Vol. 130, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 472-484 Copyright (C 1977 American Society for Microbiology

Growth and Metabolism of Inositol-Starved Saccharomyces cerevisiae SUSAN A. HENRY,* KATHARINE D. ATKINSON, ANITA I. KOLAT, AND MICHAEL R. CULBERTSON' Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461 Received for publication 5 January 1977

Upon starvation for inositol, a phospholipid precursor, an inositol-requiring mutant of Saccharomyces cerevisiae has been shown to die if all other conditions are growth supporting. The growth and metabolism of inositol-starved cells has been investigated in order to determine the physiological state leading to "inositolless death." The synthesis of the major inositol-containing phospholipid ceases within 30 min after the removal of inositol from the growth medium. The cells, however, continue to divide in an apparently normal fashion for one generation (2 h under the growth conditions used in this study). The cessation of cell division is not preceded or accompanied by any detectable change in the rate of macromolecular synthesis. When cell division ceases, the cells remain constant in volume, whereas macromolecular synthesis continues at first at an unchanged rate and eventually at a decreasing rate. Macromolecular synthesis terminates after about 4 h of inositol starvation, at approximately the time when the cells begin to die. Cell death is also accompanied by a decline in cellular potassium and adenosine triphosphate levels. The cells can be protected from inositolless death by several treatments that block cellular metabolism. It is concluded that inositol starvation results in an imbalance between the expansion of cell volume and the accumulation of cytoplasmic constituents. This imbalance is very likely the cause of inositolless death.

Inositol-requiring mutants or strains of a variety of fungi have been found to die rapidly when deprived of inositol under conditions that otherwise support growth (5, 24, 26). In contrast, most auxotrophic mutants of yeast and Neurospora, including amino acid, adenine and uracil requirers, stop growing when deprived of their respective requirements but lose viability comparatively slowly (11, 24). However, in addition to the inositol auxotrophs, biotin (13, 17), fatty acid (11), pantothenate (19), and deoxythymidine monophosphate (1)-requiring mutants of various fungi have all be reported to die when starved for their requirement. With the exception of the deoxythymidine monophosphate mutants, these auxotrophs all have defects relating to the synthesis of membrane lipids. In the yeast Saccharomyces cerevisiae both "inositolless death" and "fatty acid-less death" have been described (5, 11). In both fatty acidand inositol-starved yeast cells, electron microscopy has failed to reveal any change or damage to the cellular membranes (12; A. I Present address: Department of Genetics, Development, and Physiology, Cornell University, Ithaca, NY 14850.

472

Keith, personal communication). In addition, spin labeling of cells undergoing both inositolless and fatty acid-less death revealed no change in membrane fluidity (12; E. C. Pollard, W. Snipes, A. D. Keith, S. A. Henry, and M. R. Culbertson, Biophys. J., in press). The aqueous cytoplasm, however, increased substantially in viscosity during starvation for both compounds and in both cases the viscosity increase as well as cell death were prevented by treating the cells with cycloheximide (12; Pollard et al., in press). Shatkin and Tatum (20) originally studied Neurospora crassa hyphae deprived of inositol and ascribed growth abnormalities they observed to an imbalance between membrane synthesis and the synthesis of other cellular components. Matile (16), however, disagreed with this hypothesis, and he and others (25) have attributed inositolless death in Neurospora to disruption of the cellular membranes. Since the inositol-containing lipids are important membrane components, the failure to synthesize them in an actively metabolizing organism must inevitably lead either to membranes of abnormal composition or to a cessation of membrane growth or possibly to some combina-

VOL. 130, 1977

tion of both conditions. In the present report, we have studied the growth and metabolism of inositol-starved yeast cells to characterize the physiological state leading to inositolless death. (This work is taken in part from a dissertation to be submitted by K. D. Atkinson, in partial fulfillment of the requirements for the Ph.D. in the Sue Golding Graduate Division of the Albert Einstein College of Medicine, Bronx,

N.Y.) MATERIALS AND METHODS Yeast strains. Strain MC 13 inol-13 lys2 a, which has been previously described (4, 5), was used in all experiments. The strain grows as a haploid with virtually no "clumping." Inositol starvation medium. Medium was constructed using vitamin-free yeast nitrogen base (Difco; 6.7 g/liter) as previously described (4, 5). This basic medium, plus or minus myo-inositol (2 to 100 mg/liter), was used in all experiments described with the exception of the nitrogen and carbon source starvation experiments. Each liter of the medium used for glucose and nitrogen starvation experiments contained the following components: biotin (2 ,ug); calcium pantothenate (400 ,ug); folic acid (2 ,ug); niacin (400 ,g); paminobenzoic acid (200 ,ug); pyridoxine hydrochloride (400 ,ug); riboflavin (200 ,ug); thiamine hydrochloride (400 ;Lg); boric acid (500 ,ug); copper sulfate (40 ;Lg); potassium iodide (100 ,Lg); ferric chloride (200 jug); manganese sulfate (400 ,ug); sodium molybdate (200 ,ug); zinc sulfate (400 A.g); potassium phosphate monobasic (1 g); magnesium sulfate (0.5 g); sodium chloride (0.1 g); calcium chloride (0.1 g). In addition, since MC-13 is a lysine-requiring strain, 40 mg of L-lysine was added to each liter of medium. This basic medium was varied by the addition of glucose (20 g/liter) or ammonium sulfate (5 g/liter) depending upon whether glucose or nitrogen deprivation was required. The medium, when required, was also supplemented with myo-inositol (2 mg/ liter). Growth conditions for inositol starvation experiments. It will be shown that inositolless death is highly dependent upon the rate of metabolism of the starved cells. For this reason, all experiments described here, unless otherwise stated, were carried out under strictly comparable growth conditions, and the reproducibility of the timing of events described under these conditions has been confirmed by at least three repetitions of each experiment. In addition, at least two growth parameters were followed simultaneously in each experiment. For each inositol starvation experiment, cells were obtained from a clone recently tested for inositol auxotrophy and respiratory sufflciency. The clones were maintained on YEPD (yeast extract 1%, peptone 2%, glucose 2%, agar 2%) plates at 40C. These cells were inoculated at low density (less than 105/ml) into YEPD liquid medium (agar omitted) and allowed to grow at 250C on a shaker until they reached a density of 106 to 3 x 106 cells/ml. Cells were harvested from logarithmic-phase cultures since a high

INOSITOL STARVATION IN YEAST

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percentage of stationary inositol-requiring cells may be dead. The cells were centrifuged from YEPD medium and washed two times by centrifugation in the growth medium to be used for the experiment. The cells were then suspended in the appropriate growth medium at a density of 106 to 3 x 106 cells/ml and placed on a shaker at 30°C. Determination of cell number. Three methods were used for determination of cell number. (i) Cells were serially diluted into sterile distilled water and plated onto YEPD agar plates at an expected density of 75 to 200 colonies per plate. The plates were incubated for 2 days at 30°C and the number of colonies were counted. For each time point, 2 to 10 plates were counted. (ii) Cells were counted under the microscope on a hemacytometer. Ten standard squares were counted and the average number of cells was computed. Methylene blue staining as previously described (11) was used to distinguish living and dead cells. (iii) Cells which were grown in prefiltered growth medium were counted in a Coulter counter (model F) in Isoton (isotonic-buffered saline). Each sample was sonicated for 3 s before counting in a Branson cell disruptor. Counts were corrected for coincidence and three readings per sample were averaged. Cell volume. To obtain cell volume measurements, Coulter counter readings were taken at constant aperture and attenuation settings through a series of 10 threshold settings representing 20-kLm3 intervals. The volume of the cells was determined using the formula V = , where V = volume; K = constant for the apparatus; B = attenuation setting; T = threshold setting for a half count of particles; I = actual aperture current. To determine K, uniform 3.4-,Im-diameter latex beads (Coulter Electronics) were used. Microscopic examination of cells. Portions of culture were removed at intervals during inositol starvation and examined by phase and direct transmission light microscopy. Methylene blue stain (11) was used to determine the viability of individual cells. At 1-h intervals, 100 to 200 cells were examined and scored for viability as well as the presence or absence of a bud and the relative size of the bud to the mother cell. Dry weight. The cells were washed in prefiltered medium and suspended in filtered inositol-plus or inositol-minus medium. Duplicate 50-ml portions of each culture were collected at each time point on preweighed membrane filters (HAWP 02500; Millipore Corp.). The filters were washed twice with 10ml portions of filtered medium, dried overnight (15 to 17 h) in a 1000C drying oven, and weighed the next day. Density studies. Ludox density gradients were prepared according to the methods of Shulman et al. (21). Cells from liquid YEPD culture of 5.0 x 106 cells/ml were placed in inositol-minus or inositolplus medium for 4 h. Cells were then harvested by centrifugation and placed on separate Ludox gradients and centrifuged for 10 min at 19,000 rpm at

40C.

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[14C]glucose uptake. [14C]glucose (1 ,uCi/ml, Amersham-Searle) was added to the inositol-plus and inositol-minus cultures. Duplicate 0.5-ml samples were taken at each time point and collected on membrane filters (HAWP 02500, Millipore Corp.) and washed in 10 ml of media, dried, and then counted by scintillation counting. Macromolecular synthesis. Labeling of macromolecules was carried out according to the methods of Hartwell (8) who has demonstrated the specificity of labeling. Ribonucleic acid (RNA) synthesis was estimated by determination of total trichloracetic acid-precipitable counts in inositol-plus and inositolminus cultures containing 10 mg of cold carrier adenine per liter, labeled with 2 ,uCi of [14C]adenine per ml (54.2 mCi/mmol, Amersham-Searle). Samples of 0.1 ml from continuously labeled cultures were mixed with an equal volume of 20% (wt/vol) trichloroacetic acid and allowed to stand for 20 min on ice. Deoxyribonucleic acid (DNA) synthesis in the same culture was estimated by determining alkali-stable counts. Samples of 0.5 ml were mixed with an equal volume of hot 2 N NaOH and incubated at 60°C for 1 h. The alkali-treated samples were then mixed with an equal volume (1 ml) of cold 20% trichloroacetic acid and allowed to stand on ice for 20 min. Protein synthesis was estimated by determination of total trichloroacetic acid-precipitable counts from cultures containing 20 mg of cold carrier lysine per liter labeled with 0.1 ,tCi of [14C]lysine per liter (330 mCi/ mmol, Amersham-Searle). Samples of 0.5 ml from continuously labeled inositol-plus and inositol-minus cultures were mixed with an equal volume of 20% trichloroacetic acid and allowed to stand for 20 mm on ice. For pulse-labeling experiments, duplicate 0.5-ml samples were removed at intervals from inositolplus and inositol-minus cultures (containing cold carrier lysine and adenine as indicated above) and placed in tubes with 0.5 ,uCi of [14C]lysine or 0.5 ,uCi of ['4C]adenine. After 10 min of incubation at 30°C the samples were mixed with an equal volume of 20% trichloroacetic acid. The samples containing [14C]lysine were incubated at 100°C for 5 min and allowed to stand for an additional 15 min on ice before filtering. Samples containing ['4C]adenine were allowed to stand for 20 min on ice. RNA and protein synthesis, but not DNA synthesis, were estimated in pulse-labeling experiments. Samples were collected on membrane filters (HAWP 02500, Millipore Corp.) and washed with 20 volumes of cold 5% trichloroacetic acid. Radioactivity in the dried filters was determined by scintillation counting. Phospholipid synthesis. Lipids were pulse-labeled over 1.5-h time intervals in medium containing H332P04 (50 juCi/ml), total phosphate concentration 7.4 mM, and extracted by the method of Letters (15) as modified by Getz et al. (7). Two-dimensional thin-layer chromatography of 32P-labeled phospholipids was carried out according to the method of Getz et al. (7). Spots containing lipid were located by autoradiography and scraped from the thin-layer plates as previously described (4). Individual phospholipids were identified by comparison with au-

J. BACTERIOL.

thentic standards (General Biochemicals). The specific location of phosphatidylinositol was also confirmed by thin-layer chromatography of lipids extracted from yeast cultures labeled with [14C]inositol (215 mCi/mmol, New England Nuclear). ATP determinations. Samples of cultures at a cell concentration of 1.0 x 106 to 1.5 x 107/ml were sedimented by centrifugation and extracted according to the method reported by Scarborough (18). The ethanol extracts were centrifuged to remove debris, and a 1:10 dilution of the supernatant extract was prepared with arsenate buffer (0.5 M potassium arsenate, 0.02 M MgSO4, pH 7.4). Firefly lantern extract (purchased from Sigma Chemical Co. as a powder containing arsenate buffer ingredients) was reconstituted with water and debris removed by centrifugation at 10,000 rpm for 10 min. Adenosine triphosphate (ATP) standards were prepared by dilution of a 0.5 M ATP stock solution in arsenate buffer. Reaction mixtures were prepared which were suitable for luminescence monitoring in a Packard scintillation counter according to the methods of Ebadi et al. (6). The amount of ATP in experimental samples was determined by extrapolation from the standard curve graph. Cellular potassium ion determination. Portions of 200 ml of inositol-starved or -supplemented cultures were removed at intervals and the cells were harvested by centrifugation, washed twice in distilled water, and resuspended in water to a total volume of 2.0 ml. The sample was placed in a boiling water bath for 15 min and was then centrifuged to remove debris. The clear supernatant fluid was removed for potassium ion determination on a flame photometer (Instrumentation Laboratory Inc., model 143). Protection experiments. (i) Cells starved for inositol were shifted at intervals to inositol-minus medium containing physiological concentrations (3) (MgSO4); 5.5 mM Na+ (NaCl2); and 9.6 mM Ca2+ (CaCl2). (ii) Cycloheximide (100 ,tg/ml), as described previously (5), was added to inositol-starved cultures at intervals after the transfer of the cells to starvation medium. (iii) Cells starved for varying intervals of time in inositol-minus medium described above for nitrogen and carbon starvation experiments were shifted after two washings in deficient medium to medium lacking either glucose or ammonium sulfate as well as inositol. (iv) Cells starved for inositol in standard inositol-minus medium were shifted by centrifugation to distilled water after two washings in equal volumes of distilled water.

RESULTS Growth of the inositol-requiring strain. Yeast strain MC 13 inol lys2, a was found to require 5 mg of myo-inositol per liter of culture. At concentrations of inositol less than 5 mg/ liter, the strain grows initially at a rate of one doubling in 2 h, which is equivalent to its growth rate in the presence of 5 mg of inositol per liter (Fig. 1). However, at inositol concentrations below 5 mg/liter the strain fails to

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reach a stationary cell density equivalent to cultures containing at least 5 mg/liter. Furthermore, the cultures containing the low inositol concentrations were found to contain a large percentage of dead cells in stationary phase.

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475

The results of several experiments in which cell number was determined during the initial phases of growth in the total absence of inositol are shown in Fig. 1. For the first 2 h (one doubling of cell number) of inositol starvation, cells continue to increase in number precisely parallel to the inositol-supplemented control. At 2 h, increase in cell number abruptly ceases and cells enter a "plateau" phase in which there is no further increase in number of cells but the cells remain viable when plated onto YEPD agar medium. At 4 to 5 h cells begin to die as detected by staining with methylene blue and by plating efficiency on YEPD (Fig. 1A). However, the cells do not lyse as they die and light microscopy reveals no change in morphology. Cell counts by hemocytometer and Coulter counter remain constant throughout the period of cell death (Fig. 1B). Cells of all bud sizes were found to be distributed in the starved culture in approximately the same distribution as in the growing culture, suggesting that starved cells do not tend to collect in any given part of the cell division cycle. Figure 2 shows the distribution of cell sizes as measured by a Coulter counter in supplemented and starved cultures at 3 h. There was no significant difference in the distribution of cell sizes observed in starved and supplemented cultures at any time point and no change in either culture during the course of the experiment. Metabolism of inositol-starved cells. Incorporation of radioactive precursors into RNA, DNA, and protein continues for several hours after the cells have stopped dividing (Fig. 3 and 4). Compared with the supplemented control culture, there is no change in the rate of incorporation of [14C]lysine into trichloroacetic acidprecipitable material for the first 3 h of inositol starvation. Incorporation of lysine at a lower rate than the contol culture continues virtually until the time when the first dead cells are detected. Incorporation of [14C]adenine in alkali-stable material (DNA) similarly is unaf-

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.tidyl inositol synthesis ceases is due to incorporation into other phospholipid fractions, principally phosphatidyl serine, ethanolamine, and choline. 50% compared . with the dry mass of 15 to 18 mg/108 inositolsupplemented cells. Ludox gradients have been shown to separate yeast cells according to density of the individual cells (21). Cells with small buds are most dense and thus band lowest in the density gra_ . dient. Cells are least dense close to the time of cell separation and thus "doublets," cells having very large buds, band highest in the gradient. A logarithmic-phase culture, when cen-

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0 i 2 3 4 5 6 7 8 9 10 HOURS FIG. 3. Continuous labeling of macromolecules in inositol-minus (0) and -plus cultures (@). Each data point represents total counts accumulated by cells in 0.5 ml of culture. (A) Incorporation of ['4C]lysine into trichloroacetic acid-precipitable material. (B) Incorporation of ["4C]adenine into total trichloroacetic acid-precipitable material. (C) Incorporation of ['4C]adenine into alkali-stable trichloroacetic acidprecipitable material.

fected for 3 h. Total incorporation of [14C]adenine into acid-precipitable material, which is primarily a measure of RNA synthesis (8), is comparable to the control until 2.5 h and continues at a reduced rate until close to the time when the first dead cells are detected. The incorporation of 32p into phosphatidyl inositol in the starved culture is only 20% of the supplemented control during the first 30 min. At subsequent time points no radioactivity above background could be detected in phosphatidyl inositol in the thin-layer chromatograms (Fig. 5). The rate of incorporation of 32p into total lipids culture is compatheinsthe starved edculture culture coma rabl e totli to the supplemented for approxi-

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starting at 3 h). (A) Incorporation of ["C]lysine into trichloroacetic acid-precipitable counts. (B) Incorporation of [14C]adenine into trichloroacetic acid-precipitable counts.

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FIG. 5. Autoradiograms of thin-layer chromatograms of 32P-labeled lipids extracted from 25 ml of inositolsupplemented and -starved cultures (starting cell density, 2 x 106 cellslml). (A) Lipids extracted from an inositol-supplemented culture labeled for 4 h continuously with 32p; positions of four major phospholipids indicated by numbers: phosphatidyl inositol (1), phosphatidyl serine (2), phosphatidyl choline (3), and phosphatidyl ethanolamine (4). (This chromatogram is included for reference purposes.) (B) Lipids extracted from 50 ml ofan inositol-plus culture labeled with 32P during the interval from 0 to 30 min after transfer to the medium. Numbers refer to same lipids as above. (Labeling in phosphatidyl choline was reduced due to a short labeling period.) (C) Lipids extracted from 50 ml of an inositol-minus culture labeled with 32p during the interval from 0 to 30 min after transfer to inositol-minus medium. (Labeling in phosphatidyl inositol was much reduced compared with [B].) (D) Lipids extracted from 50 ml of inositol-minus culture labeled with 32p during the interval from 1 to 1.5 h. (Phosphatidyl inositol spot was virtually absent.)

trifuged on a Ludox gradient, forms two rather broad bands containing cells of different densities determined by their position in the cell cycle. Since it has been demonstrated that the inositol-starved cells do not increase in volume, the increase in mass must be retained in the existing cell volume, and should yield cells of increased density. Control cells from an inositol-supplemented culture at 4 h show the expected two bands in a Ludox gradient (Fig. 8). In contrast, cells in all stages of cell division from a culture at 4 h of inositol starvation band exclusively at the bottom of an identical gra-

dient, with all cells at or below the level of the more dense band observed in the supplemented culture. The inositol-starved cells thus have a density as great as or greater than the most dense cells in the growing culture. Cellular potassium ion concentration was found to increase in parallel fashion to cell number in both supplemented and starved cultures (Fig. 9). At 2 h, when the starved culture stops dividing, potassium ion concentration levels off and remains constant throughout the plateau period. Since cell volume is not increasing, the potassium ion concentration in the cy-

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HOURS OF INOSITOL STARVATION FIG. 6. Incorporation of 32p into total lipids of inositol-starved (0) and inositol-supplemented (0) cultures. Each point represents counts in lipid extracted from 5 ml of culture (starting cell density, 2 x 106 cellslml) which had been labeled with 32P over the preceding half-hour. (The 1-h data point, for example, represents labeling during the 30-min interval ending at 1 h.) Labeling, growth, and extraction procedures were as detailed in the text.

toplasm of these cells presumably remains constant during the plateau. When the cells die, potassium is lost at a rate comparable to the loss of viability. Thus, the amount of potassium per viable cell remains constant throughout the experiment. ATP levels in the inositol-starved culture were examined (Fig. 9) and found to increase at a rate comparable to the supplemented culture virtually until the time at which cells start to die. At that time, the level of ATP in the starved culture begins to fall at a rate comparable to the rate of cell death. The timing of the metabolic events described above relative to the cessation of cell division and the start of cell death is illustrated in Table 1. All the metabolic effects reported, with the exception of cessation of phosphatidyl inositol synthesis, follow the termination of cell division. During the early part of the plateau phase, macromolecular synthesis is normal, and its gradual slowing during the latter part of the plateau is not preceded or accompanied by potassium or ATP loss. Rather, the decline in levels of these molecules occurs at the end of the plateau, coincident with cell death. Protection experiments. An attempt was made to replace exogenously some of the ions that might be expected to leak from the cells at the time when potassium loss is observed. How-

ever, the exogenous administration of physiological concentrations of the ions K+, Mg2+, Ca2+, and Na+ had no effect upon the rate or extent of cell death. Interruption of protein synthesis in inositolstarved cells by the administration of the antibiotic cycloheximide, however, does substantially prevent cell death. Cycloheximide must be added at 1 h of starvation or before in order to obtain maximum protection (Fig. 10). Limited protection can be obtained by addition of cycloheximide for the next 45 min. Thus, to have any protective effect at all, cycloheximide must be added to the culture before the time when cells stops dividing. Cell death therefore, is either directly dependent upon protein synthesis, or cycloheximide the cells withindioninterference through a pcs generalprotects rectly going metabolic going metabolic processes.

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1 2 3 4 5 6 HOURS OF INOSITOL STARVATION FIG. 7. (A) Incorporation of [14C]glucose into whole cells in inositol-starved (0) and -supplemented cultures (0). Data points represent total accumulation of counts by cells from 0.5 ml of culture. (Starting culture density, 2 x 106 cellslml). (B) Dry mass of cells harvested from inositol-starved (0) and -supplemented cultures (0). Data represents the mass of cells harvested from 50 ml of culture (starting culture density, 3 x 106 cellslml).

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FIG. 8. Identical Ludox gradients containing cells taken from an inositol-minus culture (left) at 4 h and an inositol-plus culture (right) at 4 h after transfer to the medium.

In order to test the effects of generalized altered cellular metabolism on inositolless death, cells were shifted during the course of inositol starvation to distilled water. Yeast cells retain viability for extended periods of time in water as illustrated in Fig. 11. A shift to distilled water exerts a substantial protective effect even at 3 or 4 h of inositol starvation when the cells have already stopped dividing, considerably after the time when cycloheximide has an effect. Some protection of the remaining cells occurs even when cells are shifted to water as late as 7 h of starvation. Since the shift to distilled water involves simultaneous deprivation for a long list of metab-

olites, an attempt was made to dissect the components of this protection. The effects of starvation for nitrogen and carbon source were tested individually as described in Materials and Methods. In the medium specially formulated for use in these experiments, the cells had a generation time of 4 h and the timing and extent of cell death was altered (Fig. 12) compared with the experiments reported above. Cells starved for inositol but supplied with glucose and ammonium sulfate continued to divide for one generation (4 h) and started to die after 6 h (Fig. 12). The cells must be shifted at the start of the experiment to be totally protected by nitrogen starvation and intermediate protec-

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when the cells stop dividing. The plateau phase of inositol starvation following the termination of cell division is thus characterized by ongoing accumulation of cytoplasmic constituents, including macromolecules, in the absence of cell surface growth. \The lack of coupling between cell surface growth and ongoing macromolecular synthesis observed in inositol-starved cells during the plateau phase is in keeping with the model of the cell division cycle of yeast proposed by Hartwell et al. (9). The Hartwell model describes two independent "pathways" in the cell division cycle, one controlling DNA synthesis and the other controlling bud emergence. It seems likely that inositol starvation, which blocks further expansion of cell volume, involves the pathway controlling bud emergence and growth. Because of the independence of the cell division cycle pathways, a block in cell surface growth such as that caused by inositol starvation would not, according to the Hartwell model, be expected to lead to an immediate cessation of macromolecular synthesis. The imbalance in the inositol-starved cells can thus be viewed in terms of the failure to make a component needed in one independent cell division

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tion is obtained for up to 4 h of inositol starvation. Since 4 h corresponds to the start of the "plateau" in this experiment, the protection is quite comparable to the protection obtained with cycloheximide if allowance is made for the difference in the timing of events. A shift of the cells to medium deficient in carbon source, on the other hand, is totally effective for up to 4 h when the cells stop dividing and has an intermediate effect in the plateau phase (Fig. 12). This is comparable to the effect seen by shifting the cells to distilled water. DISCUSSION The termination of cell division after approximately one generation in inositol-starved cells is neither preceded nor accompanied by any change in the rate of metabolic processes, including macromolecular synthesis (Table 1). Since neither the morphology nor the volume of inositol-starved cells is detectably altered, the cell surface area presumably stops increasing

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TABLE 1. Time table of events in inositol starvation Hours of inositol starvation

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cycle pathway but not the other. Since inositol-

107

starved cells have no tendency to collect in one part of the cell cycle, they are, presumably, unable to make a component needed at all times during the cell cycle in the pathway con1016 ou -0 trolling growth of the cell surface. It is notewor-3 thy that many of the temperature-sensitive cell cycle mutants isolated by Hartwell and his col4 laborators which have the effect of blocking one or the other independent pathway also lead to 105_ X\) -5 death at the restrictive temperature (8, 10). __j The experiments involving cycloheximide \\ U suggest that inositolless death is dependent di\ \rectly or indirectly upon ongoing protein synco ~ \ \thesis. Both cycloheximide and nitrogen star104 vation, which have their most immediate ef\ \ > fects on macromolecular synthesis, protect the x -7 inositol-starved cells only when added during the period of active cell division (Table 1) and \ 103have no effect during the plateau phase. However, more effective and immediate protection of inositol-starved cells results from treatments such as glucose starvation or shift to distilled l water, which deprive the cells of an energy I I 102 source. Therefore, overall cellular metabolism 24 16 20 4 8 12 rather than macromolecular synthesis alone is HOURS OF INOSITOL STARVATION FIG. 11. Efect of transfr ofinositol-starved cells probably involved in destruction of the cells. The loss of potassium coincident with cell to sterile distilled water. Cells were transferred at the following hours of inositol starvation: 0 h (0); 1 h death suggests failure of membrane selective (0); 2 h (A); 3 h (A); 4 h (A); 5 h (V); 6 h (A); 7 h (x); permeability. It is not clear, however, whether never transferred to distilled water (0) but main- potassium loss that occurs coincident with the decline in ATP levels results from an actual tained continuously in inositol-minus medium.

e2

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A 106

105

104 LIE u.

103

u U

4

8

12

16

20

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HOURS OF INOSITOL STARVATION FIG. 12. Effect ofglucose and nitrogen starvation on inositol-starved cells. Cells were grown in the special medium designed for glucose and nitrogen starvation experiments detailed in the text. At intervals cells were transferred from the special medium containing both glucose and ammonium sulfate but lacking inositol to medium lacking either (A) glucose or (B) ammonium sulfate as well as inositol. The cells were transferred the following times of inositol starvation: 0 h (0); 1 h (0); 2 h (A); 3 h (-); 4 h (A); 5 h (V); 6 h (V); cells never transferred but maintained continuously in inositol starvation medium (0) containing both glucose and ammonium sulfate.

structural defect in the membranes or from the total failure of cellular metabolism and all energy-dependent processes. A model for the loss of selective permeability resulting in cell death exists in the case of the antibiotic nystatin which causes leakage of ions from the cytoplasm. The effects of nystatin are reportedly

mitigated by exogenous replacement of potassium (2). Exogenous replacement of potassium and several other ions had no effect upon inositol-starved cells, but this could simply be because the precise concentrations were inadequate or because there are many other molecules involved which were not replaced. Other polyene antibiotics, such as Filipin, which have a more disruptive effect upon the membranes than nystatin, cannot be counteracted by administration of exogenous potassium (2). Fatty acid starvation in yeast leads to a condition that in every way resembles inositol starvation. The fatty acid-starved cells stop dividing before any change is detected in macromolecular synthesis. The kinetics of death for fatty acid-starved cells are remarkably similar to those reported here for inositol-starved cells (11). At the time of cell death, a rise in cytoplasmic viscosity is detected in both fatty acid- and inositol-starved cells (12; Pollard et al., Biophys. J., in press). In addition, fatty acid-starved cells are protected by cycloheximide (11). Since fatty acid and inositol are both phospholipid precursors, it seems likely that the phenomena associated with inositol and fatty acid starvation are the result of failure to synthesize one or more of the phospholipids. Inositol-containing lipids in yeast include phosphatidyl inositol, diphosphoinositide, triphosphoinositide, and ceremide (inositol)2 (phosphate)2 mannose (14, 22, 23) which are found in the cell membranes and possibly also in the cell wall (22). In Neurospora, disintegration of the cellular membranes due to abnormalities of membrane composition has been invoked as the cause of inositolless death (16, 25). In yeast, no morphological changes have been observed in inositoldeprived cells, at the level of light microscopy or of electron microscopy (A. Keith, personal communication). The fluidity of the membrane hydrocarbon zones as measured by electron spin resonance is also unchanged during inositol starvation in yeast (Pollard et al., Biophys. J., in press). Thus, in yeast no evidence has yet been obtained suggestive of gross membrane disintegration during inositol starvation. Undetected structural damage to the membranes or compositional changes, of course, cannot be ruled out. The composition of the membranes of inositol-starved cells could become abnormal by continued assembly of lipid and/or protein components into the membrane in the absence of inositol-containing lipids. However, fatty aciddeprived cells which are unable to synthesize any of the phospholipids die as rapidly (11) as inositol-deprived yeast cells. Thus, it seems un-

VOL. 130, 1977

likely that an abnormal lipid composition of the membranes is solely to blame for cell death. If the cells were destroyed due to continued assembly of protein components into membrane in the absence of certain lipid components, it seems likely that cycloheximide would have a more immediate effect in preserving the cells. However, it is possible that a considerable lag occurs between the synthesis of membrane components and their assembly into membrane. The question of whether or not membrane composition becomes abnormal during inositol starvation can only be answered by isolation and analysis of the membrane fractions of the inositol-starved cells. These experiments are in progress. The hypothesis of Shatkin and Tatum (20), that inositol starvation leads to an imbalance between the rate of membrane growth and the synthesis of cytoplasmic constituents, is a plausible alternative to the hypothesis of membrane disintegration. We have reported here that an imbalance between the rate of ongoing metabolism and the rate of cell surface growth occurs in inositol-starved yeast cells. The method used here for measuring cell volume, of course, measures the volume enclosed by the most external structure, the cell wall. A subsequent report will deal with experiments on spheroplasts of the inositol mutants of yeast which have provided evidence that inositol starvation directly affects growth of the plasma membrane, in a manner consistent with the Shatkin and Tatum hypothesis. Based on these considerations we propose the following working model of inositolless death which we acknowledge as a restatement of Shatkin and Tatum's original hypothesis: inositol-less death rather than being the result of disintegration of the cellular membranes results from the failure of the plasma membrane and the cell wall to grow. Because of the independence of various sequences of events during the yeast cell division cycle, the failure of membrane growth is not recognized by the cell or coupled with a slowing of accumulation of cytoplasmic constituents. Eventually, synthetic processes do come to a halt because of the increasing cytoplasmic concentration of metabolic products. At this stage, the condition of the cytoplasm becomes incompatible with ongoing metabolism and the process becomes irreversible. This model makes a number of testable predictions concerning membrane growth in inositol-deprived cells. We believe it is consistent with all of the observations made concerning the growth and metabolism of inositoldeprived cells.

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ACKNOWLEDGMENTS This work was supported by Public Health Service grants GM19629 and GM19100 from the National Institute of General Medical Sciences and 5MOlRR50. S. A. Henry is supported by Public Health Service research career development award GM00024. K. D. Atkinson, a genetics trainee, was supported by Public Health Service grant GM00110, from the National Institute of General Medical Sciences. We wish to thank Quentin Deming, Demetrios Kambosos, Alexandra Chanas, and Elizabeth Silverman for expert advice and assistance in performing the ion determinations. LITERATURE CITED 1. Brendel, M., and U. G. Langjahr. 1974. "Thymineless death" in a strain of Saccharomyces cerevisiae auxotrophic for deoxythymidine-5-monophosphate. Mol. Gen. Genet. 131:351-358. 2. Cirillo, V. P., M. Harsch, and J. 0. Lampen. 1964. Action of the polyene antibiotics filipin, nystatin and N-acetylcandidin on the yeast cell membrane. J. Gen. Microbiol. 35:249-259. 3. Conway, E. J., and W. McD. Armstrong. 1961. The total intracellular concentration of solutes in yeast and other plant cells and the distensibility of the plant-cell wall. Biochem. J. 81:631-639. 4. Culbertson, M. R., T. F. Donahue, and S. A. Henry. 1976. Control of inositol biosynthesis in Saccharomyces cerevisiae: inositol-phosphate synthetase mutants. J. Bacteriol. 126:243-250. 5. Culbertson, M. R., and S. A. Henry. 1975. Inositol requiring mutants of Saccharomyces cerevisiae. Genetics 80:23-40. 6. Ebadi, M. S., B. Weiss, and E. Costa. 1971. Microassay of adenosine-3',5'-monophosphate (cyclic AMP) in brain and other tissues by the luciferin-luciferase system. J. Neurochem. 18:183-192. 7. Getz, G. S., S. Jakovcic, J. Heywood, J. Frank, and M. Rabinowitz. 1970. A two dimensional thin layer chromatographic system for phospholipid separation: the analysis of yeast phospholipids. Biochim. Biophys. Acta 218:441-452. 8. Hartwell, L. H. 1967. Macromolecular synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 93:1662-1670. 9. Hartwell, L. H., J. Culotti, J. R. Pringle, and B. J. Reid. 1974. Genetic control of the cell division cycle in yeast. Science 183:46-51. 10. Hartwell, L. H., J. Culotti, and B. J. Reid. 1970. Genetic control of the cell-division cycle in yeast. I. Detection of mutants. Proc. Natl. Acad. of Sci. U.S.A. 66:352-359. 11. Henry, S. A. 1973. Death resulting from fatty acid starvation in yeast. J. Bacteriol. 116:1293-1303. 12. Henry, S. A., A. D. Keith, and W. Snipes. 1976. Changes in the restriction of molecular rotational diffusion of water soluble spin labels during fatty acid starvation of yeast. Biophys. J. 16:641-653. 13. Kuraishi, H., Y. Takamura, T. Mizunaga, and T. Uemura. 1971. Factors influencing death of biotin deficient yeast cells. Gen. Appl. Microbiol. 17:29-42. 14. Lester, R. L., and M. R. Steiner. 1968. The occurrence of diphosphoinositide and triphosphoinositide in Saccharomyces cerevisiae. J. Biol. Chem. 243:4889-4893. 15. Letters, R. 1966. Phospholipids of yeast. II. Extraction, isolation, and characterization of yeast phospholipids. Biochim. Biophys. Acta 116:489-499. 16. Matile, P. 1966. Inositol deficiency resulting in death: an explanation of its occurrence in Neurospora crassa. Science 151:86. 17. Pontecorvo, G., J. A. Roger, L. M. Hemneous, K. D. MacDonald, and A. W. J. Bufton. 1953. The genetics

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of Aspergillus nidulans. Adv. Genet. 5:141. 18. Scarborough, G. 1971. Sugar Transport in Neurospora crassa. III. An inositol requirement for the function of the glucose active transport system. Biochem. Biophys. Res. Commun. 43:968-975. 19. Shimada, S., H. Kuraishi, and K. Aida. 1972. Unbalanced growth and death of yeast due to pantothenate deficiency. J. Gen. Appl. Microbiol. 18:383-397. 20. Shatkin, A. J., and E. L. Tatum. 1961. The relationship of M-inositol to morphology in Neurospora crassa. Am. J. Bot. 48:760-771. 21. Shulman, R. W., L. H. Hartwell, and J. R. Warner. 1973. Synthesis of ribosomal proteins during the yeast cell cycle. J. Mol. Biol. 83:513-525. 22. Steiner, S., and R. L. Lester. 1972. Studies on the diversity of inositol containing yeast phospholipids.

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Incorporation of 2-deoxyglucose into lipid. J. Bacteriol. 109:81-88. Steiner, S., S. Smith, C. J. Waechter, and R. L. Lester. 1969. Isolation and partial purificaton of a major inositol containing lipid in baker's yeast, mannosyl-diinositol, diphosphoryl ceremide. Proc. Natl. Acad. Sci. U.S.A. 64:1042-1048. Strauss, B. S. 1958. Cell death and unbalanced growth in Neurospora. J. Gen. Microbiol. 18:658-669. Sullivan, J. L., and A. G. Debusk. 1973. Inositolless death in Neurospora and cellular ageing. Nature (London) New Biol. 243:72-74. Thomas, P. L. 1972. Increased frequency of auxotrophic mutants of Ustilago hordei after combined UV irradiation and inositol starvation. Can. J. Genet. Cytol. 14:785-788.