Vol. 148, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1981, p. 889-8960021-9193/81/120889-08$02.00/0

Sexual Agglutination in Saccharomyces cerevisiaeKEVIN TERRANCE AND PETER N. LIPKE*

Department of Biological Sciences, Hunter College of the City University ofNew York, New York, NewYork 10021

Received 22 April 1981/Accepted 10 August 1981

Treatment of either mating type of Saccharomyces cerevisiae with the appro-priate sex pheromone increased cell-cell binding in a modified cocentrifugationassay. Constitutive agglutination of haploids was qualitatively similar to phero-mone-induced agglutination. Regardless of exposure to pheromone, agglutinablecombinations of cells exhibited maximal binding across similar ranges of ionicstrength, pH, and temperature. Binding of all combinations was inhibited by 8 Murea, 1 M pyridine, or 0.05% sodium dodecyl sulfate. From a-cells we solubilizedand partially purifed an inhibitor of a-cell agglutinability. This inhibitor reversiblymasked all a-cell adhesion sites and inactivated pheromone-treated and controlcelLs with similar kinetics. The inhibitor behaved as a homogeneous species inheat inactivation experiments. Based on these results, we proposed a model forpheromone effects on agglutination in S. cerevisiae.

In Saccharomyces cerevisiae, sexual agglutin-ability is enhanced by sex pheromones. Eachmating type synthesizes and secretes a peptidepheromone that affects the other mating type.Mating type a-celLs produce the pheromone a-factor, a-celLs produce the peptide a-factor.When the two mating types are mixed in

medium and incubated on a rotating platform,some strains begin agglutinating immediately (1,9, 10). Pheromone pretreatment of such strainsincreases the rate of agglutination. Manney andMeade (9) called these strains "constitutive ag-glutinators." Other strains, such as X2180-1A(a) and X2180-1B (a), agglutinate only after alag of 30 min. The lag can be eliminated bytreatment of either mating type with the appro-priate pheromone (1, 12). Such strains are called"inducible agglutinators" (9). Since cyclohex-imide added with the pheromone prevents in-duction of increased agglutinability in all strains,it is likely that protein synthesis is required forpheromone effects on agglutination.

In an alternate assay, the two mating typesare mixed and centrifuged. The pellet is gentlyresuspended, transferred to fresh medium, andplated (2, 5). Fornation of diploids is monitored.Hartwell (6) has improved the assay by estimat-ing agglutination from the turbidity of the cellsuspension. Such assays have yielded informa-tion on optimal conditions and have confirmedthe constitutive agglutinability of some strainsas well as the effects of the pheromones. How-ever, these assays are tedious and imprecise dueto variability in resuspension. By adding a con-trollable resuspension step we have made the

cocentrifugation assay precise enough to distin-guish agglutination systems of diverse proper-ties. We were therefore able to determimne thatagglutination is qualitatively similar in phero-mone-treated cells and in untreated cells.

MATERIALS AND METHODSStrains and growth conditions. S. cerevisiae

wild-type haploid strains X2180-1A (a) and X2180-1B(a), obtained from the Yeast Genetics Stock Center(Berkeley, Calif.), were used for most experiments.Cells were cultured in a glucose and ammonia minimalmedium that has been previously described (8). Therapid agglutinators XP300-26c (a) and XP300-29b (a)were the generous gift of T. R. Manney, Departmentof Physics, Kansas State University, Manhattan.These latter strains were grown in defined minimalmedium supplemented with (per liter): 20 mg of his-tidine, 50 mg of lysine, 30 mg of tryptophan, 90 mg ofthreonine, and 30 mg of adenine. Cultures were incu-bated on a rotary shaker at 30°C. Cell density wasmonitored as optical density at 660 nm, calibrated byhemocytometer counts.

Cycloheximide, glass beads, bovine serum albumin,Amberlite CG-50, and Amberlite XAD-2 were pur-chased from Sigma Chemical Co., St. Louis, Mo., andUltroGel AcA-34 was purchased from LKB Instru-ments, Inc., Hicksville, N.Y. Other materials were ofreagent grade from commercial suppliers.

Protein was determined by binding of Coomassiebrilliant blue G-250 (Bio-Rad Laboratories, Rich-mond, Calif.), and carbohydrate was determined bythe phenol-sulfuric acid method (3). The assay for,t/-fructofuranosidase (EC 3.2.1.26, invertase) was that ofSmith and Ballou (11).

Partial purification and assay of sex phero-mones. a-Factor was prepared by column chromatog-raphy on Amberlite CG-50 as previously described (7).

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The hormone was stored and used in the pyridineacetate buffer in which it was eluted. Activity wasassayed by ability to induce morphogenesis of a-cells(4). One unit of a-factor was defined as the minimumamount that will induce morphogenesis. This is equiv-alent to 4 U by the method of Duntze et al. (4).

a-Factor was obtained by the method of Strazdisand MacKay (personal communication, V. MacKay,Waksman Institute for Microbiology, Rutgers Univer-sity, Piscataway, N.J.). Two to five liters of X2180-1A(a) cells was grown to late log or early stationaryphase. The cells were harvested by centrifugation anddiscarded. Amberlite XAD-2 beads were washed se-quentialy in water, methanol, and again in water. Wetbeads (10 ml/100 ml of culture supernatant) wereadded, and the suspension was shaken at room tem-perature for 24 h, The beads were poured into acolumn and washed with 3 volumes of 40C water and1 volume of 4°C 20% (vol/vol) methanol. a-Factor waseluted with 40C absolute methanol. The yellow frac-tions were pooled, evaporated under reduced pressure,and redissolved in a small volume of dimethyl sulfox-ide.The activity of a-factor was determined by its ability

to induce morphogenesis of X2180-1B (a) cells. Sam-ples of a-factor were added to suspensions of a-cells at1 x 104 to 2 x 104 cels per ml in 375 td of minimalmedium containing 0.2 mg of bovine serum albuminper ml. After 4 h of incubation at 300C, the cells wereexamined by microscopy. One unit per milliliter wasdefined as the minimum concentration inducing mor-phogenesis.

Agglutination assay. Haploid a-cells and a-cellswere grown separately in minimal medium to between2.2 x 106 and 1.4 x 107 ceUls per ml. The cells wereharvested and washed at 40C in saline-phosphatebuffer (0.14 M NaCl, 0.01 M sodium phosphate, pH6.0). The ceUs were then resuspended in a mixture ofone part saline phosphate buffer and two parts freshminimal medium at a density of 2 x 107 cels per ml.When a-ceUs were to be induced, bovine serum albu-min was added to 0.2 mg/ml. For pheromone inductionof agglutinability, cells were incubated with the appro-priate pheromone (a-factor, 1 U/ml, 25 min; a-factor,0.050 to 0.1 U/ml, 70 min) on a rotary shaker at 300C.Cells that were not induced were maintained on iceduring the incubation period. After induction, all cellswere centrifuged and resuspended in an equal volumeof 0.1 M sodium acetate buffer (pH 5.0) containing 10ytg of cycloheximide per ml, which prevented furtherinduction of agglutinability.

Samples (1 ml) of each cell type were added to 13-by 100-mm test tubes. Control tubes received 2 ml ofa single cell type. The volume ofeach tube was broughtto 3 ml with 0.1 M sodium acetate (pH 5.0) containing10 ug of cycloheximide per ml. Cells in all tubes weregently compacted by centrifugation (200 x g for 5min). Hartwell's assay (6) was modified by resuspen-sion of the pellet by stirring with a 7- by 90-mm paddleat 1,000 rpm for 4 s. A stop maintained the paddle at6 mm above the bottom of the tube. A constant speedcontrol unit (Cole-Parmer Instrument Co., model4420) assured reproducible results. The suspensionswere left undisturbed for 20 min to allow settling ofthe aggregates, and the test tubes were then used as

cuvettes in a Bausch & Lomb Spectronic 20 to deter-mine optical density at 660 nm. All samples were runin duplicate, and most in triplicate. The agglutinationindex (AI) was defined as: AI = 1 - (2 x Aa+")/(Aa +A') where A is the mean optimal density of the tubescontaining the cell types or type indicated by thesuperscript. The AI varies from 0 to 1, with higherindices denoting a greater degree of agglutination.

Extraction and partial purification ofa-agglu-tinin. Midlog cultures of X2180-1B (a) were harvestedand washed once in 0.1 M sodium acetate buffer (pH5.0). The cells were resuspended at an optical densityat 660 nm of 30 to 100, and the suspension was broughtto 5 mM EDTA and dithiothreitol. The suspensionwas cooled to 0°C, and 3 g of glass beads (450 to 500,um) per ml was added. Homogenization in a SorvallOmnimixer set to the highest speed broke 80 to 90%the cells in 4 to 6 min. The extract was cleared bycentifugation at 120 x g for 5 min and then at 28,000x g for 30 min in a Sorvall SS-34 rotor. The superna-tant solution was applied to a 4- by 80-cm column ofUltrogel AcA-34 at 40C. The column was eluted with0.1 M sodium acetate buffer (pH 5.0).

Agglutinin activity was assayed by inhibition of a-cell agglutinability. Fractions were incubated with 2x 107 pheromone-treated a-cells in a total of 2 ml ofsodium acetate buffer containing 10 jig of cyclohex-imide per ml for 90 min at 300C. The incubated a-cellswere resuspended in fresh buffer and agglutinated witha-cells as in the agglutination assay. If the agglutininwas active, it inhibited agglutination, and the mixturehad a lower AI than the controls. The change in AIwas linearly dependent on the amount of extract addedat low extract concentrations (correlation coefficient,0.995).

Reversal of binding. Agglutinated mixtures werede-agglutinated by a series of washes. The cells werewashed three times in 0.05 M Tris buffer (pH 9.0)containing 10 jig of cycloheximide per ml. The washedcells were then rinsed twice in 0.1 M sodium acetate(pH 5.0) containing 10 ,ug of cycloheximide per ml.When urea was the reversal agent, the initial threewashes were in 8M urea in sodium acetate buffer (pH5.0) containing 10,tg of cycloheximide per ml. Suchwashes were also used to reverse the effects of a-agglutinin on a-cells.

RESULTSS. cerevisiae haploid cells were constitutively

agglutinable when both mating types were pres-ent. Agglutination was enhanced by treatmentof a-cells with a-factor or a-cells with a-factor(Table 1). Treatment of either mating type withits own pheromone had no effect on agglutina-bility. Neither mating type was ever selfagglutin-able, nor did either mating type agglutinate withdiploids under any combination of pheromonetreatments.

Control and pheromone-treated agglutinationmixtures had parallel responses to variations ofagglutination and resuspension conditions. AIsincreased as the centrifugation time was in-creased from 1 to 5 min. There was little change

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MODEL FOR AGGLUTINATION IN YEAST

TABLE 1. Effect ofpheromone treatment on agglutination of S. cerevisiae cellsa-Cells (X2180-1A) a-CeLls (X2180-1B) Diploid cells (X2180) AI

Control Control 0.35 to 0.42Induced with a-factor Control 0.61 to 0.82Control Induced with a-factor 0.67Control Control 0.01Control Induced with a-factor 0.04Induced with a-factor Induced with a-factor 0.85

Control + induced with a-factor 0.07Induced with a-factor Control 0.04

Control Control 0.06Control Indvced with a-factor 0.04

at longer times. AIs were dependent on the shearforce applied (Fig. 1). The AIs were independentof the time of stirring between 2 and 16 s. Understandard conditions, analysis of the supernatantafter settling revealed that the suspended parti-cles consisted of single cells and a few smallaggregates (2 to 3 cells). The fraction of agglu-tinable cells could be obtained by difference, andwas proportional to the AI.Optimal agglutination of X2180-1A (a) with

X2180-1B (a) occurred between pH 5.0 and 7.0(Fig. 2) and at an ionic strength between 0.030and 0.300. There was little difference in theeffect of different ions, including sodium acetate,NaCl, and Tris-hydrochloride. Added Ca2+ orEDTA had no effect on agglutination. Therewas little temperature dependence between 12and 370C, but a decrease in agglutination wasobserved below 100C (data not shown). Theeffects of pH, ionic strength, and temperaturewere similar for mixtures with untreated a-cellsand mixtures in which the a-cells had beentreated with a-factor.Standard assay conditions were chosen to

maximize the difference between control andinduced cells for X2180-1A (a) and X2180-1B(a). Under standard conditions, replicate assayshad a mean AI of 0.39 ± 0.11 (standard devia-tion) for mixtures ofuntreated a- and a-cells and0.72 ± 0.06 for pairs in which the a-cells werepretreated with a-factor. These results were ob-tained from 16 experiments carried out over 3months. For a single experiment, the standarderror was 0.03 AI (triplicate tubes).Comparison of weakly and strongly ad-

hering cells. Haploids X2180-1A (a) andX2180-1B (a) have been classified as inducibleagglutinators (9) because they show no consti-tutive agglutinability in rotation assays. Wecompared their agglutinability to that ofXP300-29b (a) and XP300-26c (a), strains which Man-ney and Meade isolated as excellent inducibleagglutinators (9). Substitution of either XP300haploid for the appropriate X2180 strain yieldeda mixture with increased constitutive agglutin-

0.8

0.6 F

0.4

0.2

950 1150 1350 1550Speed of ReUsnsion (RPM)

FIG. 1. Resistance of aggregates to shear. Agglu-tination mixtures were centrifuged at 20f) x g for 5min and then suspended for 4 s at several rotationspeeds. The mixtures are: (A) both mating typesinduced, (U) only a-cells induced, (*) only a-cellsinduced, and (0) neither mating type induced.

ability (Table 2). There were less marked differ-ences in agglutination after treatment of the a-cells with a-factor. Dependence of agglutinationon pH and ionic strength were similar for bothsets of cells (data not shown). In our assay,inducible and constitutive agglutinators were

qualitatively similar, differing only in thestrength of intercellular bonding.Pheromone induction of agglutinability.

a-CelLs responded to a-factor after a 10-min lag.Maximal induction was reached in 18 min (Fig.3). a-Factor was maxally effective at dosesabove 1 U/ml (5 x 10-8 U/cell). a-Factor in-duced increased agglutinability almost immedi-ately, but 60 to 90 min was needed for fullinduction (Fig. 3). The a-cells started to recoverfrom the effects of the pheromone after 90 min.a-Factor induced maximal agglutinability in a-

cells at concentrations above 0.05 U/ml.Inhibition of agglutination. The similarity

U~~~*~~~

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Os

0.6

-4"4 0.4

02

0.0

3 4 5 6 7 8 9 10pH

FIG. 2. Dependence of agglutination on pH. Hap-loid cells were agglutinated under standard condi-tions except for differences in the agglutinationbuffer: (El 0) agglutination in 0.1 M sodium acetate;(I, 0) 0.1 M This-hydrochloride; (l U) mixtures inwhich the a-cells were pretreated with a-factor; (0,0) assays with uninduced cells.

TABLE 2. Assays of strong agglutinatorsaa-Fac-

a-Cells tor a-Cells AItreat-ment

X2180-1A - X2180-1B 0.39X2180-1A + X2180-1B 0.72

X2180-1A - XP300-26c 0.63X2180-1A + XP300-26c 0.84

XP300-29b - X2180-1B 0.61XP300-29b + X2180-1B 0.79

XP300-29b - XP300-26c 0.57XP300-29b + XP300-26c 0.80a All experiments were performed at pH 5.0. The

rapid agglutinators exhibit maximal agglutination atpH 6.0. Experiments carried out at pH 6.0 yieldedsimilar results. Cells were incubated with or withoutpheromone, centrifuged, resuspended in buffer, mixed,and then agglutinated.

of the effects of pH, ionic strength, and temper-ature on agglutination of induced and uninduceda-cells suggested that only a single set of agglu-tinins was active. We subjected agglutinationmixtures to treatment with a variety of pertur-bants to test whether there were different sus-ceptibilities ofpheromone-treated and untreatedcells. Between the four agglutinable combina-tions of cells, however, there were only minordifferences in the susceptibility to a variety ofdisruptive reagents (Table 3).

0.9

0.8

0.7 F

4

0.6

0.5 F

0.4

0 20 40 60 80 100 120Time (minutes)

FIG. 3. Time course of induction of haploids. a-Cells (U) were incubated with I U of a-factor per mlfor the indicated time at 30°C. Induction was stoppedby the addition of 10 pg of cycloheximide per ml. Thecells were then washed, resuspended in agglutinationbuffer, mixed with uninduced a-cells, and agglutin-ated, a-Factor effects were determined by treating a-cells with 0.1 U of a-factor per ml for the indicatedtime. The cells were then cooled to 0°C, treated withcycloheximide (10 pg/ml), washed, and mixed withuninduced a-cells (M) or a-cells pretreated with a-factor (A).

TABLE 3. Effect ofpotential inhibitors onagglutination

% of control A.I.b

Inhibitor' Nei- a-Cells a-Cells Bothther in- in- in- in-duced duced duced duced

1 M NaCl 90 79 89 835 M NaCl 29 26 18 280.3 M KI 90 88 94 970.01% sodium dode- 61 87 90 93

cyl sulfate1% Tween 80 95 97 99 961 M pyridine 44 57 56 7120 mM EDTAc 106 101 104 9950 mM dithiothreitol

a-cells pretreatedd 45 31 26 23a-cells pretreatedd 95 88 99 103a Inhibitors were included in the agglutination buffer unless

otherwise noted.'Percent of control AI with an agglutination mixture in

which the cells were treated with pheromone under standardconditions.

'Agglutination was at pH 7.0.d The indicated cells were pretreated with dithiothreitol for

30 min at pH 7.0, washed once with buffer at pH 5.0, andagglutinated as usual.

A

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MODEL FOR AGGLUTINATION IN YEAST 893

Urea, pyridine, and sodium dodecyl sulfatewere reversible in their effects on agglutination.When the cells were washed free of such re-agents, the mixtures regained agglutinability,even in the presence of 10 ,g of cycloheximideper ml. Therefore, it was likely that the affectorshad prevented formation of intercellular bonds,but had not irreversibly disrupted molecularstructure. These three disruptive reagentsshowed single thresholds for inactivation in ti-trations of their affects on agglutination (Fig. 4).To demonstrate that other surface functions

were differentially affected by such reagents,cellular invertase was assayed in the presence oftwo inhibitors (Fig. 4). We grew X2180-1A (a)and X2180-1B (a) in miniimal medium contain-ing 2% sucrose as a carbon source. The harvestedcells were washed, and the two mating typeswere mixed in assay buffer in the presence ofperturbant. We determined invertase activity,correcting for the effect of the perturbant on thereducing sugar determination. The susceptibili-ties were clearly different from those of aggluti-nation. Invertase was more labile to urea andwas not affected by sodium dodecyl sulfate. Theincrease in activity in the latter case was prob-ably due to permeabilization of the cells and tothe consequent assay of both intra- and extra-cellular enzyme.

Solubilization ofa-agglutinin. Clarified ex-

140

120

100

60.>o6 '' u

I 2 3 456 e 0.2

tracts of a-cells did not agglutinate a-cells, butdid decrease the agglutinability of the a-cells. Aspecies mediating cell-cell binding would exhibitsuch properties if it were monovalent. We des-ignated such activity "a-agglutinin" to denote itsorigin and function in the intact cell. The prop-erties of the extracted a-agglutinin matchedthose of the cell-bound agglutinin. The solublematerial inactivated a-cells maximally at pH 5to 6. Low concentrations of extract inactivateda fraction of the a-cells proportional to theamount of extract added, showing that the in-activation was a saturable process. The inacti-vation was partially reversed by washing thesecells at pH 9 or in 8 M urea in the presence ofcycloheximide.Gel filtration yielded several active fractions

(Fig. 5). The material eluting in region I wasapproximately 80-fold more active than the ini-tial extract and exhibited the expected proper-ties of the agglutinin. It was maximally active atpH 5 to 7 and affected only a-cells. a-Cellstreated with this fraction completely recoveredtheir agglutinability after washing in pH 9 bufferor in 8 M urea. The activity eluting in regions IIand III permanently inactivated a-cells. Conse-quently, all subsequent experiments were car-ried out with material from region I.

Agglutinin-mediated inactivation of a-cells. The a-agglutinin inactivated a-cells in

0 . 0iD0.4 .6Oto. 0.001 o00 O.1

[UREAJ (M) [PYRIDINEJ M [SDI NFIG. 4. Titrations ofagglutinations by three inhibitors. Theprocesses assayed are agglutination ofmixtures

with: (A) both mating types induced, (a) only a-cells induced, (*) only alpha-cells induced, and (0) neithermating type induced. Percent activity was defined as (AI with inhibitor/AI of control) x 100. Cellularinvertase assays (x) are also presented.

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5C-

0mzO-

3

3

0o 40 so so 100

froction numberFIG. 5. Gel filtration of a-cell extract on UltroGel AcA-34. The column was 4 by 80 cm, the eluant was 0.1

M sodium acetate (pH 5.0), and 10-ml fractions were taken: (0) activity, (A) protein.

0 9

08e

07

06

~05

0-4

03

0.2

0o l I ,10 20 30 40 5 60 70 80 90 100 110 120

TIME (MIN)FIG. 6. Kinetics of a-cell inactivation. The a-cells

were incubated with AcA-34-purified a-agglutinin forthe indicated times, washed, and agglutinated withuninduced a-cells. The inactivated cells were (0)uninduced a-cells, or (a) a-factor-treated cells.

90 to 120 min (Fig. 6). Both uninduced and a-factor-treated a-cells were inactivated with sim-ilar kinetics. For a 90-min binding period, thedose-response curves were linear for change inAI of 0.3 or less (Fig. 7). We also determined theresponse of untreated and pheromone-treatedcells to various doses of concentrated alpha-ag-glutinin. The a-agglutinin contained species thatmasked all agglutination sites on a-cell surfaces(Fig. 7).Heat sensitivity assays implied that the alpha-

agglutinin preparations were functionally ho-

0.8 . . . . . .

0.6

0.4

0.2

00 200 400 1000

Extrmct Added (isI)FIG. 7. Inactivation of a-cells with a-agglutinin.

a-Agglutinin was concentrated 13-fold by dialysisagainst polyethylene glycol. Pheromone-treated (U)or uninduced (0) a-cells were incubated with a-ag-glutinin for 90 min and then washed and agglutin-ated with untreated a-cells.

mogeneous. All activity was destroyed within 10min at 550C, but the activity was stable at 40°C(Fig. 8, inset). Inactivation kinetics at 500C con-firmed the homogeneity. Extract was heated forvarious times, cooled, and incubated with a-cellsfor 1 h. The a-cells were then assayed for agglu-tinability as usual. The inactivation had no sig-nificant difference in rate between the variouscombinations of tester cells (Fig. 8).

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MODEL FOR AGGLUTINATION IN YEAST

DISCUSSIONThere are many possible models for sexual

agglutination in S. cerevisiae and for the effectsof sex pheromones on agglutination. Figure 9

5 10 15 20Tkme (minutes)

FIG. 8. Thermal lability of a-agglutinin. The ag-glutinin was incubated at 50°C for the indicatedtimes, cooled, and assayed. Assays were with bothmating types induced (A) or with neither type induced(0). Inset: inactivation threshold for a-agglutinin.Agglutinin was incubated for 10 min at the indicatedtemperature, cooled, and assayed with a-factor-treated a-cells and untreated a-cells. The percentactivity remaining was calculated from the ratio ofthe remaining activity to that of the unheated agglu-tinin with the same tester cells.

A B

shows three of these models. Consistent withobservations on S. cerevisiae (1, 12), thesemodels show pheromone induction of eithermating type causing increased agglutination.The pheromones induce increased densities ofcell surface agglutinins. Such increases wouldlead to increased probability of adhesion aftercollision in rotation assays (1, 12) as well as tothe increased strength of cell-cell binding thatwe observed.

In modelA there is only one kind of agglutininon each mating type. Pheromone treatment in-creases the effective concentration of these ag-glutinins at the cell surface. In models B and Cthere are two pairs of complementary aggluti-nins. In model B, induction of either cell in-creases adhesions because the constitutive ag-glutinin is induced as well as a new type ofagglutinin. In model C, pheromone treatmentincreases the concentration of one or both typesof agglutinin, but both types are constitutivelyexpressed. There are other models that fit theconditions listed above, but they have three ormore complementary sets of agglutinins.Whereas in the first model all intercellular

bonds are the same, models B and C predict thatinduced pairs of cells have heterogeneous adhe-sive bonds. We found that under all tested con-ditions, the cells adhered as if there were onlyone type of adhesive bond. Untreated and in-duced cells adhered over the same range of pH,ionic strength, and temperature. Agglutinationof treated and untreated pairs was prevented bythe same perturbants. For three dissimilar per-turbants, all combinations showed identicalthresholds for disruption of intercellular bonds.Experiments with the a-agglutinin were also

consistent with model A. The a-agglutinin wasextracted from untreated a-cells, but it com-

C

0011FIG. 9. Models of sexual agglutination in S. cerevisiae: a, a-cells; a, a-cells. Arrows denote pheromone

treatment of the cells. a-Agglutinins are represented by -c and-<, and a-agglutinins are represented by-oand +.

90

70

50 F

40 _

430 F

A A

0

0

AA

SA40 45 50 55

Tepa ("C)

20 F

1I

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896 TERRANCE AND LIPKE

pletely inactivated untreated and pheromone-induced a-cells. a-Agglutinin was therefore com-plementary to all expressed a-cell agglutinins.The thermal inactivation profile of this materialwas consistent with a single species of macro-molecule being responsible for its activity.

All of our experiments therefore support themodel of Fig. 9A for agglutination of S. cerevi-siae haploids. The data imply that in yeastdevelopmental induction of altered adhesiveproperties is dependent on quantitative ratherthan qualitative alterations of the cell surface.

ACKNOWLEDGMENTSMaritza Alvarado camed out the experiments with the XP-

300 strains. We thank Anne Lipke for helpful discussions andediting.

This work was supported by National Science Foundationgrant PCM8004732 and by City University ofNew York PSC-BHE grant RF13323. K.T. was supported by a Public HealthService MinorityBiomedical Support Grant from the NationalInstitutes of Health to the Division of Sciences and Mathe-matics of Hunter College.

LITERATURE CITED

1. Betz, RI, W. Duntse, and T. R. Manney. 1978. Matingfactor-mediated seual agglutination in Saccharomycescerevisuae. FEMS Microbiol. Lett. 4:107-110.

2. Campbell, D. A. 1973. Kinetics of mating specific aggre-gation in Saccharomyces cerevisiae. J. Bacteriol. 116:323-330.

3. Dubois,M, K.A. Gilles, J. K. Hamilton, and F. Smith.1956. Colorimetric method for determination of sugarsand related substances. Anal. Chem. 28:350-356.

4. Duntze, W., D. Stotzler, E. Bucking-Throm, and S.Kalbitzer. 1973. Purification and partial characteriza-tion of alpha-factor, a mating-type specific inhibitor ofcell reproduction in Saccharomyces cerevisiae. Eur. J.Biochem. 35:357-365.

5. Fehrenbacher, G., K. Perry, and J. Thorner. 1978.Cell-cell recognition in Saccharomyces cerevissae. reg-ulation of mating specific adhesion. J. Bacteriol 134:893-901.

6. Hartwell, L H. 1980. Mutants of Saccharomyces cere-visiae unresponsive to cell division control by polypep-tide mating hormone. J. Cell Biol. 85:811-822.

7. Lipke, P. N., and C. E. Ballou. 1980. Altered immuno-chemical reactivity ofSaccharomyces cerevisiae a-cellsafter alpha-factor-induced morphogenesis J. Bacteriol.141:1170-1177.

8. Lipke, P. N., A. Taylor, and C. E. Ballou. 1976. Mor-phogenic effects of alpha-factor on Saccharomyces cer-eviswie a-cells. J. Bacteriol. 127:610-618.

9. Manney, T. R., and J. H. Meade. 1977. Cell-cell inter-actions during mating in Saccharomyces cerevisiae, p.281-321. In J. L. Reissig, (ed.), Microbial interactions,Chapman and Hall, London.

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