Vol. 132, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 462-472 Copyright t 1977 American Society for Microbiology

a-Factor from Saccharomyces cerevisiae: Partial Characterization of a Mating Hormone Produced by Cells of Mating Type a RICHARD BETZ,' VIVIAN L. MAcKAY,2* AND WOLFGANG DUNTZE2 Institut fur Physiologische Chemie, Ruhr-Universitat Bochum, 4630 Bochum, Germany,' and Waksman Institute of Microbiology, Rutgers Unitersity, Piscataway, New Jersey 088542

Received for publication 13 April 1977

Conjugation between haploid cells of Saccharomnyces ceretisiae is mediated through the action of diffusible mating hormones, two of which have been designated as a-factor and a-factor. Partially purified fractions exhibiting afactor activity have been obtained from culture filtrates of a cells by ultrafiltration, ion-exchange chromatography, and gel filtration. The a-factor preparations specifically caused both Gi arrest and morphological alterations in cells of a-mating type, whereas a cells, a/a diploids, and nonmating a mutants were not affected. The a-factor activity was found in the culture filtrates of all a strains tested, but not in filtrates of a or a/a cell cultures. The hormone is sensitive to various proteases, showing that it is associated with a peptide or protein. Gel filtration studies suggest an apparent molecular weight >600,000; however, this result may be due to aggregation with carbohydrate present in the preparations. Although the biological activities of a-factor are analogous to those described previously for a-factor, the chemical properties of these two hormones appear to be quite different. In the yeast Saccharornvces cerecisiae, sexual conjugation is achieved through a complex pathway of intra- and intercellular reactions, involving specific mating hormones (2, 5, 10) and a number of genes (21, 22). Conjugation is regulated by the mating-type locus, which is a single, probably multicistronic genetic site (12, 14, 22) that confers the a or a phenotype on haploid cells. Within 2 or 3 h after mixing, a and a cells fuse, forming characteristic dumbbell-shaped diploid zygotes, which subsequently bud off diploid cells. Before fusion, both a and a cells accumulate as single, unbudded cells in the Gl phase of the cell cycle (2, 10), with mating apparently occurring optimally between such Gl-arrested cells (3, 11, 24). If cell fusion in liquid medium is inhibited by constant agitation, the unbudded cells further develop into elongated, pear-shaped forms (2, 6, 10, 17, 21) that have been called "shmoos" (21). Both a and a shmoos are formed when conjugation is prevented (2). Development of the shmoo morphology in a cells is accompanied by substantial alteration in the structure of newly synthesized cell wall material (18). at least some of which is localized at the growing end of the shmoo (J. Tkacz and V. L. MacKay, in press). Similar changes in the surface of' a shmoos and at the conjugation bridge of zygotes f'rom normal mating mixtures also were de-

tected. At least in part, these preconjugational interactions are mediated by diffusible substances generally referred to as mating hormones (5, 8). Although synthesis of and response to the hormones are strictly mating-type dependent, there is as yet no evidence to suggest that these hormones are gene products of the mating-type locus itself (22). The hormone secreted by mating-type a cells, which has been termed a-factor, has been purified and characterized extensively (6, 7, 25). It is a linear oligopeptide, for which the primary structure has been determined (26). Mating-type a cells exposed to the hormone exhibit the GI arrest and morphological changes characteristic of cells in mating mixtures (2, 6, 27). When concentrated filtrates of a cultures were examined for an analogous a-factor, an activity was found that transiently arrested ai cells in G1 but did not evoke a shmoo formation (29). However, when a cells were placed close to a heavy growth of a cells on rich solid medium, limited morphological changes could be detected after several hours; these apparently were caused by a diffusible activity secreted by the a cells (21). The possible involvement of this substance in conjugation was suggested by the absence of any detectable response to the activity in nonmating mutants derived from a parents (21). In addition, the 462

VOL. 132, 1977

a-FACTOR FROM S. CEREVISIAE

majority of the nonmating a mutants isolated did not cause any response in the a assay cells. In this paper, we report experiments demonstrating that culture filtrates from a cells contain one or more substances that specifically inhibit deoxyribonucleic acid (DNA) synthesis and cell division in a cells and, furthermore, induce a cells to form shmoos at high frequency. The activities responsible for Gl arrest and morphological changes copurify and are indistinguishable in their chemical properties and biological effects. Thus, a single matingtype-specific hormone appears to bring about both Gi arrest and shmoo formation in a cells, analogous to the action of a-factor on a cells. MATERIALS AND METHODS Yeast strains. The strains used in this study and

463

their genotypes and sources are listed in Table 1. X2180 is an a/a diploid that is assumed to be homozygous at all loci except the mating-type locus (R. K. Mortimer, personal communication). X21801A and X2180-1B are haploid a and a spore clones obtained by dissection of asci derived from X2180 and are presumed to be otherwise isogenic. These strains were used in all experiments, unless noted otherwise. Culture conditions. The culture medium in all experiments was a minimal glucose medium (MV) containing 0.67% yeast nitrogen base without amino acids (Difco) and 2% glucose (28). When necessary, MV was supplemented with the following nutrients (milligrams per liter): adenine, 30; arginine, 30; histidine, 20; leucine, 40; lysine, 40; methionine, 20; tryptophan, 30; uracil, 20 (21). All cultures were incubated at 30°C with aeration. Preparation and concentration of a-factor activity. Mating-type a cells were inoculated at a density

TABLE 1. Yeast strains and their genotypes X2180 XT1172 XV320

XV321 XV323 XV326

a gal2 a gal2 a hom2 thrl arol trp5-1 leul ade6 lysl his6 ural arg4 CUP1 gal2 + + + + + + + + + gal2 + a + + ainol-13ino4-8gal2canl + + + + a + + gal2 canl ade6 his6 leul metl trp5-1 a leu2-1 ade8 thr4 ura3 lys2 gal2 + + + a + + + gal2 his4-12 + + a leu2-1 ade8 thr4 ura3 Iys2 gal2 + + + + gal2 his4-290 ade2-1 a + + a leu2-27 ade8 lys2 + a + + + gal2 his4-1111

X2180-1A

agal2

XT1 177-S47c XJ24-17a A4703-7C XY506-6D X2180-1B

a ade2-1 his2 lysl-1 trp5-18 gal2 canl a ade6 Iys2 arg4-17 a his4-12 a his5-2 lysl-l ura4-1 umr7 a gal2

XT1172-S185 A4703-9C 4571-1B XY505-18C XT1 172-S245c VC2b VC3 b VC8b VC73 b

Source

Genotypea

Strain

a ade6 leul gal2

T s Yeast Genetics Stock Center T. R. Manney X

1-

I-

V. L. MacKay V. L. MacKay

V. L. MacKay V. L. MacKay Yeast Genetics Stock Center T. R. Manney I. Herskowitz G. Fink J. Lemontt Yeast Genetics Stock Center T. R. Manney G. Fink R. Roth J. Lemontt T. R. Manney V. L. MacKay V. L. MacKay V. L. MacKay V. L. MacKay V. L. MacKay V. L. MacKay V. L. MacKay V. L. MacKay

his4-12 leu2-1 ade2-1 his4 his5-2 lysl-l ura4-1 ade6 his6 leul metl trp5-1 gal2 canl stel-2 (class 1) a ste3-3 (class 4) a ste (class 8) a ste (class 10) a stel-5 (class 2) VN33b a ste3-1 (class 4) VQ3b a ste (unclassified) ade2-1 lysl-l trp5-18 ural gal2 canl VB5 a ste (class 12) ade2-1 lysl-l trp5-18 ural gal2 canl VB7 a Gene symbols: Mating type-a or (haploid) or a/a (diploid). Mutations that lead to nutritional requirements: ade (adenine), arg (arginine), aro (aromatic amino acids), his (histidine), hom (homoserine), ino (inositol), leu (leucine), lys (lysine), met (methionine), thr (threonine), trp (tryptophan), ura (uracil); other mutations: can (canavanine resistance), CUP (resistance to copper), gal (inability to ferment galactose), umr (resistance to mutagenesis by ultraviolet light). t These mutant strains were derived from XT1172-S245c and have the same genotype. a a a a a

a

464

BETZ, MAcKAY, AND DUNTZE

of 107 cells per ml and grown aerobically for 24 h at 30°C on a reciprocal shaker at approximately 200 rpm or in 20-liter carboys that were vigorously aerated by bubbling air through the medium. The cells were removed by centrifugation or, if large cultures were grown, by filtration through porcelain filter candles as described previously (7). The cellfree filtrates were concentrated 10- to 100-fold by rotary evaporation or by hollow-fiber ultrafiltration, using an Amicon HIDP 10 hollow-fiber membrane cartridge, which retained more than 90% of the biological activity. Determination of a-factor activity. Two assay procedures were used to determine a-factor activity: low concentrations were tested by following the transient inhibition of exponentially growing cultures of a cells, whereas higher concentrations could be more conveniently assayed by monitoring the formation of shmoos. For growth inhibition tests, 4.0 ml of the sample to be assayed (adjusted to pH 5) and 1.0 ml of 5x-concentrated MV medium were added to 5.0 ml of an exponentially growing culture of a cells at a density of 2 x 106 to 4 x 106 cells per ml. The test cultures were incubated at 30'C on a reciprocal shaker. Samples of 0.2 ml were removed at intervals, and the cell numbers were determined in a model FN Coulter Counter, as described previously (2). High concentrations of a-factor were assayed by looking for morphological changes in a cells. Assay mixtures contained 1.5 ml of the sample, 0.5 ml of 1.0 M KPO4, pH 5.0, 0.5 ml of 5x-concentrated MV (with supplements, if necessary), and 2.5 ml of exponentially growing a cells at a final concentration of 106 cells per ml. The assay cultures were incubated with aeration at 30'C for 4 to 5 h and examined microscopically for shmoos. By testing twofold serial dilutions of the active fractions, a semiquantitative determination of a-factor was possible. The most dilute test culture that had a shmoos was defined as containing 1 U of a-factor per ml. Chromatographic methods. (i) SP-Sephadex. Concentrated (100-fold) filtrate containing a-factor (200 ml) was subjected to diafiltration in an Amicon DC2 ultrafiltration system equipped with an HlDP 10 hollow-fiber cartridge, using 20 mM sodium citrate, pH 3.0 (1,000 ml), as a buffer. The sample was then concentrated to 30 ml and applied to a column (2.5 by 25 cm) of SP-Sephadex C-25, previously equilibrated with the same buffer. Elution was carried out at a flow rate of 50 ml/h with 100 ml of the buffer, followed by a linear gradient (800 ml) of 0 to 0.5 M NaCl in 50 mM sodium phosphate, pH 5.0. (ii) Phosphocellulose. Cell-free unconcentrated culture filtrate (at approximately pH 2.6) was applied directly to a column of phosphocellulose, previously equilibrated with 0.05 M KPO4, pH 6.0. For 4 liters of filtrate, a 50-ml column (2.5 by 10 cm) was used. After washing with 75 ml of 0.05 M KPO4, pH 6.0, the activity was eluted with 0.5 M KPO4, pH 6.0. The active fractions were adjusted to pH 5.0, 10% glycerol, and 1 mM ethylenediaminetetraacetate and stored at either -70 or -20'C, where they are stable for at least 1 year. The ability of different strains to produce a-factor

J. BACTERIOL. was determined by assaying fractions from phosphocellulose chromatography of their culture fluids, as described above. Cultures (50 ml) were prepared in MV, supplemented as necessary; in all cases, the standard a strain (X2180-1A) was grown simultaneously in the same medium to eliminate the possibility of medium effects. For each culture, the supernatant fluid was applied to a 1-ml column (0.5 by 5 cm) of phosphocellulose, which was washed with 1.5 ml of 0.05 M KPO4, pH 6.0, and eluted with 10 ml of 0.5 M KPO4, pH 6.0. Either 1- or 2.5-ml fractions were collected, adjusted to pH 5, and assayed for activity that caused a cells to form shmoos. (iii) Gel filtration. Sephadex G-50 and G-200 or Bio-Gel P-100, equilibrated with 0.05 M sodium acetate (pH 5.0)-0.2 M sodium chloride, were used for gel filtration of active fractions. Determination of DNA and RNA synthesis. Incorporation of 1I'Cluracil into DNA and ribonucleic acid (RNA) was measured according to the method of Hartwell (9). The final concentration of uracil in the cultures was 0.1 mM, with a specific radioactivity of 10 mCi/mmol. l)etermination of protein and carbohydrate. Protein was determined by the procedure of Lowry et al. (19), and total carbohydrate was determined by the anthrone method of Hodge and Hofreiter (15). Enzymatic digestions. (i) Pronase treatment. The reaction mixture contained 1.0 ml of an active a-factor preparation in 0.1 M KPO4, pH 5.0, and 1.5 mg of Pronase per ml. After incubation for 1 h at 37°C, the mixture was boiled for 5 min and then assayed for a-factor. The a-factor activity is not sensitive to boiling. (ii) Trypsin treatment. An a-factor sample was incubated in 0.1 M triethanolamine buffer, pH 7.7, with 0.5 mg of trypsin per ml for 3 h at 37°C and then boiled for 5 min and assayed. (iii) Ribonuclease and deoxyribonuclease treatment. A 1.0-ml sample of a-factor was adjusted to pH 5.0 with 0.1 ml of 1.0 M sodium acetate and incubated for 30 min at 37°C with either 17 ,ug of ribonuclease per ml or 17 4g of deoxyribonuclease per ml with 3 mM MgCl2. Activity of the ribonuclease was verified by adding to the reaction {H-labeled viral RNA (from vesicular stomatitis virus) which was rendered almost totally acid soluble. Activity of the deoxyribonuclease was demonstrated by its ability to digest to acid solubility sH-labeled Escherichia (c0oi DNA in the reaction mixture. Chemicals. SP-Sephadex C-25 and Sephadex G50 and G-200 were obtained from Pharmacia Fine Chemicals, Inc. (Piscataway, N.J.) and from Deutsche Pharmacia GmbH (Frankfurt am Main, Germany). Phosphocellulose (Whatman P-11) was purchased from Reeve Angel (Clifton, N.J.), and Bio-Gel P-100 was from Bio-Rad Laboratories (Richmond, Calif.). Pancreatic deoxyribonuclease, pancreatic ribonuclease, and trypsin were obtained from Worthington Biochemicals Corp. (Freehold, N.J.), and Pronase was from Calbiochem (La Jolla, Calif.). IHIRNA from vesicular stomatitis virus was the gift of E. Hefti (Rutgers University, New Brunswick, N.J.) and PHIDNA from E. coli was prepared by the method of Lehman (16).

VOL. 132, 1977

a-FACTOR FROM S. CEREVISIAE

465

portional to the logarithm of the added amount of a-factor (Fig. 2, insert). This relation allowed a quantitative comparison of different preparations of the inhibitory activity under strictly controlled conditions. At a-factor concentrations for which the period of inhibition exceeded one generation time, the arrested a cells developed the shmoo morphology (Fig. 3). Thus, the same type of morphological alteration observed in a cells treated with a-factor can also be induced in a cells exposed to concentrations of a-factor that are greater than the minimal amount needed to evoke GI arrest. Like the inhibition of a cell division induced by a-factor, the morphological response to afactor is unique to a cells. Only a cells (X21801B) developed into shmoos; a cells (X2180-1A) and a/a diploids (X2180) were resistant to the activity even at high concentrations (6 to 8 U/ ml). The ability of a cells to form shmoos is highly pH dependent, occurring only between pH 4 and 6, although the a-factor activity itself is stable over a wide range of pH values (at least from pH 2 to 7.6). It is of interest that the optimal pH for conjugation has been reported to be 4.5 under certain incubation conditions (1). Partial purification of a-factor. Whereas afactor can be concentrated by chromatography on Amberlite CG50 (6), the a-factor activity

RESULTS Detection of a-factor activity. The presence of an activity that specifically inhibits cell division of mating-type a cells was demonstrated in filtrates of cultures of wild-type a cells (strain X2180-1A) that had been grown to stationary phase. Concentration of the cell-free filtrates by ultrafiltration yielded preparations that caused a transient inhibition of cell division when added to an exponentially growing culture of a cells (Fig. 1). Neither haploid a cells nor a/a diploid cells were inhibited by the same preparations. Furthermore, analogous preparations from cultures of a cells (X21801B) or of a/a diploids (X2180) did not inhibit cell division of the a cells. It was, therefore, assumed that the inhibitory activity is identical to the a-factor described by Wilkinson and Pringle (29). Depending on the amount of the a-factor preparation added, a cells are arrested for 0.5 to 5 h and then resume growth at the same rate as the uninhibited controls (Fig. 2). The extent of inhibition is also dependent on the concentration of a cells and is somewhat influenced by assay conditions, such as aeration and pH of the cultures. Under identical conditions, however, the period of inhibition is pro 10' X 2180-1B

X 2180-1A

8-

6-

4-

0

2-

E

0

2

4

6

8 0

2

4

6

0

2

4

6

time (hours)

FIG. 1. Mating type specificity of a-factor inhibition. To cultures of isogenic strains growing exponentially at 30'C in MV medium, 0.1 ml of 200-fold-concentrated culture filtrate of X2180-1A (a) cells was added per ml of culture at 0 time. Closed symbols: Control cultures (no addition); open symbols: filtrate added. (0, 0) X2180-1B (a); (-, O) X2180-1A (a); (A, A) X2180 (a/a).

466 _

12

E

_

16-

0/

-

10 -

l _1 °s1150.5

8-

6

9~~~~~~~*

AAb

2

'

1i2 L

3 45

/

2

t (hotu rs)

(.0

.D

J. BACTERIOL.

BETZ, MACKAY, AND DUNTZE

U,

5-

'"

4 U

z

3-

I~~

n

t-

1

0

2

9

4

6

8

10

time (hours)

FIG. 2. Inhibition of a cell diuision with

increas-

concentrations of partially purified a-factor (SPSephadex eluate). At 0 time, a-factor (diluted in 0.1 M NaPO4, pH 5.0) was added to X2180-IB (a) cells ing

exponentially in MV (1.2 x 106 cells per ml). The cultures were incubated aerobically at 30°C, and the cell density was monitored. Symbols: (@a control, no addition; (A) 0.5 U/ml; (*) 2 U/ml; (0) 4 U/ml; (A) 8 U/ml; (L) 16 U/ml. The insert shows the length of the inhibition period (At) as a function of the concentration of a-factor. Similar results were obtained with less purified preparations of a-tactor.

growing

does not bind to this resin. However, concentration of large volumes of a culture filtrate could be achieved by ultrafiltration through Amicon HlDP 10 membranes, which retain a-factor. Without increasing the salt concentration of the active preparation, this method permitted a 100-fold concentration of 15 liters of filtrate within 10 h. When added to growing cultures of cells, the resulting concentrates induced the formation of characteristic shmoos with high efficiency. Thus, the amount of a-factor activity could be determined semiquantitatively by microscopic examination of treated at cells, as described in Materials and Methods. The results of a typical ultrafiltration experiment are summarized in Table 2. Ten liters of' filtrate from a culture of X2180-1A was concentrated approximately 300-fold, yielding a preparation containing 2,000 U of a-factor, 39 mg of protein, and 153 mg of total carbohydrate. Further purification was achieved by chromaa

tography on a column of' SP-Sephadex C-25. The a-factor activity was eluted with a linear NaCl gradient from 0 to 0.5 M NaCl (Fig. 4). The activity eluted between 0.1 and 0.3 M together with the bulk of the bound protein. Active fractions were pooled and assayed for protein, carbohydrate, and a-factor. Approximately 60% of the total activity was recovered after chromatography on SP-Sephadex (Table 2). The specific activity (units per milligram of protein) increased approximately threefold, whereas the total carbohydrate content was reduced by 97%. Fractionation of unconcentrated a culture filtrates by phosphocellulose chromatography yielded recoveries of a-factor activity and protein similac to those obtained by SP-Sephadex (data not shown). However, the active fractions from phosphocellulose contained much more carbohvdrate. which eluted as a peak with the activitv. Chemical characterization of a-factor. The biological activritv in a-factor preparations is sensitive to the action of various proteases. Incubation with Pronase or try-psin. as well as with pepsin. thermolysin, or chymotrypsin, completely destroyed the activity; treatment with deoxyribonuclease or ribonuclease had no effect on the activity. These results indicate that the a-factor activity is associated with a peptide or protein. Upon gel filtration with either Bio-Gel P-100 or Sephadex G-200, the activity was consistently found in the fractions eluting with the void volume (Fig. 5). This elution pattern was independent of the fraction and purity of the afactor applied to the column; i.e., the activity always eluted with the void volume whether the applied sample had been concentrated by rotary evaporation or ultrafiltration or had been partially purified by chromatography on SP-Sephadex or phosphocellulose. Even after boiling in 8 M urea, the activity was excluded from Sephadex G-50. These results suggested that a-factor is quite large (greater than 600,000 daltons if a globular protein), in contrast to the small oligopeptide a-factor (ca. 1,800 daltons). However, this apparent molecular weight could result from aggregation of smaller polypeptides to each other or to the large amounts of carbohydrate present in culture filtrates. Further purification will be required to ascertain whether the carbohydrate is an integral part of the active structure and to detail the chemical nature of the a-factor. Biological activity of partiallv purified afactor. The active fractions of the SP-Sephadex eluate specifically inhibited cell division in exponentially growing cultures of a cells. Approx-

VOL. 132, 1977

a-FACTOR FROM S. CEREVISIAE

467

FIG. 3. Cells of mating type a treated with a-factor. Exponentially growing cells of X2180-1B (a) were incubated in MV with partially purified a-factor for 5 h at 30°C. (a) Control, no addition; (b) 0.5 UIml; (c) 1 U/ml; (d) 4 UIml. TABLE 2. Concentration and partial purification of a-factor from culture filtrates of strain X2180-1A Fraction

Culture filtrate .......... Amicon concentrate ........ SP-Sephadex eluate .......

.......... ........ .........

Vol

Total activity (U)

Total protein (mg)

10 liters 30 ml 55 ml

2,000 2,000 1,200

1,300 39

imately 0.4 generation time preceding the inhibition of a cell division, DNA synthesis was blocked, indicating that the a cells are arrested before the S phase of the cell cycle (Fig. 6). Furthermore, cell division resumed about 35 min after DNA synthesis had started again. The a-factor preparation did not exert any detectable effect on RNA synthesis (Fig. 6).

8

Total carbolhydrate (mg)

1,700 153 4.5

Sp act (U tein)

1.5 51 150

These observations indicate that the hormone might affect in a cells the same cell cycle function that is blocked by a-factor treatment of mating type a cells (2, 11). The quantitative relationship between Gl arrest and shmoo formation is represented in Fig. 7 and Table 3. Increasing concentrations of a-factor were added to a cells growing expo-

J. BACTERIOL.

BETZ, MACKAY, AND DUNTZE

468

nentially in MV, and the period of inhibition (At) was obtained as in Fig. 2. The percentage of arrested cells that developed into shmoos was determined after 6 h by direct counting in 020 a hemacytometer. Table 2 shows that a cells begin to develop into shmoos after they have been arrested for one generation time or longer. (In this experiment, the generation time was 105 min.) As the period of inhiapproximately 015bition is lengthened with increasing concentrations of a-factor, the percentage of shmoos that E developed in the population also increases, reaching an apparent plateau at approximately 3.3 h of inhibition (Fig. 7). Thus, both the 010 length of the inhibition period and the percenta age of shmoos appear to be functions of the afactor concentration. Studies using other strains. In the experi005ments using the isogenic strains X2180-1A (a), X2180-1B (a), and X2180 (a/a), the a-factor activity was found only in culture filtrates of a cells and was effective only against a cells (see a tactor Fig. 1). However, the results might be unique ?,2 to this set of isogenic strains. Therefore, exper40 30 20 50 iments to determine the mating-type specificity traction number of a-factor production and response were exFIG. 4. Elution of a-factor from SP-Sephadex Ctended to additional strains. All of the strains 25. A sample (50 ml) of 300-fold-concentrated culture filtrate of X2180-1A (a) cells was applied to a listed in Table 1 were tested for the presence of column (2.5 by 25 cm) of SP-Sephadex C-25. The diffusible a-factor in the cultures. After the cultures had been grown to stationary phase activity was eluted at a flow rate of 50 ml/h with a linear gradient of NaCl, as described in the text. (approximately 108 cells per ml), the cell-free

Ie I~

-,

activity

10

E

c 0

a 0

n 0

a

50

eluate( ml)

FIG. 5. Gel filtration of a-factor on Sephadex G-200. A sample (2.0 ml) of partially purified a-factor was applied to a column (1.5 by 25 cm) of Sephadex G-200 equilibrated with 0.05 M sodium acetate (pH 5.0, 0.2 M NaCl. The column was eluted with the same buffer at a flow rate of 12 ml/h.

VOL. 132, 1977

a-FACTOR FROM S. CEREVISIAE 100

-

80

-

60

-

469

D

C,

IL 0

E

c3

E

40-

=

1: 20

0

Q

2

E

8

012

16

0-

a- factor(units/ml culture) z C]

z :r

0

1

2

3

4

5

time (hours)

FIG. 6. Inhibition of DNA synthesis and cell dicells treated with low concentrations of afactor. Exponentially growing cells of X2180-1B (a) in MV medium were incubated with 1 U of partially purified a-factor per ml. Cell density and the incorporation of [I4C]uracil into DNA and RNA were monitored. Open symbols: Controls (without addition of a-factor); closed symbols: a-factor added. (0, 0) Cell density; (A, A) DNA; (El, *) RNA. vision in a

supernatant culture fluids were applied to phosphocellulose columns and eluted, as described in Materials and Methods. (For a series of samples of small volume, phosphocellulose chromatography is more practical than ultrafiltration and SP-Sephadex purification.) All of the fractions were assayed for shmoo development in X2180-1B assay cells. For the fractionated and a/a culture fluids, there was no detectable activity in any of the phosphocellulose fractions. However, for all of the a culture fluids, a-factor activity was present in the first 2 ml of the 0.5 M KPO, elution. A comparison of the activity recovered for the five strains is shown in Table 4, although these values must be considered approximate because of the small a

culture volumes used. It is clear that all of the a strains tested secrete a-factor. However, more a-factor activity was obtained consistently from the cultures of X2180-1A. To determine if the response to a-factor is

FIG. 7. Shmoo formation in a cells as a function of the a-factor concentration. Culture filtrate from X2180-1A (a) cells was partially purified through SP-Sephadex chromatography, yielding a fraction containing approximately 50 UIml. At 0 time, 4 ml of an appropriate dilution (in 0.1 M NaPO4, pH 5.0) was added with 1 ml of 5 x MV to 5 ml of X2180-1B (a) cells growing exponentially in MV (final volume, 10 ml with 106 cells per ml in all samples). The cultures were incubated aerobically at 30°C, and the period of inhibition (At) was determined as in Fig. 2. After 6 h, the number of shmoos per milliliter in each sample was obtained by counting in a hemacytometer. To obtain the actual percentage of arrested cells that developed into shmoos, the concentration of shmoos must be related to the concentration of cells in the culture during the inhibition period (= 1.9 x 106/ml), since at 6 h the cells in most of the assays have already resumed growth. See Table 3 for the experimental data. TABLE 3. Quantitative relationship between a-factor concentration and its effects on a cellsa Concn of afactor in assays (U/ml)

Period of inhibition (h)

0.5 1 2 4 8 16

0.75 2.0 2.5 3.3 4.1 4.8

9o% Arrested Shmoos/ml cells that deinto 106) veloped (X

at 6 h

0 0.59 0.92 1.53 1.42 1.43

shmoos b 0 31 48 80 75 75

See the legend to Fig. 7 for experimental detail. bShmoos per milliliter divided by the total cells per milliliter during the inhibition period (= 1.9 x a

106).

470

BETZ, MAcKAY, AND DUNTZE

TABLE 4. Production of a-factor by five independent a strains Strain

Units of a-factora

X2180-1A ......... XT1177-S47c .. ...... XJ24-17a ........ A4703-7C ........ XY506-6D .........

6 2 2 4 3

a Values refer to total units of a-factor recovered after phosphocellulose chromatography of cell-free supernatant fluids from 50-ml cultures of the a strains listed.

mating-type specific and characteristic of all a strains, a number of strains (including all of those in Table 1) were tested for the ability to form shmoos in response to 4 U of a-factor per ml. After 4 to 5 h of incubation, none of the six a/a diploid and eight a strains studied had undergone any morphological changes or obvious Gl arrest, whereas all of the 15 a strains had developed the shmoo morphology. In addition, we have examined eight of the nonmating a mutants, representing six of the original classes of a mutants (22), for the appearance of shmoos in the presence of a-factor (4 U/ml). Although the parent strain formed numerous large unbudded shmoos, none of the mutants exhibited this response. Therefore, both the production of and response to a-factor seem to be specific properties regulated by the mating type of the cell and may be related to the conjugation process.

DISCUSSION The unequivocal demonstration of a mating hormone specifically produced by a cells of S. cereuisiae and effective exclusively in a cells has long been hampered by the lack of a simple and reproducible assay system, although several lines of evidence argued for the existence of such an a-factor. For example, a cells undergo a characteristic morphological alteration when opposed on agar to a heavy overnight growth of a cells (21). However, only a low percentage of the a cells formed shmoos. thereby prohibiting the use of this method as a reliable assay procedure. Wilkinson and Pringle (29) observed that filtrates of a cultures contain an activity that arrests, as unbudded cells, asynchronously growing a cells in the GI phase of the cell cycle. By determining the percentage of unbudded cells, the authors could detect a-factor in concentrated culture filtrates. However, this procedure was very tedious and was not used for quantitation of the activity. In contrast, determination of the transient inhibition of cell division in exponentially

J. BACTERIOL.

growing cultures of

a cells permits the detection of a-factor activity in less-concentrated culture filtrates and may be used under controlled conditions for an approximate quantitation of the a-factor concentration. Using this assay, we have obtained concentrated and partially purified a-factor preparations. which also reproducibly induce shmoo formation in a cells in a suitable medium. Unfortunately, development of a shmoos is poor when the cells are exposed to the a-factor on agar plates, and, even in liquid medium, the morphological alterations are less pronounced than those observed in a cells after exposure to comparable concentrations of a-factor. For comparing the production of a-factor by a number of different strains, the formation of a shmoos was the assay of choice, because many samples and dilutions could be conducted simultaneously. In all experiments, the prototrophic a strain X2180-1A clearly secreted more active a-factor than the other a strains (Table 4). However, it should be noted that the amount of a-factor produced by an a strain appears to be inversely related to the number of its nutritional requirements (Table 1). Thus, the higher recovery of a-factor from X2180-1A cultures may be a function of its general metabolism rather than of any significant difference in its mating characteristics. Only low activity was recovered from cultures of XT1177-S47c, the parent strain of the nonmating a mutants previously described (21, 22). The production of afactor by the parent must be increased before we can determine quantitatively the ability of the various a mutants to secrete a-factor. The reason why the a mating hormone is apparently less active than the corresponding a-factor is not yet understood. Several alternatives have to be considered in addition to the possibility that a-factor might possess a higher intrinsic activity. For example, in a cells treated with a-factor, the inhibition of cell division is readily reversible, and biologically active a-factor is no longer detectable in the a culture when cell division resumes (R. Betz and W. Duntze, unpublished data). Hicks and Herskowitz (13) and Chan (4) have previously shown that a cells also remove, inactivate, or inhibit a-factor in the medium. (A substance secreted by a cells that appears to inhibit afactor activity [13] has been separated from afactor by phosphocellulose chromatography and is distinct from a-factor [V. L. MacKay, unpublished datal). Thus, it is possible that a cell.. might be more resistant to a-factor because of more rapid depletion of the hormone activity. Alternatively, constitutive production or activity of a-factor may be low in most a strains and

a-FACTOR FROM S. CEREVISIAE

VOL. 132, 1977

be increased in mixtures with a cells (20). This possibility is consistent with the presence of a shmoos in mating mixtures (2) but not in populations of a cells incubated with unconcentrated filtrates from pure a cultures. As long as the a-factor has not yet been purified to homogeneity, it is difficult to decide whether the observed effects on a cell morphology and cell division result from the action of the same molecule. However, during the course of the purification, it has not been possible to separate the two activities. Furthermore, with purified a-factor, it has been shown that both effects are brought about in a cells by the same molecular species. Therefore, in the absence of contradictory evidence, we would prefer the hypothesis that both the inhibition of cell division and the induction of morphological changes are caused by the same compound and probably result from the same molecular mechanism of action. The results presented in this paper indicate that the a-factor activity is associated with a protein or peptide, as has been demonstrated for a-factor (7). However, the chemical properties of the hormones differ remarkably. In contrast to the very hydrophobic a-factor, the a-factor activity is not retained on the cation exchanger Amberlite CG50 and is poorly soluble in methanol (R. Betz, unpublished data). Moreover, whereas a-factor readily penetrates standard dialysis membranes, a-factor is retained by membranes that normally do not retain molecules below 10,000 molecular weight. However, after prolonged dialysis of partially purified a-factor preparations, significant amounts of the hormone are found in the dialysate (R. Betz, unpublished data). This observation suggests that the apparent high molecular weight may result from association with other material from which it is only slowly released. It is tempting to propose that the carbohydrate found in partially purified preparations represents such high-molecular-weight impurities. However, as long as there is no more information on the chemical structure of the a-factor, it cannot be excluded that the carbohydrate represents a structural component of the active molecule. ACKNOWLEDGMENTS We thank Madeline van de Burgt-Gozzi and Marie-Luise Petersen for technical assistance and Joan Strazdis for helpful discussion. This research was supported by grants (to V. M.) from the U.S. Public Health Service (GM22149, from the National Institute of General Medical Sciences) and from the Biological Sciences Support Grant awarded to Rutgers University and (to W. D.) by the Deutsche Forschungsgemeinschaft.

471

LITERATURE CITED 1. Bilinski, T., J. Litwinska, J. Zuk, and W. Gajewski. 1973. Synchronization of zygote production in Saccharomyces cerevisiae. J. Gen. Microbiol. 79:285-292. 2. Bucking-Throm, E., W. Duntze, L. H. Hartwell, and T. R. Manney. 1973. Reversible arrest of haploid cells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76:99-110. 3. Campbell, I). A. 1973. Kinetics of the mating-specific aggregation in Saccharomyces cerevisiae. J. Bacteriol. 116:323-330. 4. Chan, R. K. 1977. Recovery of Saccharomyces cereuisiae mating-type a cells from GI arrest by a factor. J. Bacteriol. 130:766-774. 5. Duntze, W. 1974. Control of intercellular cooperation in yeast by hormone-like substances. Postepy Mikrobiol. 13:41-51. 6. Duntze, W., V. L. MacKay, and T. R. Manney. 1970. Saccharomyces cerevisiae: a diffusible sex factor. Science 168:1472-1473. 7. Duntze, W., I). 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 cereuisiae. Eur. J. Biochem. 35:357-365. 8. Gooday, G. W. 1974. Fungal sex hormones. Annu. Rev. Biochem. 43:35-49. 9. Hartwell, L. H. 1967. Macromolecule synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 93:1662-1670. 10. Hartwell, L. H. 1973. Synchronization of haploid yeast cell cycles, a prelude to conjugation. Exp. Cell Res. 76:111-117. 11. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 12. Hawthorne, D. C. 1963. A deletion in yeast and its bearing on the structure of the mating type locus. Genetics 48:1727-1729. 13. Hicks, J. B., and I. Herskowitz. 1976. Evidence for a new diffusible element of mating pheromones in yeast. Nature (London) 260:246-248. 14. Hicks, J. B., and I. Herskowitz. 1976. Interconversion of yeast mating types. I. Direct observations of the action of the homothallism (HO) gene. Genetics 83:245-258. 15. Hodge, J. E., and B. T. Hofreiter. 1962. Determination of reducing sugars and carbohydrates, p. 380-394. In R. L. Whistler and M. L. Wolfrom (ed.), Methods in carbohydrate chemistry, vol. 1. Academic Press Inc., New York. 16. Lehman, I. R. 1960. The deoxyribonucleases of Escherichia coli. I. Purification and properties of a phosphodiesterase. J. Biol. Chem. 235:1479-1487. 17. Levi, J. D. 1956. Mating reaction in yeast. Nature (London) 177:753-754. 18. 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. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 20. MacKay, V. L. 1976. Mating-type specific pheromones as mediators of sexual conjugation in yeast. Proc. Soc. Dev. Biol. 35:000-000. 21. MacKay, V. L., and T. R. Manney. 1974. Mutations affecting sexual conjugation in Saccharomyces cerevisiae. I. Isolation and phenotypic characterization of non-mating mutants. Genetics 76:255-271. 22. MacKay, V. L., and T. R. Manney. 1974. Mutations affecting sexual conjugation in Saccharomyces cerevisiae. II. Genetic analysis of non-mating mutants. Genetics 76:273-288. 23. Scherer, G., G. Haag, and W. Duntze. 1974. Mechanism

472

BETZ, MACKAY, AND DUNTZE

of a-factor biosynthesis in Saccharomyces cerevisiae. J. Bacteriol. 199:386-393. 24. Sena, E. P., D. N. Radin, and S. Fogel. 1973. Synchronous mating in yeast. Proc. Natl. Acad. Sci. U.S.A. 70:1373-1377. 25. Stotzler, D., and W. Duntze. 1976. Isolation and characterization of four related peptides exhibiting afactor activity from Saccharomyces cerevisiae. Eur. J. Biochem. 65:257-262. 26. Stotzler, D., H-H. Kiltz, and W. Duntze. 1976. Primary structure of a-factor peptides from Saccharomyces cerevisiae. Eur. J. Biochem. 69:397-400.

J. BACTERIOL. 27. Throm, E., and W. Duntze. 1970. Mating-type dependent inhibition of deoxyribonucleic acid synthesis in

Saccharomyces cerevisiae. J. Bacteriol. 104:13881390. 28. Wickerham, L. J. 1946. A critical evaluation of the nitrogen assimilation tests commonly used in classification of yeasts. J. Bacteriol. 52:293-301. 29. Wilkinson, L. E., and J. R. Pringle. 1974. Transient GI arrest of S. cerevisiae cells of mating type a by a factor produced by cells of mating type a. Exp. Cell Res. 89:175-187.

a-Factor from Saccharomyces cerevisiae: partial characterization of a mating hormone produced by cells of mating type a.

Vol. 132, No. 2 Printed in U.S.A. JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 462-472 Copyright t 1977 American Society for Microbiology a-Factor from Sa...
2MB Sizes 0 Downloads 0 Views