JOURNAL OF BACTERIOLOGY, Apr. 1983, p. 161-1690021-9193/83/040161-09$02.00/0Copyright C 1983, American Society for Microbiology

Vol. 154, No. 1

Cell Wall Receptor for Yeast Killer Toxin: Involvement of(1 -- 6)-p-D-Glucan

KENDRICK HUTCHINS AND HOWARD BUSSEY*Department ofBiology, McGill University, Montreal, Quebec, Canada H3A IBI

Received 19 October 1982/Accepted 17 January 1983

The linear (1 -+ 6)-p-D-glucans pustulan and luteose were effective competitiveinhibitors of killer toxin action. Affinity chromatography of killer toxin on apustulan-Sepharose column showed that toxin bound directly to a (1 6)-p-linked polysaccharide. Other polysaccharides found in yeast cell walls, including(1 -- 3)-p-D-glucan, mannan, chitin, and glycogen, were not effective as inhibitorsof toxin. Fractionation of yeast cell walls was attempted to identify the toxinreceptor in sensitive Saccharomyces cerevisiae. The receptor activity wasretained among the insoluble glucans in alkali-washed cells; yeast mannan andalkali-soluble glucan had little receptor activity. A minor fraction of receptoractivity was removed from alkali-washed cells by hot acetic acid extraction, aprocedure which solubilized some (1 -* 6)-3-D-glucan and glycogen. The majorfraction (>70%o) of receptor activity remained with the acid-insoluble (1 6)-+ -

and (1 -- 3)-p-glucans. Zymolyase, an endo-(1 -- 3)-3-D-glucanase, solubilized asubstantial fraction of the receptor activity in the acid-insoluble glucans. Thereceptor activity in yeast cell walls was periodate and (1 -* 6)-p-D-glucanasesensitive, but was resistant to (1 -. 3)-p-D-glucanase and a-amylase. The acid-soluble glucan fractions of a sensitive strain and a krel-I receptor-defective toxin-resistant mutant were examined. The krel-l strain had a reduced amount (ca.50%) of (1 -. 6)-3-D-glucan compared with the sensitive parent strain. A sensitiverevertant of the krel-I strain regained the parental level of glucan. These resultsimplicate (1 -. 6)-3-D-glucan as a component of the yeast cell wall receptor forkiller toxin.

The role of eucaryotic cell surface polysac-charides as receptors for proteins in many cellevents is widely appreciated, but the mechanismof their action is poorly understood. As well asacting as receptors for bacteria, viruses, andtoxins, surface polysaccharides are thought tobe vitally involved in cell interactions such ascell association, distribution, and turnover (8,14, 22).

In an attempt to understand a simple polysac-charide receptor-mediated process, we have ex-amined the way in which a small yeast protein,killer toxin, interacts with a cell wall receptor onsensitive cells. Wall receptor binding of toxinappears to be a necessary in vivo prelude tokilling (1, 6). Killer toxin will insert in vitro intoa lipid bilayer system (B. Kagan, personal com-munication), and this, probably membrane re-ceptor augmented, is likely the mechanism oftoxin action in vivo also. Previous work defineda specific cell wall receptor for killer toxin bymeasuring binding of toxin to sensitive cells andto resistant mutants with defective receptors.The receptor, probably a polysaccharide or gly-coprotein, was solubilized from yeast cell walls

by endo-(1 -- 3)-3-D-glucanase action, and itwas heat and pronase resistant but periodatesensitive (1, 5).We report here the involvement of a yeast cell

wall (1 -+ 6)-p3-D-glucan as a component of thereceptor for killer toxin. This work defines a rolefor a yeast cell wall polysaccharide and providesa system amenable to genetic analysis to studythe interaction of a protein with a eucaryotic cellsurface receptbr.

MATERIALS AND METHODS

StrWi, media, and c als. Strains of Saccharo-myces cerevisiae used were S14a, krel-l(S14.%),S14.96r, A69, A8207NK, and X2180-1A, X2180-1A-5(mnn2), and X2180-1A-4 (mnnl) provided by C. Bal-lou. Bacillus circulans WL-12 was provided by H. J.Phaff. The yeast growth medium was either YCM(0.5% yeast extract [Difco Laboratories]-0.5% pep-tone [Difcol-2% dextrose) or YCM in pH 4.7 Halvor-son buffer (YEPD) (11). Solid yeast growth mediumwas YEPD-agar, which consisted of 1% yeast extract,2% agar (Difco), and 2% peptone in pH 4.7 Halvorsonbuffer. Yeast cultures were grown at 30"C with shak-ing. Luteose and elicitor from Phytophthora mega-sperma were gifts from D. J. Manners and P. Alber-

161

162 HUTCHINS AND BUSSEY

sheim, respectively. Pustulan and laminarin wereobtained from Calbiochem. Other saccharides, conca-navalin A, and lectins from jequirity bean, garden pea,wheat germ, and Lens culinaris were from SigmaChemical Co.Enzymes. Zymolyase 60,000 was from the Kirin

Brewery Co. and was further purified by dialysisagainst 0.01 M phosphate buffer (pH 6.0) followed bygel chromatography on CM-100 (15). Nonlytic (1 -- 6)-P-D-glucanase was purified from the culture fluid of B.circulans WL-12 grown on 0.25% pustulan (28). Theenzyme preparation was purified through the G-100chromatography step as described (28). a-Amylasewas from the Boehringer Mannheim Corp. Degrada-tion of (1 -- 3)-p-D-glucan in the acid-insoluble yeastcell wall fraction was effected with Zymolyase 60,000in Tris (pH 7.5) buffer at 37°C for variable times.Conditions for other polysaccharide degradations byZymolyase 60,000 and by (1 -. 6)-p-D-glucanase anda-amylase were, respectively, pH 5.0, 100 mM sodiumsuccinate for 3 h at 37°C; pH 5.0, 100 mM sodiumsuccinate for 2 h at 30°C; and pH 6.8, 50 mM potassi-um phosphate for 2 h at 30°C.

Preparation of ovalbumin glycopeptides and gentio-oligosaccharides. Ovalbumin (Sigma) (2 g) was digestedwith pronase, and the digest was fractionated on aSephadex G-25 column as described (13). The hexose-containing fraction was freeze-dried. Pustulan washydrolyzed (80°C in 1 M HCI for 50 min), and theoligosaccharides were separated on a P-2 (Bio-RadLaboratories) column (90 by 1.5 cm).

Assays. Polysaccharide degradation was determinedby measurement of the reducing sugar content, usingthe Nelson-Somogyi procedure described by Naka-jima et al. (20). The spectrophotometric method ofAspinall and Ferrier was used in the quantitativeoxidation of carbohydrates by periodate (3). Totalcarbohydrate was measured by the borosulfuric acidmethod (4).Chromatography. Preparation of glucan-coupled Se-

pharose 6B was as suggested by the manufacturer.Epoxy-activated Sepharose 6B, 2.25 ml of preswollengel, was added to 40 mg of glucan in 0.1 M NaOH andincubated on a roller drum at 37°C for 19 h. The gelwas washed consecutively with 0.1 M NaOH, 0.1 Macetate buffer (pH 4.0), and 0.1 M sodium bicarbonatebuffer (pH 8.0) containing 0.5 M sodium chloride. Thegel was left overnight at room temperature in 1 Methanolamine and washed in 0.1 M acetate buffer (pH4.7) with 15% glycerol before use.Concanavalin A-Sepharose chromatography was as

described (10).Thin-layer chromatography, as described by Hotta

and Kurokawa (12) using the solvent butanol-acetone-water (5:4:1), was used to test the purity of the mannanpreparation isolated by Fehlings precipitation. Glu-cose and mannose were run simultaneously as stan-dards. The carbohydrates were detected by anisidinephthalate spray (26).

Competition assay. Saccharide inhibition of toxin-dependent sensitive-cell death was measured by amodification of the procedure described previously(5). A 175-,ul amount of a sensitive A8207NK YEPDculture was grown to early log phase, estimated spec-trophotometrically, and added to 25 ,l containingkiller toxin and variable amounts of saccharide. Thismixture was incubated 1.5 h at 18°C with shaking.

Appropriate YEP dilutions of the cell mixture at thestart and after 1.5 h were plated on YEPD agar andincubated for 2 days at 30°C. Colonies were counted,and the percent survival after 1.5 h was calculated.The assay was used to compare relative competitive

activities of carbohydrates by analysis of dose-re-sponse curves obtained in a single assay (e.g., Fig. 1).Assay-to-assay variation in percent survival of controlcultures precluded quantitative comparisons betweenassays.Mannan isolation. Mannan was isolated according to

the method of Peat et al. (24) and partially purified byFehlings precipitation of autoclaved whole cells asdescribed by Kocourek and Ballou (16). After hydroly-sis of this material, only glucose and mannose weredetected by thin-layer chromatography (12). Mannancomprised at least 90% of the material, judged bystandards of various amounts of glucose and mannose(results not shown).

Cel wall fractionation. Strains were grown on YCMand harvested at late stationary phase. Cell walls wereprepared (9) and extracted with 3% NaOH by heatingat 75°C for 1 h. This mixture was centrifuged and alkaliextracted twice more (18). Mannan and alkali-soluble(1 - 3)-3-D-glucan were separated from the pooledalkali-soluble fractions (9). Acid extractions were thenperformed on the alkali-insoluble residue by using 0.5M or 1 M acetic acid at 75 or 90°C for 3 h (18). Aftercentrifugation the acid-soluble fraction was dialyzedagainst double-distilled water, and an aliquot of theacid-insoluble residue was washed with double-dis-tilled water; both samples were freeze-dried. Theremainder of the acid-insoluble fraction was furtherextracted with acetic acid.The (1 -- 6)-p-D-glucan remaining in the acid-insolu-

ble fraction was isolated by extensive Zymolyase60,000 digestion. Acid-insoluble material (300 mg) wassuspended in 30 ml of 0.1 M Tris-hydrochloride (pH7.5) buffer containing Zymolyase 60,000 (0.2 mg/ml)and incubated at 37°C for 24 h. After centrifugation,the supernatant was heated at 100°C for 30 min,dialyzed against double-distilled water, and freeze-dried. This material was dissolved in 5 ml of double-distilled water and contained 41 mg as glucose by theborosulfuric carbohydrate assay (see above). Thepellet, after initial digestion, was resuspended in 10 mlof 0.1 M Tris (pH 7.5) with 0.5 mg of Zymolyase 60,000per ml and further incubated at 37°C for 18 h. Thesupernatant after centrifugation was treated as previ-ously described.Whole cells were similarly alkali and acid extracted,

but due to contamination of the alkali-soluble fractionwith intracellular material, neither mannan nor alkali-soluble glucan could be isolated.

RESULTS

Competitive inhibition of killer toxin action bypolysaccharides. A powerful approach to recep-tor identification involves the use of competitiveinhibitors of the ligand, which inhibit ligandinteraction with receptors in vivo. We have usedsugars and polysaccharides of known composi-tion as competitive inhibitors of killer toxinbinding with the polysaccharide cell wall recep-tor.

J. BACTERIOL.

CELL WALL RECEPTOR FOR YEAST KILLER TOXIN

200

0

I.

0

oo

10080

60

40

20

1086

4

2 t . . .

100 200 300 400

ug/ml Carbohydrate

FIG. 1. Effects of luteose, pustulan, and S14a acid-soluble material on toxin-mediated cell death. Percentsurvival of A8207NK was measured in the presence of increasing concentrations of luteose (U), pustulan (0),and S14a acid-soluble material (V). Error is expressed as the standard deviation of surviving colonies asdetermined by plate counts.

The linear (1 -* 6)-3-D-glucans, pustulan andluteose, were effective competitive inhibitors oftoxin action (Fig. 1). These polysaccharideswere specific inhibitors of toxin action, as poly-saccharides with other linkages (2) failed to

compete with toxin (Table 1). The mixed-linkage(1 -- 3)-3-D- and (1 -+ 6)-3-D-glucan from P.

megasperma (10) was tested, and this showedsignificant competition. No mono- or disaccha-rides tested competed for toxin action; these

TABLE 1. Carbohydrates lacking killer toxin receptor activitya

Amt (mg/ml) % Survival of A8207NKCarbohydrate Main glucosidic linkage in competi- With carbo- Without car-

tion assay hydrate bohydrate

D-Mannose 0.6 24 ± 2 26 ± 5D-Glucose 0.5 0.8 t 0.1 1.0 ± 0.1Gentiobiose 1.5 1.0 t 0.3 6.3 ± 0.8Glycogen (1 4)-a 0.2 5.6 + 0.6 3.9 ± 0.2Amylopectin (1 - 4)-a 0.5 0.7 + 0.1 1.0 ± 0.1Amylose (1 - 4)-a 1.5 3.0 + 0.2 6.3 ± 0.8Chitin (1 - 4)-p-N-acetylglucosamine 2.5 0.6 + 0.2 1.0 ± 0.1Laminarin (1-44)-0 1.5 3.5 0.5 6.3 ± 0.8Lichenin (1- 4)-P and (1 3)-P 1.5 2.9 + 0.2 6.3 ± 0.8Pullulan (1 6)-a 1.5 1.9 + 0.3 6.3 ± 0.8Celiotriose (1 4)-P 20.0 3.0 6.9Ovalbumin glycopeptide 1.7 0.17 0.10Gentiotriose 0.4 0.07 + 0.01 0.24 + 0.02Gentiotetraose 0.4 0.29 + 0.03 0.24 + 0.02Gentiopentaose (1 6)- 0.4 0.16 t 0.01 0.24 + 0.02Methyl-a-D-glucoside 0.5 17 9 18 3Methyl-a-D-mannoside 0.5 10 3 10 3Melibiose 1.5 3.9 t 0.5 6.3 + 0.8

a The various carbohydrates were incubated at the indicated concentrations in the competition assay asdescribed in the text. Variation in the percent survival of A8207NK without carbohydrate occurs because thetable is a composite of many assays with toxin preparations of varying activity.

163VOL. 154, 1983

164 HUTCHINS AND BUSSEY

included gentiobiose, melibiose, methyl-a-D-mannoside, methyl-a-D-glucoside, D-mannose,and D-glucose. Several lectins (listed above)were tested for their ability to compete with thekiller toxin for toxin receptors on sensitiveA8207NK; none competed. This result was con-sistent with a ,-glucan toxin receptor since thelectins tested had no affinity for the P-glucosidiclinkage.Binding of toxin to pustulan-coupled Sepha-

rose. The competition assay for receptor wasindirect, and rather than binding the toxin, thecompetitor polysaccharide could bind directly tothe receptor, blocking toxin action. We testedthis possibility using an affinity column in whichthe linear (1 -+ 6)-p-D-glucan pustulan was cou-pled to an epoxy-activated Sepharose 6B col-umn. The pustulan-Sepharose 6B bound puretoxin in 0.1 M sodium actate-acetic acid buffer(pH 4.7) with 15% (wt/vol) glycerol with a capac-ity of approximately 200 F±g per ml of packedcolumn. The toxin activity could be quantita-tively eluted from the column with 0.1 M Trisbuffer (pH 7.5) with 15% (wt/vol) glycerol. Killertoxin in 0.1 M sodium acetate-acetic acid bufferacetic acid buffer (pH 4.7) with 15% (wt/vol)glycerol to Sepharose 6B or to epoxy-activatedSepharose 6B that had been through the cou-

250

200-Ao A

150

0

c100 1,:2 ~~~~~~~~120

I-_._

50-

1 20 22 24 26 28 30 32

Fraction number

FIG. 2. Elution pattern of killer toxin from a pustu-lan-Sepharose 6B column. A crude killer toxin extractwas applied to a pustulan column (1.9 by 3.8 cm) in 0.1M sodium acetate buffer (pH 4.7) with 15% glycerol(point A). Fractions (1 ml each) were collected andassayed for toxin activity. At point B, the eluant waschanged to 0.1 M Tris buffer (pH 7.5) with 15%glycerol. The insert shows a Coomassie blue-stainedsodium dodecyl sulfate gel (8 to 18% linear polyacryl-amide gradient) of crude killer toxin before passageover the pustulan column (lane 2) and the materialwhich eluted with 0.1 M Tris buffer (pH 7.5) with 15%glycerol (lane 1), showing a single band of 11,000-molecular-weight killer toxin.

pling procedure without a ligand and to whichunreacted groups were coupled with ethanol-amine. We examined pustulan-Sepharose as anaffinity adsorbant for toxin from crude extracts.A 1-liter amount of growth medium from a toxin-producing strain, T1S8C/S14a, was concentratedto 45 ml by ultrafiltration and pumped over apustulan-Sepharose column (0.9 by 3.8 cm) (2.4ml). Toxin activity was retained by the column(greater than 99.5%). After washing with 0.1 Macetate buffer (pH 4.7) with 15% glycerol, whicheluted no activity, toxin activity was quantita-tively eluted with 0.1 M Tris buffer (pH 7.5) with15% glycerol (Fig. 2). Fractions containing mostof the activity were pooled, dialyzed, and con-centrated, and the material was analyzed bysodium dodecyl sulfate-polyacrylamide gel elec-trophoresis. Only one band was visible by Coo-massie blue staining, and it corresponded to the11,000-molecular-weight toxin protein as puri-fied by the glycerol-controlled pore glass chro-matography method (23) (Fig. 2).The competition data strongly suggest that a

(1 -+ 6)-p-D-glucan is involved in the toxinreceptor on the yeast cell wall. We attempted toexamine the possible receptor nature of theyeast cell wall (1 -* 6)-p-D-glucan by a series ofstudies on yeast cell wall fractions.

Alkali fractionation of cell wall polysaccharidesand toxin receptor. In yeasts, the major cell wallpolysaccharides are mannan and glucans (25).The conventional procedure for separating thesecomponents involves repeated hot-alkali, fol-lowed by hot-acid, extractions of whole cells ormechanically isolated cell walls. Structural anal-ysis shows that mannan and a class of (1 3)-* -

D-glucans are enriched for by alkali extraction(9). Exhaustive acid extraction of the alkali-insoluble material removes glycogen and onetype of (1 -> 6)-p-D-glucan (19). The bulk of theacid-insoluble fraction consists of (1 3)-,-D-glucan structurally distinct from the alkali-solu-ble type and a second type of (1 -* 6)-3-D-glucan(18, 19).

Fractionation of S14a cells by the above pro-cedure indicated that the alkali-insoluble glucanfraction retained the major portion of the toxinreceptor found in whole cells (Fig. 3). The alkali-insoluble fraction contained 79% of the totalwhole-cell carbohydrate. This suggested that themajor receptor was a ,-glucan.

Yeast mannan was not a competitive inhibitor.To examine the role of mannan in toxin recep-tion, we purified mannan by the Fehlings proce-dure, followed by concanavalin A-Sepharosechromatography. This material was tested as acompetitive inhibitor of toxin (Fig. 4). The mate-rial isolated by alkali extraction or Fehlingsprecipitation retained some activity, though witha specific activity at least fourfold less than that

J. BACTERIOL.

CELL WALL RECEPTOR FOR YEAST KILLER TOXIN

0/> 10 r86~~~~

4

2

1-00-80-60-4

100 20 300 400

mg/ml Carbohydrate

FIG. 3. Effects of Sl4a heat-inactivated whole cells and Sl4a alkali-insoluble material on toxin-mediated celldeath. Percent survival of A8207NK was measured in the presence of killer toxin and increasing concentrationsof S14a whole cells killed by incubation at 82°C for 1.5 h (e) and S14a alkali-insoluble material (0). Error isexpressed as the standard deviation of the surviving colonies as determined by plate counts.

200

1001-80 ' I60 /I

0

to._;

64

2

1.00-80-60-4

200 400 600 800 1000

ag/mI CarbohydrateFIG. 4. Effects of pustulan and S14a mannan and alkali-insoluble glucan on toxin-mediated cell death.

Percent survival of A8207NK was measured in the presence of pustulan (0), S14a alkali-insoluble glucan (U),and S14a mannan (V). Mannan was isolated by cell wall fractionation and purified by Fehlings precipitation andconcanavalin A chromatography. Error is expressed as the standard deviation of the surviving colonies asdetermined by plate counts.

165VOL. 154, 1983

I.

iI

166 HUTCHINS AND BUSSEY

TABLE 2. Periodate sensitivity of killer toxinreceptor in sensitive S14a cell wall fractionsa

% Survival of sensitive A8207NKin the presence of killer toxin and

Cell wall S14a cell wall fractionsfraction

Untreated treated

None 0.21 ± 0.05Acid soluble 37 ± 1 0.44 ± 0.05Acid insoluble 41 ± 6 0.6 ± 0.2

a S14a cell wall fractions were prepared as de-scribed in the text and suspended at 0.17 M as anhy-droglucose in 0.4 M sodium metaperiodate, incubatedin the dark at 22'C for 48 h, extensively dialyzed, andthen freeze-dried. Each fraction, treated or untreated,was suspended at 400 ,ug/ml in the competition assay.

of the alkali-insoluble fraction. The residual ac-tivity was almost completely removed upon fur-ther purification of the mannan by adsorptionand elution from a concanavalin A-Sepharosecolumn. This suggested that yeast mannan wasnot involved as a toxin receptor.

Several mannan mutants have been isolated(27). These strains vary in their structure of thecell wall mannan component, and the variationsrange from slight alterations in side chain struc-ture to the complete absence of the outer sidechains.The wild-type X2180 and each of the mannan

mutants derived from it, including mnn2-lA,were equally sensitive to killer toxin (results notshown). The mannan core polysaccharide struc-ture is similar to that found in many glycopro-teins containing N-asparagine-linked oligosac-charides. We purified the glycopeptide fromovalbumin, which contains such a structure (21);it did not compete with toxin binding in acompetition assay (Table 1).The sensitivity of the mannan mutants, which

have altered mannan structures, and the lowactivity of Fehlings-precipitated, concanavalinA-Sepharose-purified mannan suggest that man-nan is not a toxin receptor.Acid fractionation of alkali-insoluble cell wall

glucans. For further identification of the sourceof receptor activity, the alkali-insoluble materialwas acid extracted. The alkali-insoluble cell wallfraction contains mostly (1 -- 3)-p-D-glucan and(1 - 6)-p-D-glucan, glycogen, and a smallamount of chitin (7, 18, 19, 25). Multiple hot-acidextractions of this material release glycogen andone type of (1 -> 6)-p-D-glucan (18). After foursuccessive hot-acid extractions, the acid-solublefraction had a twofold-higher specific activitythan the acid-insoluble fraction, but 81% of therecovered receptor remained associated with theacid-insoluble material. In a further attempt toremove the toxin receptor from alkali-washed

cells, 30 hot-acid extractions were performed.By the 30th extraction, very little material wasreleased, yet 74% of the total receptor activityremained with the acid-insoluble fraction. Theseresults indicate that although material with ahigh specific activity was obtained by acid ex-traction, most of the receptor activity was notsolubilized by this procedure.Enzymatic removal of (1 -+ 6)-p-D-glucan from

exhaustively acid-washed, alkali-insoluble glucan.Endo-(1 -- 3)-j3-D-glucanase digestion of ex-

haustively acid-extracted, alkali-insoluble glu-can releases an acid-insoluble (1 6)-P-D-glU-can which is similar, but not structurallyidentical, to acid-soluble (1 -+ 6)-f-D-glucanreleased by acid extraction of alkali-washedcells (19). This procedure was used to furtherfractionate the toxin receptor. The material solu-bilized by the endo-(1 -* 3)-p-D-glucanase zymo-lyase contained 69o of the recovered activity,with the remainder residing in the zymolyase-insoluble pellet. Total recovery of activity was64%. These results suggest that the receptorcontains a component solubilized by zymolyasedigestion which is stable to endo-(1 -3)--D-glucanase attack. Such a component could be a(1 6)-,3-D-glucan. Pustulan incubated with

zymolyase under similar conditions retained itsactivity as a competitive inhibitor, suggestingthat the enzyme preparation contains little if any(1 -+ 6)-p-D-glucanase activity.

Periodate sensitivity of the toxin receptor. Ofthe main yeast cell wall glycosidic linkages, onlyintrachain (1 -- 3)-P-linked residues are resistant

TABLE 3. Substrate specificity of a-amylase,zymolyase, and (1 -. 6)-3-D-glucanasea

Ratio of glucose releasedEnzyme Substrate to total carbohydrate as

anhydroglucose

a-Amylase Pustulan No detectable activityLaminarin Not testedGlycogen 1:7.3

Zymolyase Pustulan No detectable activityLaminarin 1:5.6Glycogen Not tested

(1 -. 6)-p-D- Pustulan 1:7.6Glucanase Laminarin 1:120

Glycogen 1:90a The carbohydrates were incubated at 3.5, 0.63,

and 3.5 mg/ml in the respective enzyme preparations,o-amylase, zymolyase, and (1 -* 6)-3-D-glucanase.The incubation conditions are described in the text.The Nelson-Somogyi procedure was used to deter-mine the reducing sugar content of the carbohydratesbefore and after enzyme incubation. The amount ofdegradation was determined by the reducing sugarcontent at the end of the enzyme incubation less theequivalent figure for the native carbohydrates. Thisfigure is expressed as the indicated ratio.

J. BACTERIOL.

CELL WALL RECEPTOR FOR YEAST KILLER TOXIN 167

TABLE 4. (1 -. 6)-p-D-Glucanase sensitivity ofkiller toxin receptor in S14a cell wall fractions and

pustulan'

% Survival of A8207NK

Celi wall frac- Amt (mg/ml) in the receptor assaybtion or pustulan i6toxin e- (1-6-D-ceptor assay Untreated Glucanase

treatedAlkali-soluble 0.218 14 ± 1 0.8 ± 0.3glucan

Mannan 1.600 9 2 0.7 ± 0.2Acid insoluble 0.218 32 ± 3 2.6 ± 0.3Acid soluble 0.156 40 + 5 1.0 ± 0.2Pustulan 0.200 119 ± 5 4.0 ± 0.4

a S14a cell wall fractions were prepared and assayedfor receptor activity as described in the text. Each ofthe fractions and pustulan were incubated with 19 U of(1 -. 6)-o-E-glucanase in 0.1 M sodium succinate (pH5.0) at 300C with shaking for 4 h, followed by enzymicheat inactivation (100°C for 5 min) (6). Cell wallfractions were diluted 4-fold and pustulan 17-fold inthe toxin receptor assay mixture to give the indicatedconcentrations.

b Percent survival of A8207NK in the presence ofkiller toxin alone was 1.3 ± 0.2 for the cell walfraction experiments and 3.9 ± 0.2 for the pustulanexperiment.

to periodate oxidation. It was known from earli-er work that the receptor in whole cells wasperiodate sensitive. This sensitivity was exam-ined in the fractionated cell wall polysaccha-rides. All toxin receptor activity was penodatesensitive (Table 2), suggesting that (1 -3)_,-D_glucans or other periodate-resistant polysaccha-rides such as chitin were not involved in toxinbinding.

Glucanase degradation of the toxin receptor.To examine further the nature of the toxinreceptor, (1 -+ 6)-p-D-glucanase inactivation ofthe toxin receptor activity was measured. The (1-* 6)-3-D-glucanase preparation from B. circu-lans had the highest hydrolytic activity towardthe linear (1 -- 6)-3-D-glUcan, pustulan, withminor, presumably contaminating, activities forlaminarin and glycogen (Table 3). Pure nonlytic(1-+ 6)-o-D-glucanase acts as an endoglucanase,and upon extensive degradation of pustulan,glucose and gentiobiose are formed (28). The (1

6)-p-D-glucanase preparation destroyed toxinreceptor activity in pustulan and acid-solubleand -insoluble yeast glucan as well as the minoractivity in the other yeast cell wall fractions(Table 4). To show that this degradation ofreceptor activity was caused by the (1 6)-p-D-glucanase activity in the enzyme preparation, a-amylase and zymolyase, both devoid of (1 -- 6)-3-D-glucanase activity, were tested for theirability to degrade receptor activity. With thelatter two enzymes, no loss of receptor activity

was found in cell walls, acid-soluble yeast glu-can, or pustulan (Table 5). The simplest explana-tion of these results is that (1 -- 6)-p-D-glucan-ase degraded the toxin receptor by hydrolysis ofa polysaccharide containing (1 6)-1-linked

glucose residues.Acid-soluble glucan from receptor-defective re-

sistant mutants. It was found that there was lessmaterial in the acid-soluble extract of the recep-tor-defective resistant mutant S14.96 than in thewild-type S14a. Of five separate extractions byvarious procedures (0.5 M acetic acid at 75°C,1.0 M acetic acid at 75 or 90°C), the mean

amount found in S14.96 was 54.2 + 14.6%(standard deviation) of the wild type. Althoughthe acid-soluble extract is not pure (1 -6)-,-D-glucan, but also contains glycogen, this suggest-ed that glucan synthesis might be defective inthe mutant. For two acid extractions whereglycogen contamination was estimated by pas-sage of the material through a concanavalin Acolumn, the (1 -- 6)-43-D-glucan component was43 and 47% of that present in the wild type.Quantitative periodate oxidation indicated thatfor the S14a acid-soluble material eluted througha concanavalin A column, 2.1 ± 0.3 mol ofperiodate was reduced per mol of carbohydrateas glucose. The S14.96 mutant had normal, wild-type amounts of other major cell wall polysac-charides (data not shown). Acid-soluble glucanor zymolyase-digested glucan from alkali-washed yeast cells of strain S14a was coupled toepoxy-activated Sepharose. Both glucan-cou-pled columns retained pure killer toxin when itwas applied in 0.1 M acetate buffer (pH 4.6) with15% glycerol, and no activity was removed bywashing with acetate buffer. Toxin could be

TABLE 5. Toxin receptor activity in sensitive cellwall fractions and pustulan treated with zymolyase

and a-amylase'

Sl4a cell % Survival of A8207NKbwall frac- Enzymatic Control

tion or pus- treatment Un- Enzyme (no carolotulan treated treated (nodcarbo-

Cell wall Zymolyase 39 ± 9 31 ± 5 6.4 ± 0.5Acid Zymolyase 48 ± 3 60 ± 8 6.4 ± 0.5

solublea-Amylase 30 ± 1 18 ± 1 3.9 ± 0.2

Pustulan a-Amylase 141 ± 6 136 ± 14 3.9 ± 0.2

a S14a cell walls were obtained by mechanical dis-ruption of whole cells and extracted with alkali fol-lowed by acid (see the text). Cell walls, acid-insolublematerial, and pustulan were treated with zymolyase ora-amylase as described in the text. The enzyme-treated and untreated carbohydrates were suspendedat 400 p.g/ml in the competition assay.

b Error is expressed as the standard deviation ofsurviving colonies as determined by plate counts.

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168 HUTCHINS AND BUSSEY

eluted quantitatively with 0.1 M Tris buffer (pH7.5) with 15% glycerol. Zymolyase-digested glu-can from alkali-washed cells of resistant strainS14.96 was also coupled to epoxy-activated Se-pharose. Pure toxin was applied to such a col-umn under conditions identical to those used forthe S14a-coupled column. Some 77% of thetoxin activity bound, and further activity elutedin washing with 0.1 M acetate buffer (pH 4.6)with 15% glycerol; 70% of the applied activitywas eluted after a 15-ml buffer wash. The re-maining activity was eluted with 0.1 M Trisbuffer (pH 7.5) with 15% glycerol.A separate set of resistant mutants was isolat-

ed from A8209NK by ethyl methane sulfonatemutagenesis. One of these, A69, with a singlegene mutation by tetrad analysis, was in thesame complementation group as S14.96 and hadno detectable toxin receptor activity in cells oralkali-washed cell wall polysaccharides. Acid-extractable glucan from this resistant mutantand parent was obtained, and the (1 -* 6)-p-D-glucan was estimated. The parent had 1.13 mgper g (wet weight) of cells, and the mutant had0.40 mg per g (wet weight) of cells.

In a further test, the acid-soluble extract froma same-locus revertant of S14.96, S14.96r (1),which regained receptor activity, was measured.It contained 2.47 mg/g (wet weight), comparedwith 2.88 ± 0.40 (standard deviation) for S14aand 1.61 ± 0.45 (standard deviation) for S14.96.This would be consistent with the coreversion ofacid-soluble glucan synthesis with receptor ac-tivity.

DISCUSSIONEvidence from competition studies with pure

polysaccharides, from cell wall fractionation andchemical and enzymatic degradation, and fromreceptor-defective resistant mutants suggeststhat a yeast (1 -+ 6)-,3-D-glucan is involved as akiller toxin receptor in the cell wall of S. cerevis-iae.The fungal (1 -- 6)-p-D-glucans, pustulan and

luteose, were both potent competitive inhibitorsof toxin action. Toxin competition was specificfor the (1 -. 6)-p-linkage, as none of the polysac-charides tested composed of (1 -* 3)-p-, (1 -3 4)-,B-, (1 -- 6)-a-, or (1 -* 4)-a-linkages or a mixtureof these linkages showed competitive inhibition.

Studies of the enzymatic degradation of killertoxin receptor on yeast cell walls again implicat-ed (1 -- 6)-p-D-glucan in killer toxin binding.The (1 -* 6)-,B-D-glucanase from B. circulansinactivated the receptor in all cell wall fractionsand in pustulan. The endo-(1 -+ 3)-p-D-glucan-ase, zymolyase, and a-amylase had no effect onreceptor activity. Periodate oxidation of recep-tor activity, while not having the specificity ofthe above two approaches, also gave a clear

result; i.e., the receptor was inactivated in allcell wall fractions. Periodate-resistant wall com-ponents such as intrachain (1 -) 3)-p-linkedglucose residues and chitin are thus ruled out asreceptors by this test.Wall fractionation studies of the receptor are

less readily interpretable, because of limitationsin our knowledge of glucan structure, but theyremain consistent with (1 -+ 6)-p-D-glucan in-volvement. Alkali extraction of isolated cellwalls or yeast cells indicated that the bulk of thetoxin receptor was alkali insoluble. Alkali-solu-ble polysaccharides ruled out as receptors in-clude mannan and a mixed-linkage (1 3)-,-D-and (1 -+ 6)-13-D-glucan (9). Alkali insolubilitytaken with the degradation and competition testswas consistent only with a (1-+ 6)-p-D-glucancomponent. The procedure for isolation of (1 -*6)-13-D-glucan from the alkali-insoluble yeast cellwall involves exhaustive extractions with hot,dilute acetic acid (19). This procedure does notremove all (1-+ 6)-13-D-glucan and defines oper-ationally two classes, acid-soluble and acid-insoluble (1 -* 6)-p-D-glucans. Fractionation ofthe toxin receptor indicated that most of thereceptor activity remained with the acid-insolu-ble glucan. Significantly, however, the minorreceptor activity present in the acid-soluble (1 -*6)-p-D-glucan-enriched fraction had a higherspecific activity than other yeast cell wall frac-tions. The evidence that the majority of thereceptor activity was associated with an acid-insoluble (1 -- 6)-p-D-glucan that remained withthe matrix (1 -- 3)-p-D-glucan was indirect butcompelling. Quantitative periodate oxidation in-dicated that (1 -* 6)-p-D-glucan was present inthe acid-insoluble fraction (19; this study). Peri-odate and (1 -3 6)-p-D-glucanase sensitivity ofthe receptor activity in the acid-insoluble frac-tion indicated its nature. Endo-(1 -- 3)-p-D-glucanase digestion of the acid-insoluble fractionreleases the acid-insoluble (1 -- 6)-p-D-glucan(19). A similar digestion performed with zymo-lyase solubilized 69o of the recovered receptoractivity, suggesting that most of the acid-insolu-ble receptor activity was caused by (1 -) 6)-p-D-glucan.These studies indicated that the acid-insoluble

(1 -- 6)-p-D-glucan possessed a major portion ofthe total receptor activity and the acid-soluble (1-- 6)-p-D-glucan a minor fraction. Although link-age analysis by Lindberg (17) and Manners et al.(18, 19) substantiates such a difference, the finestructure of these two glucans remains unclear.The krel resistant mutants make less acid-

soluble (1 -- 6)-p-D-glucan and, based on ab-sence of receptor, presumably less acid-insolu-ble (1 -- 6)-13-D-glucan, though this remains tobe determined. Such mutants appear to be de-fective in some aspect of (1 -- 6)-p-D-glucan

J. BACTERIOL.

CELL WALL RECEPTOR FOR YEAST KILLER TOXIN 169

synthesis. The krel mutants are resistant tokiller toxins from a range of yeasts (1), suggest-ing that (1 -+ 6)-p-D-glucan is a common recep-tor for these proteins. The structure of themutant (1 -* 6)-p-D-glucan has not been exam-ined, though the reduction of receptor specificactivity is greater than the reduction in amountof acid-soluble glucan, suggesting some alteredstructure rather than mere reduction in amountof (1 -- 6)-3-D-glucan synthesized. We have asyet no information on the kre2 receptor.The exact site of binding of the toxin to a (1

6)-3-D-glucan has not been established. Toxinbinds to the linear (1 -+ 6)-p-D-glucan polymers,pustulan and luteose, but not to the gentio-oligosaccharides with chain lengths of two tofive residues. The mixed-linkage (1 - 6)--D-and (1 -* 3)-3-D-glucan elicitor from P. mega-sperma had a much lower specific activity thanpustulan or luteose. These data suggest that alinear (1 -- 6)-p-D-glucan of at least six residuesis necessary for toxin binding.

ACKNOWLEDGMENTSWe thank D. Saville, A. Tamarkin, M. Ahmad, and R.

Gordon for help at various stages of this work.This work was supported by the Natural Sciences and

Engineering Research Council of Canada and The NationalCancer Institute of Canada.

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