JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1501-1507

Vol. 133, No. 3

0021-9193/78/0133-1501$02.00/0 Copyright ( 1978 American Society for Microbiology

Printed in U.S.A.

Reaction Order of Saccharomyces cerevisiae Alpha-FactorMediated Cell Cycle Arrest and Mating Inhibition MARK M. UDDENt AND DAVID B. FINKELSTEIN* Department of Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235 Received for publication 10 November 1977

Alpha-factor-mediated cell cycle arrest and mating inhibition of a mating-type of Saccharomyces cerevisiae have been examined in liquid cultures. Cell cycle arrest may be monitored unambiguously by the appearance of morphologically abnormal cells after administration of alpha factor, whereas mating inhibition is determined by comparing the mating efficiency in the absence or presence of added alpha factor. For both cell cycle arrest and mating inhibition, a dosedependent response may be observed at limiting concentrations of the pheromone. If cell cycle arrest and mating inhibition require a small number of alpha-factor molecules, one might expect that responsive/nonresponsive cells = K(alpha factor)N where N is the order of dependence of cell cycle arrest (or mating inhibition) on alpha-factor concentration. The value of N has been determined to be 0.98 ± 0.18 (standard error of the mean) for cell cycle arrest and 1.08 ± 0.32 for mating inhibition. These results support the notion that saturation of a single site by alpha factor is sufficient to cause cell cycle arrest or mating inhibition of a mating-type cells.

cells

The yeast Saccharomyces cerevisiae can exist in either the haploid or diploid state. Haploid cells of opposite mating type can conjugate to form a diploid zygote that, by mitotic division, will give rise to a clone of stable diploid cells which are heterozygous for mating type (a/a). The conjugation process of yeast appears to require a mutual arrest of haploid cells early in the Gl phase of their cell cycles as a prelude to successful mating (6). This mutual arrest in the mating mixture is accomplished by the mutual secretion of diffusible factors by the haploid cells that are capable of causing arrest of cells of the opposite mating type at the "start" point of their cell cycles (3, 14). The arrest of a mating-type cells is accomplished in the mating mixture by a small polypeptide "alpha factor," which is excreted by cells of the a mating type (4). While a cells are themselves immune to the effects of alpha factor, a mating-type cells are arrested as unbudded mononucleate cells at the same point in the cell cycle as arrest by Hartwell's cdc28 mutant (7). As arrest at this point in the cell cycle by alpha factor continues, one sees appearance of morphological alterations in the cell wall (8) that manifest as mycelial-type processes. The arrest of a mating-type cells is a reversible process. After removal of alpha factor from the growth medium, the cells immediately begin a

synchronous round of division. The time course of alpha-factor arrest is related to the concentration of alpha factor in the medium: low concentrations of alpha factor cause only transient cell cycle arrest (2, 13). As the cell wall of the parent cell remains morphologically altered during the recovery from alpha-factor arrest, one has an unambiguous method of scoring whether or not an individual cell has been arrested by alpha factor. This possibility of examining the response of individual cells to alpha factor should allow one to determine the dose response of submaximal levels of alpha factor in order to discern the reaction order of alpha-factor arrest. We report results that are consistent with the notion that saturation of a single target by alpha factor is sufficient for the cell cycle arrest of an a mating-type cell. MATERIALS AND METHODS

Strains. The haploid grande strain of S. cerevisiae X2180-1B a gal2 (obtained from the Yeast Genetic Stock Center, Berkeley, Calif.) was used for the preparation of alpha factor. The tester strain for alpha factor was 55-R5-3C a ura. For mating experiments, 55R5-3C was crossed with 650-2C a his trp. All cultures were maintained and routinely subcultured on YPD agar plates containing 1% yeast extract (Difco Laboratories, Detroit, Mich.), 2% peptone (Difco), 2% glucose, and 2% agar (Difco). All cultures were routinely grown at 30°C in a Lab Line Orbital shaker in flasks t Present address: Department of Internal Medicine, Bay- filled to less than 20% of their stated capacity in either lor College of Medicine, Houston, TX 77030. YPD or on modified G medium (5). 1501

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Partial purification of alpha factor. Alpha factor was purified from 60-liter cultures of X2180-1B by the procedure of Duntze et al. (4) to the stage of neutralized eluent III. This material was then sterile filtered through a 0.2-jm porosity membrane filter (Millipore Corp., Bedford, Mass.) and stored at 4°C. Alpha-factor activity is expressed as units per milliliter, as defined by the diffusion assay of Duntze et al. (4) on YPD agar plates using strain 55-R5-3C as a tester. Liquid assay for alpha factor. Liquid assays for alpha factor were performed in a volume of 200 jil in glass test tubes (12 by 75 mm), using cells at a density of 5 x 10' to 1 x 107 cells/ml and a twofold serial dilution of alpha factor. The cultures were allowed to incubate at 23°C with occasional shaking. The reactions were terminated after 4 h by addition of NaN3 to a final concentration of 20 mM. Cells were counted in a hemacytometer, and cell morphology was scored. Unless stated otherwise, the following conventions were used: all buds were counted as cells, and morphologically abnormal cells with a normal bud (recovering cell) were scored as one abnormal cell plus one normal cell. In plotting the dose response of alphafactor-mediated cell cycle arrest, a correction was made for the one-generation lag in the appearance of morphologically abnormal cells by dividing the number of morphologically normal cells by two. The best-fit line and slope for log-log plots was calculated by use of a linear regression analysis using a Monroe calculator (model 1785). Micrographs were taken by using a Leitz Diavert microscope equipped with Smith Differential Interference Contrast optics at an instrument magnification of x600. Mating. Cells were grown with shaking at 30°C to mid-log phase in liquid YPD to a density of 2 x 107 cells/ml. One-half milliliter of each culture plus 1 ml of a suitable dilution of alpha factor (in water) were combined in a conical centrifuge tube and centrifuged for 5 min. at 800 x g at room temperature (ca. 22°C), using a Clay Adams bench-top centrifuge. The cell pellets were allowed to incubate at room temperature for 15 min. After a brief sonic treatment the tubes were incubated on a gyratory shaker at 30°C for 4 h. The mating reaction was terminated by diluting the mixture 1:1,000 with cold sterile water. Appropriate dilutions of this mating mixture were plated onto minimal medium, 0.67% yeast nitrogen base without amino acids [Difco]-2% glucose-2% agar) to score for diploid cells by prototrophic selection. The mating efficiency (relative to the a cell input) varied from 2 to 10% depending on the experiment. Mating inhibition due to alpha factor is expressed relative to a control mating performed at the same time without alpha factor taken as 100%.

RESULTS Response to alpha factor as a function of alpha-factor concentration. To study the dose response of a mating-type cells to alpha factor, it is necessary to have some unambiguous method of distinguishing a cell that has responded to the pheromone. Whereas the measure of the increase of unbudded cells in an

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alpha-factor-treated culture realtive to an untreated culture has been used by others to detect alpha-factor arrest (1, 2), this method suffers from the presence of a background of 20 to 40% unbudded cells in a normal asynchronous logphase culture, thus making the detection of a small number of arrested cells impossible. We have chosen instead to examine alpha-factor arrest by the examination of cell morphology. After arrest by alpha factor, cells of a matingtype become morphologically abnormal. This morphological alteration can be readily scored microscopically. Figure 1 presents the results of an experiment where log-phase cells were exposed to various concentrations of alpha factor for a period of 4 h, and the cell morphology was scored for the presence of abnormal cells. Figure 1A shows a picture of cells that were exposed to a dose of 64 U of alpha factor per ml. It may be seen that the result of this dose of alpha factor is that virtually all cells assume a distinctive morphology (cf. Fig. 1C, which represents a normal log-phase culture untreated with alpha factor). Even at this very high concentration of alpha factor one can still detect an occasional normally budding cell. In Fig. 1B a culture of cells has been exposed to 1 U of alpha factor per ml for 4 h. It may be seen that with this dose of pheromone at least four different cell morphologies are apparent: (i) normally growing budded cells, (ii) morphologically abnormal unbudded cells (arrested cells), (iii) morphologically abnormal budded cells (cells which are recovering from alpha-factor arrest; D. Finkelstein and L. McAlister, unpublished observations), and (iv) unbudded apparently normal cells. This last morphology is somewhat ambiguous since, as noted above, it could represent either a normally growing cell or a cell which has become arrested but has not yet become morphologically abnormal. This ambiguity of unbudded cells becomes apparent if we examine the time course of appearance of morphologically abnormal cells. If cells were exposed to 64 U of alpha factor per ml and their morphology examined with time, one obtained the results presented in Fig. 2. Under the conditions of this experiment, greater than 90% of the cells became unbudded by 2 h, yet the appearance of abnormal cells was not complete until 4 h. Hence, an apparent onegeneration lag exists between the arrest of cells in an unbudded state and the appearance of morphologically abnormal cells. This delay in the appearance of detectable abnormal cell morphology complicates a quantitative study of alpha-factor arrest. It leads to an underestimate of the degree of cell cycle arrest, as we can only score cell cycle arrest one generation after the

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FIG. 1. Effect of alpha factor on growing yeast cells. Strain 55-R5-3C, actively growing on YPD at a cell density of 5 X it'P cells/ml, was exposed to either 64 U (A), 1 U (B), or no alpha factor (C) per ml and allowed to continue growth for 4 h at 230C. After termnination of growth by addition of sodium azide to 0.02 M, the cells were photographed under Smith Interference Optics. Normally growing budded cell (a), morphologically abnormal unbudded cell (b), morphologically abnormnal budded cell (c), morphologically normal unbudded cell (d). Bar = 5 um. arrest has occurred, whereas unarrested cells have doubled during this timne period. It thus becomes necessary to correct the number of unarrested cells by one generation (the last point at which a cell could become arrested and still be detected as morphologically abnormnal in our

assay).

To quantitate the dose response of alpha-fac-

tor-mediated cell cycle arrest, cells were treated with alpha factor at concentrations varying over a 4,000-fold range, and the cultures were scored for abnormnal morphology at 4 h (Fig. 3). Though it can be seen that the cell morphology changes from predominantly normnal to predominantly

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FIG. 2. Time course of appearance of abnormal cells. An actively growing culture of 55-R5-3C at a density of 5 x 10i cells/ml was exposed to 64 U of alpha factor per ml and allowed to incubate at 23°C. At various times, samples were removed and growth was inhibited by addition of sodium azide to 0.02 M. Morphologically abnormal cells were scored as described in the text.

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abnormal over a relatively narrow range of alpha-factor concentrations, it is important to note that both normal and abnormal cells were observed over the entire range of alpha-factor concentrations examined. Reaction order of alpha-factor arrest. The results of Fig. 3 demonstrate that the arrest of a mating-type cells by alpha factor, as detected by alterations in cell morphology, is related to the dose of alpha factor administered. Since the arrest of cells by alpha factor is a reversible reaction (2, 13; see also Fig. 1B), it is possible to define alpha-factor-mediated cell cycle arrest by the following equation: unarrested cell + N (alpha factor) arrested cell (1), where N is the number of alpha-factor molecules required to achieve cell cycle arrest. Under equilibrium conditions we may write: (arrested cell)/(unarrested cell) (alpha factor)N = K (2). As shown above it may be seen that we can determine the number of arrested cells by the measurement of abnormal cell morphology. Thus, if equation 2 holds, it follows that a loglog plot of the ratio of abnormal to normal cells versus alpha-factor concentration should give a straight line with a slope of N. If the data of Fig. 3 is recast to give a log-log format, we get a straight line as shown in Fig. 4. The slope of this line, as determined by a linear-regression anal-

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FIG. 3. Dose response of a mating-type cells to alpha factor. Actively growing cells were exposed to twofold serial dilutions of alpha factor of 4 h and treated as described in the legend to Fig. 2. Abnormal cells (0), morphologically normal cells (corrected as described in the text), (0) and corrected total cell count (5).

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FIG. 4. Reaction order of alpha-factor-mediated cycle arrest. The data is reduced from the experiment presented in Fig. 3. The line represents the best fit by a linear-regression analysis. Slope is 1.07; correlation coefficient is 0.97. cell

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ysis, is 1.07. The results of 10 such experiments performed under varying conditions are presented in Table 1. It may be seen that under all growth conditions examined the value of N in equation 2 is very close to or equal to the integer 1 (N = 0.98 + 0.18 standard error of the mean). As would be expected from the above equation, the experimentally determined value of N is independent of cell density. This result supports the view that the saturation of a single target by alpha factor is required to arrest an a matingtype cell. Mating inhibition by alpha factor. In the preceding experiments we showed that cell cycle arrest mediated by alpha factor is a first-order reaction with respect to alpha factor. Sena et al. (12) have reported that the addition of exogenous alpha factor to a population of mating yeast cells results in an inhibition of the mating reaction. If the mechanism of this mating inhibition is the same as that causing the prolonged inhibition of the cell cycle, then one would expect to see the same reaction order for mating inhibition as for cell cycle arrest. However, it could also be possible that newly formed zygotes are arrested by alpha factor. Therefore, to test this hypothesis a mixture of a and a cells were permitted to mate, and, at various times during the mating reaction, samples were removed and plated on minimal medium and also on miniimal medium containing alpha factor. (The concentration of alpha factor used was sufficient to inhibit the appearance of a mating-type colonies on solid medium.) The results of this experiment (Fig. 5) show that, once formed, zygotes can give rise to a normal diploid colony of cells even in the presence of alpha factor and, therefore, must be TABLE 1. Reaction order of alpha-factor-mediated cell cycle arrest Growth Correlation Growth phase denpitycl medium of input coefficient (el/i cells YPD Stationaryb 5 x 106 0.72 0.995 YPD Stationary 5 x 106 0.90 0.95 G Stationary 5 x 106 1.05 0.990 YPD Stationary 1 x 107 0.48 0.97 YPD Log 5 x 106 1.07 0.97 1 x 107 1.33 YPD Log 0.94 5 x 106 1.39 G 0.96 Log 5 x 106 0.97 YPD Log 0.97 YPD Stationary 5 x 105 0.92 0.97 YPD Log 5 x 105 0.93 0.98 a N, the reaction order of alpha-factor-mediated cell cycle arrest, represents the slope of the line of log (abnormal cells/normal cells) = N log (alpha factor) + log K. b Stationary-phase cells were freshly diluted into new growth medium.

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FIG. 5. Time course of mating. A mating reaction between 55-R5-3C and 650-2C was carried out as described in the text. At various times, samples were plated to select for diploid cells on either minimal medium (0) or minimal medium containing 50 U of alpha factor (0) per ml.

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If mating inhibition by alpha factor involves a small number of alpha-factor molecules, then one can construct the following equation: mating cell + N(alpha factor) = nonmating cell (3). Since the mating efficiency in this cross is only about 5 to 10% (relative to the a cell input), one can neglect those cells that did not mate in the absence of alpha factor. Thus, for a given dose of alpha factor, the nonmating cells are taken to be equal to the number of mating cells in the

absence of alpha factor minus the number of mating in the presence of alpha factor. Reasoning by analogy with equation 1 above it may be seen that if equation 3 is valid, then it follows that a log-log plot of the ratio of nonmating cells to mating cells versus alpha-factor concentration should give a straight line with a slope of N. To test the validity of this relationship, matings were performed, using a graded series of alpha-factor concentrations as well as a control mating that did not receive exogenous alpha factor (Fig. 6). The slope of this best-fit line is 0.925. From four such experiments one can see that mating inhibition by exogenous alpha factor is a first-order reaction (N = 1.08 + 0.32 standard error of the mean). cells

DISCUSSION The experiments reported in this paper were designed to determine the reaction order of a

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FIG. 6. Reaction order of alpha-factor inhibition of mating. Strain 55-R5-3C and 650-2C were allowed to mate for 4 h in the presence of various concentrations of alpha factor. After plating, diploids were scored and the data was reduced as described in the text. The line was fitted as for Fig. 4. The slope is 0.925; correlation coefficient is 0.995.

mating-type cell cycle arrest by alpha factor. The two criteria we measured are the inhibition of the mating reaction and the alterations in cell morphology after alpha-factor arrest. Though neither parameter appears to be capable of measuring a transient elongation of the Gl phase of the cell cycle, both methods allow the advantage of low background measurement that is much more sensitive than the measure of the change in the percentage of unbudded cells in a cell culture exposed to alpha factor. Indeed, whereas the measurements of cell cycle arrest as monitored by inhibition of DNA synthesis or arrest in the increase in cell density show no apparent arrest at doses of alpha factor of less than 1 U/ml (Udden and Finkelstein, unpublished), examination of cellular morphology or mating inhibition allow one to obtain a measureable response at these doses of pheromone. A first-order reaction for cell cycle arrest was concluded from the measurement of the slope of a log-log plot of responsive/nonresponsive cells versus alpha-factor concentration. It should be noted that such a determination requires only a knowledge of the relative concentration of alpha factor rather than the absolute concentration of the pheromone. Thus, though alpha factor is inactivated by proteolysis upon exposure to a mating-type cells, this does not interfere with the determination of the reaction order, as the

rate of alpha-factor inactivation is proportional to the input of the pheromone over the concentration ranges used in these studies (D. Finkelstein, unpublished observations). It has been implied that the yeast mating pheromones are necessary to mutually synchronize cells as a prelude to a successful mating reaction (6), yet we as well as others (12) find the apparently contradictory results that exogenously added mating pheromones inhibit mating. Presumably the optimal alpha-factor concentration necessary for mating is well below that which produces major morphological changes in a culture. From the data of Scherer et al. (11), who examined the time course of alpha factor-production by a mating-type cells, it is possible to estimate that the alpha-factor concentration in a normal mating mixture would be less than 0.1 U/ml, which would cause less than a 5% inhibition of mating due to prolonged cell cycle arrest (Fig. 6). Presumably, at this low concentration of alpha factor, one sees primarily a slight elongation in the G1 phase of the cell due to the reversible nature of alpha-factor arrest. Thus it would appear that as a transient response to alpha factor is necessary for mating, so the changes in the structure of the cell wall (or some other cell constituent) resulting from prolonged cell cycle arrest are inhibitory to the mating reaction of a mating-type cells. First-order dependence of cell cycle arrest and mating inhibition yields information about the mechanism of action of alpha factor. The presence of a first-order reaction implies that there is only a single target per cell that must be saturated for alpha-factor action. This would rule out any mechanism for alpha-factor action involving the stoichiometric inhibition of an enzyme or a species of tRNA, for example, as all of these molecules are present at more than one copy per cell. It is interesting to note that the possibility of alpha-factor arrest being mediated via a mechanism involving a single pheromone molecule binding to a chromosome (or a chromosomal protein) to activate (or inactivate) transcription at a given site is not ruled out by a first-order-reaction mechanism, but alpha-factor action in analogy with the inducer of the Escherichia coli lac operon would not be consistent with a first-order-reaction mechanism. The action of many mammalian polypeptide hormones appears to involve the activation of a membrane-bound adenyl cyclase and the increase in the level of a second messenger, cyclic AMP (9). A first-order reaction of alpha factor would be consistent with such a mechanism if only a single receptor needed to be occupied to trigger such a response. This would be consistent

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with the threshold model proposed by Rodbard (10). Unfortunately, there have been no experiments performed with hormone-stimulated mammalian cells to demonstrate that a submaximal response represents a complete response of only a portion of the cells. Though we have demonstrated an all-or-none response for individual yeast cells arrested with alpha factor, it will require future study to determine if there is a saturable, mating-type-specific, class of alphafactor receptors on the yeast cell membrane. ACKNOWLEDGMENTS We thank Susan Strausberg for preparing the alpha factor used in these experiments and Robert 0. McAlister for allowing us to use his interference microscope. This investigation was supported by grant PCM 76-17208 from the National Science Foundation and a Public Health Service, National Cancer Institute Specialized Cancer Center grant (CA 17065) from the National Cancer Institute. LITERATURE CITED 1. Bucking-Throm, E., W. Duntze, L. H. Hartwell, and

T. R. Manney. 1973. Reversible arrest of haploid yeastcells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76:99-110. 2. Chan, R. K. 1977. Recovery of Saccharomyces cerevisiae mating type a cells from G1 arrest by a factor. J. Bacteriol. 130:766-774. 3. Duntze, W., V. MacKay, and T. R. Manney. 1970. Saccharomyces cerevisiae: a diffusible sex factor. Sci-

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ence 168:1472-1473. 4. Duntze, W., D. Stotzler, E. Bucking-Throm, and S. Kalbitzer. 1973. Purification and partial characterization of a-factor, a mating-type specific inhibitor of cell

reproduction from Saccharomyces cerevisiae. Eur. J. Biochem. 35:357-365. 5. Finkelstein, D. B., and R. A. Butow. 1976. DNA-binding proteins in yeast. Effect of growth phase and mitochondrial function. Arch. Biochem. Biophys. 174:52-65. 6. Hartwell, L. H. 1973. Synchronization of haploid yeast cell cycles, a prelude to conjugation. Exp. Cell Res. 76:111-117. 7. Hereford, L. M., and L. H. Hartwell. 1974. Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis. J. Mol. Biol. 84:445-461. 8. Lipke, P. N., A. Taylor, and C. E. Ballou. 1976. Morphogenic effects of a-factor on Saccharomyces cerevisiae a cells. J. Bacteriol. 127:610-618. 9. Robison, G. A., R. W. Butcher, and E. W. Sutherland. 1971. Cyclic AMP, p. 17-47. Academic Press Inc., New York. 10. Rodbard, D. 1973. Theory of hormone-receptor interaction. III. The endocrine target cell as a quantal response unit: a general control mechanism, p. 342-364. In B. W. O'Malley and A. R. Means (ed.), Receptors for reproductive hormones. Plenum Publishing Corp., New York. 11. Scherer, G., G. Haag, and W. Duntze. 1974. Mechanism of a factor biosynthesis in Saccharomyces cerevisiae. J. Bacteriol. 119:386-393. 12. Sena, E. P., D. N. Radin, and S. Fogel. 1973. Synchronous mating in yeast. Proc. Natl. Acad. Sci. U.S.A. 70:1373-1377. 13. Throm, E., and W. Duntze. 1970. Mating-type-dependent inhibition of deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J. Bacteriol. 104:1388-1390.

Reaction order of Saccharomyces cerevisiae alpha-factor-mediated cell cycle arrest and mating inhibition.

JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1501-1507 Vol. 133, No. 3 0021-9193/78/0133-1501$02.00/0 Copyright ( 1978 American Society for Microbiology...
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