Mol Gen Genet (1982) 185:506-509

© Springer-Verlag 1982

Short Communication

Cell Cycle Inhibition of Yeast Spheroplasts

Seishi Murakami and Dennis M. Livingston Department of Biochemistry, 4-225 Millard Hall, University of Minnesota, Minneapolis, Minnesota 55455, USA

Summary. Osmotically stabilized yeast spheroplasts are capable of extensive DNA synthesis. Although the rate of DNA synthesis in spheroplasts is approximately one-third that of intact cells, the relative amounts of nuclear and mitochondrial DNA synthe- sized by spheroplasts is very similar to the relative amounts synthesized by intact cells. Furthermore, nuclear but not mito- chondrial DNA synthesis is inhibited in M A T a spheroplasts by the application of the yeast mating pheromone, e-factor. Similar- ly, D NA synthesis is reversibly temperature-sensitive in sphero- plasts created from cdc7 and cdc8 mutant cells.

Yeast spheroplasts have been used to investigate aspects of mac- romolecular biosynthesis, including DNA replication, in the yeast, Saccharomyces cerevisiae (Hutchinson and Hartwell 1976; Oertel and Goulian 1977; Doi and Doi 1979). More recently yeast spheroplasts have been used for transformation by chimeric plasmid DNAs (Beggs 1978; Hinnen etal . 1978). The ability of osmotically fragile spheroplasts to regenerate into intact cells after transformation implies that spheroplasts are competent to carry out many of the same metabolic processes as intact cells. In this regard we were interested in learning whether spheroplasts are capable of initiating rounds of nuclear DNA synthesis and whether this synthesis is sensitive to cell division cycle inhibitors.

We began our investigation of the cell division cycle of yeast spheroplasts by confirming that cultures of yeast spheroplasts are capable of carrying out DNA synthesis (Hutchinson and Hartwell 1967). Synthesis is dependent on the presence of an osmotic stabilizer because spheroplast cultures without an os- motic stabilizer exhibit less than 0.1% of the amount of DNA synthesis measured in a stabilized culture (data not shown). We next compared the rate of DNA synthesis in spheroplasts to that in intact cells. To make this comparison, cells were grown in a medium with 3H-adenine for five generations, spheroplasts were prepared from a portion of the culture, and the spheroplasts were returned to an osmotically stabilized medium with 3H- adenine of the same specific activity as before. As shown in Fig. 1, after an approximately 2 h lag period the spheroplasts are able to double their amount of DNA in 5-6 h while the intact cells do so in 2 h. This rate difference indicates that sphero- plasts synthesize DNA at one-third the rate synthesized by intact cells. In other experiments (Murakami et al. 1982) we have found that the rate of spheroplast protein synthesis is also one-third that of intact cells.

We have also examined two other properties of spheroplast

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Time (hr.) Fig. 1. A comparison of the rate of DNA synthesis in intact cells and spheroplasts. Cells of strain A364A (Hartwell 1967) were grown at 25 ° C in synthetic medium (Hartwell 1970) containing 20/aCi/5 rag/ ml 3H-adenine (Amersham) until they reached a density of 1 x 107 cells/ ml. A portion of cells were removed and made into spheroplasts by the method of Beggs (1978). The spheroplasts were returned at a density of 5x 106 spheroplasts/ml to regeneration medium (0.17% Difco-Yeast Nitrogen Base without amino acids and ammonium sul- fate, 0.15% ammonium sulfate, 2% w/v glucose, 1.0 M sorbitol) con- taining the required amino acids, nucleosides, and 3H-adenine of the same specific activity. Aliquots were removed at the times indicated and analyzed for DNA (Hartwel11967). The values have been normal- ized so that the initial value is 1,000 cpm (approximately 3 x 105 cells). The rate of DNA accumulation in the intact cells corresponds closely to the 140 min division time which we measured. - e - - e - intact cells; - o - - o - spheroplasts

DNA synthesis. First, we found that DNA synthesis in a sphero- plast culture occurs continuously in a linear fashion for more than 24 h at which time the spheroplasts begin to regenerate into osmotically stable intact cells (data not shown). Second, we determined the ratio of nuclear to mitochondrial DNA of

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Fig. 2. DNA synthesis in e-factor treated spheroplasts. Cell of strains X2180-1 a (MA ira), X2180-1 b (MATe) and + D 5 (MA Ta/e) (Hartwell 1970) were grown in YM-1 medium (Hartwell 1967) at 25 ° C with a division time between 100 and 120 min. Spheroplasts were made from samples of exponentially growing cultures and were resuspended in regeneration medium (see Fig. 1) with [8-3H] adenine (20 gCi/5 mg/ ml) at 25 ° C. For each strain identical samples were incubated in the presence or absence of e-factor. The e-factor was purified by the method of Bucking-Throm et al. (1973) and sufficient e-factor was used to arrest a culture of intact MATa cells for greater than two division cycles, o © , • • - DNA synthesis in X2180-1a spheroplasts in the absence and presence of c~-factor, respectively; - D - - u - , - w - - K - DNA synthesis in X2180-1b spheroplasts in the absence and presence of e-factor, respectively; --ZX--A--, --A • DNA synthesis in ÷ D5 spheroplasts in the absence and presence of e-factor, respectively

the newly synthesized DNA by using isopycnic centrifugation and found that spheroplasts like intact cells make approximately four times more nuclear than mitochondrial DNA (see Fig. 3).

The prolonged ability of spheroplasts to synthesize nuclear DNA suggests that spheroplasts may be able to initiate rounds of nuclear DNA synthesis and that this synthesis may be under cell division cycle control. To examine these possibilities, we applied e-factor to yeast spheroplasts. The polypeptide mating pheromone, e-factor, is secreted by yeast cells of the MATe mating type and specifically inhibits cell division of yeast cells of the MATa mating type (Throm and Duntze 1970). The phero- mone arrests cell division at a point very close to the start of the division cycle. As such, the pheromone inhibits synthesis of nuclear DNA replication which occurs during S phase. In contrast mitochondrial DNA replication is unaffected by e-factor (Petes and Fangman 1973; Cryer et al. 1973).

We tested whether c~-factor inhibits yeast spheroplasts by assaying for inhibition of DNA synthesis in the spheroplasts. Figure 2 shows that spheroplasts made from MATa cells are sensitive to e-factor inhibition. Spheroplasts made from MAT:~

or diploids (MATa/MATe) are insensitive. Furthermore, RNA and protein synthesis in arrested MATa spheroplasts is unaf- fected during the same time period (data not shown). This pattern of inhibition is similar to that found for intact cells (Throm and Duntze 1970).

In another experiment not shown, e-factor was applied to different cultures of MATa spheroplasts 0, 4, 20, and 24 h after spheroplast preparation. In all cases inhibition comparable to that shown in Fig. 2 was observed. This result suggests that spheroplasts may be capable of undergoing more than one round of nuclear DNA replication. If only a single round of nuclear DNA replication occured in each spheroplast, sensitivity to the factor would only be observed very early after spheroplast forma- tion.

The observed lag time of 4-5 h between the time of e-factor application and decline in the rate of DNA synthesis may be the result of two independent phenomena. First, the spheroplasts undergo an approximately 2 h lag period in reaching a steady state rate of DNA synthesis which is one-third that of intact cells. Thus, the lag in response to e-factor may be a reflection both of this initial lag and of the overall diminished rate. Second, residual DNA synthesis is observed after e-factor inhibition of intact cells. This results from rounds of nuclear DNA replication in progress at the point of pheromone application which are insensitive to e-factor (Bucking-Throm et al. 1973) and from mitochondrial DNA replication which is unaffected by the pheromone (Petes and Fangman 1973 ; Cryer et al. 1973).

To examine the type of DNA made during e-factor arrest of spheroplasts, the spheroplasts were given 3H-adenine for half- hour pulses at various times after e-factor arrest, and the species of DNA were then separated by isopycnic centrifugation. Fig- ure 3 shows that the pattern of DNA synthesized in e-factor arrested spheroplasts is different from that of unarrested sphero- plasts. Early after application of e-factor, both nuclear and mito- chondrial DNA are made by the e-factor inhibited spheroplasts in the same proportion (approximately 4:1) as that of unarrested spheroplasts. At later times nuclear DNA synthesis is drastically decreased while mitochondrial DNA synthesis continues un- abaited. This decrease of nuclear DNA synthesis after arrest and the continual synthesis of mitochondrial DNA throughout pheromone arrest are similar to the results obtained previously from intact cells (Petes and Fangman 1973; Cryer et al. 1973). The contrast between the continual nuclear DNA synthesis in unarrested spheroplasts and the inhibition of nuclear DNA syn- thesis in arrested spheroplasts suggests that spheroplasts are able to enter and complete S phase.

In addition to their sensitivity to e-factor, spheroplasts should also exhibit sensitivity to inhibition by cell division cycle (cdc) mutations which affect DNA replication (Hartwell 1971; Hart- well 1973). We tested whether DNA replication in spheroplasts was affected by the cdc7 and cdc8 mutations. Both mutations confer temperature-sensitive DNA synthesis to yeast cells. One difference between the two mutations is that the cdc8 mutation inhibits nuclear DNA replication immediately upon temperature shift while the cdc7 mutation only affects the initiation of new rounds of nuclear DNA replication but does not affect comple- tion of rounds of replication in progress at the time of tempera- ture shift (Hartwell 1971 ; Hartwell 1973). Furthermore, the cdc7 mutation does not inhibit mitochondrial DNA replication as does the cdc8 mutation (Cottrell et al. 1973; Cryer et al. 1973; Newlon and Fangman 1975). The mode of action of the cdc7 mutation on DNA replication is therefore very much like the application of e-factor to cells. Residual DNA synthesis occurs after a shift to the restrictive temperature, and this residual

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Fig. 3 a-f. Isopycnic centrifugation of DNA from spheroplasts. Sphero- plasts of strain A364A were grown in osmotically stabilized medium either in the presence or absence of e-factor. At times after the onset of incubation 3H-adenine was added to the medium and incubation was continued for 30 min. The spheroplasts were harvested, osmoti- cally lysed, and the extract was subjected to isopycnic centrifugation along with purified 32p-yeast nuclear DNA. After centrifugation frac- tions were collected and analyzed for DNA. The gradients have been aligned to place the peak of the 32P-marker DNA in the same fraction. Panels a, e, and e show the analysis from spheroplasts incubated in the absence of e-factor which were given pulses of 3H-adenine at 2, 4.5, 7 h, respectively. Panels b, d, and f show the same analysis from e-factor inhibited spheroplasts. - 0 - - © - 3H; • • 32p

synthesis is a combina t ion of rounds of nuclear synthesis in progress at the time of arrest and of cont inual mi tochondr ia l D N A synthesis.

Figure 4 shows studies on D N A replication in cdc7 and cdc8 m u t a n t spheroplasts. M u t a n t cells were grown either entirely at the permissive temperature or were shifted to the restrictive temperature 2.5 h before spheroplast preparat ion. Spheroplasts were prepared at the same temperature at which the cells were grown prior to harvest. Spheroplast D N A synthesis was mea- sured at bo th the permissive and restrictive temperatures. Panels a and c of Fig. 4 show the pa t tern of the D N A replication in cdc7 and cdc8 spheroplasts prepared from cells grown at the permissive temperature. The spheroplasts exhibit temperature sensitive D N A replication. The residual D N A replication in cdc7 spheroplasts at 36 ° C was analyzed by isopycnic centr ifugation as described for c~-factor arrested cells in Fig. 3. This analysis showed that bo th nuclear and mi tochondr ia l D N A synthesis occur early after the temperature shift but the synthesis becomes almost exclusively mi tochondr ia l by later times (5-7 h). This pa t tern of D N A synthesis is similar to tha t seen in intact cdc7 mutan t cells (Newlon and Fangm an 1975).

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Fig. 4a-d. DNA synthesis in spheroplasts made from edc7 and cdc8 mutant cells. Strains 201.14.4 (cdc7) and 198 (cdc8) (Hartwell et al. 1973) were grown in YM-1 medium at 25 ° C. Under these conditions the mutant strains had a division time between 130 and i50 rain. Exponentially growing cells at a density of 5 x 106 cells/ml were either harvested directly or were incubated at 36 ° C for 2.5 h before harvest- ing. Spheroplast formation procedures were either carried out at 25 ° C or in a 36°C temperature controlled room. The spheroplasts were incubated in regeneration medium containing 3H-adenine (20 gCi/ 5 mg/ml) as before. At the times indicated aliquots were removed and analyzed for DNA. Panel a shows the analysis of cdc7 spheroplasts made from cells grown at 25°C before spheroplast formation and incubated at either 25 ° C ( - o - - o ) or 36 ° C ( - o - - e @ Panel b shows the analysis of cdc7 spheroplasts made from cells grown for 2.5 h at 36 ° C before spheroplast formation which was carried out at 36 ° C. The spheroplasts were incubated at either 25°C ( - o - - o @ or 36°C ( - e - - o ) . Panels e and d are the analogous analyses to panels a and b carried out on cdc8 spheroplasts

Panels b and d of Fig. 4 show the pat tern of D N A replication in spheroplasts prepared from mutan t cells incubated at the restrictive temperature before harvesting. Spheroplast prepara- t ion was also carried out at the restrictive temperature. The mutan t spheroplasts are incapable of nuclear D N A synthesis if they are fur ther incubated at the restrictive temperature. The residual D N A synthesis observed in the cdc7 spheroplasts at 36 ° C again has been analyzed by isopycnic centr ifugation and shown to be mi tochondr ia l D N A synthesis as expected (Newlon and Fangman 1975). Thus, inhibit ion of D N A synthesis is not lost dur ing the manipula t ions needed to make the spheroplasts. U p o n incubat ion of the spheroplasts at the permissive tempera- ture D N A synthesis resumes. Thus, the spheroplasts are capable of quickly recovering from the inhibition. These results show that spheroplasts, like intact cells, exhibit reversible, temperature- sensitive D N A synthesis when made from appropriate mu tan t strains.

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Our experiments thus demonstrate that spheroplasts exhibit some of the cell division cycle controls found in mitotically dividing intact cells. Our data also suggests that spheroplasts are capable of undergoing more than one round of nuclear D N A synthesis during the 24 to 48 h period during which they regener- ate their cell wall. This suggestion further implies that sphero- plasts may be capable of continued cell division. In this regard we have observed microscopically that the number of cell bodies increases in a spheroplast culture during the first 24 h of liquid regeneration at a rate comparable to that of D N A and protein synthesis (data not shown). Whether spheroplasts are actually capable of undergoing cell division will require further studies. Nevertheless, the results of our study show that osmotically fragile yeast spheroplasts have not lost their sensitivity to cell division specific inhibition.

Acknowledgements. This work was supported by NSF grant PCM 80-21893.

References

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Communicated by G. Fink

Received December 29, 1981 / February 17, 1982