Inhibitory Effect of 2-Deoxy-D-Glucose on the Formation of the Cell Wall in

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1 JOURNAL OF BACTERIOLOGY, May 1969, p Copyright 1969 American Society for Microbiology Vol. 98, No. 2 Printed in U.S.A. Inhibitory Effect of 2-Deoxy-D-Glucose on the Formation of the Cell Wall in Yeast Protoplasts V. FARKA9, A. SVOBODA,1 AND S. BAUER Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Czechoslovakia Received for publication 2 December Deoxy-D-glucose (2DG) acted as a competitive inhibitor of the synthesis of cell wall components in Saccharomyces cerevisiae protoplasts. The synthesis of fibrillar glucan cell wall component was inhibited at a glucose to 2DG ratio of 4:1 in the cultivating medium. The completion of the formation of cell wall by the synthesis of the amorphous mannan-protein cell wall component was inhibited at a glucose to 2DG ratio of about 20:1. The inhibition could be reversed by increasing the glucose to 2DG ratio in the nutrient medium. No incorporation of 2DG into fibrillar glucan cell wall component was observed. 2-Deoxy-D-glucose (2DG) is known as a strong inhibitor of metabolism and growth in cells of diverse origin (1, 3, 5, 14). With yeast cells, 2DG seems to interfere primarily with the processes connected with cell wall synthesis (5, 6, 8). Yeast protoplasts are a very suitable model system for the study of the inhibitory effect of 2DG on the formation of cell wall components. The protoplasts may regenerate into new yeast cells, forming a complete cell wall de novo when cultivated in a medium with a high concentration of gelatin (9). When cultivating the protoplasts in a liquid, osmotically stabilized medium containing glucose, a fibrillar network composed mainly of glucan (Svoboda and Ne6as, International Symposium on Yeast Protoplasts, 2nd, Brno, 1968) is formed on their surface (2, 10). The process of cell wall regeneration may be investigated by means of phase-contrast and electron microscopy. The results presented in this paper give experimental evidence of the inhibitory effect of 2DG on the synthesis of yeast cell wall components in regenerating yeast protoplasts. MATERIALS AND METHODS Saccharomyces cerevisiae strain C-7, obtained from VULKP Prague, was grown in the wort medium, 12 Balling, at ph 5.4. The protoplasts were prepared by treating log-phase cells with snail gut juice from Helix pomatia in a nonbuffered medium containing 1 M MgSO4 as an osmotic stabilizer (4). Cultivation of the protoplasts in liquid medium. The naked protoplasts were washed twice with 1 M MgSO4 I Present adress: Department of Biology, Faculty of Medicine, Purkyne University, Brno, Czechoslovakia. and were cultivated in liquid synthetic media containing 0.1 to 10% (w/v) glucose and 2DG in various ratios to glucose in Mclllvaine citrate-phosphate buffer (ph 5.3) osmotically stabilized with 0.7 M mannitol. In some experiments, glucose was replaced by mannose. The samples were incubated at 28 C for 2.5 to 16 hr. The protoplasts were then collected by centrifugation, lysed by dilution with distilled water, washed three times with distilled water, and transferred on Formvar-coated grids for electron microscopy. Cultivation of the protoplasts in 30% gelatin. Regeneration of cell wall by yeast protoplasts as a function of the ratio of glucose to 2DG in the medium was investigated. Gelatin media were prepared by adding gelatin (Difco) to the appropriate liquid nutrient medium. After being washed with 1 M MgSO4, the protoplasts were embedded in the gelatin media and cultivated at 22 C for 16 to 24 hr. The gelatin was then dissolved in water at 37 C, and liberated "cells" were prepared for electron microscopy as described above. The specimens were shadowed with platinum and were examined with a Tesla table electron microscope, type BS 242 A. RESULTS As was already mentioned, during cultivation of yeast protoplasts in a liquid, osmotically stabilized medium containing glucose, a fibrillar glucan network was formed on their surface (Fig. 1). When 2DG, in addition to glucose, was present in the cultivating medium, considerably less fibrillar glucan was synthesized. The extent of this inhibition apparently depended only on the glucose to 2DG ratio in the nutrient medium. The inhibitory effect of 2DG on the synthesis of the fibrillar glucan network was observed at a glucose 744

2 VOL. 98, DEOXYGLUCOSE IN YEAST PROTOPLASTS 745 /f - : ~~3 FIG. 1. Isolated fibrillar network formed on the surface ofprotoplasts cultivatedfor 4 hr in liquid medium with 0.5S glucose. Bundles ofelementary fibrils can be seen both on the original protoplast and on the hernia. X 8,000. FIG. 2. Isolated surface structures from the protoplasts cultivatedfor 16 hr in the presence of 2% glucose plus 0.5S% 2DG (4:1) in liquid medium. Note the reduced amount and density of the fibrillar network. X 10,000. FIG. 3. Osmotic lysis of protoplast after 16 hr of cultivation in liquid medium in the presence of 1% glucose plus 0.5% 2DG (2: 1). No rigid cell wall components are observed; only remnants of cytoplasm andfragments of plasma membrane are apparent. X 10,000.

3 FARKAS, SVOBODA, AND BAUER 746 J. BACTERIOL. to 2DG ratio of 4:1 in the nutrient medium. The fibrils formed on the surface of protoplasts cultivated in such a medium were less dense and less extensive than those formed in control experiments (Fig. 2). When the glucose to 2DG ratio was 2:1 or lower, the synthesis of the fibrillar network was totally blocked (Fig. 3). Moreover, the protoplasts did not grow in such a medium, but survived more than 24 hr. By transferring the protoplasts cultivated in the medium containing 2DG into medium containing glucose only, or simply by increasing the glucose to 2DG ratio in the cultivating medium to greater than 4:1, the synthesis of the fibrillar network was renewed completely, even after 24 hr of cultivation of the protoplasts in the medium with 2DG. No 2D0 could be detected, either chromatographically (13) or by the thiobarbituric acid assay (12) of the hydrolysate of the fibrils formed on the surface of the protoplasts cultivated in liquid media containing 2DG in addition to glucose or mannose. Complete regeneration of cell walls by the protoplasts could be achieved by cultivating them in the presence of glucose in highly concentrated gelatin (9). Smooth-surfaced membranes, with an inner structure very similar to that of the normal cell wall, were formed on the surface of the protoplasts (Fig. 4). When the gelatin media contained 2DG as well as glucose, regeneration of normal cell wall was inhibited at a glucose to 2DG ratio of about 20:1. The membranes formed on the surface of the protoplasts cultivated in such a medium were of variable thickness and had an apparently fibrillar texture. Amorphous mannanprotein material, which masks the fibrillar glucan groundwork in the normal cell wall, was formed incompletely (Fig. 5). However, regenerated "cells" remained osmotically sensitive. The formation of the mannan-protein matrix material was almost completely blocked at a glucose to 2DG ratio of 4:1. Only a very fine fibrillar network was formed on the surface of the protoplasts in this case (Fig. 6). At a glucose to 2DG ratio lower than 2:1, the formation of the cell wall was completely blocked, with no fibrillar network being formed. No signs of the forming cell wall could be observed on the surface of the protoplasts. When glucose was replaced by mannose in similar experiments, the results, from a morphological point of view, were the same. Addition of mannose to the media containing glucose and 2DG gave results corresponding to a final ratio of the concentrations of glucose plus mannose to 2DG. DISCUSSION Comparison of the effect of 2DG on yeast growth with that on fermentation showed that 2DG was a much more potent inhibitor of yeast growth than of yeast fermentation (5, 14). The selective killing of growing cells by 2DG (6, 8) indicates that 2DG or its metabolites primarily attack the processes involved in cell wall synthesis. The action of 2DG has been found to cause both lysis of yeast cells at the sites of extensive cell wall synthesis (6, 9) and dissolution of cell wall glucans (7). The results presented here give experimental evidence for the inhibitory effect of 2DG on the synthesis of cell wall components. However, the mechanism of this inhibition remains unexplained. According to Heredia et al. (5), trapping of uridine nucleotides by phosphorylated derivatives of 2DG seems to be the main cause of inhibition of cell wall synthesis, whereas Johnson (6) considered it to be the result of the greater accumulation of uridine diphosphate-2dg which might interfere in the synthesis of cell wall glucans. The inhibition of glucan and mannan-protein synthesis by 2DG which we observed suggests that the final effect of 2DG on cell wall synthesis may be a cumulative result of several individual effects at different steps of glucose utilization. The fact that the inhibitory effect of 2DG on cell wall synthesis did not depend on its absolute concentration, but only on the glucose to 2DG ratio in the cultivating medium, indicates the competitive nature of this inhibition. From the protoplast cultivation in 30% gelatin, it seems that 2DG inhibits the synthesis of the mannan-protein cell wall component to a greater extent than the glucan synthesis or interferes in some way with its binding to the glucan fibrillar groundwork. The resulting inhibition of cell wall completion appears to be more effective than the inhibition of formation of glucan fibrils only. Blocking of cell wall completion by 2DG is probably one of the reasons why the protoplasts did not regenerate in 30% gelatin. It is known from earlier studies (11) that formation of a complete new cell wall on the surface of the protoplasts is one of the decisive factors for the restoration of morphogenesis. The fact that the protoplasts failed to grow and did not lyse spontaneously, but remained viable for a relatively long period of time in the cultivating medium containing 2DG, indicates that the inhibitory effect of 2DG or its metabolites affects the cell metabolism as a whole. Moreover,

4 *:::.. e * *....*. : :: :.: :: :.::.:.S.::t.: z. :ss79us 5 r4 6 FIG. 4. Newly formed cell walls on the surface of protoplasts after 24 hr of cultivation in 30% gelatin with 0.5% glucose. The cell walls were isolated after mechanically destroying the regenerating protoplasts. They roughly correspond to those of normal yeast cells. X 10,000. FIG. 5. Isolated cell walls from growing protoplasts after 24 hr of cultivation in 30% gelatin with 10% glucose plus 0.5% 2DG. (20:1). Note the reduced density and the differences on the surface of the cell walls. X 10,000. FIG. 6. Surface structures from protoplasts after 24 hr of cultivation in 30% gelatin containing 2% glucose plus 0.5% 2DG (4:1). Note the fibrillar character of these thin membranes. X 8,

5 748 FARKAS, SVOBODA, AND BAUER J. BACTERIOL. with regard to reversibility of the inhibition, it seems that 2DG causes no serious damage to metabolic processes occurring in the cell. LITERATURE CITED 1. Barban, S., and H. 0. Schulze Effect of 2-deoxyglucose on the growth and metabolism of cultured human cells. J. Biol. Chem. 236: Eddy, A. A., and D. H. Williamson Formation of aberrant cell walls and of spores by the growing yeast protoplasts. Nature 183: Ely, J. O., F. A. Tull, and J. A. Hard Influence of 2-deoxy-D-glucose on the growth of embryonic chickenheart fibroplasts in tissue culture. J. Franklin Inst. 253: Gascon, S., and J. R. Villanueva Magnesium sulphate as a stabilizer during liberation of yeast and mold protoplasts. Nature 205: Heredia, C. F., G. de la Fuente, and A. Sols Metabolic studies with 2-deoxyhexoses. L. Mechanism of inhibition of growth and fermentation in baker's yeast. Biochim. Biophys. Acta 86: Johnson, B. F Lysis of yeast ceu walls induced by 2-deoxyglucose at their sites of glucan synthesis. J. Bacteriol. 95: Johnson, B. F Dissolution of yeast glucans induced by 2-deoxyglucose. Exp. Cell Res. 50: Megnet, R Effect of 2-deoxyglucose on Schizosaccharomyces pombe. J. Bacteriol. 90: NeZas, Physical condtions as important factors for the regeneration of yeast protoplasts. Nature 192: Necas, Mechanism of regeneration of yeast protoplasts. II. Formation of the cell wall de novo. Folia Biol. (Praha) 11: t. Necas, O., and A. Svoboda Relation between the biosynthesis of the cell wall and regeneration in yeast protoplasts, p In R. MUller (ed.), Symposium Uber Hefeprotoplasten, Jena, Akademie Verlag, BerUn. 12. Waravdekar, V. S., and L. D. Saslaw A method of estimation of 2-dexoyribose. Blochim. Biophys. Acta 24: Weidemann, G., and W. Fischer Zum papier chromatographischen Nachweis von 2-Desoxyzuckern. Z. Phys. Chem. 336: Woodward, G. E., F. B. Cramer, and M. T. Hudson Carbohydrate analogs as antagonists of glucose in carbohydrate metabolism of yeast. J. Franklin Inst. 256: