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The effect of cycloheximide (actidione) on cell wall synthesis in yeast protoplasts
6.
JiLEINSMITII,
1..
J.,
ALLPREY,
\'.
(;.
and I\ln~srtu, A. E., I’roc. xnfl dcrtrl. Sci. IIS 55, 11X2
(1966). T., in KOSIXGSBERGER, \‘. \‘. and BOSCH, L. (eds), Regulation of nucleic acid and r;rotcin synthesis, p. 241. Elsevier, Amsterdam; 1965. 8. LANGAN, T. an& SMITH, i., Information Exchange Group No. 7, 8113 (1965). 9. SAKAMOTO, T., Fox, C. I;. and \V~rss, S. B., .I. Rid. Chem. 239, 167 (1964). 10. 1’HOMAS, .J!i., c. A., BERXS, K. I. and KELLI', JR., .J., in C.\STOSI, G. L. and DAVIES, D. K. (eds), Procedures in nucleic acid research, p. 535. Harper & HOW, Sew York, 1966. 11. \V,\NG, T. Y., in The cell nucleus: metaholisni and radiosensitivity, p, 213. Taylor and ITran&, London, 1966. i,
12. 13.
I,.~NGAN,
--
.J. Bid.
\YAXn,
T.
Chem. Y,
and
THE EFFECT
242, .JOHXS,
1220 (196i). E. \T'., Arch.
h’iochrm.
OF CYCLOHEXIMIDE SYNTHESIS 0.
Department
NE8AS,
IN YEAST A. SVOBODli
124, Ii6 (1968).
JSiophys.
(ACTIDIONE)
ON CELL WALL
PROTOPLASTS and
M.
KOPECKA
of General Biology, Medical Faculty, University, Bmo, Czechoslovakia
J. E. Purkynee
Received May 7, 1968
T HE
yeast protoplasts are capable of synthetizing a new cell wall and regenerate into new cells [4, 71. The protoplasts of Saccharomyces cereuisiae build a complete new cell wall only when cultivated in high concentrated gelatin media [5]. In liquid media only a fibrillar network, composed probably of glucan, is formed on the surface of protoplasts [2, 71. Thus decoupling of the formation of fibrillar groundwork and that of the matrix takes place in liquid media. It has been proved in previous papers [6, 81 that the gelatin creates certain physical conditions in the surroundings of protoplasts, necessary for the structural organization of the wall matrix. During a systematic study of the effect of antibiotics on the regeneration ability of protoplasts Sogkova ef al. [IO] found that cycloheximide (Actidione) prevented the reversion of Saccharomyces cerevisiae protoplasts into normal cells, Thus the question arose whether this antibiotic also interferes with the synthesis of cell wall components. This investigation has been concerned with the effect of cycloheximide on the formation of fibrillar network in liquid media and on the synthesis of a complete cell wall in concentrated gelatin. The protoplasts of Saccharomyces cerevisiae (lab. strain No. 7) were prepared using the snail enzymes. Cycloheximide (Upjohn Company, Mich.) was added in a concentration of 20 y/ml both into the liquid and gelatin media (30 per cent of Difco gelatin) whose basic component was the synthetic nutrient medium. The formation of a fibrillar network on the surface of protoplasts in liquid media was not affected by cycloheximide. A fibrillar network composed of intermeshed bundles of elementary fibrils comparable with those of controls was produced after 1-2 h incubation. The fibrils were formed even when the yeast cells were treated with cycloheximide 2-4 h before their conversion into protoplasts. In gelatin media in presence of cycloheximide only a very fine fibrillar network was formed; the matrix masking the fibrils, however, could not be detected in contrast to 19*
- 681813
Experimental
Cell
Research
53
0. Setas, A. Svoboda and M. Kopecka’
292
control experiments (Figs I, 2). This fine network represents the fibrillar groundwork of the newly synthetized walls. When the yeast cells were incubated in the presence of cycloheximide 2-4 h before conversion to protoplasts and these were then embedded into the gelatin, the results were the same, i.e. only fibrils without matrix were found. From these experiments it appears that action of cycloheximide on the formation of fibrils and matrix walls is different. Cycloheximide affects the proteosynthesis on the level of ribosomes [I, 9, 10). Thus it may be presumed that in freshly prepared yeast protoplasts all structural precursors of fibrils and the respective polymerizing enzymes are already at hand thus making the construction of extracellular fibrils independent of proteosynthesis. The pretreatment of cells before their conversion into protoplasts stops the growth entirely after 2 h and the whole “fabric” involved in the formation of fibrils cannot be exhausted. The formation of the wall matrix is in close dependence on proteosynthesis. At present, there is no experimental evidence as to whether only synthesis of structural proteins (peptides) of the matrix is affected by cycloheximide and thus the construction of a matrix structure is impossible, or whether cycloheximide also interferes with the synthesis of the polysaccharides (mannans) of the wall matrix. It has been reported by Morris [3] in Chlorella that cycloheximide not only inhibits the synthesis of proteins but also interfers, after several hours, with the synthesis of polysaccharides as well. Morris suggests that this inhibition is a secondary effectfor instance the failure of resynthesis of enzymes involved in the polysaccharide synthesis. It is possible that the enzymes engaged in the synthesis of fibrillar network in yeast protoplasts have a slow rate of turnover and this is the cause of their relative independence on proteosynthesis. Summarizing our results we can state that the formation of cell wall matrix, in yeast protoplasts can be blocked with cycloheximide in contrast to the formation of fibrillar groundwork of cell wall. This indicates that biosynthesis of these two structural components follows different pathways and may be controlled by different mechanisms. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
DE KI,OET, S. R., Biochem. J. 99, 582 (1966). EDDY, A. A., WrLLrAn~sox, D. H., Nature 183, 1101 (1959). MORRIS, I., J. Exptl Rot. 18, 54 (1967). i%d!AS, O., Nature 177, 898 (1956). ~ ibid. 192, 580 (1961). -Folia biol. (Praha) 8, 256 (1962). ~ ibid. 11, 97 (1965). NE&S, O., SVOBODA, A., in R. M~;LLER (ed.), Symposium iiber Hefe-Protoplasten, Verlag Akademie, Berlin, 1967. 9. SIEGEL, hf. R. and SISLER, H. D., Biochim. Biophys. Acta 103, 558 (1965). 10. SoBxovA, L., Svoso~a, A. and SO&A, J., Folia microbial. 13, 240 (1968). Fig. l.-The new cell wall formed on the surface of protoplast gelatin. The protoplasts were desintegrated, washed in distilled supporting membranes. Shadowed by platinum. Fig. 2.-Fibrillar network formed on the surface of protoplast gelatin in presence of 20y/ml cycloheximide. The protoplasts prepared as shown in Fig. 1. Experimental
Cell
Research
53
after water
24 h cultivation and transferred
p. 67.
in 30% onto the
after 24 h cultivation in 30% were osmotically disrupted and
Effect
of cycloheximide
(crctidione)
293
Experimental
Cell Research
53
JiLEINSMITII,
1..
J.,
ALLPREY,
\'.
(;.
and I\ln~srtu, A. E., I’roc. xnfl dcrtrl. Sci. IIS 55, 11X2
(1966). T., in KOSIXGSBERGER, \‘. \‘. and BOSCH, L. (eds), Regulation of nucleic acid and r;rotcin synthesis, p. 241. Elsevier, Amsterdam; 1965. 8. LANGAN, T. an& SMITH, i., Information Exchange Group No. 7, 8113 (1965). 9. SAKAMOTO, T., Fox, C. I;. and \V~rss, S. B., .I. Rid. Chem. 239, 167 (1964). 10. 1’HOMAS, .J!i., c. A., BERXS, K. I. and KELLI', JR., .J., in C.\STOSI, G. L. and DAVIES, D. K. (eds), Procedures in nucleic acid research, p. 535. Harper & HOW, Sew York, 1966. 11. \V,\NG, T. Y., in The cell nucleus: metaholisni and radiosensitivity, p, 213. Taylor and ITran&, London, 1966. i,
12. 13.
I,.~NGAN,
--
.J. Bid.
\YAXn,
T.
Chem. Y,
and
THE EFFECT
242, .JOHXS,
1220 (196i). E. \T'., Arch.
h’iochrm.
OF CYCLOHEXIMIDE SYNTHESIS 0.
Department
NE8AS,
IN YEAST A. SVOBODli
124, Ii6 (1968).
JSiophys.
(ACTIDIONE)
ON CELL WALL
PROTOPLASTS and
M.
KOPECKA
of General Biology, Medical Faculty, University, Bmo, Czechoslovakia
J. E. Purkynee
Received May 7, 1968
T HE
yeast protoplasts are capable of synthetizing a new cell wall and regenerate into new cells [4, 71. The protoplasts of Saccharomyces cereuisiae build a complete new cell wall only when cultivated in high concentrated gelatin media [5]. In liquid media only a fibrillar network, composed probably of glucan, is formed on the surface of protoplasts [2, 71. Thus decoupling of the formation of fibrillar groundwork and that of the matrix takes place in liquid media. It has been proved in previous papers [6, 81 that the gelatin creates certain physical conditions in the surroundings of protoplasts, necessary for the structural organization of the wall matrix. During a systematic study of the effect of antibiotics on the regeneration ability of protoplasts Sogkova ef al. [IO] found that cycloheximide (Actidione) prevented the reversion of Saccharomyces cerevisiae protoplasts into normal cells, Thus the question arose whether this antibiotic also interferes with the synthesis of cell wall components. This investigation has been concerned with the effect of cycloheximide on the formation of fibrillar network in liquid media and on the synthesis of a complete cell wall in concentrated gelatin. The protoplasts of Saccharomyces cerevisiae (lab. strain No. 7) were prepared using the snail enzymes. Cycloheximide (Upjohn Company, Mich.) was added in a concentration of 20 y/ml both into the liquid and gelatin media (30 per cent of Difco gelatin) whose basic component was the synthetic nutrient medium. The formation of a fibrillar network on the surface of protoplasts in liquid media was not affected by cycloheximide. A fibrillar network composed of intermeshed bundles of elementary fibrils comparable with those of controls was produced after 1-2 h incubation. The fibrils were formed even when the yeast cells were treated with cycloheximide 2-4 h before their conversion into protoplasts. In gelatin media in presence of cycloheximide only a very fine fibrillar network was formed; the matrix masking the fibrils, however, could not be detected in contrast to 19*
- 681813
Experimental
Cell
Research
53
0. Setas, A. Svoboda and M. Kopecka’
292
control experiments (Figs I, 2). This fine network represents the fibrillar groundwork of the newly synthetized walls. When the yeast cells were incubated in the presence of cycloheximide 2-4 h before conversion to protoplasts and these were then embedded into the gelatin, the results were the same, i.e. only fibrils without matrix were found. From these experiments it appears that action of cycloheximide on the formation of fibrils and matrix walls is different. Cycloheximide affects the proteosynthesis on the level of ribosomes [I, 9, 10). Thus it may be presumed that in freshly prepared yeast protoplasts all structural precursors of fibrils and the respective polymerizing enzymes are already at hand thus making the construction of extracellular fibrils independent of proteosynthesis. The pretreatment of cells before their conversion into protoplasts stops the growth entirely after 2 h and the whole “fabric” involved in the formation of fibrils cannot be exhausted. The formation of the wall matrix is in close dependence on proteosynthesis. At present, there is no experimental evidence as to whether only synthesis of structural proteins (peptides) of the matrix is affected by cycloheximide and thus the construction of a matrix structure is impossible, or whether cycloheximide also interferes with the synthesis of the polysaccharides (mannans) of the wall matrix. It has been reported by Morris [3] in Chlorella that cycloheximide not only inhibits the synthesis of proteins but also interfers, after several hours, with the synthesis of polysaccharides as well. Morris suggests that this inhibition is a secondary effectfor instance the failure of resynthesis of enzymes involved in the polysaccharide synthesis. It is possible that the enzymes engaged in the synthesis of fibrillar network in yeast protoplasts have a slow rate of turnover and this is the cause of their relative independence on proteosynthesis. Summarizing our results we can state that the formation of cell wall matrix, in yeast protoplasts can be blocked with cycloheximide in contrast to the formation of fibrillar groundwork of cell wall. This indicates that biosynthesis of these two structural components follows different pathways and may be controlled by different mechanisms. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
DE KI,OET, S. R., Biochem. J. 99, 582 (1966). EDDY, A. A., WrLLrAn~sox, D. H., Nature 183, 1101 (1959). MORRIS, I., J. Exptl Rot. 18, 54 (1967). i%d!AS, O., Nature 177, 898 (1956). ~ ibid. 192, 580 (1961). -Folia biol. (Praha) 8, 256 (1962). ~ ibid. 11, 97 (1965). NE&S, O., SVOBODA, A., in R. M~;LLER (ed.), Symposium iiber Hefe-Protoplasten, Verlag Akademie, Berlin, 1967. 9. SIEGEL, hf. R. and SISLER, H. D., Biochim. Biophys. Acta 103, 558 (1965). 10. SoBxovA, L., Svoso~a, A. and SO&A, J., Folia microbial. 13, 240 (1968). Fig. l.-The new cell wall formed on the surface of protoplast gelatin. The protoplasts were desintegrated, washed in distilled supporting membranes. Shadowed by platinum. Fig. 2.-Fibrillar network formed on the surface of protoplast gelatin in presence of 20y/ml cycloheximide. The protoplasts prepared as shown in Fig. 1. Experimental
Cell
Research
53
after water
24 h cultivation and transferred
p. 67.
in 30% onto the
after 24 h cultivation in 30% were osmotically disrupted and
Effect
of cycloheximide
(crctidione)
293
Experimental
Cell Research
53