osmotic imbalance in inositol-starved spheroplasts

JOURNAL. OF BACTIERIOI()(G.Y, Dec. 1977, p. 806-81ICopyright ©) 1977 American Society for Microbiology

Vol. 132, No. :3Prin te(d int U. .S.A.

Osmotic Imbalance in Inositol-Starved Spheroplasts ofSaccharomyces cerevisiae

KATHARINE D. ATKINSON,* ANITA I. KOLAT, AND SUSAN A. HENRYDepartment of Genetics, Albert Einstein College of Medicine, Bronx, New York 1046!

Received for publication 8 September 1977

Physiological states associated with inositol starvation of spheroplasts of Sac-charomyces cerevisiae were investigated and compared with conditions precedingdeath of starved whole cells. In the absence of synthesis of inositol-containinglipids, cell surface expansion terminated after one doubling of whole cells. Inspheroplasts, cessation of membrane expansion was apparently followed by rapiddevelopment of an osmotic imbalance, causing lysis. Continued synthesis andaccumulation of cytoplasmic constituents within the limited cell volume wereimplicated as a cause of the osmotic imbalance. In whole cells, an increase ininternal osmotic pressure also follows termination of membrane and cell wallexpansion. The cell wall prevents lysis, allowing a state of increasing cytoplasmicosmotic pressure to persist in the period preceding onset of inositol-less death.

Rapid metabolism-dependent cell death in theabsence of the phosopholipid component inositoloccurs in a variety of inositol-requiring fungalmutants and strains (2, 17-19). Similarly rapidcell death has been observed during fatty acidstarvation (6), biotin deficiency (11, 15), andpantothenate deficiency (17). These lethal star-vation conditions all involve interference withthe synthesis of one or more membrane lipids.Two different explanations for the lethality re-sulting from defective lipid synthesis in inositol-starved fungi have been proposed. Shatkin andTatum (16) proposed a failure of membraneexpansion leading to an imbalance between thegrowth of cell surface and of intracellular com-ponents. Ma';ile (13) favored the hypothesis thatdevelopment of an abnormality in membranecomposition and structural integrity leads todisruptior. of cellular membranous organelles,particularly lysosomes.

In an 'nositol-requiring mutant of the yeastSaccharomyces cerevisiae, the physiologicalstages preceding inositol-less death have beenexamined (7). After removal of exogenous ino-sitol, synthesis of the major inositol-containinglipid, phosphatidylinositol, ceases rapidly. Celldivision and cell volume expansion cease withinone generation time, whereas intracellular mac-romolecular synthesis continues for another gen-eration time until the onset of logarithmic celldeath. The finding of continued metabolismwithin a no-longer-expanding cell volume sup-ports Shatkin and Tatum's hypothesis of animbalance between synthesis of intracellularcomponents and cell surface growth (16).

Continuation of macromolecular synthesiswithin a limited cell volume suggests that an

increase in cytoplasmic osmotic pressure maybe one of the abnormal cellular conditions pro-duced by inositol starvation. The rigid yeast cellwall protects the cell against osmotic stress.Since the wall is the outermost surface of wholeyeast cells, the cessation of its expansion is di-rectly implicated in the previous finding of in-terrupted cell volume expansion (7). To learnthe effects of inositol starvation on the expansionof the plasma membrane and to investigate cy-toplasmic osmotic pressure, we studied inositolstarvation of yeast spheroplasts, which are cellsstripped of their cell walls. Yeast spheroplasts,after a lag in metabolic activity immediatelyupon removal of the cell walls, continue metab-olism and cell volume growth without cell divi-sion for two or three doublings of deoxyribonu-cleic acid content (10). If inositol starvation leadsto the cessation of plasma membrane expansion,this should be reflected in a premature cessationof spheroplast volume growth. If the growth andmetabolism of inositol-starved spheroplasts par-allel those of starved whole cells, a cessation ofvolume growth may be expected to lead to in-creased osmotic sensitivity of the spheroplasts.

(This work was taken in part from a disserta-tion to be submitted by K.D.A., in partial fulfill-ment of the requirements for the Ph.D. degreein the Sue Golding Graduate Division of theAlbert Einstein College of Medicine, Bronx,N.Y.)

MATERIALS AND METHODSYeast strains. An inositol-requiring strain of S.

cerevisiae, MC13 inol-13 lys2 canr a (2) was used inall experiments. This strain was used previously inphysiological studies of inositol starvation in wholecells (7).

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INOSITOL-STARVED YEAST SPHEROPLASTS 807

Culture media. Overnight precultures were grownin YEPD (1% yeast extract, 2% peptone, 2% glucose).Synthetic complete medium for whole cells, with andwithout 2 jLg of inositol per ml, has been describedpreviously (7). Isotonic spheroplast medium was com-posed of the same synthetic complete medium osmot-ically stabilized by addition of 0.4 M MgSO4, 1%(wt/vol) succinic acid, and 0.6% (wt/vol) NaOH (pH5.2; 4, 10). Isotonic spheroplast medium lacking lysinewas used in some experiments.

Cycloheximide (100 yg/ml) or inositol (2 yg/ml)was added to cultures from a 100-fold-concentratedsterile stock solution.

Osmotic supplements, containing all ingredients ofisotonic spheroplast medium, were prepared with alarge amount of water displaced by addition of either2 M MgSO4 (in addition to the isotonic content of 0.4M), 2 M sucrose, 3 M glucose (in addition to theisotonic content of 2%), 6 M glycerol, 2 M NaCl, or 2M KCI. After addition of one of these supplements toa spheroplast culture, the increased culture volume,still occupied by the same number of spheroplasts,was calculated into all subsequent measurements. Ad-dition of one of these supplements effectively addedthe indicated compound with no change in the con-centration of any other medium component exceptfor the displaced water.

Preparation of spheroplasts. Spheroplasts wereprepared by the methods of Hutchison and Hartwell(10). Cells were harvested from a YEPD preculturegrown at 24°C to a density of 1 x 107 to 3 x 107 cellsper ml. Cells were washed twice in distilled water andsuspended in 0.05 volume of 1 M glycerol. Glusulase(Endo Laboratories Garden City, N. Y.) was added(10 1l/ml of cells in glycerol), and the suspension wasincubated for 20 to 30 min at 30°C. Efficiency ofspheroplasting was estimated by testing for osmoticsensitivity in 0.1% (wt/vol) sodium dodecyl sulfate,visually observed as a rapid loss of optical density.Spheroplasts were also examined microscopically forspherical shape and absence of buds. Sorbitol (10)was avoided in the preparation and growth of spher-oplasts, since it has been found to support growth ofthe inositol-requiring strain, presumably because ofcontaminating inositol.Growth conditions. Portions of the spheroplast

preparation in glycerol were added to isotonic spher-oplast medium to a density of 1 x 107 to 5 x 107 cellsper ml and incubated at 30°C. At intervals, subcultureswere prepared by mixing with prewarmed portions ofthe various medium supplements.To determine the relationship between physiologi-

cal stages in whole cells and in spheroplasts duringinositol starvation, cells were converted to sphero-plasts at various times during inositol starvation. Cellsgrown in YEPD were transferred to inositol-contain-ing complete synthetic medium at a density of 2.5 x10" cells per ml and incubated at 30°C for 4 h beforespheroplasts were prepared. At intervals, subcultureswere transferred to inositol-less medium. Cell densitywas adjusted at the time of transfer so that all culturesreached a density of 107 cells per ml at the time ofspheroplast preparation. Spheroplasts were preparedfrom each culture and placed in inositol-less isotonicspheroplast medium at 30°C.

Cell viability. Whole cells in osmotically supple-

mented medium, with and without inositol, wereplated at appropriate dilutions onto YEPD agar platesto determine viability, as described previously (7).Dilutions were prepared in the osmotically bufferedculture medium, rather than in water, to avoid possibleosmotic shock.Counting spheroplasts. The number of intact

spheroplasts per milliliter was determined at intervalsby placing a portion of a culture in the chamber of aSpencer hemacytometer and counting microscopicallyvisible spheroplasts. Small vesicles and cell ghosts canbe visually distinguished from intact spheroplasts, andwere not counted.Osmotic sensitivity. The stability of spheroplasts

in hypotonic medium was investigated by the methodsof Alterthum and Rose (1). Spheroplasts were col-lected from isotonic cultures by centrifugation at 6,000x g for 7 min. The pellets were gently suspended inequal volumes of buffer (0.05 M citrate, 0.04 M dibasicsodium phosphate, pH 6.0) containing 0.02 to 0.4 MMgSO4. The optical density of each suspension wasread in a Klett spectrophotometer with a red filter(660 nm) immediately after mixing and again after 15min at 25°C. Changes in optical density were calcu-lated as a percentage of the initial optical density ofeach suspension. Microscopic examination was usedto confirm that maintenance of the optical density ofsuspensions indeed reflected maintenance of intactspheroplasts.

Spheroplast cell volume. The cell volume distri-bution of spheroplast cultures was determined in aCoulter Counter, model F, as previously described (7).The samples of spheroplast cultures were withdrawnfrom the main culture flask and used immediately forvolume determinations. In place of the standard elec-trolyte Isoton, which is not isotonic to yeast sphero-plasts, medium was used as the electrolyte solution,with 0.1% sodium azide (the concentration used inIsoton to protect the Coulter Counter from bacterialcontamination). Azide was present only in sampledilutions used for Coulter Counter measurements, andnot in the culture from which samples were drawn.The electrical resistance of our isotonic spheroplastmedium and of medium supplemented with 1 M su-crose was found to be within an acceptable range foruse with a 100-Mm1-aperture tube. Spheroplast me-dium supplemented with an additional 1 M MgSO4proved to be an unacceptable electrolyte. Volumedeterminations were standardized by calibrationagainst monosized latex beads as described previously(7).Macromolecular synthesis. Protein and nucleic

acid synthesis in spheroplasts under different growthconditions was estimated by determination of trichlo-roacetic acid-precipitable [14C]lysine and [14C]uracilcounts, as previously described (7). All media usedcontained 40 mg of lysine per liter and 20 mg of uracilper ml as cold carrier. _-[UL4C]lysine (348 ,uCi/mmol)and [2-14C]uracil (59 jiCi/mmol) were purchased fromAmersham/Searle, Arlington Heights, Ill. For contin-uous-labeling experiments, corrections for the changein cell density after the addition of an osmotic supple-ment were accomplished by including the sameamount of isotope (0.2 MCi/ml) in the additive andcorrecting counts recovered by the factor to whichthe culture volume was increased. For pulse-labeling

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808 ATKINSON, KOLAT, AND HENRY

experiments, corrections after addition of an osmoticsupplement were accomplished by an appropriate in-crease in sample volume, to maintain spheroplastcount, and an increase in the amount of isotope, tomaintain the same isotope concentration of 1 jiCi/ml.

Total protein determinations. The total proteincontent of spheroplasts cultured in isotonic mediumwith and without inositol, and in subcultures with anadditional 1 M MgSO4, was estimated by the methodof Lowry et al. (12). Spheroplasts in isotonic mediumwere harvested by centrifugation at 12,000 x g for 10min. Most of the osmotically supplemented media aretoo viscous or too dense to allow removal of thespheroplasts by centrifugation. Spheroplasts in me-dium supplemented with 1 M MgSO4 were mixed with3 volumes of medium with 1.5 M NaCl to reduce theviscosity of the suspension without reducing osmoticsupport. After dilution, these spheroplasts were har-vested by centrifugation at 19,000 x g for 15 min.Spheroplasts were suspended in 0.1 volume of buffer[0.1 M tris(hydroxymethyl)aminomethane-sulfate (pH7.5), 0.005 M MgSO4, and 0.001 M ethylenediamine-tetraacetate] and cooled in an ice bath until all sampleswere collected.Potassium ion determinations. Retention of po-

tassium ions by spheroplasts cultured in isotonic me-dium with and without inositol, and in subcultureswith an additional 1 M MgSO4, was determined.Spheroplasts were harvested by filtration through dis-posable 0.45-,um filters (Falcon Plastics, Oxnard,Calif.). Filtration required 20 to 30 min for 75-mlculture samples. Filters were dried and then boiled in2.0 ml of distilled water for 10 min. Samples werecentrifuged to remove debris, and the clear superna-tant fluids were analyzed for potassium content asdescribed previously (7). Values obtained for sphero-plasts in isotonic inositol-supplemented medium wereidentical when spheroplasts were harvested by eitherfiltration or centrifugation, suggesting that filtrationdoes not produce substantial breakage of the sphero-plasts. Filtration collection of spheroplasts in osmoti-cally supplemented medium was preferred to centrif-ugation, which requires altering the medium beforecentrifugation.

RESULTSLysis in the absence of inositol. Inositol-

supplemented spheroplasts of strain MC13 ex-hibited the normal growth and metabolism de-scribed by Hutchison and Hartwell (10), withno detectable cell division or loss of intact spher-oplasts over a period of 6 h or more (Fig. 1). Bycontrast, inositol-starved spheroplasts lysed(Fig. 1). Lysis is not characteristic of all formsof nutrient starvation. Strain MC13 was addi-tionally auxotrophic for lysine. Starvation forthis amino acid requirement did not lead to anylysis over a period of 8 h or more (data notshown).Immediately after spheroplast preparation,

metabolic activity, as indicated by protein andnucleic acid synthesis (Fig. 2), was very low, butit recovered to a rate comparable to that of

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HOURSFIG. 1. Lysis of inositol-starved spheroplasts.

Spheroplasts of the inositol-requiring strain werepre-pared from YEPDprecultures and incubated at 300Cin isotonic medium with (0) and without (0) 2 jug ofinositolper ml. Intact spheroplasts per milliliter werecounted in a hemacytometer at intervals.

intact cells in approximately 2.5 h. Macromolec-ular synthesis in inositol-starved spheroplastsexactly paralleled synthesis in supplementedspheroplasts until the time of lysis and thendeclined with cell disruption (Fig. 2). Freshlyprepared spheroplasts placed in inositol-less me-dium began rapid lysis after 2.5 h. However,spheroplasts cultured in inositol-supplementedmedium for 2.5 h to recover optimal metabolicactivity and then transferred to inositol-less me-dium, began to lyse 2 h after the transfer. Mac-romolecular synthesis in "recovered" sphero-plasts showed no slowing before lysis (data notshown).Relation of inositol-starved cells and

spheroplasts. Inositol-starved cells abruptlyceased dividing at 2 h of starvation (7). Sphero-plasts prepared from whole cells that had beenstarved for inositol 2 or more hours before spher-oplasting, began to lyse immediately in inositol-less isotonic medium (Fig. 3). Spheroplasts thatwere prepared from whole cells deprived of ino-sitol for less than 2 h before spheroplastingbegan to lyse after a total of 2.5 h in inositol-less medium. For instance, cells starved for ino-sitol 1.5 h and then converted to spheroplastsbegan to lyse after 1 h in inositol-less isotonicmedium.Spheroplast cell volume. The volume dis-

tribution of spheroplasts after growth in isotonicmedium with or without inositol is shown in

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INOSITOL-STARVED YEAST SPHEROPLASTS

Fig. 4. Inositol-starved spheroplasts doubledtheir initial mean cell volume in 2.5 h, from ca.30 to 60,m3, as did inositol-supplemented spher-oplasts. Thereafter, the supplemented sphero-plasts increased in volume by 30 ,um3 every 2 h,in parallel with their linear rate of protein syn-thesis. However, no further increase in cell vol-ume of inositol-starved spheroplasts was de-tected after 2.5 h. Spheroplast lysis after 2.5 hwas detected as an increase in small particles,presumably the cellular debris resulting fromlysis.Prevention of lysis. Lysis was prevented by

restoration of inositol to a spheroplast culturebefore 2 h of starvation (Fig. 5). Restoration ofinositol after 2 or more h resulted in incompleteprevention of lysis. Lysis began at 2.5 h, followedby complete rescue of those spheroplasts stillintact at a time corresponding to 15 to 30 minafter the addition of inositol.Cycloheximide prevented lysis when added

very shortly after spheroplasts were placed ininositol-less isotonic medium (Fig. 6). Addition

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FIG. 2. Pulse-labeling of macromolecules. Spher-oplasts were placed in isotonic medium with (0) andwithout (0) inositol at a density of 2 x 107 sphero-plasts per ml. At intervals, duplicate 0.5-ml sampleswere mixed with 0.5 uCi of ["4Clysine or f44Cluraciland incubated for 10 min. (A) Incorporation off14C]lysine into trichloroacetic acid-precipitablecounts. (B) Incorporation of ['4C]uracil into trichlo-roacetic acid-precipitable counts.

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FIG. 3. Spheroplasts ofstarved whole cells. Wholecells were starved for inositol, converted to sphero-plasts, and placed in isotonic inositol-less medium.Intact spheroplasts were counted in a hemacytometer.Length of whole-cell starvation: none (0), 1.5 h (A),1.75 h (A), 2 h (0), 2.25 h (A), and 2.5 h (El).

of cycloheximide after 30 min of growth in ino-sitol-less medium resulted in incomplete preven-tion of lysis. Lysis began at 2.5 h in all cases,but the rate of lysis was greatly retarded withearlier additions of cycloheximide.Osmotic delay of lysis. Supplementing the

medium with MgSO4, sucrose, glucose, glycerol,NaCl, or KCl delayed the lysis of inositol-starvedspheroplasts. With all the osmotic supplementsexamined, two different patterns of delayed lysiswere observed that were typified by MgSO4 andNaCl (Fig. 7). Addition of different concentra-tions ofMgSO4 shortly before lysis was expectedresulted in a longer delay of lysis as each higherconcentration was added. The rate of eventuallysis in the presence of different concentrationsof MgSO4 closely paralleled the rate of lysis inisotonic inositol-less medium (Fig. 7A). A similarpattern of delayed lysis was produced by theaddition of sucrose or glucose. Equivalent con-centrations that delayed lysis by 1.5 h (up to 4h of total starvation) were 1 M MgSO4 (abovethe isotonic content), 0.9 M sucrose, and 1.4 Mglucose (above the isotonic content). Additionof NaCl to inositol-starved cultures also resulted

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in delayed lysis. However, increasing concentra-tions of NaCl not only delayed the onset of lysis,but also significantly retarded the rate of lysis(Fig. 5B). A similar pattern of delayed lysis wasproduced by additions of KCl and glycerol.Equivalent concentrations that delayed lysis by1.5 h were 1 M NaCl, 1 M KCl, and 2 M glycerol.Delay of lysis caused by an increase of the

medium osmolarity with any of the compoundsstudied was instantaneous. Figure 8 shows thelysis of inositol-starved spheroplasts when 1 MMgSO4 was added at different times. All spher-oplasts that had not already lysed at the timeof addition remained intact until the 4-h point

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HOURSFi;(-. 5. Lysits prevention by restoration of inositol.

Spheroplasts were placed in inositol- less isotonic me-dium. At intervals, subcultures were withdrawn, and

B 2 ug of inositol per ml was added. Intact spheroplastsper milliliter were counted in a hemacytometer. Timeat which inositol was restored to the culture: 0.5 h(0), 1 h (A), 1.5 h (A), 2 h (-), 2.5 h (O), and 3 h(x). (O), The primary culture with no inositol.

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FIG. 4. Spheroplast cell uolume in

dium. Spheroplasts were placed in isotwith (0) and without (-) inositol at a a

107 spheroplasts per ml. At intervals, s

removed and the cell volume distributiomined in a Coulter Counter. (A, B, and Crepresents the midpoint of a 17- um

Points represent the midpoint of a 34-,(A) Volume distribution immediately afting spheroplast cultures; (B) after 2.5h; and (D) after 4 h.

C of delayed lysis characteristic of 1 M MgSO4supplementation.Absence of osmotic sensitivity before

lysis. In parallel with metabolic "recovery,"spheroplasts lost osmotic sensitivity. Freshlyprepared spheroplasts required the osmotic sup-port of 0.4 M MgSO4 to remain intact. After 2

D h of growth in isotonic medium, 0.2 M MgSO4provided sufficient osmotic support to preventany lysis over a period of 15 min, and 0.05 MMgSO4 was completely sufficient by 2.5 h. Be-

-_ fore the 2.5 h point, when lysis in isotonic me-dium began, there was no detectable increase

160 200 in the osmotic sensitivity of inositol-starvedspheroplasts. Starved spheroplasts became in-creasingly insensitive to osmotic shock during

isotonic me- the first 2.5 h of growth, exactly as did inositol-onic medium supplemented spheroplasts. When transferredlensity of 2 x to a buffer which presumably did not support,amples were further metabolism, spheroplasts starved of ino-in was deter- sitol for 2.5 h required only 0.05 M MgSO4 to

interaali(D) remain intact. In growth medium, osmotic sen-um3 interval sitivity abruptly appeared after this point. Toter establish- delay lysis of inositol-starved spheroplasts byi; (C) after 3 only a few minutes after 2.5 h, the osmotic

support of 1 M MgSO4 was required (0.4 M

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isotonic medium content plus additional 0.6 Msupplementation, indicated in Fig. 7A).Osmotic shrinkage. Increasing the osmolar- 7

ity of the medium produced rapid spheroplast 1 Dshrinkage that could be quantitated, as shown (A)in Fig. 9. Addition of 1 M sucrose caused spher-oplasts to shrink to one-half their size. Inositol-supplemented and -starved spheroplasts thenprogressively increased in size. At 4.5 h, inositol- 106starved spheroplasts in 1 M sucrose medium 2 106began delayed lysis. Between 4 and 5 h theinositol-starved spheroplasts reached a maximal _imean volume of approximately 60 [Lm3, whereas osupplemented spheroplasts continued to grow. O *A similar cell-volume study with the addition Iof 0.8 M sucrose revealed less initial shrinkage, ,but expansion to the same critical volume of 60 . (B)Am3 by the time of delayed lysis (data not shown).Metabolic effects of osmotic supple- i

ments. The addition of osmotic supplements Z 106produced transitory inhibition of protein synthe-sis in addition to the other effects describedabove. To the extent to which protein synthesiswas inhibited by the addition of osmotic supple-ments to spheroplasts adapted to isotonic me-dium, osmotic supplementation partially repro- 0 2 4 6 8 10

I: X1 X1HOURSFIG. 7. Osmotic delay of lysis. Spheroplasts were

107 a a6 placed in inositol-less isotonic medium. (A) After 210 AA^^ i * h, subcultures were prepared containing 0.6 M (A),

A* ~^ 0.8 M (U), 1.0 M (0), 1.2 M (A), 1.4 M (O), or no (0)additional MgSO4. (B) After 2.5 h, subcultures wereprepared containing 0.2 M (A), 0.6 M (U), 0.8 M(0), 1.0 M (A), 1.4 M (O), or no (0) added NaCl. In

Ln \ \ Keach experiment, intact spheroplasts per milliliterV) \ \ Na were counted in a hemacytometer in each subculture<L \ \ at intervals. Counts were corrected by the factor byO which the initial culture volume was altered by ad-ui 106- \ ditions of osmotic supplements.0-Ln\

duced the effects of cycloheximide. In compari-son with the rate of protein synthesis in isotonic

Z cultures, the addition of MgSO4, sucrose, andglucose produced minimal and transient inhibi-tion of protein synthesis while producing animmediate and complete delay of lysis. There-

105 I fore, the effects of these supplements in delaying0 1 2 3 4 5 6 lysis are most likely not due simply to inhibition

HOURS of protein synthesis. Continuous labeling of pro-teins before and after additions of the different

FIG. 6. Partial lysis prevention by cycloheximide osmotic supplements indicated that addition oftreatment. Spheroplasts were placed in inositol-less 1 M MgSO4, 1 M sucrose, or 1.4 M glucose doesisotonic medium. At intervals, subcultures were with- not significantly alter the ongoing accumulationdrawn and 100 pg ofcycloheximideper ml was added. of radioactive precursor into protein by inositol-Intact spheroplasts per milliliter were counted in a

hemacytometer. Primary inositol-less culture with no supplemented (not shown) or -starved (Fig. l0)cycloheximide (0). Subcultures with cycloheximide spheroplasts. However, addition of 1 M NaCl, 1added immediately (A); after 0.5 h (U), after 1 h (0), M KCI, or 2 M glycerol to spheroplasts adaptedafter 1.5 h (A). and after 2 h (O). to isotonic medium produced a significant tran-

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sitory inhibition of protein synthesis (Fig. 10).The differing metabolic effects of these twogroups of osmotic supplements may explain thedifferences observed in the kinetics of delayedlysis (Fig. 7) discussed above. The latter groupof osmotic supplements (NaCl, KCI, and glyc-erol) that significantly inhibit metabolism notonly delayed lysis, but also reduced the rates ofeventual lysis (Fig. 7B). These retarded rates oflysis resembled those observed in cyclohexi-mide-treated cultures (Fig. 6), suggesting thatNaCl, KCI, and glycerol delay lysis of inositol-starved spheroplasts by superimposed osmoticand metabolic effects. Even with the transitoryinhibition produced by several of the osmoticadditives, all allowed the accumulation of ap-proximately 50% more labeled precursor intoprotein before delayed lysis than was accumu-lated in the unchanged isotonic inositol-less cul-ture.

Protein synthesis, estimated by pulse-labeling,was briefly and partially inhibited after addition

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FIG. 8. Immediate osmotic delay of lysis. Sphero-plasts were placed in inositol-less isotonic medium.At intervals, subcultures were withdrawn and an

additional 1 M MgSO4 was added. Intact sphero-plasts per milliliter were counted in a hemacytometerin each culture at intervals. Counts were correctedby the factor by which the initial culture volume was

altered by the osmotic additions. (0) The startinginositol-less culture. Subcultures with 1 M MgSO4:added immediately (A), after 1 h (-), 2 h (0), 2.5 h(A), and 3 h (OI).

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FI(G. 9. Osmotic shrinkage of spheroplast cell vol-ume. Spheroplasts were placed in isotonic mediumwith (0) and without (0) inositol at a density of 2 x107 spheroplasts per ml. After 2 h, 1 M sucrose wasadded to each culture. Cell volume distribution wasdetermined in a Coulter Counter immediately before(A) and after (B) addition of sucrose at 2 h, andafter a total of 3 h (C), 4 h (D), and 5 (E). (A) Eachpoint represents the midpoint of a 17-,um:1 intervalfor volume determinations in isotonic medium. (B-E)Points represent a 16-am3 interval for calibrationsin medium with 1 M sucrose.

of 1 M MgSO4 (Fig. 11) or 1 M sucrose (notshown). As compared with isotonic cultures withand without inositol, an instantaneous 50% re-duction in the rate of protein synthesis wasobserved in subcultures to which 1 M MgSO4was added at 2 h. Synthesis returned to the

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same rate as in the isotonic inositol-supple-mented culture within 1 h. In media supple-mented with an additional 1 M MgSO4, inositol-supplemented spheroplasts eventually achieveda higher steady-state rate of protein synthesisthan did the isotonic-supplemented culture. Incomparison, in the osmotically rescued inositol-starved spheroplasts, the rate of protein synthe-sis never increased above the steady-state levelobserved in isotonic cultures and leveled off a

10,0007r

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HOURSFIG. 11. Pulse-labeling ofproteins in the presence

of 1 M MgSO4. Spheroplasts were placed in isotonicmedium with (0) and without (0) inositol at a densityof2 x 107 spheroplastsper ml. After 2 h, an additionalI M MgSO4 was added to subcultures with (A) andwithout (A) inositol. At intervals, duplicate 0.5-mlsamples of isotonic cultures and 1.0-ml samples of 1M MgSO4 cultures were incubated for 10 min with I/uCi of f4C]lysine per ml and precipitated with tri-chloroacetic acid.

full hour before the sharp decline associatedwith lysis.

Total protein contained by inositol-starvedspheroplasts after the addition of 1 M MgSO4

I I increased beyond that accumulated by starved2 3 4 5 6 7 8 spheroplasts in isotonic medium. Figure 12 in-

HOURS dicates total protein contained by spheroplastsin isotonic medium with and without inositolContinuous labeling of proteins in the and in subculltures with 1 M MgSO4 added at 2

osmotic supplements. Spheroplasts weresotonic inositol-less medium with 0.2 fiCi h. Total protein accumulation continued in os-ne per ml at a density of 2 x 10i sphero- motically rescued starved spheroplasts fbr annl. After 2 h, subcultures were mixed with additional hour after addition of the osmotic)plements also containing isotope. Dupli- supplement. This resulted in approximately 15%samples were withdrawn from each cul- more protein per spheroplast than was containedprecipitated with trichloroacetic acid. by inositol-starved spheroplasts in isotonic me--e corrected by the factor by which each dium before lysis.ume was increased by the osmotic addi- Potassium ion retention. The potassiumlch panel, dotted lines indicate labeling ion content of inositol-supplemented sphero-'asts in isotonic medium with no osmoticic* M* -

'ation, with (upper curve) and without plasts in lsotonlc or 1 M MgSo4medlum in-e) inositol. Cultures (A) with an additional creased roughly in parallel With macromolecular)4 (O) or 1 M NaCl (0); (B) with 1 M synthesis and cell volume. Each time point rep-or 2 M glycerol (0); and (C) with an resents nearly a half-hour interval, due to the

1.4 Mglucose (O) or 1 M KCI (A). time required for harvesting of spheroplasts by

FIG. 10.presence ofplaced in isof [4C]lysiiplasts per nosmotic supcate 0.5-mlture andCounts werculture volltions. In ecof spheroplsupplement(lower curv41 M MgSCsucrose (A)additional

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DISCUSSIONWe believe that the data reported here are

consistent with the hypothesis that lysis of yeastspheroplasts during inositol starvation is theresult of an increase in cytoplasmic osmotic pres-sure caused by continued accumulation of met-abolic products within a limited cell volume.This interpretation is supported principally bytwo lines of evidence: the timing of lysis relativeto termination of volume expansion, and theefficacy of various treatments in preventing lysis.Additionally, the failure to obtain evidence sup-porting several alternative hypotheses will bediscussed.The termination of division in whole cells and

the start of lysis in spheroplasts apparently co-incide in time. This contention is supported bythe observation that spheroplasts made frominositol-starved cells that were no longer divid-ing (i.e., after 2 h) lysed instantly, whereas spher-oplasts made from cells that were still dividing

j (before 2 h) required an additional period of6 incubation before lysis (Fig. 3). Furthermore,

spheroplasts made from unstarved cells lysed

FIG. 12. Total protein content of spheroplasts.Spheroplasts were placed in isotonic medium with(0) and without (0) inositol at a density of 4 x 107spheroplasts per ml. After 2 h, an additional I M

MgSO4 was added to subcultures with (A) and with-out (A) inositol. (A) Spheroplasts were harvested at

intervals, and total protein content was determined.(B) Intact spheroplasts per milliliter were counted in

a hemacytometer in the same cultures.

filtration. Within the limitations of the experi-mental procedure, inositol-starved spheroplastsin isotonic or 1 M MgSO4 medium contained as

much potassium as their supplemented counter-parts until the time of lysis (Fig. 13). Potassiumion loss roughly paralleled lysis in both cases.

Effects of osmotic additives on wholecells. The kinetics of inositol-less death ofwholecells was virtually unaffected by any of the os-

motic supplements studied. Figure 14 indicateswhole-cell viability in cultures with (Fig. 14A)and without (Fig. 14B) inositol in the variousosmotically supplemented growth media. Syn-thetic complete and isotonic spheroplast mediasupplemented with inositol supported an iden-tical rate of growth of strain MC13. All of theosmotically supplemented media produced a

considerable lag in the growth of inositol-supple-mented cells (Fig. 14A). However, in the same

media lacking inositol, rapid cell death followednearly identical kinetics in all cultures (Fig.14B). Osmotic supplementation did not reduceinositol-less death.

10-3

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1 2 3HOURS

4 5 6

FIG. 13. Spheroplast potassium ion retention.Spheroplasts were placed in isotonic medium with(0) and without (0) inositol at a density of 2.5 x 10ispheroplasts per ml. After 2 h, parallel cultures at a

density of 5 x 107 spheroplasts per ml were mixedwith an equal volume of 2 M MgSO4 osmotic supple-ment, to establish comparable 1 M MgSO4 culturesuwith (A) and without (A) inositol. At intervals, 75-ml samples of each culture were filtered, and potas-sium ion content of the collected spheroplasts was

determined. Brackets surrounding each point indi-cate the time required to filter that sample.

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INOSITOL-STARVED YEAST SPHEROPLASTS 815

108

107

106

__ 105

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0 2 6 10 12 16 24

HOURSFIG. 14. Whole-cell viability with osmotic supple-

ments. Cells from a YEPD culture were washed twicewith complete synthetic medium and incubated incomplete synthetic medium (0), isotonic spheroplastmedium (A), and spheroplast media with additional1 MMgSO4 (V), 1 M sucrose (V), 1.4 Mglucose (O), 2M glycerol (El) 1 M KCl (A), or 1 M NaCl (0). Atintervals, appropriate dilutions were made in thesame medium for each culture, and cells were platedonto YEPD plates. (A) Inositol-supplemented cul-tures. (B) Inositol-less cultures.

after an equivalent 2 h of inositol starvation (2.5h if the spheroplasts had not been allowed a

period of metabolic recovery). The amount ofgrowth that occurs in inositol-starved sphero-plasts immediately before lysis is apparentlyequivalent to the growth of inositol-starvedwhole cells at the time when they stop dividing.

Spheroplasts reached an average limiting vol-ume before lysis that was approximately doubletheir starting volume (Fig. 4). Since spheroplastsdo not divide, this doubling in volume is equiv-alent to the doubling in cell number that occursby 2 h of inositol starvation in the intact cells(7). Inositol-starved whole cells do not increasein volume after they cease dividing, but macro-molecular synthesis and other metabolic proc-esses continue unabated for a period of timeduring which the cells increase in mass anddensity (7). Continued accumulation of cyto-plasmic metabolic products within a limited cellvolume may be expected to increase cytoplasmicosmotic pressure. In intact cells, this situationdoes not result in lysis, due to the presence ofthe cell wall. We propose that it is this rise incytoplasmic osmotic pressure, caused by cessa-tion of surface expansion without a coordinateslowing of cytoplasmic metabolism, that is thecause of lysis in the osmotically sensitive spher-oplasts.The treatments that interfere with sphero-

plast lysis lend support to this hypothesis. Cy-cloheximide treatment, according to this hy-pothesis, prevents the occurrence of an osmoticimbalance by interrupting further synthesis ofcytoplasmic components, whereas restoration ofinositol allows resumption of cell surface expan-sion. Elevation of the osmotic content of themedium shrinks spheroplasts (Fig. 9), whichmay be expected to reduce stress on the cellsurface from the increasing pressure within. Asintracellular metabolism continues in osmoti-cally rescued cells (Fig. 10-13), the osmotic pres-sure within the cell would be expected to con-tinue to increase, leading to delayed lysis whenthe same limiting cell volume (Fig. 9) is againfilled. The instantaneous nature of osmotic res-cue (Fig. 8), even well into the progress of lysis,as compared with only partial rescue by cyclo-heximide (Fig. 6) and the lag in effecting rescueby restoring inositol (Fig. 5), indicates that theosmotic treatment most rapidly alleviates thecondition responsible for lysis. This finding isconsistent with our interpretation of the natureof the problem and the mode of rescue: physicalstress on the cell surface is most rapidly relievedby physical (osmotic) support. Restoring thecellular balance of cytoplasm and surface growthby either stopping one or resuming the otherrequires some time.

In the case of whole cells, the intact cell wallis evidently sufficient physical support to with-stand the increased osmotic pressure indicatedby the spheroplast studies. Additional support,provided by growing whole cells in osmoticallyelevated medium (Fig. 14), does not significantly

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816 ATKINSON, KOLAT, ANI) HENRY

alter the rate of inositol-less death despite sub-stantial effects on the growth of inositol-supple-mented cells. This result indicates that osmoticrescue of spheroplasts is not due to "osmoticremediation" (5) restoring activity to the geneproduct of the inol-13 allele.

Several alternative theories as to causes oflysis, such as abnormal ion retention or progres-sive development of membrane fragility, are notsupported by the data. Potassium ion retentionis normal in inositol-starved whole cells for atleast 4 h (7), whereas spheroplasts made fromcells starved for only 2 h lyse instantly. Potas-sium ion retention in inositol-starved sphero-plasts is normal until lysis begins (Fig. 13). Nor-mal retention in osmotically supplementedspheroplasts argues that potassium ion loss doesnot occur when the osmotic imbalance begins.At the time of lysis, ions and other cytoplasmiccomponents are, of course, released, regardlessof the cause of lysis.

Likewise, lysis is not preceded by any detect-able development of an osmotically fragile state.In other studies, increased osmotic sensitivityof yeast spheroplasts has been associated withalterations in membrane lipid composition (1, 3,8, 9). Alterations of membrane composition maypossibly occur during inositol starvation. It has,in fact, been shown that synthesis of phospha-tidylinositol ceases within 30 min after removalof inositol, and concomitant increases in thesynthesis of the other major phospholipids occur(7). However, if these changes in lipid synthesisresult in membranes of abnormal composition,these alterations do not lead to a gradual in-crease in membrane fragility during the periodin which cell volume continues to expand. Onthe contrary, during this period, resistance toosmotic shock gradually develops in inositol-starved spheroplasts, exactly as in supplementedspheroplasts, most likely due to the elaborationof fibrillar surface materials as reviewed byNecas (14). Coincident with the cessation of cellvolume expansion, osmotic sensitivity of inosi-tol-starved spheroplasts develops abruptly. Theappearance of osmotic sensitivity after a periodof progressively developing osmotic resistancepresents a paradox that we believe is more ade-quately explained by the hypothesis of an im-balance between cytoplasmic and surface growththan by development of abnormal membranecomposition and integrity. Before the cessationof net surface expansion, coordinate growth ofcytoplasm and surface maintains a suitable bal-ance. The cell surface constructed during thisperiod posesses the same osmotic sensitivity orresistance as normal cell surfaces. In inositol-starved cells, net surface expansion ceases ab-

ruptly (7), at precisely the time that a changein osmotic sensitivity of inositol-starved spher-oplasts develops. We propose that the contin-uation of cellular metabolism beyond this pointthen creates an imbalance: the cytoplasm accu-mulates metabolic products more rapidly thansurface expansion is able to accommodate. Afterthe termination of net surface expansion, alter-ations in surface membranes may develop, al-though such membrane properties and func-tions, other than maintenance of potassium ionbalance, have not been examined in this study.Studies in progress involving the isolation ofplasma membranes should determine whethermembrane composition becomes abnormal dur-ing the course of inositol starvation.We believe that the data presented here and

in our previous report (7) are consistent with,and most readily explained by, the hypothesisoriginally proposed by Shatkin and Tatum (16)that inositol starvation results in an imbalancebetween the rate of membrane synthesis andthe synthesis of other cellular components. Inwhole cells, the cell wall apparently stops growthcoordinately with the plasma membrane, thuspreventing lysis. In both whole cells and spher-oplasts, the termination of surface membranegrowth is not coupled to an immediate slowingof general metabolism. It is this imbalance whichwe propose causes both the immediate lysis ofinositol-starved spheroplasts and the ultimatedeath of intact cells. It is not clear why inositol-starved cells die several hours after cell divisionceases, but it is likely that there are physiologicallimitations to the accumulation of metabolicproducts in a limited cytoplasmic volume.

ACKNOWLEDGMENTS

'ihis work was supported by Public Health Service grantsGM-19629 and GM-19i00 from the National Institute of Gen-eral Medical Sciences and grant 5 MOIl RR50 from the GeneralResearch Support Branch, Division of Research Resources.S.A.H. is supported by Public Health Service research careerdevelopment award GM-00024 from the National Institute ofGeneral Medical Sciences. K.D.A., a genetics trainee, wassupported by Public Health Service grant GM-00110 fromthe National Institute of General Medical Sciences.We thank Quentin Deming, Demetrios Kambosos, Alex-

andra Chanas, and Elizabeth Silverman for expert advice andassistance in performing the ion determinations. We alsothank Jonathan Warner and Richard Shulman for advice onspheroplast preparation.

LITERATURE CITED

1. Alterthum, F., and A. H. Rose. 1973. Osmotic lysis ofspheroplasts from Saccharomyces ceretcisiae grown an-aerobically in media containing different unsaturatedfatty acids. J. Gen. Microbiol. 77:371-382.

2. Culbertson, M. R., and S. A. Henry. 1975. Inositol-requiring mutanls of Saccharomyces cereuisiae. Ge-netics 80:23-40.

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3. Diamond, R. J., and A. H. Rose. 1970. Osmotic prop-erties of spheroplasts from Saccharomyces cerevisiaegrown at different temperatures. J. Bacteriol.102:311-319.

4. Hartwell, L. H., 1970. Periodic density fluctuation duringthe yeast cell cycle and the selection of synchronouscultures. J. Bacteriol. 104:1280-1285.

5. Hawthorne, D. C., and J. Friis. 1964. Osmotic-remedialmutants. A new classification for nutritional mutantsin yeast. Genetics 50:829-839.

6. Henry S. A. 1973. Death resulting from fatty acid star-vation in yeast. J. Bacteriol. 116:1293-1303.

7. Henry, S. A., K. D. Atkinson, A. I. Kolat, and M. R.Culbertson. 1977. Growth and metabolism of inositol-starved Saccharomyces cerevisiae. J. Bacteriol.130:472-484.

8. Hossack, J. A., and A. H. Rose. 1976. Fragility ofplasma membranes in Saccharomyces cerevisiae en-

riched with different sterols. J. Bacteriol. 127:67-75.9. Hossack, J. A., V. J. Sharpe, and A. H. Rose. 1977.

Stability of the plasma membrane in Saccharomycescerevisiae enriched with phosphatidylcholine or phos-phatidylethanolamine. J. Bacteriol. 129:1144-1147.

10. Hutchison, H. T., and L. H. Hartwell. 1967. Macro-molecule synthesis in yeast spheroplasts. J. Bacteriol.94:1697-1705.

11. Kuraishi, H., Y. Takamura, T. Mizunaga, and T.Uemura. 1971. Factors influencing death of biotin de-ficient yeast cells. J. Gen. Appl. Microbiol. 17:29-42.

12. Lowry, O., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

13. Matile, P. 1966. Inositol deficiency resulting in death: an

explanation of its occurrence in Neurospora*rassa.Science 151:86-88.

14. Necas, 0. 1971. Cell wall synthesis in yeast protoplasts.Bacteriol. Rev. 35:149-170.

15. Pontecorvo, G., J. A. Roger, L. M. Hemneous, K. D.Mac Donald, and A. W. J. Bufton. 1953. The geneticsof Aspeprgillus nidulans. Adv. Genet. 5:141-238.

16. Shatkin, A. J., and E. L. Tatum. 1961. The relationshipof m-inositol to morphology in Neurospora crassa. Am.J. Bot. 48:760-771.

17. Shimada, S., H. Kuraishi, and K. Aida. 1972. Unbal-anced growth and death of yeast due to pantothenatedeficiency. J. Gen. Appl. Microbiol. 18:383-397.

18. Strauss, B. S. 1958. Cell death and unbalanced growthin Neurospora. J. Gen. Microbiol. 18:658-669.

19. Thomas, P. L. 1972. Increased frequency of auxotrophicmutants of Ustilago hordei after combined UV irradia-tion and inositol starvation. Can. J. Genet. Cytol.14:785-788.

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osmotic imbalance in inositol-starved spheroplasts

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