THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 22, Issue of November 25, pp. 13849-13856,1983 Printedin U.S.A.

Comparison of Dose-Response Curves for ar Factor-induced Cell Division Arrest, Agglutination, and Projection Formation of Yeast Cells IMPLICATION FOR THE MECHANISM OF a FACTOR ACTION*

(Received for publication, March 8, 1983)

Susan A. Moore$ From the Department of Genetics, University of Washington, Seattle, Washington 98195

MATa cells of the yeast Saccharomyces cerevisiae produce a polypeptide mating pheromone, a factor. MATa cells respond to the pheromone by undergoing several inducible responses: the arrest of cell division, the production of a cell surface agglutinin, and the formation of one or more projections on the cell surface commonly termed the “shmoo” morphology. Dose-re- sponse curves were determined for each of these in- ducible responses as a function of (Y factor concentra- tion. It is shown that under conditions commonly em- ployed in previous studies, the dose-response for cell division arrest is determined by the rate at which cells inactivate the a factor. In order to achieve conditions where inactivation would not be the dominant param- eter, the cell division response to a factor was moni- tored at low cell densities. Under conditions of essen- tially no a factor destruction, the dose of a factor at which cells exhibit a half-maximal response for cell division arrest (2.5 X M) is nearly the same as that at which cells exhibit a half-maximal response for agglutination induction (1.0 X M). On the con- trary, the half-maximal response for projection for- mation was obtained at doses of a factor 2 orders of magnitude higher (1.4 % lo-’ M). These results are consistent with the same high affinity a factor receptor mediating both cell division arrest and agglutination induction. A different system of lower affinity must mediate projection formation. Alternatively, if the same system and receptor are used, then a much higher occupancy is required for the induction of projections compared to division arrest and agglutination induc- tion.

Polypeptide pheromones are secreted into the medium by each of the two haploid mating types of Saccharomyces cere- uisiue yeast cells, a-factor by MATa cells (1) and (Y factor by MATa cells (2). The purpose of these pheromones is to bring about the initial stages of conjugation (for recent reviews see Refs. 3-6).

a factor has been purified (7) and sequenced (8,9). (Y factor causes MATa cells to arrest cell division at the highly signif-

*Leland H. Hartwell was supported by National Institutes of Health Grant GM-17709. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Postdoctoral Fellowships DRG-171-F from Damon Runyon-Walter Winchell and 1-F32-GM07581-01 from the National Institutes of Health. Present address, Department of Chemistry, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.

icant “start” step which has been characterized genetically (10,11). Cells arrest at start as unbudded mononucleated cells prior to DNA synthesis (12). Cells grow up to 30 times their normal size when arrested (13). Arrested cells eventually recover to resume cell division; longer arrest times ( i e . later recovery) occur a t higher LY factor concentrations (14). Recov- ery from division arrest has been attributed to the inactivation of a factor by a cell-associated protease which cleaves a factor between Leu-6 and Lys-7 (15-17). Consistent with this are the independently reported observations that faster a factor inactivation (16) and earlier recovery from arrest (14) both occur a t higher cell densities.

a factor has many other effects on the cell. It induces the appearance of an agglutinin on the surface of MATa cells which mediates agglutination with MATa cells. Agglutination aggregates are formed of 100-500 cells with an &:cy ratio of approximately 1 (18,19). LY factor also induces a characteristic morphological change in MATa cells called the “shmoo” (20). Shmoo cells typically have one or two narrow projections extending from the cell body. The function of the projection appears to be that of a copulation tube since cell fusion occurs at the tip of the projection (20, 21). Biochemical changes in the cell wall of the projection compared to the remainder of the cell or untreated cells (22, 23) presumably mediate fusion at the projection tip during conjugation.

In addition to the physiological effects mentioned above, a factor induces intracellular protein degradation and vacuole permeability (24), alters the molecular weight of a specific phosphoprotein in MATa but not MAT& cells (25), and at high concentrations inhibits adenylate cyclase in isolated yeast cell membranes (26). a factor induces the formation of a folded chromosome structure which is distinctly different from stationary phase Go and exponential phase G, structures (27). It has been concluded that a factor induces a microdif- ferentiated pathway in the cell (27).

In sorting out this plethora of responses to a factor, we would like to know the sequence of events which are necessary and sufficient for each response and what common elements are used in the mechanisms of the various responses. A quantitative kinetic approach to this problem is described here, in which dose-response curves for various changes in- duced by a factor are compared under an identical set of conditions. This type of quantitative comparison can help to elucidate whether the different responses are mediated through a common element, and in fact the data are consistent with arrest and agglutinin induction occurring through a common a factor saturable site, or receptor.

Several assays for a factor have been reported but there are difficulties associated with them. The most common involves a measure of shmoo cells (i.e. unbudded cells containing

13849

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13850 a Factor-induced Responses in Yeast

projections) versus a factor concentration (7, 16). The shmoo cell assays are semiquantitative (3), and the average pure a factor concentration which I can estimate to produce a half- maximal response has varied from 2 X lo-" M (9, 28, 29) to 2 X M (16), with a variety of intermediate values (4, 7, 17, 30). No dose-response curves of this assay have been shown in the literature. Similarly the average a factor con- centration to produce a half-maximal agglutination response has varied from 6 X 10"' M (31) to 4 X M (32), with a variety of intermediate values (4, 33, 34). There is only one reported dose-response curve for cell division arrest, which is based on a factor inhibition of DNA synthesis (31). It shows an a factor concentration to produce a half-maximal response of 10-6-10-7 M (31, 33). These large variations in the amount of a factor to produce half-maximal responses may be related to strain and other assay condition differences, but much of the discrepancy is almost certainly due to uncontrolled a factor destruction by the MATa cell-associated protease, as will be demonstrated in this paper. It was felt that there was a need to make the assays more rigorous so that they could be quantitatively compared, and this was done for agglutina- tion and projection formation. Two new cell division arrest assays were devised here and are the N4/NO and the % u B ~ h assays.

MATERIAL AND METHODS'

RESULTS

Cell Division Arrest Assays-Two quantitative assays for the induction of cell division arrest by a factor were developed in this study. The first is based on the kinetics of cell number increase over time in the presence of a factor (Fig. 5A). This kinetics has been described previously (14). The cell number at 4 h (where the cells have doubled at least once) divided by the initial cell number (N4/NO) is plotted against initial a factor concentrations in Fig. 1A to provide a quantitative assay for a factor-induced cell division arrest at high cell concentrations (?IO5 cells/ml).

The second assay procedure, used at both high and low cell concentrations (10-107 cells/ml), is based on the kinetics of the increase in per cent unbudded cells over time after the addition of a factor (Fig. 5B). These kinetics have also been previously described (3,14). The assay based on these kinetics is a measure of the per cent unbudded cells at 3 h (%uB3h) after the addition of a factor (Fig. 1B). The Kb0 is defined as the a factor concentration which produces 50% of the maxi- mum response in a given assay. The value of -1/K50 is given by the x axis intercept of the double reciprocal plots. The K50 of the N4/NO assay was compared to that for the % u B ~ h assay at 6 X lo5 cells/ml and found to be identical under identical conditions (see Fig. 2). This is expected because upon recovery from 100% arrest, cell number begins to change about 1 h after the %UB (14). Thus, cell number measurements are made 1 h later than %UB in order to obtain proportional changes in the two parameters at identical a factor concen- trations.

Effect of Cell Concentration and a Factor Inactivation on K50 for Cell Division Arrest-The K50 for cell division arrest

Portions of this paper (including "Materials and Methods," part of "Results", Figs. 5-7, and Tables I1 and 111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 83M-602, cite the authors, and include a check or money order for $5.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

A

B

4 4

,d'O/C.fl

0 2 4 6 8 ' t " Z M

1010 Caf 1 FIG. 1. Dose-response curve for a factor-induced cell divi-

sion arrest. A, the N4/No arrest assay. Varying concentrations of a factor were added to cell cultures. Final conditions at zero time were 1.1 X lo6 381G cells/ml in YM1 + 2% glucose, pH 3.6, 5 ml, 23 "C. At zero time and 4 h after the addition of a factor, 0.2-ml aliquots were added to quenching solution and counted for cell density. The value of N4/N0 represents the cell density at 4 h divided by that at zero time. The inset is the double reciprocal plot of this data where A = N4/No (zero af) - N4/No (plus af). The plot of A versus [afl is a rectangular hyperbola (data not shown). B, the %U&haSSay. Varying concentrations of a factor were added to cell cultures. Final conditions at zero time were 90 381G cells/ml in 100 ml of prefiltered YM1 + 2% glucose, pH 5.8, 23 "C. At 3 h, 10 ml of 37% prefiltered formal- dehyde were added to each culture. Cells were collected, sonicated, and assayed for per cent unhudded cells (%U&h). The inset is the double reciprocal plot of this data where A = %UB3h (plus af) - %U&h (zero af). The plot of A versus [afl is a rectangular hyperbola (data not shown).

by CY factor was found to depend on cell concentration above lo5 cells/ml, but not below lo3 cells/ml (Fig. 2).

It is likely that the dependence of K50 on cell concentration is due to the cell-associated inactivation of a factor by prote- olysis (15-17) because a factor proteolysis occurs more rapidly at higher cell concentrations (16). To determine the exact extent of a factor inactivation, the disappearance of a factor activity was measured under the conditions of Fig. 2 using the agglutination assay. The disappearance of a factor activity was first order in all cases for at least three half-lives (Fig. 6). Such kinetics are expected for an enzymic reaction where the substrate concentration is at least 100 times smaller than the K, value and where there are no cooperativity effects (43). The initial concentration of a factor used in these experiments (lo-' M) is well below the reported K, of 2.4 X M for a

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CY Factor-induced Responses in Yeast 13851 TABLE I

a factor KN values

10" KW arrest" 10" Km agglutinationb 10'' KW agglutination + 10 mM TAME*

10'' Km projections"

M M M M

YM1 (pH 5.8) + 2% glucose 381G 2.5 f 0.1 1.0 f 0.2' 1.2 f 0.4 1400 A364a 0.7 f 0.2d

7041a 1.4 f 0.1 14 f 5 5 f 1 X2180-1A 1.5 f 0.1 13 f 6 2 f 0.5

1.0 k 0.2 220 f 70 40 f 10 YM1 (pH 3.4) + 2% glucose

MIN (pH 3.5) + 2% glucose 381G 0.2 f 0.05' 0.2 f 0.05

X2180-1A 2.5 k 1.0 Assay at 10' cells/ml, 23 "C. Assay at 4 X lo6 cells/ml, 23 "C.

e No effect was observed on the dose-response curve, including the KW value, upon adding into the assay with a factor 10 mM CAMP or 1.7 PM trypsin or pepsin-cleaved and -inactivated a factor preincubated with the cells 1 h and included in the assay.

dAssay at 1 X lo6 cells/ml, 23 "C. e No effect was observed on the dose-response curve, including the Ks, value, upon adding into the assay with a

factor 10 mM Ne-p-tosyl-L-lysine methyl ester, 10 mM CAMP, 45 p M leuteinizing hormone, or 4 pg/ml of tunicamycin.

cells/ml FIG. 2. The dependence of Kao for cell division arrest on

cell concentration. Kso is defined in the text and was determined by the N4/No (0) or %UB3h (0) assay. Final conditions were 381G cells in YM1 + 2% glucose, pH 5.8, 23 "C, and cell concentrations at zero time as indicated.

factor proteolysis by X2180-1A cells in YPD medium (16). First order rate constants for a factor inactivation at var-

ious average cell concentrations were calculated as described under "Materials and Methods." The plot of kobs against cell density was linear, and the slope yielded the second order rate constant for cy factor inactivation, k, = 6.5 X lo-' C-* min" where C = cells/ml.

The fraction (F) of a factor remaining was calculated for the experiment of Fig. 2 using this second order rate constant and Equation 1,

where t is the time of assay in minutes and C is the average cell concentration during the assay. At initial cell concentra- tions of lo2 and lo3 cells/ml, there is 0.9998 and 0.998 a factor remaining, respectively, after 3 h. That is, there is essentially no a factor destruction during the assay where the &O(arrest) is constant uersus cell concentration. At lo5, IO6, and lo7 cells/ ml, there is 0.86, 0.21, and 2 X cy factor remaining, respectively, after 4 h. Thus, there is a significant loss of a factor activity at the time of assay where the K50(arrest) increases with increasing cell concentration.

These data are consistent with the &O(srrest) values above lo5 cells/ml being dominated by the kinetics of a factor inactivation. The K50(arrest) value below lo3 cells/ml must re- flect some other process because cy factor destruction is insig- nificant at these low cell concentrations. The 1(50(arr&) values measured at 10' cells/ml, where there is no a factor inactiva- tion during the assay, are given in Table I for various cell strains and culture conditions.

K50 Values for a Factor-induced Agglutination of MATa Cells-Preincubation of MATa cells with a factor increased their agglutinability with nongrowing tester MATa cells in all cell strains tested (Table 111). The dose-response curve for a factor induction of agglutinability is shown in Fig. 3. The &O(aggl) values obtained from dose-response curves for various cell strains and assay conditions are given in Table I. The independence of &(aggl) for 381G cells on the change in cell density between 1 and 4 X IO6 cells/ml and the lack of any effect by the addition of 10 mM TAME' into the assay (Table I) indicate that no significant a factor inactivation is occur- ring in the assay for this particular cell strain. TAME is a protease inhibitor which inhibits a factor destruction (15). The &(eggl) values for other cell strains are significantly lowered by the addition of 10 mM TAME with a factor (Table I), indicating that a factor inactivation is occurring in these cases. TAME at 10 mM had no effect on the cell doubling time.

The addition of CAMP, trypsin- and pepsin-inactivated a factor, luteinizing hormone, and tunicamycin had no effect on the K50(&) (Table I, footnotes). Therefore, no effector of the agglutination response has yet been found, whereas sev- eral of the above compounds do effect the shmoo cell response (26, 44).

Ks0 for a Factor-induced Projection Formation-Cells which are arrested for division by a factor grow large and form unusual shapes consisting of one or more large pointed pro- jections extending from the cell body. This is referred to as the shmoo morphology (20, 22, 23). I t was found here that projections are formed a t high (210-* M) but not low (<lo-' M) a factor concentrations (see Fig. 7). At 4 h in 4 nM a factor, cell division is arrested and 295% of the cells are

* The abbreviations used are: TAME, NO-p-tosyl-L-arginine methyl ester; af, a factor; LH, luteinizing hormone; TLME, N"-p-tosyl-L- lysine methyl ester; YNB, yeast nitrogen base.

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13852 a Factor-induced Responses in Yeast

r

2or 1o1o Car 1

FIG. 3. Dose-response curve for (Y factor-induced cellular agglutination. Varying concentrations of a factor were added to cell cultures. Final conditions at zero time were 4 X lo6 381G cells/ml in 5-ml of YM1 + 2% glucose, pH 5.8,23 "C. After 60 min, 100 pg/ml of cycloheximide were added to stop the agglutination induction reaction and cell growth. Cells were then assayed for their ability to agglutinate with EMS63 MATa cells and the per cent agglutination (% Aggl.) was calculated as described under "Material and Methods." Errors are &3% agglutination on each point. The inset is the double recip- rocal plot of this data.

unbudded IO0 p'-Q--"-

% cells 8

i W 1 I I I

0 2 4 6 E M

108 C o f l FIG. 4. Dose-response curve for a factor-induced projection

formation. Final conditions at zero time in each culture were lo2 381G cells/ml in 100 ml of prefiltered YMl + 2% glucose, pH 5.8, 23 "C, and varying concentrations of a factor. At 7 h, 5 ml of 37% prefiltered formaldehyde were added to each culture. Cells were then collected, sonicated, and assayed for per cent of cells containing projections (0) and per cent unbudded cells (0). The inset is the double reciprocal plot of this data where [afleorr = [ a f l , d u - [aflnoresponse. The highest value of a factor which did not induce a projection formation response, [afln0 was 5 nM.

unbudded. Under these conditions, the cells retain a round symmetrical shape. After 12.5 h in 4 nM a factor, the cells assume large ovoid shapes lacking projections. In contrast, at 100 nM a factor, the unbudded cells at 4 h have a sharp pointed projection at one end. After 12.5 h, the cells have varying shapes with typically one or two projections protrud- ing from a rounded end. Thus, cell division arrest and shmoo formation are not obligatorily coupled events. After 12.5 h, the total cell volume is significantly lower for cells in 100 nM compared to 4 nM a factor (Fig. 7), indicating that high a factor concentrations inhibit the rate of increase in cell vol- ume.

The per cent of cells in a population which contain projec- tions after 7 h in a factor is dependent on the a factor concentration (Fig. 4). A &,(proj) of 1.4 X lo-* M a factor is obtained from Fig. 4 and is listed in Table I for 381G cells in YM1, pH 5.8, medium. This value was obtained at 10' cells/

ml where no a factor destruction occurs during the assay. A similar KSO(proj) value was obtained when projection formation was assayed at 4 h after the addition of a factor, although the experimental error in this case was larger because projections were smaller and more difficult to monitor.

a factor concentrations of less than 5 nM gave no observable projections after 7 h (Fig. 4). This means either there is a threshold a factor concentration below which projection for- mation cannot occur, or the assay is not sensitive enough to detect small projections or associated morphological changes which may occur below 5 nM a factor. In order to obtain a linear double reciprocal plot for projection formation, it was necessary to correct the a factor concentration as described in the legend of Fig. 4.

DISCUSSION

The Mechanistic Determinant of KSO(arreat) at Different Cell Densities-The &(arrest) is dependent on cell concentration above lo5 cells/ml, and independent of this parameter below lo3 cells/ml (Fig. 2). This is because different biochemical effects determine the kinetics of cell division arrest and re- covery, and therefore the observed &(arrest) value, at high and low cell densities.

At lo7 cells/ml, the K50(arrest) is very high at 2 X loT7 M (Fig. 2). From the second order rate constant for a factor inacti- vation, k, = 6.5 X lo-' C-' min", it can be calculated that at the 4-h time of assay there is 2 X of the original a factor concentration remaining (8 X 1O"j at 3 h). That is, nearly all of the a factor has been destroyed at the time of assay. This indicates that the quantitative value of the K50(arrest) at IO7 cells/ml is determined predominately by the mechanism of inactivation of a factor by the cell-associated protease previ- ously reported (15-17). It is noteworthy that the value of ki, which was determined here by the loss of a factor activity in the agglutination assay, is in good agreement with the values of ki which I was able to calculate from published data which directly measure the proteolytic cleavage of a factor. These values are 2.3 X lo-' C-' min" for X2180-1A cells in SD medium, 25 "C (15) and 2.6 x 10-l' C" min-l for X2180-1A cells in YPD medium, 23 "C (16).

Below lo3 cells/ml, the of 2.5 X 10"' M is inde- pendent of cell density (Fig. 2). This observation alone strongly suggests that a factor inactivation by the cell-asso- ciated protease is not a dominant parameter in the kinetics of arrest below lo3 cells/ml because decreasing the cell (and therefore the cell-associated protease) concentration has no effect on the observed K50 value. Consistent with this is the calculation from the ki value that 0.998 or more of the original a factor concentration is present at the 3-h time of assay at cell densities below lo3 cells/ml. In fact, at 1 nM a factor and 10' cells/ml when most or all of the cells had recovered and recommenced cell division, the old "conditioned" medium minus the original cells was capable of arresting fresh cells with identical kinetics of arrest and recovery as the original cell population (45). This demonstrates unequivocally that there was no significant inactivation of a factor activity at 10' cells/ml under these conditions. It is concluded that cells can recover from arrest without a factor inactivation by becoming insensitive, or desensitizing, to a factor (45). Thus, the kinetics of arrest and recovery at 10' cells/ml represents this slow, rate-determining recovery by desensitization (45). The K50(arreat) values in the intermediate range of 104-106 cells/ ml are expected to be dependent on both recovery by a factor inactivation and recovery by desensitization, with a factor inactivation predominating at the higher and desensitization predominating at the lower cell concentrations.

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01 Factor-induced Responses in Yeast 13853

Protease and Protease Inhibitors-Studies on cell division arrest in the absence of significant a factor destruction can probably be achieved at higher cell densities using the burl (39) or sstl (40) mutant that is deficient in the protease which inactivates CY factor. Alternatively, where it is desirable to use strains previously reported, or wild type strains, the modified forms of a factor in which Lys-7 is replaced by D-LYS or Arg may be used. These forms of CY factor are inactivated very slowly or not at all (28, 41).

The protease inhibitor TAME was found (data not shown) to potentiate the division arrest response of a factor (i.e. lower the K5,)(srrest))r consistent with previous reports (5, 15). How- ever, TAME at concentrations where growth was unaffected (510 mM) did not completely eliminate a factor destruction in the arrest assay which takes 3-4 h. The inability of TAME to eliminate a factor destruction a t these concentrations is due in part to the fact that TAME is hydrolyzed by the proteases it inhibits (see Ref. 15).

On the other hand, TAME does appear to completely inhibit a factor destruction in the agglutination assay which takes only 1 h and has a half-time for induction of agglutin- ation of only 20 min (31, 32, 34). The varying effects of 10 mM TAME in the agglutination assay (Table I) may be caused by different amounts of active protease in the various cell strains. By this criterion, 381G would have the lowest level of active protease because the K50(aggl) is not affected by TAME. Nevertheless, 381G does destroy a factor (Fig. 6).

Independence and Interdependence of Mechanisms-It is clear that arrest and agglutinin induction by CY factor must involve mechanisms which are independent of both significant CY factor inactivation and a-factor-induced and fully formed projections because these are eliminated in the assays where complete arrest and agglutination induction are observed. The data do not rule out the possibility that a very small amount of CY factor inactivation and the occurrence of small scale biochemical changes associated with projection formation are required for complete arrest and agglutinin induction. Shmoo formation has been reported to be independent of CY factor inactivation based on the observation that the inhibition of a factor inactivation by chloroquine produces a proportional increase in the production of shmoo cells (16).

The observation that arrest and agglutination induction by CY factor occur with nearly identical Kso values for 381G cells (Table I) is consistent with a common a factor saturable site, or receptor, inducing both arrest and agglutinin production in MATa cells. Recently binding studies have indicated the existence of a receptor for CY factor on the surface of MATa cells (42), and there is genetic evidence for a receptor (35). On the other hand, the observation that the K50(pro,ections) is approximately 2 orders of magnitude higher than the Kb0 value for arrest and agglutination induction in the absence of inactivation of the CY factor suggests that a different system of lower affinity mediates projection formation. If the same system and receptor are used, a much higher occupancy is required for the induction of projection formation compared to the induction of division arrest and agglutination.

In contrast to the results described here, it has been re- ported that t.he concentration of CY factor needed to induce agglutination in normal MATa cells is 103-105-fold lower than that needed to inhibit DNA synthesis (31) or arrest cell division (4). The results described here indicate that the previous results on cell division arrest required higher a factor concentrations either because of the occurrence of a factor inactivation in the assay at the high cell density used (31) or because the a factor concentration which induced shmoo formation was erroneously taken to be identical with that

needed to induce cell division arrest (4). The Temporal Sequence of Events Induced by a Factor-It

has been claimed that the earliest detectable response of MATa cells to CY factor is the increase in agglutinability towards MATa cells, after which cell division arrest occurs (4, 5, 31). Other workers (3) have made the opposite claim. It was found here at saturating a factor concentrations that whole populations of exponentially growing cells complete the induction of agglutinin in response to CY factor (t1/2 20 min) (31, 32, 34) before all of the cells traverse the cell cycle and display cell division arrest (tsO = 90 min) (Fig. 5B), as previ- ously described (6). However, individual cells just prior to the CY factor execution point arrest cell division and induce agglu- tination in response to a factor nearly simultaneously.

The half-time for induction of agglutination at saturating a factor concentrations is about 20 min (31, 32, 34). In comparison, individual cells near the a factor execution point (the cdc28 step (10)) arrest cell division a t saturating CY factor concentrations with an estimated apparent half-time of 510 min because the per cent unbudded cells begins to increase after a lag of only 10-30 min after the addition of a factor (3) (Fig. 5B). Ten minutes is an upper limit on this value because at least part of the lag is not due to arrest, but occurs because unbudded cells located after the CY factor execution point continue through the cell cycle. I t can be concluded that the half-times and K,, values (Table I) for arrest and agglutina- tion induction are similar, and therefore arrest and agglutin- ation induction will occur nearly simultaneously for individual cells at all CY factor concentrations.

Acknowledgments-I am grateful to Professor Leland H. Hartwell for helpful discussions, laboratory space, and equipment, Dr. Michael J . Gresser for support during the writing of the manuscript, and Janice Abbott for help in preparation of the manuscript.

REFERENCES 1. Wilkinson, L. E., and Pringle, J . R. (1974) Exp. Cell Res. 89,

175-187 2. Duntze, W., MacKay, V. L., and Manney, T. R. (1970) Science

(Wash. D. C.) 168, 1472-1473 3. Manney, T. R., Duntze, W., and Betz, R. (1981) in Sexual

Interactions in Eukaryotic Microbes (O'Day, D. H., and Horgan, P. A., eds) pp. 21-29, Academic Press, New York

4. Thorner, J. (1980) in The Molecular Genetics of Development (Leighton, T. J., and Loomis, W. A., Jr., eds) pp. 119-178, Academic Press, New York

5. Thorner, J. (1981) in The Molecular Biology of the Yeast Saccha- romyces cereuisiae (Broach, J., Jones, E., and Strathern, J., eds) pp. 143-180, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

6. Yanagishima, N., and Yoshida, K. (1981) in Sexual Interactions in Eukaryotic Microbes (O'Day, D. H., and Horgan, P. A., eds) pp. 261-295, Academic Press, New York

7. Duntze, W., Stotzler, D., Bucking-Throm, E., and Kalbitzer, S. (1973) Eur. J. Biochem. 35,357-365

8. Stotzler, D., Kiltz, H.-H., and Duntze, W. (1976) Eur. J. Biochem.

9. Tanaka, T., Kita, H., Murakami, T., and Narita, K. (1977) J .

10. Hereford, L. M., and Hartwell, L. H. (1974) J. Mol. Biol. 84,

11. Reed, S. I. (1980) Genetics 95, 561-577 12. Bucking-Throm, E., Duntze, W., Hartwell, L. H., and Manney,

T. R. (1973) Exp. Cell Res. 76,99-110 13. Throm, E., and Duntze, W. (1970) J. Bucteriol. 104, 1388-1390 14. Chan, R. K. (1977) J. Bucteriol. 130, 766-774 15. Ciejek, E., and Thorner, J. (1979) Cell 18, 623-635 16. Finkelstein, D. B., and Strausberg, S. (1979) J. Biol. Chem. 254,

17. Maness, P. F., and Edelman, G. M. (1978) Proc. Natl. Acud. Sei.

18. Yoshida, K., and Yanagishima, N. (1978) Plant Cell Physiol. 19,

69,397-400

Biochem. (Tokyo) 82, 1681-1687

445-456

796-803

U. S. A. 75, 1304-1308

1519-1533

by guest on October 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

13854 a Factor-induced Responses in Yeast

19. Kawanabe, Y., Yoshida, K., and Yanagishima, N. (1979) Plant

20. Tkacz, J . S., and MacKay, V. L. (1978) J. Cell Biol. 80, 326-333 21. Lipke, P. N., Taylor, A., and Ballou, C. E. (1976) J. Bacteriol.

22. Schekman, R., and Brawley, V. (1979) Proc. Natl. Acad. Sci. U.

23. Lipke, P. N., and Ballou, C. E. (1980) J. Bacteriol. 141, 1170-

24. Sumrada, R., and Cooper, T. G. (1978) J. Bacteriol. 136, 234-

25. Finkelstein, D. B., and McAlister, L. (1981) J. Biol. Chem. 256,

26. Liao, H., and Thorner, J . (1980) Proc. Natl. Acad. Sci. U. S. A.

27. Pinon, R., and Pratt, D. (1979) Chromosoma (Berl.) 73, 117-129 28. Masui, T., Chino, N., Sakakibara, S., Tanaka, T., Murakami, T.,

and Kita, H. (1979) Biochem. Biophys. Res. Commun. 86,982- 987

29. Masui, T., Chino, N., Sakakibara, S., Tanaka, T., Murakami, T., and Kita, H. (1977) Biochem. Biophys. Res. Commun. 78,534- 538

30. Stotzler, D., and Duntze, W. (1976) Eur. J. Biochem. 65, 257- 262

31. Shimoda, C., Yanagishima, N., Sakurai, A., andTamura, S. (1978)

Cell Physiol. 20, 423-433

127,610-618

S. A . 76,645-649

1177

246

2561-2566

77,1898-1902

Plant Cell Physiol. 19, 513-517 32. Yanagishima, N., Yoshida, K., Hamada, K., Hagiya, M., Ka-

Physiol. 17,439450 wanbe, Y., Sakurai, A., and Tamura, S. (1976) Plant Cell

33. Sakurai, A., Tamura, S., Yanagishima, N., and Shimoda, C. (1977) Agric. Biol. Chem. 41,395-398

34. Doi, S., Suzuki, Y., and Yoshimura, M. (1979) Biochem. Biophys. Res. Commun. 91,849-853

35. Hartwell, L. H. (1980) J. Cell Biol. 85, 811-822 36. Hartwell, L. H. (1967) J. Bacteriol. 93, 1662-1670 37. Johnston, G. C., Pringle, J. R., and Hartwell, L. H. (1977) Exp.

38. Hartwell, L. H. (1970) J. Bacteriol. 104, 1280-1285 39. Sprague, G. F., Jr., and Herskowitz, 1. (1981) J. Mol. Biol. 153,

40. Chan, R., and Otte, C. A. (1982) Mol. Cell Biol. 2, 11-29 41. Samokhin, G. P., Lizlova, L. V., Bespalova, J. D., Titov, M. I.,

and Smirnov, V. N. (1981) Exp. Cell Res. 131,267-275 42. Merkel, G. J., Naider, F., and Becker, J. M. (1981) Fed. Proc. 40,

1759 43. Plowman, K. M. (1972) Enzyme Kinetics, McGraw-Hill, Publi-

cations, New York 44. Stotzler, D., Betz, R., and Duntze, W. (1977) J. Bacteriol. 132,

45. Moore, S. A. (1984) J. Biol. Chem. 259, in press

Cell Res. 105,79-98

305-321

28-35

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a Factor-induced Responses in Yeast 13855

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S A Mooremechanism of alpha factor action.

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1983, 258:13849-13856.J. Biol. Chem. 

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