leucine as a standard. Con-tents of ninhydrin-positive compounds in cells and ex-tracts are expressed as micrograms of leucine equiva-lent per milligram (dry weight) of cells.

Viability measurements. The viability of yeast popu-lations was measured by using the slide-culture methodof Postgate et al. (30) and malt extract-yeast extract-glucose-peptone medium (39). Before use, the mediumcontaining 1.5% agar was filtered hot through cellulosepulp, adjusted to pH 5.0, sterilized at 120 C for 15min, and then filtered hot through a membrane filter(Oxoid Ltd., London, England; 5-cm diameter;standard grade) to remove particulate matter. Threeslide cultures were made from each sample. Viabilityvalues are quoted as the percentage ratio of the numberof microcolonies to the total number of objects presentin a total of at least 300 objects.

Measurements of the rate of solute accumulation.Accumulation of glucosamine, a compound which isnot utilized as a carbon or energy source by the yeast,was measured by suspending cells in a reaction mixture(pH 4.5) consisting of 10 mm KH2PO4 containing 5mM [1- 12C]glucosamine-hydrochloride with 41.6nmoles of [1-_4C]glucosamine-hydrochloride (specificactivity, 4.05 mCi per mmole) per ml. The reactionmixture containing [1-14C]glucosamine had an activityof about 0.0167 mCi per mole of glucosamine. Reactionmixtures were dispensed into 50-ml conical flasks andwere incubated in a shaker-water bath at 30 C. Cells [1mg (dry weight) per ml] were suspended in prewarmedreaction mixtures, and the rate of glucosamine accumu-

lation was measured by removing duplicate portions(1.0 ml) which were filtered through membrane filters(2.5-cm diameter; 1.2-Am pore size; Millipore Corp.,Bedford, Mass.) that had been washed with 10 ml ofcool (5 C) [1-_2C]glucosamine-hydrochloride (10 mmin 10 mm KH2PO4). At the beginning and end of eachexperiment, portions of suspension were filteredthrough two filters on the same base. The lower filterwas then removed after washing and used as a controlfor measuring background radioactivity. Immediatelyafter the portion had been filtered, the filter and cellswere washed with 10 ml of cool (5 C) glucosamine-hydrochloride solution (10 mm in 10 mM KH2PO4).Using forceps, the filters were then transferred to vials(20-ml capacity; Beckman Instruments Co. Ltd., Glen-rothes, Fife, Scotland) made of low potassium glass,each of which was then charged with 5 ml of precooled(10 C) scintillation fluid which consisted of a mixtureof 3 volumes of toluene containing 0.5% 2,5-diphenyl-oxazole and 2 volumes of 2-methoxyethanol. The ra-

dioactivity of the contents of the vials was measured ina Beckman liquid scintillation spectrometer (CPM200). Samples were counted for 20 min. Readings were

corrected for average background count during the pe-

riod of the experiment. The number of micromoles ofglucosamine accumulated per gram (dry weight) ofcells per minute was calculated from the regressioncoefficient of the time-course plot. After the filters hadbeen removed, the sintered glass bases were washedwith water. Preliminary experiments showed that, using

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this washing procedure, there was no detectable reten-tion of radioactivity in the filtration apparatus.

The procedure for measuring the rate of accumula-tion of 2-aminoisobutyrate (AIB) was similar to thatfor measuring the rate of glucosamine accumulation. Itwas shown that AIB, an analogue of alanine, was notused as a carbon or nitrogen source for growth of theyeast. The reaction mixture (pH 4.5) consisted of 5 mm[1-12C]AIB in 10 mm KH2PO4 containing [1-14C]AIB(3.8 to 10.4 mmoles/ml, depending on the specific ac-tivity of the batch of AIB). In experiments on AIBaccumulation, the filters and sintered glass bases werewashed with 10-ml portions of cool (5 C) water.

Enzyme assays. Invertase (fi-fructofuranosidase; EC3.2.1.26) activity was assayed by a method based onthat of Sutton and Lampen (38). The reaction mixturecontained nystatin (50 ug/ml) to inhibit the action ofglycolytic enzymes. The reaction was carried out at 30C and was stopped by adding a spatula tipful of solidcalcium carbonate and placing the tube on a bath ofboiling water for 5 min. Glucose produced was assayedusing the glucose oxidase method (Boehringer bloodsugar kit) in tris(hydroxymethyl)aminomethane (Tris)buffer (pH 7). Activity of L-leucyl-B-naphthylamidase(EC 3.4.1.1.) was determined at 30 C by the method ofGoldbarg and Rutenburg (8). Acid and alkaline phos-phatase activities (EC 3.1.3.1. and EC 3.1.3.2.) wereassayed colorimetrically at 30 C by using p-nitrophenyl-phosphate as a substrate (1). The acid phosphatasewas assayed at pH 4.0, and the alkaline phosphatasewas measured at pH 8.0 in the presence of 10 mMMg2

Chemicals. All chemicals used were reagent grade orof the highest purity available commercially. [1-14C]Glucosamine-hydrochloride and [1-14C]AIB were fromThe Radiochemical Centre, Amersham, Berks, Eng-land. 2,5-Diphenyloxazole was obtained fromBeckman Instruments Ltd., Glenrothes, Fife, Scotland.

RESULTSEffect of shock treatments on the rates of

solute accumulation and on viability. Initially, anexamination was made of the effect of a full coldosmotic shock treatment on the rate at whichyeast cells accumulate glucosamine and AIB,and on their viability. The shock procedure was amodification of that used with bacteria (I 1).Phosphate buffer (pH 4.5) was used in stage Iinstead of Tris-hydrochloride (pH 7.3) becauseof the preference of yeasts for an environment atacidic pH values. The hyperosmolar solutioncontained mannitol instead of sucrose becausestrains of S. cerevisiae possess a periplasmic in-vertase (14); it was also unbuffered. The EDTAconcentration was 10 mm because this concentra-tion was shown by Diamond and Rose (5) to bemost effective in potentiating osmotic lysis ofspheroplasts from this yeast. Preliminary experi-ments with cells grown in batch culture at 30 Cindicated that submitting cells to the standardshock caused them to accumulate glucosamineat a much slower rate than that shown by un-

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shocked cells. Since the time required to measurethe rate of solute uptake by individual batches ofcells was about 20 min, it was necessary to storecells in buffer at 10 C. Storage under these con-ditions caused a progressive increase in the rateat which the cells accumulated glucosamine (Ta-ble 1), thereby making comparisons of accumu-lation rate impossible. It was therefore decided torestrict experiments to yeast grown in a chemo-stat from which batches of cells sufficient formeasurement of the rate of solute accumulationcould be removed immediately before use, therebyobviating the need for storing cells.Table 2 shows the results of subjecting a sus-

pension of chemostat-grown cells to the full coldosmotic shock treatment and to various modifiedshock treatments. The viability of the suspensionwas little affected by the complete shock treat-ment, although when the cells were suspended inwater instead of 0.5 mM MgCl2 at 0 C in stage 3,the viability declined appreciably. The rate ofaccumulation of glucosamine and AIB by cellsthat had been subjected to the full treatment wasalways slower than with unshocked cells, al-though the percentage of decrease in the rate ofaccumulation showed some variation betweenexperiments. Varying the shock treatment byomitting EDTA from the hyperosmolar solution,

TABLE 1. Effect ofstorage at 10 C on the rate atwhich cells accumulate glucosamine before and after

cold osmotic shocka

Rate of accumulationPeriod of storage of glucosamine5

at 10 C(min) Unshocked Shocked

cells cellsc

35 6.155 10.478 7.195 12.4113 11.1135 13.7155 11.9173 16.1

aCells were grown in batch culture at 30 C and har-vested when the culture contained 0.24 mg (dry weight)of cells per ml. After washing in 10 mm KH2PO4 at 10C, they were suspended in this buffer and maintainedat 10 C.

b Rate of accumulation of glucosamine was meas-ured as described in the text. Rates are expressed asmicromoles of glucosamine accumulated per gram (dryweight) of cells per minute. The values given are thoseobtained in a typical experiment.

c Shock treatment given to cells [1 mg (dry weight)/ml] was that described in the text and was performedimmediately before the rate of glucosamine accumula-tion was measured.

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TABLE 2. Viability and rate of accumulation ofglucosamine and 2-aminoisobutyrate (A IB) by cells subjected tofull and modified cold osmotic shock treatmentsa

Glucosamine AIB

Treatment Viability Rate of Per cent Rate of Per cent(%) accumu- of full accumu- of full

lationb rate" lationd rate"

Cells taken directly from chemostat ...... .......... 99Cells washed and suspended in buffer at 30 C ....... 90 5.4 100 0.45 100Full cold osmotic shock ......... ................. 87 3.9 68 0.23 44Full shock but omitting EDTAe ....... ............ 89 5.2 95 0.22 45Full shock but omitting 0.8 M mannitol ...... ......... 91 5.5 98 0.18 37Full shock but omitting MgCI2......... ............ 62 4.2 80 0.24 49Full shock but using MgCl2 solution at 30 C ........ 87 4.8 88 0.36 83

a Cells were grown in a chemostat at 30 C and a dilution rate of 0. 10 hr- 1. Shock procedures were carried outas described in the text. The cell concentration was I mg (dry weight) per ml.

b Rates of glucosamine accumulation are expressed as micromoles of glucosamine accumulated per gram (dryweight) of cells per minute. Values quoted are the averages of at least four separate determinations. The controlvalue is the average of 18 determinations and is 5.41 4 0.78.

c Percentage values quoted are the averages of percentages calculated in individual experiments.d Rates of accumulation of AIB are expressed as micromoles of AIB accumulated per gram (dry weight) of

cells per minute. Values quoted are averages of at least four separate determinations. The control value is an av-erage of five separate determinations and is 0.45 4 0.11.

e Ethylenediaminetetraacetic acid.

or suspending cells in an EDTA solution lackingmannitol in stage 2, had little effect on the rateof glucosamine accumulation. Suspending cells inwarm (30 C) instead of cold (O C) MgCl2 solu-tion or in cold water in stage 3 led to a smallerdecline in the rate of glucosamine accumulation.The rates at which AIB was accumulated by cellswere considerably slower than the rates of glu-cosamine accumulation. The full cold osmoticshock treatment again led to a marked decreasein the rate at which cells accumulated AIB,whereas using warm instead of cold MgCl2 solu-tion led to a smaller decline in the rate of AIBuptake. In contrast to the findings on glucosa-mine accumulation, omitting EDTA or mannitolfrom stage I of the complete treatment hardlyaffected the loss of accumulating ability causedby the full treatment. Using cold water instead ofcold 0.5 mm MgCl2 did not prevent the loss ofAIB-accumulating ability.An attempt to assess more fully the metabolic

damage done to the cells as a result of cold os-motic shock was made by incubating shockedand unshocked cells in malt extract-yeast ex-tract-glucose-peptone medium (39) and in achemically defined medium (31) and measuringthe time-course of growth. When shocked cellswere suspended in defined medium, there was notusually an increased lag or difference in growthrate compared with unshocked cells (Fig. 1). Bycontrast, shocked cells always had a longer lagphase and initially showed a slower rate ofgrowth compared with unshocked cells whensuspended in the complex medium (Fig. 1.)

~~0-%TIM"E (MIIN) TIMEllMIN)

IG. 1. Time course of growth of unshocked cells(solid line) and cells submitted to the complete coldosmotic shock treatment (broken line) in defined me-dium (a; 31) and in malt extract-yeast extract-glucose-peptone medium (b; 39). The cells were harvested froma chemostat operating at 30 C and a dilution rate of0.10hr-0.After the cells were submitted to cold os-motic shock, they were suspended in the appropriatemedium and incubated in 100-ml portions of mediumin 350-m1 conical flasks in a shaker-water bath (100rotations per min) at 30 C.

Release of cell components as a result of coldosmotic shock. Cold osmotic shock in bacteriacauses the release from cells of low-molecular-weight nucleotides and ninhydrin-positive com-pounds and of proteins (I11). A search was there-fore made for release of certain of these com-pounds when yeast cells were subjected to com-plete and modified cold osmotic shock treat-ments. Complete shock (stages I to 3) of cellsled to the release of over half of the pool of in-tracellular low-molecular-weight ninhydrin-posi-tive compounds and about 6 to 8% of the cellprotein (Table 3). Most of the low-molecular-

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TABLE 3. Release of low-molecular-weight ninhydrin-positive compounds and protein from cells as a result offulland modified cold osmotic shock treatmentsa

Release of

Low-molecular-weightninhydrin-positive Protein

Treatment Stage" compounds

Per cent Per centAmtc of total Amtd of total

lost lost

Full cold osmotic shock .......... ................ 1 0.32 40 Trace2 0.0 0 Trace3 0.19 24 25 6

EDTA omitted in stage 2 ......... ................ 2 0.0 0 9 23 0.07 9 13 3

Mannitol omitted in stage 2 ........ .............. 2 0.02 2 9 33 0.0 0 Trace

Water (0 C) used instead of 0.5 mM MgCI2 in stage 3. 3 0.13 16 5

MgCI2 (0.5 mM) used at 30 C ....... .............. 3 0.19 24 11 2

Contents in unshocked cells ....................... 0.80 100 384 100

a Cells were grown in a chemostat at 30 C and a dilution rate of 0.10 hr- 1. The suspensions contained 2.5 mg(dry weight) of cells per ml.

Individual stages of the complete cold osmotic shock treatment are described in the text.Micromoles of leucine equivalent per milligram (dry weight) of cells. Values quoted are averages of at least

four separate determinations.d Micrograms per milligram (dry weight) of cells. Values quoted are averages of at least four separate determi-

nations. "Trace" indicates a concentration of less than 5 gg of protein per mg (dry weight) of cells; with superna-tant fluids from suspensions in mannitol + ethylenediaminetetraacetic acid (EDTA), "trace" indicates less than10 ,gg of protein per mg (dry weight) of cells.

weight compounds lost by cells were released instage 1 of the shock treatment, and the re-mainder were released in stage 3. OmittingEDTA from stage 2 or MgCl2 from stage 3 ledto release of smaller amounts of low-molecular-weight ninhydrin-positive compounds in stage 3.Omission of mannitol prevented completely therelease of these compounds in stage 3. Using 0.5mM MgCl2 at 30 C instead of 0 C did not affectrelease of these compounds. During completeshock, a trace of protein was released into bufferin stage 1 and in stage 2, but much more wasreleased in stage 3. A somewhat similar patternof effects was observed with release of protein aswith release of low-molecular-weight ninhydrin-positive compounds when the various modifiedshock treatments were applied, except that using0.5 mM MgCl2 at 30 C instead of 0 C led to therelease of less protein.

Release of protein into cold 0.5 mm MgCl2 atstage 3 in the complete shock treatment was usedto study the effect of other conditions on coldosmotic shock of yeast cells. Changing the dilu-

tion rate in the chemostat from 0.1 to 0.05 hr-at 30 C led to a slight increase in the proteincontent of cells, but the amount of protein re-leased in stage 3 of the complete shock treatmentwas about the same. When the chemostat wasoperated at 15 C instead of 30 C, with a dilutionrate of 0.05 hr- , there was a slight increase inthe amount of the total cell protein released instage 3 of the complete shock treatment. Theconcentration of cells in the suspension used inthe complete shock treatment was also shown toaffect the proportions of total cell protein andintracellular low-molecular-weight ninhydrin-positive compounds released (Table 4). Whenthe cell concentration was lowered from 2.5 to1.0 mg (dry weight) per ml, there was a markedincrease in the proportion of cell protein releasedinto cold 0.5 mm MgCI2 and of ninhydrin-posi-tive compounds released into buffer. However,this change in the cell concentration had littleeffect on the amounts of ninhydrin-positive com-pounds released into cold 0.5 mm MgCI2.

Effect of cold osmotic shock on the enzyme

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TABLE 4. Effect of cell concentration on release of cellcomponents from yeast during cold osmotic shocka

Component released

. ~~~Low-molecular--Low-molecular- weihnnb-Cell suspen- weight ninhy- weight ninhy- Protein re-sion concn1 drin-positive drinnpositive leased into

compounds compounds 0 . mM MgCl,released into released into a

bufferc 0.5 mm MgCI2 at0at 0 C

2.5 0.32 0.19 25.01.0 0.52 0.16 35.0

a Cells were grown in a chemostat at 30 C and a di-lution rate of 0.10 hr- 1.

° Milligrams (dry weight) per milliliter.c Contents expressed as micromoles of leucine equiv-

alent per milligram (dry weight) of cells. Unshockedcells contained 0.80 i 0.15 tsmole of leucine equiva-lent per mg (dry weight) of cells.

d Contents expressed as micrograms per milligram(dry weight) of cells. Unshocked cells contained 38413 Ag of protein per mg (dry weight).

contents of cells. In view of the reported release ofperiplasmic enzymes from bacteria as a result ofcold osmotic shock (11), an examination wasmade of shock treatment supernatant fluids; inparticular, the supernatant from stage 3 wasexamined for the presence of invertase, acidphosphatase, and L-leucine-f,-naphthylamidase,all of which are enzymes located in the cell enve-lope layers of S. cerevisiae (14, 18), and for al-kaline phosphatase which is a cytoplasmic en-zyme in S. cerevisiae (19). No evidence was ob-tained for the release of detectable amounts ofalkaline phosphatase or L-leucine-f,-naphthylam-idase. Small amounts of invertase were releasedat each of the three stages in the complete shocktreatment, but these amounted to only about 1%of the total invertase activity of intact or homog-enized unshocked cells in each stage. Amountsof invertase of this order were also released fromcells when the modifications to the shock treat-ment indicated in Table 3 were carried out. Acidphosphatase activity was not detected in the su-pernatant fluids, but the total activity of thisenzyme in cells and cell homogenates was low,approximately 0.06 ;mole of substrate per mg(dry weight) of cells per hr.

DISCUSSIONCold osmotic shock in S. cerevisiae NCYC

366 is similar in many respects to related phe-nomena previously described for gram-negativebacteria (11, 23). The main similarities are re-tention of viability accompanied by a decrease inthe rate at which cells accumulate solutes, a re-lease of 6 to 8% of the cell protein, and a pro-

portion of the intracellular pool of low-molec-ular-weight ninhydrin-positive compounds, andby an increase in the duration of the lag phase ofgrowth when shocked cells are suspended incomplex nutrient medium. The only other eukary-otic microbe in which cold osmotic shock hasbeen reported is N. crassa (40), although thisreport did not include extensive data on the re-lease of cell constituents. Cold osmotic shock isused to extract transport proteins from microor-ganisms. Release of transport proteins fromyeast was not demonstrated in the present studyand can only be inferred from the decline in therate at which shocked cells accumulate glucosa-mine and AIB. If transport proteins are indeedlost when S. cerevisiae is subjected to cold os-motic shock, this technique will furnish a valu-able experimental tool to workers interested inthe physiology of solute uptake in yeast, particu-larly as the plasma membrane in this yeast isamong the most extensively studied of microbialplasma membranes (12, 15).

In S. cerevisiae, the requirements for coldosmotic shock-induced loss of glucosamine-ac-cumulating ability and AIB-accumulating abilitydiffer. Loss of glucosamine-accumulating abilityoccurs only when cells are subject to both anosmotic stress and to EDTA treatment; omis-sion, on the other hand, of either of these treat-ments does not prevent the loss or reduce theloss of AIB-accumulating ability. These findingscan be explained by assuming that the proteinsinvolved in glucosamine transport are morefirmly held in the membrane than those involvedin AIB transport. The ability to accumulate bothsolutes is largely retained when the magnesiumchloride solution in stage 3 is used at 30 C in-stead of 0 C, indicating the need for a cold stressfor the release of transport proteins. One possi-bility is that, when the cells are rapidly cooled,freezing of the membrane lipids facilitates therelease of transport proteins from the membrane.It might be expected, therefore, that when theproportion of unsaturation in the membranelipids is increased, and therefore the meltingpoint is decreased, as a result of growing thecells at a suboptimal temperature (5), loss ofmembrane proteins should not be so extensive.That this was not so suggests that the effect ofcooling on the cells may not be related to thefreezing of membrane lipids. Growth of S. cere-visiae at 15 C instead of 30 C also leads to syn-thesis of an increased proportion of phosphati-dylcholine (5, 12), and our data indicate that thegreater cation-binding power which this mayconfer on the membrane (5) also does not affectretention of membrane proteins when the cellsare subjected to cold osmotic shock. The ability

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of S. cerevisiae cells to lose membrane proteinsas a result of cold osmotic shock may be relatedto the phenomena reported by Schlenk and hiscolleagues (32, 33); they found that Candidautilis and S. cerevisiae, in the absence of saltsand buffers, become sensitive to several proteinsof moderate size, including ribonuclease, prota-mine, lysozyme, and bovine plasma albumin. Thesensitivity of these cells to proteins could be ex-plained by the ability of the proteins to replacemembrane-bound proteins in cation-free suspen-sions and might explain the need for EDTA iithe cold osmotic shock-induced loss of glucosa-mine-accumulating ability.

Loss of cell protein followed a pattern similarto that observed for loss of glucosamine-accu-mulating ability, rather than loss of AIB-accu-mulating ability. However, loss of low-molec-ular-weight ninhydrin-positive compounds duringcold osmotic shock would not appear to be re-lated to loss of cell protein or of solute-accumu-lating ability. Thus, the bulk of these compoundsare released into buffer in stage 1, whereas themain requirements for release of the remainderof the compounds in stage 3 of the treatmentappear to be an osmotic stress and treatment ofthe cells with EDTA.Our finding that Mg2+ ions are required in

stage 3 to retain viability in cells subjected tocold osmotic shock agrees with data reported forbacteria (11). Magnesium ions may be requiredto retain certain components such as phospho-lipids or proteins in the membrane by acting asionic bridges. Curiously, however, omitting theMg2+ ions from stage 3 decreased the amountsof glucosamine-transporting proteins released.The need for Mg2+ to obtain maximal release ofglucosamine-transporting proteins is not easilyexplained.The lag in growth observed when shocked cells

were suspended in complex nutrient medium issimilar to the lag described by Neu and Chou(23) with Citrobacter freundii; it is probably ex-plained by the need to synthesize membrane pro-teins that were lost during the shock treatment.Our inability to demonstrate a lag in growth ofshocked cells in defined medium might be ex-plained by assuming that only a small proportionof the transport proteins were lost from the cellsas a result of the shock, and that, because thecell possesses an excess of these proteins, growthin defined medium which may well require fewertransport proteins is not affected. In this respect,S. cerevisiae would appear to differ from N.crassa (40). If this assumption is correct, use ofcold osmotic shock could provide a way of as-sessing the role of transport processes in regu-lating the rate of growth of yeast cells.

One of the most interesting findings from ourexperiments on cold osmotic shock in S. cerevi-siae was the discovery that the cell envelope en-zymes, invertase, acid phosphatase, and L-leu-cine- 3-naphthylamidase, remain cell bound whenthe cells are shocked. Failure of the cells to losealkaline phosphatase indicates that cytoplasmicproteins are probably not released to any greatextent. One reason for the retention of the cellenvelope enzymes is that these may be covalentlybound to wall components (4). However, evi-dence that this is not so in our strain of yeastcomes from the finding (J. W. Patching and A.H. Rose, unpublished data) that, when cells aredisrupted in a Braun homogenizer, a large pro-portion of the total invertase and L-leucine-,B-naphthylamidase activity is found in the particle-free supernatant fluid. Another possible reasonfor the retention of cell envelope enzymes whenS. cerevisiae is subjected to cold osmotic shock isthat these enzymes are glycoproteins (2, 25) un-like those that are located in the periplasm ofgram-negative bacteria. It is conceivable that thepresence of a polysaccharide moiety on the pro-tein molecule allows the molecule to becomemore firmly bound by secondary bonds to thewall or membrane, thereby preventing it frombeing released during cold osmotic shock. Molec-ular size would not appear to be an importantfactor. A recent estimate of the molecular weightof the periplasmic invertase from S. cerevisiaewas in the range 4,000 to 10,000 (26), which isprobably lowep than the molecular weight ofmembrane-bound transport proteins and approx-imately the threshold value reported by Gerhardtand Judge (7) for the size of molecules that canpass across the wall of S. cerevisiae.Our need to use yeast cells grown in a chemo-

stat stemmed from the finding that storage ofbatch-grown cells at 10 C led to an increase inthe rate at which these cells accumulated glucos-amine. We are not aware of any previous reportof this effect, although workers who study thekinetics of solute accumulation particularly inyeast should be aware of the existence of thisstorage phenomenon.

ACKNOWLEDGMENTSJ.W.P. thanks the Science Research Council (England) for

the award of a studentship which was held during the course ofthis work. The Science Research Council (England) also kindlyprovided funds for equipment and supplies under grantB/SR/5724. The competent technical assistance of SarahMiles is also acknowledged. G. Wheeler gave valuable assist-ance in the assays for L-leucine-jt-naphthylamidase activity.

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